Principles and Methods for Assessing Direct Immunotoxicity
    Associated with Exposure to Chemicals

    This report contains the collective views of an international group of
    experts and does not necessarily represent the decisions or the stated
    policy of the United Nations Environment Programme, the International
    Labour Organisation, or the World Health Organization.

    First draft prepared at the National Institute of Health Sciences,
    Tokyo, Japan, and the Institute of Terrestrial Ecology, Monk's Wood,
    United Kingdom

    Published under the joint sponsorship of the United Nations
    Environment Programme, the International Labour Organisation, and the
    World Health Organization

    World Health Organization
    Geneva, 1996

          The International Programme on Chemical Safety (IPCS) is a joint
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    of the biological action of chemicals.

    WHO Library Cataloguing in Publication Data

    Principles and methods for assessing direct immunotoxicity
      associated with exposure to chamicals

    (Environmental health criteria ; 180)

    1.Immunotoxins  2.Immune system  3.Risk assessment  I.Series

    ISBN 92 4 157180 2                 (NLM Classification: QW 630.5.13)
    ISSN 0250-863X

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         1.1. Historical overview
         1.2. The immune system; functions, system regulation, and
               modifying factors; histophysiology of lymphoid organs
               1.2.1. Function of the immune system
                 Encounter and recognition
                 Choice of effector reaction; diversity
                                  of the answer
                 Modifying factors outside the immune
                 Immunological memory
               1.2.2. Histophysiology of lymphoid organs
                 Overview: structure of the immune system
                 Bone marrow
                 Lymph nodes
                 Mucosa-associated lymphoid tissue
                 Skin immune system or skin-associated
                                  lymphoid tissue
         1.3. Pathophysiology
               1.3.1. Susceptibility to toxic action
               1.3.2. Regeneration
               1.3.3. Changes in lymphoid organs


         2.1. Description of consequences on human health
               2.1.1. Consequences of immunosuppression
                 Infectious diseases
               2.1.2. Consequences of immunostimulation

         2.2. Direct immunotoxicity in laboratory animals
               2.2.1. Azathioprine and cyclosporin A
                 Cyclosporin A
               2.2.2. Halogenated hydrocarbons
    2,3,7,8-Tetrachlorodibenzo- para-dioxin
                 Polychlorinated biphenyls
               2.2.3. Pesticides
                 Organochlorine pesticides
                 Organophosphate compounds
               2.2.4. Polycyclic aromatic hydrocarbons
               2.2.5. Solvents
                 Other solvents
               2.2.6. Metals
                 Gallium arsenide
               2.2.7. Air pollutants
               2.2.8. Mycotoxins
               2.2.9. Particles
               2.2.10. Substances of abuse
               2.2.11. Ultraviolet B radiation
               2.2.12. Food additives
         2.3. Immunotoxicity of environmental chemicals in wildlife and
               domesticated species
               2.3.1. Fish and other marine species
                 Marine mammals
               2.3.2. Cattle and swine
               2.3.3. Chickens
         2.4. Immunotoxicity of environmental chemicals in humans
               2.4.1. Case reports
               2.4.2. Air pollutants
               2.4.3. Pesticides
               2.4.4. Halogenated aromatic hydrocarbons
               2.4.5. Metals
               2.4.6. Solvents
               2.4.7. Ultraviolet radiation
               2.4.8. Others


         3.1. General testing of the toxicity of chemicals
         3.2. Organization of tests in tiers
               3.2.1. US National Toxicology Program panel
               3.2.2. Dutch National Institute of Public Health and
                       Environmental Protection panel
               3.2.3. US Environmental Protection Agency, Office of
                       Pesticides panel
               3.2.4. US Food and Drug Administration, Center for Food
                       Safety and Applied Nutrition panel
         3.3. Considerations in evaluating systemic and local
               3.3.1. Species selection
               3.3.2. Systemic immunosuppression
               3.3.3. Local suppression


         4.1. Nonfunctional tests
               4.1.1. Organ weights
               4.1.2. Pathology
               4.1.3. Basal immunoglobulin level
               4.1.4. Bone marrow
               4.1.5. Enumeration of leukocytes in bronchoalveolar lavage
                       fluid, peritoneal cavity, and skin
               4.1.6. Flow cytometric analysis
         4.2. Functional tests
               4.2.1. Macrophage activity
               4.2.2. Natural killer activity
               4.2.3. Antigen-specific antibody responses
               4.2.4. Antibody responses to sheep red blood cells
                 Spleen immunoglobulin M and
                                  immunoglobulin G plaque-forming cell
                                  assay to the T-dependent antigen, sheep
                                  red blood cells
                 Enzyme-linked immunosorbent assay of
                                  anti-sheep red blood cell antibodies of
                                  classes M, G, and A in rats
               4.2.5. Responsiveness to B-cell mitogens
               4.2.6. Responsiveness to T-cell mitogens
               4.2.7. Mixed lymphocyte reaction
               4.2.8. Cytotoxic T lymphocyte assay
               4.2.9. Delayed-type hypersensitivity responses
               4.2.10. Host resistance models
                 Listeria monocytogenes
                 Streptococcus infectivity models
                Viral infection model with mouse and rat
                Influenza virus model

                Parasitic infection model with
                                   Trichinella spiralis
                 Plasmodium model
                B16F10 Melanoma model
                PYB6 Carcinoma model
                MADB106 Adenocarcinoma model
               4.2.11. Autoimmune models
         4.3. Assessment of immunotoxicity in non-rodent species
               4.3.1. Non-human primates
               4.3.2. Dogs
               4.3.3. Non-mammalian species
         4.4. Approaches to assessing immunosuppression  in vitro
         4.5. Future directions
               4.5.1. Molecular approaches in immunotoxicology
               4.5.2. Transgenic mice
               4.5.3. Severe combined immunodeficient mice
         4.6. Biomarkers in epidemiological studies and monitoring
         4.7. Quality assurance for immunotoxicology studies
         4.8. Validation


         5.1. Introduction: Immunocompetence and immunosuppression
         5.2. Considerations in assessing human immune status related to
         5.3. Confounding variables
         5.4. Considerations in the design of epidemiological studies
         5.5. Proposed testing regimen
         5.6. Assays for assessing immune status
               5.6.1. Total blood count and differential
               5.6.2. Tests of the antibody-mediated immune system
                 Immunoglobulin concentration
                 Specific antibodies
               5.6.3. Tests for inflammation and autoantibodies
                 C-Reactive protein
                 Antinuclear antibody
                 Rheumatoid factor
                 Thyroglobulin antibody
               5.6.4. Tests for cellular immunity
                 Flow cytometry
                 Delayed-type hypersensitivity
                 Proliferation of mononuclear cells in
               5.6.5. Tests for nonspecific immunity
                 Natural killer cells
                 Polymorphonuclear granulocytes
               5.6.6. Clinical chemistry
               5.6.7. Additional confirmatory tests


         6.1. Introduction
         6.2. Complements to extrapolating experimental data
               6.2.1. In-vitro approaches
               6.2.2. Parallellograms
               6.2.3. Severe combined immunodeficient mice
         6.3. Host resistance and clinical disease






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    International Register of Potentially Toxic Chemicals, Case postale
    356, 1219 Châtelaine, Geneva, Switzerland (Telephone No. 979 9111).

                                    * * *

          Funding and support for the preparation and finalization of this
    monograph were provided by the United States Environmental Protection
    Agency under Cooperative Agreement with the World Health Organization
    No. CR 821767-01-0, by the German Federal Ministry for the
    Environment, Nature Conservation and Nuclear Safety, and by the
    Netherlands National Institute for Public Health and Environmental

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    FIGURE 1

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    Dr A. Emmendörffer, Department of Immunobiology, Fraunhofer Institute
    of Toxicology & Aerosol Research, Hanover, Germany

    Dr H.S. Koren, Health Effects Research Laboratory, US Environmental
    Protection Agency, Chapel Hill, NC, USA  (Vice-Chairman)

    Dr R.W. Luebke, Immunotoxicology Branch, Health Effects Research
    Laboratory, US Environmental Protection Agency, Research Triangle
    Park, NC, USA  (Joint Rapporteur)

    Dr M. Luster, National Institute of Environmental Health Sciences,
    Research Triangle Park, NC, USA

    Dr C. Madsen, Institute of Toxicology, National Food Agency of
    Denmark, Ministry of Health, Soborg, Denmark

    Dr P. Ross, Dalhousie University, Halifax, Nova Scotia, Canada
    (c/o Seal Rehabilitation and Research Centre, Pieterburen,

    Dr H.J. Schuurman, Preclinical Research/Immunology, Sandoz Pharma Ltd,
    Basel, Switzerland

    Dr H. Van Loveren, Laboratory for Pathology, National Institute of
    Public Health and Environmental Protection, Bilthoven, Netherlands
     (Joint Rapporteur)

    Dr J.G. Vos, National Institute of Public Health and Environmental
    Protection, Bilthoven, Netherlands  (Chairman)

    Dr K.L. White, Jr, Immunotoxicology Group, Medical College of
    Virginia, Virginia Commonwealth University, Richmond, VA, USA



    Dr P. Montuschi, Department of Pharmacology, Catholic University of
    the Sacred Heart, Rome, Italy


    Dr R.W.R. Crevel, Environmental Safety Laboratory, Unilever Research
    and Engineering, Sharnbrook, Bedfordshire, United Kingdom


    Dr J.H. Dean, Sanofi Winthrop, Inc., Sanofi Research Division,
    Collegeville, PA, USA

    Mr V. Quarg, Federal Ministry for Environment, Nature Conservation &
    Nuclear Safety, Bonn, Germany

    Dr E. Smith, International Programme on Chemical Safety, World Health
    Organization, Geneva, Switzerland


          A WHO Task Group on Principles and Methods for Assessing Direct
    Immunotoxicity Associated with Exposure to Chemicals met at the World
    Health Organization, Geneva, from 10 to 14 October 1994. Dr E. Smith,
    IPCS, welcomed the participants on behalf of Dr M. Mercier, Director
    IPCS, and the cooperating organizations. The Task Group reviewed and
    revised the draft monograph and prepared the final text.

          The first draft of the monograph was prepared by a group of
    authors (listed below) under the coordination of Dr J.G. Vos and
    Dr H. Van Loveren of the Dutch National Institute for Public Health
    and Environmental Protection (RIVM), an IPCS Collaborating Centre for
    Immunotoxicology and Allergic Hypersensitization. The second draft,
    incorporating comments received after international circulation to
    national experts of the first draft to IPCS contact points for
     Environmental Health Criteria monographs, was prepared by Dr J.G.
    Vos and Dr H. Van Loveren of the Netherlands and Dr Kimber White, USA.

          Dr E. Smith of the IPCS Unit for the Assessment of Risk and
    Methods was responsible for the scientific content of the monograph
    and Mrs E. Heseltine for the editing.

          The efforts of all who helped in the preparation and finalization
    of the monograph are gratefully acknowledged.

          The contributing authors were:

    Dr J.H. Dean, Collegeville, PA, USA
    Professor J. Descotes, Lyon, France
    Dr F. Kuper, Zeist, Netherlands
    Dr M. Luster, Research Triangle Park, NC, USA
    Dr P.S. Ross, Bilthoven, Netherlands
    Dr H.J. Schuurman, Basel, Switzerland
    Dr M.J. Selgrade, Research Triangle Park, NC, USA
    Dr R.L. de Swart, Bilthoven, Netherlands
    Dr H. Van Loveren, Bilthoven, Netherlands
    Professor J.G. Vos, Bilthoven, Netherlands
    Dr P.W. Wester, Bilthoven, Netherlands
    Professor A.G. Zapata, Madrid, Spain


    ACTH         adrenocorticotrophic hormone
    Ah           aromatic hydrocarbon
    AIDS         acquired immunodeficiency syndrome
    B            bursa-dependent
    CALLA        common acute lymphoblastic leukaemia antigen
    CD           cluster of differentiation
    CEC          Commission of the European Communities
    CH50         haemolytic complement
    CML          cell-mediated lympholysis
    DMBA         7,12-dimethylbenz[ a]anthracene
    DNCB         dinitrochlorobenzene
    ELISA        enzyme-linked immunosorbent assay
    EPO          erythrocyte lineage differentiation factor
    FACS         fluorescence activated cell sorter
    GALT         gut-associated lymphoid tissue
    G-CSF        granulocyte colony-stimulating factor
    GM-CSF       granulocyte-macrophage colony-stimulating factor
    GVH          graft-versus-host
    HCB          hexaclorobenzene
    HEV          high endothelial venule
    HIV          human immunodeficiency virus
    HPCA         human progenitor cell antigen
    HSA          heat-stable antigen
    ICAM         intercellular adhesion molecule
    IFN          interferon
    Ig           immunoglobulin
    IL           interleukin
    IPCS         International Programme on Chemical Safety
    LFA          lymphocyte function-related antigen
    LIF          leukaemia inhibitory factor
    LOAEL        lowest-observed-adverse-effect level
    LOEL         lowest-observed-effect level
    M            microfold
    MALT         mucosa-associated lymphoid tissue
    MARE         monoclonal anti-rat immunoglobulin E
    MARK         monoclonal antibody anti-kappa
    M-CSF        macrophage colony-stimulating factor
    MED          minimal erythemal dose
    MHC          major histocompatibility complex
    NCAM         neural cell adhesion molecule
    NK           natural killer
    NOAEL        no-observed-adverse-effect level
    NOEL         no-observed-effect level
    NTP          National Toxicology Program
    PAH          polycyclic aromatic hydrocarbon
    PCB          polychlorinated biphenyl
    PG           prostaglandin
    QCA          quiescent cell antigen

    RIVM         Dutch National Institute of Public Health and
                 Environmental Protection
    S9           9000 x g supernatant
    SCF          stem-cell factor
    SCID         severe combined immunodeficiency
    SIS          skin immune system
    STM           Salmonella typhimurium mitogen
    TBTO         tri- n-butyltin oxide
    Tc           cytotoxic T cell
    TCDD         2,3,7,8-tetrachlorodibenzo- para-dioxin
    TCR          T-cell receptor
    Tdth         delayed-type hypersensitivity T cell
    TGF          transforming growth factor
    Th           T helper-inducer cell
    THAM         T-cell activation molecule
    THI          2-acetyl-4(5)-tetrahydroxybutylimidazole
    O,O,S-TMP     O,O,S-trimethylphosphorothiate
    TNF          tumour necrosis factor
    UVB          ultraviolet B
    UVR          ultraviolet radiation
    VCAM         vascular cell adhesion molecule
    VLA          very late antigen


    1. The immune system has evolved to counter challenges to the
    integrity of self from either microorganisms or cells that have
    escaped the organism's control mechanisms. Recognition that
    xenobiotics can impair the function of the immune system has led to
    progress in immunotoxicology over the last two decades. Experimental
    approaches (mainly in rodent species) have been developed and
    validated in multilaboratory studies. In this monograph, the function
    and histophysiology of the immune system are reviewed, and the
    information necessary to understand and interpret the pathological
    changes caused by immunotoxic insults is provided. Emphasis is laid on
    the immune systems of humans and rodent species, but reference is made
    to other species, including fish, that have been the object of
    immunotoxicological studies. The pathophysiology of the immune system,
    including the variable susceptibility of its components, alterations
    to the lymphoid organs, and the reversibility of changes are important
    for understanding the impact of immunotoxicity.

    2. Immunosuppression and immunostimulation both have clinical
    consequences. Immunodeficiency states and severe immunosuppression,
    such as can occur during transplantion and cytostatic therapy, have
    both been associated with increased incidences of infectious diseases
    (particularly opportunistic ones) and cancer. Exposure to immunotoxic
    chemicals in the environment, however, may be expected to result in
    more subtle forms of immunosuppression which may be difficult to
    detect, leading to increased incidences of infections such as
    influenza and the common cold. Studies of experimental animals and
    humans have shown that many environmental chemicals suppress the
    immune response. Immunotoxic xenobiotics are not restricted to a
    particular chemical class. Compounds that adversely affect the immune
    system are found among drugs, pesticides, solvents, halogenated and
    aromatic hydrocarbons, and metals; ultraviolet radiation can also be
    immunotoxic. Therapeutic administration of immunostimulating agents
    can have adverse effects, and a few environmental chemicals that have
    immunostimulating properties (beryllium, silica, hexachlorobenzene)
    can have clinical consequences.

    3. The complexity of the immune system results in multiple potential
    target sites and pathological sequelae. The initial strategies devised
    by immunotoxicologists working in toxicology and safety assessment
    were to select and apply a tiered panel of assays to identify
    immunosuppressive and immunostimulatory agents in laboratory animals.
    Although the configuration of these testing panels may vary depending
    on which agency or laboratory is conducting the test and on the animal
    species employed, they all include measurement of one or more of the
    following: altered lymphoid organ weights and histology; changes in
    the cellularity of lymphoid tissue, peripheral blood leukocytes,
    and/or bone marrow; impairment of cell function at the effector or
    regulatory level; and altered susceptibility to challenge with
    infectious agents or tumour cells.

          The original test guideline No. 407 of the Organisation for
    Economic Co-operation and Development, published in 1981, was not
    designed to detect potential immunotoxicity, and modifications have
    been proposed to make the guideline more useful for identifying
    immunotoxicants. Tiered testing systems have been designed for more
    extensive investigation of potential immunotoxicity, by the US
    National Toxicology Program, the Dutch National Institute of Public
    Health and Environmental Protection, the US Environmental Protection
    Agency Office of Pesticides, and the US Food and Drug Administration
    Center for Food Safety and Applied Nutrition.

          Studies have been conducted in mice, and to a lesser extent in
    rats, to investigate the specificity, precision (reproducibility),
    sensitivity, accuracy, and relevance for the assessment of risk to
    human health of a variety of measures of immune status. International,
    interlaboratory validations of methods have been carried out within
    the International Collaborative Immunotoxicity Study of IPCS and the
    European Union, the Bundesinstitut für Gesundheitlichen
    Verbraucherschutz, und Veterinärmedizin, and in studies of cyclosporin
    A in Fischer 344 rats.

    4. The tests used in the tiered testing schemes are described in
    Section 3, which indicates the rationale for their selection and the
    complexities involved in their performance. Although these protocols
    were designed for studies of rats and mice, some have been applied
    successfully for studying immunotoxicity in other animal species,
    including non-human primates, marine mammals, dogs, birds, and fish.

          A variety of factors must be considered in evaluating the
    potential of an environmental agent or drug to influence the immune
    system of experimental animals adversely. These include selection of
    the appropriate animal models and exposure variables, inclusion of
    general toxicological parameters, an understanding of the biological
    relevance of the end-points being measured, use of validated measures,
    and quality assurance. The experimental conditions should take into
    account the potential route and level of human exposure and any
    available information on toxicodynamics and toxicokinetics. The doses
    and  sample sizes should be selected so as to generate clear dose-
    response curves, in addition to no-observed-adverse-effect or
    no-observed-effect levels. The strategies are continually refined to
    allow better prediction of conditions that may lead to disease. In
    addition, techniques should be developed that would help to identify
    mechanisms of action; these might include methods  in vitro,
    examination of local immune responses (such as in the skin, lung, and
    intestines), and use of the techniques of molecular biology and
    genetically modified animals.

    5. The detection of immune changes after exposure to potentially
    immunotoxic compounds is more complicated in humans than in
    experimental animals. The testing possibilities are limited, levels of
    exposure to the agent (i.e. dose) are difficult to establish, and the
    immune status of populations is extremely heterogeneous. Age, race,
    gender, pregnancy, acute stress and the ability to cope with stress,
    coexistent disease and infections, nutritional status, tobacco smoke,
    and some medications contribute to this heterogeneity.

          An important factor in assessing the usefulness of a particular
    study for risk assessment is epidemiological study design. The
    commonest design used in immunotoxicity is the cross-sectional study,
    in which exposure status and disease status are measured at one time
    or over a short period. The immune function of 'exposed' subjects is
    then compared with that of a comparable group of 'unexposed'
    individuals. There are possible pitfalls in this study design.

          Because many of the immune changes seen in humans after exposure
    to a chemical may be sporadic and subtle, recently exposed populations
    must be studied and sensitive tests be used for assessing the immune
    system. Conclusions about immunotoxic effects should be based on
    changes not in a single parameter but in the immune profile of an
    individual or population.

          Most of the tests for specific immunity (cell-mediated and
    humoral), nonspecific immunity and inflammation were developed to
    detect immune alterations in patients with immunodeficiency disease
    and are not always adequate to detect subtle alterations induced by
    environmental chemicals. IPCS, the Centers for Disease Control, and
    the US National Academy of Sciences have each described procedures for
    evaluating changes in the human immune system resulting from exposure
    to immunotoxicants, but the tests described require evaluation for
    this purpose.

    6. Risk assessment is a process in which relevant data on the
    biological effects, dose-response relationships, and exposure for a
    particular agent are analysed in an attempt to establish qualitative
    and quantitative estimates of adverse outcomes. Typically, risk
    assessment comprises four major steps: hazard identification, dose-
    response assessment, exposure assessment, and risk characterization.
    Up until now, immunotoxicology has focused mainly on hazard
    identification, and to some extent on dose-response assessment, and
    very few studies have included exposure assessment or risk

          As in other areas of toxicology, uncertainties exist which may
    affect the interpretation of data on immunotoxicity with regard to
    human health risk. The two most problematic issues -- extrapolating
    effects from individual cells to a whole organ or beyond and
    extrapolating data from experimental animals to humans -- are common
    to most non-cancer end-points. The first issue is due to uncertainties

    associated with establishing a quantitative relationship between
    changes in individual immune function and altered resistance to
    infections and neoplastic disease. The second issue is due to
    uncertainties associated with assessing risk to human health on the
    basis of studies in laboratory animals.

          The ultimate purpose of risk assessment is to protect human
    health and the environment. Suitable model systems must therefore be
    chosen. The toxicokinetics of the test material and the nature and
    magnitude of the immune response generated in the model should be
    comparable to that of humans.

          Conventionally, empirical uncertainty factors are used in risk
    assessment to derive an acceptable exposure limit from experimental
    results. This approach does not take into account the functional
    reserve or redundancy of the immune system. A more recent development
    in risk assessment is use of in-vitro models as an adjunct to studies
    of experimental animals. The advantages of this approach are that it
    improves the accuracy of extrapolation of data from animals to man and
    minimizes the use of animals; it also bridges the gap between those
    data, particularly when human experimentation is limited for ethical
    considerations. Chapter 6 cites two examples in which in-vitro data
    make it possible to reduce the uncertainties in risk assessment
    associated with exposure to ozone and ultraviolet radiation. The
    difficulty in establishing quantitative relationships between
    immunosuppression and clinical disease has limited the use of
    immunotoxicological data in risk assessment.


     Recommendations for the protection of human health

    1. Chemicals should be screened to determine if they are potentially
    immunotoxic to humans. If immunotoxicity is detected, the chemicals
    should be investigated further as part of the risk assessment process.

    2. Chemicals for which little or no information is available on
    toxicity should be screened for potential immunotoxicity following a
    protocol based on, for example, the revised OECD guideline No. 407.
    When some information is available on the test material (e.g.
    physicochemical properties, toxicokinetics, structure-activity
    relationships), a flexible approach to testing is recommended which
    permits a rational selection of test procedures.

    3. The immunotoxic risk of mixtures of environmental pollutants, in,
    for example, fish, to certain human consumer groups (e.g. fishermen)
    should be assessed.

     Recommendations for protection of the environment

    1. Chemicals should be screened to determine if they are potentially
    immunotoxic to wildlife species. If immunotoxicity is detected, the
    chemicals should be investigated further as part of the risk
    assessment process.

    2. The immunotoxic risk of environmental pollution to the health of
    the ecosystem should be assessed in laboratory, semi-field, and field
    studies of the wildlife occupying high trophic levels or those species
    judged to be sensitive.

     Recommendations for further research

    1. The panels of tests suggested for evaluating xenobiotic-induced
    immunotoxicity in humans should be investigated to determine their
    ability to detect subtle alterations in immune status.

    2. The relationships between alterations in immune function and human
    health should be established for use in immunotoxic risk assessment.
    Epidemiological studies should be carried out that include assessment
    of exposure, in order to establish dose-response relationships.

    3. The relationship between immunotoxicity and the development of
    neoplasia should be investigated.

    4. Baseline immunological data should be established for the general
    population and for subpopulations such as ethnic minorities, children,
    the aged, and pregnant and lactating women in order to assess their
    immune status.

    5. Immunotoxicological assessment should be conducted for
    subpopulations potentially susceptible to the effects of immunotoxic
    compounds, including those at the extremes of age and those with
    deficient nutritional status.

    6. Biomarkers of exposure, effect, and susceptibility should be
    identified, developed, and validated for use in epidemiological
    studies of immunotoxicity in both humans and wildlife.

    7. The quantitative relationship between immune function and host
    resistance in animal models, including the nature, magnitude, and
    significance of functional reserve and redundancy, should be explored
    for risk assessment.

    8. Since chemicals and biological agents enter the body via the
    respiratory and alimentary tracts and the skin, more research should
    be carried out on local immunity.

    9. Preliminary observations in laboratory animals that suggest that
    primary immunization does not compromise testing for subacute toxicity
    should be substantiated by further research, so that functional
    testing can be incorporated into toxicology testing.

    10. Methods and reagents should be developed in order to characterize
    the immune system of wildlife species and to assess their immune
    status for immunotoxicological studies.

    11. The mechanisms of the immunotoxic action of xenobiotics in humans
    should be elucidated by a combination of studies in laboratory animals
     in vivo and experiments with human and animal tissues and cell lines
     in vitro.

    12. In view of the sensitivity of the developing immune system to
    immunotoxic injury, more emphasis should be placed on studies
    involving perinatal exposure to a chemical or mixture of chemicals.

    13. Studies should be conducted to establish whether exposure to
    xenobiotics that are not themselves sensitizing adds to the risk of
    allergic disease in general.

    14. Autoimmune models in laboratory animals should be used to assess
    whether xenobiotics can modulate autoimmune disease in humans.

    15. The effects on immune function of confounding factors in humans
    and animals, including age, race, sex, gender, nutritional status,
    acute stress, and underlying disease, should be evaluated further in
    order to determine their effects in tests for the immunotoxicity of
    environmental chemicals.

    16. Methods for assessing cytokines and their production in different
    body compartments, including plasma, bronchoalveolar lavage fluid, and
    nasal lavage fluid, and by cells isolated from various anatomical
    sites should be validated for humans and laboratory animals, and their
    applicability for assessing the risk of chemicals should be

    17. Data from clinical trials should be made more widely available;
    and patients undergoing therapy with immunomodulatory drugs should be
    monitored clinically and immunologically in a systematic way.

    18. The toxicokinetics of immunotoxic chemicals should be further
    investigated, particularly with regard to whether their concentrations
    in human biological fluids indicate levels of environmental exposure.

    19. The interactions between the immune system, the nervous system,
    and the endocrine system should be further investigated, with
    particular emphasis on how xenobiotics adversely affect them.

    20. The significance of ultraviolet radiation-induced
    immunosuppression for public health and the health of ecosystems
    should be evaluated.


    1.1  Historical overview

          It is well established that each individual has an intrinsic
    capacity to defend itself against pathogens in the environment, with a
    defence known as the immune system. By general definition, the immune
    system serves the body by neutralizating, inactivating, or eliminating
    potentially pathogenic invaders such as microorganisms (bacteria and
    viruses); it also guards against uncontrolled growth of cells into
    neoplasms, or tumours. The major features of the structure and
    function of the immune system have been elucidated over the last three
    decades; in parallel, awareness grew of toxicological manifestations
    after exposure to xenobiotic chemicals. Recognition of the interplay
    between toxicology and immunology is relatively recent: A
    comprehensive review, published in 1977 (Vos, 1977), was the first
    survey of a large series of xenobiotics that affect immune reactivity
    in laboratory animals and hence may influence the health of exposed
    individuals. Most research groups focusing on toxicity to the immune
    system started their activities during the last decade. Textbooks of
    immunotoxicology date only from the early 1980s (Gibson et al., 1983;
    Dean et al., 1985; Descotes, 1986), while one on clinical
    immunotoxicology is more recent (Newcombe et al., 1992).

          Immunotoxicology is the study of the interactions of chemicals
    and drugs with the immune system. A major focus of immunotoxicology is
    the detection and evaluation of undesired effects of substances by
    means of tests on rodents. The prime concern is to assess the
    importance of these interactions in regard to human health. Toxic
    responses may occur when the immune system is the target of chemical
    insults, resulting in altered immune function; this in turn can result
    in decreased resistance to infection, certain forms of neoplasia, or
    immune dysregulation or stimulation which exacerbates allergy or
    autoimmunity. Alternatively, toxicity may arise when the immune system
    responds to the antigenic specificity of the chemical as part of a
    specific immune response (i.e. allergy or autoimmunity). Certain drugs
    induce autoimmunity (Kammüller et al., 1989; Kammüller & Bloksma,
    1994). The differentiation between direct toxicity and toxicity due to
    an immune response to a compound is to a certain extent artificial.
    Some compounds can exert a direct toxic action on the immune system as
    well as altering the immune response. Heavy metals like lead and
    mercury, for instance, manifest immunosuppressive activity,
    hypersensitivity, and autoimmunity (Lawrence et al., 1987).

          This monograph is concerned mainly with one aspect of
    immunotoxicology: the direct or indirect effect of xenobiotic
    compounds (or their biotransformation products) on the immune system.
    This effect is usually immunosuppression, or the induction of a state
    of deficiency or unresponsiveness. Allergy and autoimmunity will be
    dealt with in a future  Environmental Health Criteria monograph.

          Toxicological research over the past decade has indicated that
    the immune system is a potential 'target organ' for toxic damage. This
    finding was the basis for a number of large scientific conferences on
    immunotoxicology and sparked the active interest of national and
    international organizations in this field. One of the milestones in
    the development of the discipline was the international seminar on
    'The Immunological System as a Target for Toxic Damage', held in
    Luxembourg in 1984 and organized by the International Programme on
    Chemical Safety (IPCS), and the Commission of the European Communities
    (CEC). At the seminar, immunotoxicology was defined as 'the discipline
    concerned with the study of the events that can lead to undesired
    effects as a result of interaction of xenobiotics with the immune
    system. These undesired events may result as a consequence of: (1) a
    direct and/or indirect effect of the xenobiotic (and/or its
    biotransformation product) on the immune system; or, (2) an
    immunologically-based host response to the compound and/or its
    metabolite(s) or host antigens modified by the compound or its
    metabolites' (Berlin et al., 1987). Recommendations were made
    concerning the significance to public health of immunotoxicology,
    immunotoxicity testing, research and development in immunotoxicology,
    the development of databases, and training and education. A subsequent
    workshop on 'Immunotoxicity of Metals and Immunotoxicology', organized
    by IPCS and the CEC, in collaboration with the International Union for
    Pure and Applied Chemistry and German governmental agencies, was held
    in Hanover, Germany, in 1989 (Dayan et al., 1990). A meeting on risk
    assessment in immunotoxicology was organized by the United States
    National Institute for Environmental Health Sciences in 1990. A
    meeting on human immunotoxicology tests was organized by the Agency
    for Toxic Substances and Disease Registry and the Centers for Disease
    Control, in Atlanta, Georgia, United States of America, in 1992. In
    1994, two meetings were held: one in Oxford, United Kingdom, organized
    by IPCS, on risk assessment in human imunotoxicity, and one in
    Washington DC, United States, organized by the International Life
    Sciences Institute, on methods in immunotoxicology.

          In parallel to these meetings, activities were started within
    IPCS for the development and validation of methods for assessing
    toxicity to the immune system. In this regard, a hallmark event was
    the meeting in 1986 of a technical review and working group, in
    London, United Kingdom (IPCS, 1986).

          A number of tiered approaches to immunotoxicity testing have been
    proposed, in rats (Vos, 1980; Van Loveren & Vos, 1989) and subsequently
    in mice (Luster et al., 1988). These approaches have been evaluated
    for their capacity to identify chemicals as immunosuppressive. Of a
    group of 18 pesticides evaluated in rats, six were identified as
    inducing immunotoxicity at doses similar to those that cause other
    toxic effects, and five were immunotoxic at lower doses (Vos & Krajnc,
    1983; Vos et al., 1983a). Effects were seen on different parameters

    with different compounds and included lymphocytopenia, reduced thymic
    and spleen weights, and increased levels of serum immunoglobulin (Ig)
    G. One of the compounds identified was hexachlorobenzene (Vos, 1986),
    which is further described in Section 2. In mice, the tiered approach
    was used to assess the immunotoxicity of 51 chemicals, selected on the
    basis of factors including structure-activity relationships with
    previously identified immunotoxic substances, and use (Luster et al.,
    1992). Of the spectrum of assays applied, the strongest associations
    with immunotoxic potential were observed with the splenic IgM antibody
    plaque-forming cell response and cell surface marker analysis; somewhat 
    weaker associations were found for natural killer (NK) cell activity,
    cytotoxic T-lymphocyte cytolytic activity, lymphocyte proliferation
     in vitro after mitogen stimulation, and thymus:body weight ratio.
    The tiered approach in immunotoxicity testing is further described in
    Section 3.

          Multi-laboratory studies have been initiated to validate the
    screening of immunotoxic compounds, including the IPCS-European Union
    International Collaborative Immunotoxicity Study, a study in Fischer
    344 rats, and the international study of the Bundesinstitut für
    Gesundheitlichen Verbraucherschutz, und Veterinärmedizin, which were
    designed to determine interlaboratory reproducibility. The
    experimental animal used in these studies is the rat, and some
    functional tests are included. Test methods are also being developed
    and validated within the National Toxicology Program (NTP) in the
    United States. This programme includes studies of carcinogenicity in
    rats and mice, but because the immune system of mice is better
    characterized than that of rats, the NTP chose the mouse as the
    experimental animal for immunotoxicity assessment. The immunotoxicity
    database of the NTP has been evaluated to determine the predictability
    (sensitivity and specificity) of the assays. In the Netherlands, a
    Committee for Immunotoxicology of the Dutch Health Council reviewed
    methods that could be used to assess the immunotoxic properties of a
    compound and for deriving information about risks to humans on the
    basis of the results of laboratory experiments. The Committee also
    examined the relationship between the immunotoxic properties of a
    substance and its mutagenic and carcinogenic properties (Dutch Health
    Council, Committee for Immunotoxicology, 1991).

          The immune system was reviewed by the United States National
    Research Council in order to identify the kinds of basic research that
    might reveal markers of environmental exposure and disease. Major
    emphasis was placed on biological markers of three types: those
    originating from the immune system, those related to exposure to
    immunosuppressive toxicants, and those of effects of environmental
    pollutants. Markers of susceptibility to environmental materials were
    also considered to be important, especially if they are of a genetic

    nature and can be used to identify individuals susceptible to
    autoimmune diseases. The National Research Council subcommittees on
    pulmonary toxicology and on immunotoxicology, have published their
    reports (US National Research Council, 1989, 1992).

          Interest in immunotoxicology within the scientific community is
    reflected by the existence of a special section on immunotoxicology
    within the Society of Toxicology. An immunotoxicology discussion group
    initiated in the United States has an international composition. The
    European Union has a programme on science and technology for
    environmental protection that includes immunotoxicology as an
    important aspect.

          There is growing concern in society about the effects of
    xenobiotics, such as environmental pollutants, on public health; the
    immune system is one of the targets of such effects. Some chemicals
    present in the environment that have been reported to influence the
    immune system are listed in Table 1 (IPCS, 1986). Immunotoxicity can
    result in e.g. reduced resistance towards infection or generation of
    tumours that escape immune surveillance. A number of substances 
    affect immunological parameters; these include halogenated 
    hydrocarbons such as polychlorinated biphenyls, polybrominated 
    biphenyls, polychlorinated dibenzo- para-dioxins, and polychlorinated
    dibenzofurans (Elo et al., 1985; Lu & Wu, 1985; Bekesi et al., 1987;
    Kimbrough, 1987; Hoffman 1992); pesticides and precursors (Fiore et
    al., 1986; Deo et al., 1987; Nigam et al., 1993); organic solvents
    (Capurro, 1980; Denkhaus et al., 1986); asbestos (Lew et al., 1986);
    silica (Uber & McReynolds, 1982); and metals like lead (Ewers et al.,
    1982; Reigart & Graber, 1976). Oxidant air pollutants, like sulfur
    dioxide, nitrogen dioxide, and ozone, and particles in airborne dust
    may affect immune function (Koren et al., 1989; Van Loveren et al.,

          Immunotoxicity in humans is further discussed in Section 2. Few
    epidemiological data have been published that indicate suppression or
    altered resistance to infection and tumours. In general, the
    usefulness of the epidemiological studies that have been published is
    limited by the following: exposure is usually uncontrolled, mainly
    occurring during accidents; the magnitude and pattern of exposure are
    not known, and the exposure is often too low to alter the immune
    system measurably; exposure is often not to one xenobiotic but to a
    mixture; it is almost impossible to control for confounding
    parameters, such as age, sex, genetic background, health status, and
    nutritional status; and it is not always possible to define and
    analyse appropriate control groups (US National Research Council,
    1992). Environmental pollution and its effect on health status are
    currently subjects of concern in eastern European countries and have
    generated much interest in the worldwide environmental health science
    community. Recent epidemiological studies have compared the possible
    relationship between exposure to air pollutants and health effects in
    the former German Democratic Republic and Federal Republic of Germany,

    Table 1.  Examples of compounds that are immunotoxic for humans or

    Chemical                                     Immune toxicity
                                                 Rodent       Human

    2,3,7,8-Tetrachlorodibenzo-para-dioxin       +            +
    Polychlorinated biphenyls                    +            +
    Polybrominated biphenyls                     +            +
    Hexachlorobenzene                            +            Unknown
    Lead                                         +            Unknown
    Cadmium                                      +            Unknown
    Methyl mercury compounds                     +            Unknown
    7,12-Dimethylbenz[a]anthracene               +            Unknown
    Benzo[a]pyrene                               +            Unknown
    Di-n-octyltindichloride                      +            Unknown
    Di-n-butyltindichloride                      +            Unknown
    Benzidine                                    +            +
    Nitrogen dioxide and ozone                   +            +
    Benzene, toluene, and xylene                 +            +
    Asbestos                                     +            +
    N-Nitrosodimethylamine                       +            Unknown
    Diethylstilboestrol                          +            +
    Vanadium                                     +            +

    From IPCS (1986)

    and some of these studies included immunological data or end-points.
    For instance, von Mutius et al. (1992) found a higher prevalence of
    asthma among schoolchildren in western than eastern Germany, and
    Behrendt et al. (1993) observed, surprisingly, that total serum IgE
    levels were higher in schoolchildren in eastern than in western
    Germany. Several factors were found to influence total IgE: history of
    parasitic disease, number of persons per dwelling, and passive
    smoking. Sex and passive smoking were the only variables that had a
    significant effect in western German children. Air pollutants and
    parasitic infections were suggested to be the major contributing
    factors to increased IgE production in children in eastern Germany.
    Remarkable differences in air quality were seen between eastern and
    western Germany, and Behrendt et al. (1995) distinguished two types of
    air pollution: type I, composed of sulfur dioxide particles and dust,
    occurring predominantly in eastern Europe, is associated with
    respiratory infections and other chronic inflammatory airway
    reactions; type II, occurring both indoors and outdoors in the
    environment in industrialized western countries, is composed mainly of

    nitric oxide, nitrogen dioxide, ozone, volatile organic compounds, and
    fine particles. The latter type of air pollution is associated with
    allergic diseases and allergic sensitization, indicating that air
    pollutants interfere with parameters of allergy at the level of
    sensitization, elicitation of symptoms, and exacerbation of disease.

          Until further epidemiological studies are conducted in humans,
    assessment of immunotoxicity in rodents, with subsequent extrapolation
    to the human situation, is still a good indicator of toxicity and can
    serve as a basis for subsequent decisions and regulations by
    authorities to reduce or prevent the risk of human exposure. This
    aspect is discussed further in Section 5.

          Humans are exposed to environmental contaminants mainly via food,
    water, and air. Open water (e.g. rivers, lakes, and coastal areas) and
    sediments often act as sinks for environmental pollution. This global
    problem can be deduced from disease manifestations in fish that live
    in coastal areas, especially those species that live in close contact
    with contaminated silt. High levels of contaminants and the diseases
    associated with them are not only of economic importance (i.e. to
    fisheries) but also affect people who consume the fish, as seen in
    studies showing increased levels of contaminants in people eating fish
    from the contaminated Baltic Sea (Svensson et al., 1991) and in Inuit
    and Indian populations in Canada who consume large quantities of fish
    and marine mammals (DeWailly et al., 1992). There is now some evidence
    that wildlife aquatic species have decreased resistance and enhanced
    incidences of infection and tumours that may be linked to
    environmental pollution (Vos et al., 1989; Wester et al., 1994).
    Because the immune system of fish has not been characterized in such
    detail as that of mammals, immunotoxicological studies have not been
    extensively included in ecotoxicology, although a number of reports of
    direct toxic actions of xenobiotics on fish species have been
    published in this developing field (Wester & Canton, 1987; Payne &
    Fancey, 1989; Anderson, 1990; Wester et al., 1990; Khangarot &
    Tripathi, 1991; Secombes et al., 1992; Anderson & Brubacher, 1993;
    Faisal & Hugget, 1993).

    1.2  The immune system: functions, system regulation, and modifying
         factors; histophysiology of lymphoid organs

    1.2.1  Function of the immune system

          In order to interpret pathological alterations of the immune
    system in terms of altered function, the physiology of the system must
    be understood. Since knowledge of the structure and function of the
    immune system is growing rapidly, a review of this subject, focusing
    on histophysiology, is presented. This section is not meant to serve
    as a textbook on immunology but to provide sufficient information for
    an understanding of pathological changes due to immunotoxic action.
    For general textbooks on immunology, reference may be made to Sell

    (1987), Klein (1990), Brostoff et al. (1991), Roitt (1991), Paul
    (1993), and Roitt et al. (1993). The section covers mainly humans and
    rodents, but reference is made to other species that are relevant in
    immunotoxicity assessment, e.g. fish in ecotoxicology. It should be
    noted that species differences can be large, despite fundamental
    similarities between the immune systems of animals. It is therefore
    difficult to conduct immunotoxicological studies in immunologically
    less well characterized animal species, although comparative studies
    that are under way may lessen the problems. Zapata and Cooper (1990)
    have written a comprehensive textbook on phylogenetic aspects of
    immunology. Phylogenetic data, from primitive fish to mammals, are
    presented in Table 2 (Cooper, 1982; Klein, 1986; Du Pasquier, 1989;
    Zapata & Cooper, 1990; Sima & Vetvicka, 1992). Relevant phylogenetic
    aspects of the immune system are described below.

          In mammals, the immune system and its reactions consist of a
    finely tuned, complex interplay between various cell types and soluble
    mediators secreted by those cells (Figure 1), some of which are listed
    in Section 7.

          Immune responses can be classified roughly as innate (natural and
    nonspecific) and acquired (adaptive) responses, in which the reaction
    is directed to a specific determinant (antigenic determinant or
    epitope). The nonspecific response involves effector cells such as
    macrophages (Vetvicka & Fornusek, 1992), NK cells (Herberman &
    Ortaldo, 1981), granulocytes (Ross, 1992), and mediator systems
    including the complement system (Tomlinson, 1993). Specificity is
    based on recognition by specific receptors on lymphocytes or by
    antibodies: The classical reaction to bacterial infection, resulting
    in antibacterial antibody formation and antibody-mediated destruction
    of the pathogen, is only one part of the intrinsic capacity of the
    system. Further attributes of the system (Nossal, 1987) are summarized
    below.  Encounter and recognition

          The initiation of an immune response requires adequate
    recognition of the pathogen. This recognition often occurs immediately
    after entry, e.g. during or after passage through the epithelial
    barrier of the body (skin or mucus-secreting epithelia in the
    respiratory and gastrointestinal tract). The first defence includes
    nonspecific inactivation, e.g. by nonspecific killer cells,
    neutrophilic granulocytes, and cells of the mononuclear phagocyte
    system (formerly called the reticuloendothelial system). It also
    includes antigen processing and presentation to cells such as
    lymphocytes of the T-helper-inducer type (Th) which can generate a
    specific response.

        Table 2.  Evolution of immunologically important traits among vertebrates

    A.  Major histocompatibility complex (MHC) and transplantation

    Species                                           Graft       MLR       CML     MHC control    Serologically
                                                      rejection   and/or            of immune      detectable
                                                                  GVH               response       MHC antigens

    Tunicata (sea squirts)                            +           ?         ?       ?              ?
         Hagfish (Hyperotreti)                        +           ?         -       ?              ?
         Lamprey (Hyperoartii)                        +           ?         -       ?              ?
    Chondrichthyes (cartilaginous fish)
         Shark, ray                                   +           ?         +       ?              +
    Osteichthyes (bony fish)
         Sturgeon (Chondrostei)                       +           ?         ?       ?              ?
         Bony fish (Teleostei)                        +           ?         +       ?              +
         Lungfish (Dipnoi)                            +           ?         ?       ?              ?
    Amphibia (amphibians)
         Salamanders (Urodela)                        +           ?         ?       ?              +
         Frogs, toads (Anura)                         +           +         +       +              +
    Reptilia (reptiles)
         Turtles (Chelonia)                           +           +         ?       ?              ?
         Lizards, snakes (Squamata)                   +           +         +       ?              ?
         Crocodiles, alligators (Crocodilia)          +           ?         ?       ?              ?
    Aves (birds)                                      +           +         +       +              +
    Mammalia (mammals)                                +           +         +       +              +

    Table 2 (cont'd)

    B. Complement and immunoglobulins (Ig)

    Species                                           Complement     Immunoglobulins
                                                                     IgM    IgG-like    IgA     IgD     IgE

    Tunicata (sea squirts)                            ?              -      -           -       -       -
         Hagfish (Hyperotreti)                        ?              ?      -           -       -       -
         Lamprey (Hyperoartii)                        ?              ?      -           -       -       -
    Chondrichthyes (cartilaginous fish)
         Shark, ray                                   +              +      +           -       -       -
    Osteichthyes (bony fish)
         Sturgeon (Chondrostei)                       +              +      +           -       -       -
         Bony fish (Teleostei)                        +              +      +           -       -       -
         Lungfish (Dipnoi)                            +              +      +           -       -       -
    Amphibia (amphibians)
         Salamanders (Urodela)                        +              +      ?           -       -       -
         Frogs, toads (Anura)                         +              +      +           +       -       -
    Reptilia (reptiles)
         Turtles (Chelonia)                           +              +      +           -       -       -
         Lizards, snakes (Squamata)                   +              +      +           -       -       -
         Crocodiles, alligators (Crocodilia)          +              +      +           ?       -       -
    Aves (birds)                                      +              +      ?           +       -       -
    Mammalia (mammals)                                +              +      +           +       +       +

    Table 2 (cont'd)

    C. Leukocytes

    Species                                           Lymphocytes                   Plasma    Macrophages
                                                      Small     T         B

    Tunicata (sea squirts)                            +         -         -         -         +
         Hagfish (Hyperotreti)                        +         -         -         +         +
         Lamprey (Hyperoartii)                        +         -         -         +         +
    Chondrichthyes (cartilaginous fish)
         Shark, ray                                   +         -         ?         +         +
    Osteichthyes (bony fish)
         Sturgeon (Chondrostei)                       +         -         ?         +         +
         Bony fish (Teleostei)                        +         +         +         +         +
         Lungfish (Dipnoi)                            +         ?         ?         +         +
    Amphibia (amphibians)
         Salamanders (Urodela)                        +         +         +         +         +
         Frogs, toads (Anura)                         +         +         +         +         +
    Reptilia (reptiles)
         Turtles (Chelonia)                           +         +         +         +         +
         Lizards, snakes (Squamata)                   +         +         +         +         +
         Crocodiles, alligators (Crocodilia)          +         +         +         +         +
    Aves (birds)                                      +         +         +         +         +
    Mammalia (mammals)                                +         +         +         +         +

    Table 2 (cont'd)

    D. Lymphoid organs

    Species                                           Bone      Thymus    Spleen    Lymph glands
                                                      marrow                        or nodes

    Tunicata (sea squirts)                            -         -         -         -
         Hagfish (Hyperotreti)                        -         -         -         GALT
         Lamprey (Hyperoartii)                        -         -         -         GALT
    Chondrichthyes (cartilaginous fish)
         Shark, ray                                   -         +         +         GALT
    Osteichthyes (bony fish)
         Sturgeon (Chondrostei)                       -         +         +         GALT
         Bony fish (Teleostei)                        -         +         +         GALT
         Lungfish (Dipnoi)                            -         +         +         GALT
    Amphibia (amphibians)
         Salamanders (Urodela)                        +         +         +         -
         Frogs, toads (Anura)                         +         +         +         ?
    Reptilia (reptiles)
         Turtles (Chelonia)                           +         +         +         ?
         Lizards, snakes (Squamata)                   +         +         +         ?
         Crocodiles, alligators (Crocodilia)          +         +         +         ?
    Aves (birds)                                      +         +         +         +
    Mammalia (mammals)                                +         +         +         +

    +, positive; ±, to be confirmed; -, negative; ?, not investigated.
    MLR, mixed leukocyte response; GVH, graft-versus-host; CML, cell-mediated lympholysis;
    GALT, gut-associated lymphoid tissue
    FIGURE 1

          The importance of an epithelial barrier for primitive defence
    mechanisms is clear in fish, in which a specific mucosal immune system
    with local production of antibodies associated with mucus secretion
    has been recognized. The gills are the main entry for both antigens
    and pathogens living in water, and both migrating macrophages and gill
    epithelial cells are involved in the process.  Specificity

          The immune system can distinguish one particular determinant in
    an immense spectrum of determinants. The discrimination between 'self'
    and 'non-self' (i.e. the avoidance of autoreactivity) is an example of
    this specificity.

          Antigens can be polypeptides, carbohydrates, or lipids (lipopoly-
    saccharides and lectins). A polypeptide antigen epitope is made up of
    about 10 amino acids. Lymphocytes are central to antigen specificity,
    as they express receptors for a single, distinct antigenic determinant
    on their surface. On B lymphocytes, this antigen receptor is
    essentially an antibody (immunoglobulin) molecule (Hasemann & Capra,
    1989). The antigen-binding fragment of the surface receptor and the
    antibodies produced after differentiation of B cells into plasma cells
    have virtually identical structures -- a quaternary structure
    comprising the dual heavy and light chains of the immunoglobulin
    molecule (the so-called variable part of these protein chains). B-Cell
    surface immunoglobulin and the immunoglobulin product of the plasma
    cell progeny may differ in the constant part of the heavy chain. Like
    the T-cell receptor (TCR), the B-cell receptor has a hetero-oligomeric
    structure. After the antigen is bound to the surface immunoglobulin,
    signal transduction occurs, in which one alpha-ß chain is linked to
    the surface immunoglobulin. Biological responses involving tyrosine
    phosphorylation and calcium mobilization are then induced, including
    activation, tolerance, and differentiation, depending on the
    differentiation stage of the B cell (Pleiman et al., 1994). On virgin
    B cells, the surface receptor is an IgM or IgD molecule (with µ or
    delta heavy chains, respectively). After so-called immunoglobulin
    class switching (Vercelli & Geha, 1992; Harriman et al., 1993), IgG
    (gamma chain), IgA (alpha chain), and IgE (epsilon chain) molecules
    can be synthesized. Associated with the immunoglobulin molecule on the
    cell surface is a dimeric transmembrane molecule, the Igalpha and
    Igßchain, which functions in signal transduction and intracellular
    activation of kinases of the  src family (Pleiman et al., 1994). This
    alpha-ß dimeric molecule is now used in identifying B cells.

          For T cells, the antigen receptor is a heterodimeric molecule
    (either the alpha-ß or the gamma-delta heterodimer), which has a
    constant and a variable part, like those of immunoglobulin molecules
    (Hedrick, 1989). Transmembrane signalling (tyrosine phosphorylation)
    after antigen contact occurs when this heterodimer is linked on the
    cell surface to the T3 (or CD3) molecule, which consists of at least
    five invariant chains (Clevers et al., 1988; Chan et al., 1992).

          The structural differences between the TCR for antigens and the
    B-cell receptor (i.e. antibody) arise from differences in the gene
    segments that encode the receptors. It is thus not surprising that
    T cells recognize other determinants on the antigenic compound than
    those recognized by B cells. For large antigens such as proteins,
    distinct T-cell and B-cell epitopes can be identified, as illustrated
    by the fact that the alpha-ß heterodimeric receptor on T cells
    recognizes the antigenic determinant in the context of polymorphic
    determinants of the major histocompatibility complex (MHC) antigens.
    The antigenic determinant is either a small peptide, produced during
    processing of antigen in the antigen-presenting cell and located in a
    'groove' formed by the quaternary structure of the MHC molecule
    (Adorini, 1990; Rothbard & Gefter, 1991; Germain & Margulies, 1993),
    or a larger molecule associated with the MHC molecule outside the
    groove. The latter is found for so-called 'superantigens', like
     Staphylococcus enterotoxin A (Herman et al., 1991). Thus, with
    either type of antigen, Th cells and delayed-type hypersensitivity
    T cells (see below) recognize the antigenic determinant only when it
    is presented together with the individual's own (self) determinant of
    class II MHC. This phenomenon is called MHC class II restriction. In
    contrast, T cells of the suppressor (Ts) and cytotoxic (Tc)
    populations are MHC class I restricted. The processing of antigen by
    antigen-presenting cells and subsequent complexing with MHC molecules
    occur intracellularly, but with different pathways for MHC class
    I-associated and MHC class II-associated complexing. In addition, MHC
    class II-associated complexing may occur with proteins present at very
    high concentrations in the extracellular environment (Neefjes &
    Momburg, 1993; Engelhard, 1994). MHC restriction does not necessarily
    apply to T-cell subsets within the pool expressing the alpha-ß
    heterodimeric TCR (Haas et al., 1993). B Lymphocytes do not function
    in an MHC restricted manner but recognize nominal antigen with their
    surface immunoglobulin receptor. Therefore, recognition of antigens by
    B cells and by most gamma-delta T cells, does not require antigen
    presentation on cells carrying their own MHC class I or class II
    determinants. The total repertoire of antigen recognition
    specificities is about 107 for antibodies and somewhat less for the
    TCR (about 106) (Roitt et al., 1993)

          Phylogenetically, the capacity to reject an allograft is acquired
    very early (e.g. sponges), and this has been interpreted as evidence
    for an MHC complex. Information on the molecular features of MHC
    antigens (Hughes & Nei, 1993) is available, however, only for the toad
     Xenopus (Flajnik et al., 1991; Sato et al., 1993) and for fish
    species (Hashimoto et al., 1990; Kasahara et al., 1992; Ono et al.,
    1992; Hordvik et al., 1993). All vertebrate species produce specific
    antibodies and typical cell-mediated immunity indicative of specific
    responses, demonstrating that both T and B cells exist, as in birds
    and mammals. Few data are available, however, because there are
    virtually no reagents for lymphocyte identification and
    classification. In chickens, monoclonal antibodies recognize alpha-ß
    and gamma-delta TCR and a third TCR with a configuration ß-ß'; various

    T-cell subsets have also been identified in this species with
    appropriate antibodies (CD3, CD4, CD8; see below). MHC restriction of
    antigen recognition by T cells has been demonstrated in  Xenopus. In
    fish, as in mammals, antigens are processed and presented by accessory
    antigen-presenting cells, such as monocytes, to specific lymphocytes
    in a seemingly alloantigen (presumably MHC or MHC-like) restricted
    fashion (Vallejo et al., 1990; Stet & Egberts, 1991; Vallejo et al.,
    1992). 'B-like' cells expressing immunoglobulins occur in all of the
    fish and amphibian species that have been studied so far; however,
    there is no IgD molecule in lower vertebrates. Presumably, all B-like
    cells express on their surface an immunoglobulin molecule of high
    relative molecular mass, similar to IgM in mammals.

          All non-mammalian vertebrates produce immunoglobulins of high
    relative molecular mass (Fellah et al., 1992; Wilson & Warr, 1992;
    Wilson et al., 1992; Litman et al., 1993; Marchalonis et al., 1993).
    There is probably also an IgG-like immunoglobulin of low relative
    molecular mass that is functionally but not structurally equivalent to
    mammalian IgG, but it has been observed in only a few species of bony
    fish. There is also evidence for the presence of an IgA-like
    immunoglobulin in some lower vertebrates. Non-mammalian vertebrates
    have no IgD or IgE. The total repertoire of antigen recognition
    specificities for antibodies is smaller in non-mammalian vertebrates
    than in mammals.  Choice of effector reaction; diversity of the answer

          After activation of Th cells, an immune response develops in
    order to eliminate the antigen; in practical terms, the response
    results in inactivation of the pathogen. The response is humoral
    (antibody-mediated) and/or cellular (cell-mediated).

          In the humoral response, Th cells together with antigen activate
    specified B cells to become antibody-producing plasma cells. The
    antibodies produced mediate the subsequent inactivation of the foreign
    substance in a number of ways. When present in the form of immune
    complexes, IgG and IgM either activate the complement system and
    induce complement-mediated cytotoxicity (Hansch, 1992; Tomlinson,
    1993) or activate secondary effects, which include: (i)
    vasodilatation, increased vascular permeability, and attraction of
    granulocytes, with subsequent release of lysosomal proteolytic enzymes
    (i.e. components of acute inflammation); (ii) IgG antibody-mediated
    cellular cytotoxicity, in which the antibody forms the antigen-
    specific bridge between the killer cell (macrophage, binding of the Fc
    fragment of IgG) and the target; (iii) IgG- and IgM-induced
    opsonization and ingestion by phagocytic cells (granulocytes,
    macrophages), with involvement of receptors for immunoglobulin Fc and
    the complement split product C3d; (iv) binding of IgE to IgE receptors
    on the surface of mast cells and basophilic granulocytes, which
    induces degranulation with release of mediators after antigen binding.

          Complement is phylogenetically very old, as genes that encode
    complement components and complement proteins have been identified in
    hagfish (Hanley et al., 1992; Ishiguro et al., 1992), and C3-like
    activity exists in invertebrates and in all vertebrates. Complement C3
    has been purified from all classes of vertebrates, including fish
    (e.g. hagfish); in primitive fish like lampreys, it shows 30% homology
    with human C3, whereas that in rodents is about 80% homologous with
    that in humans (Lambris, 1993). Complement activation by immune
    complexes occurs in most primitive vertebrates, but the secondary
    effects emerged later in phylogeny. Antibody-mediated cellular
    cytotoxicity involving NK cells has been documented in some bony fish.
    Little is known about inflammatory reactions in lower vertebrates,
    except for some histopathological data obtained in fish.

          In the cellular response, Th cells activate precursors of Tc
    cells, which subsequently kill the target after antigen-specific
    recognition. Furthermore, precursors of lymphokine-producing cells
    (for example, delayed-type hypersensitivity T cells) can be activated,
    and the lymphokines thus secreted subsequently activate macrophages to
    kill the target. Studies of Th clones  in vitro have revealed the
    existence of different types (Mosmann & Coffman, 1989). Th1 cells
    synthesize interleukin (IL)-2, IL-3, tumour necrosis factors alpha and
    ß, and interferon (IFN)gamma(cytokines are discussed below), provide
    help to B cells (especially in IgG2a synthesis), activate macrophages,
    and initiate delayed-type hypersensitivity reactions. Th2 cells secrete
    IL-3, IL-4, IL-5, IL-6, IL-10, IL-13, and tumour necrosis factor alpha,
    providing extensive help to B cells (for IgM, IgG1, IgA, and IgE
    synthesis), and activate eosinophilic granulocytes. Th2 cells do not
    play a role in the initiation of delayed-type hypersensitivity. Various
    cytokines are involved in the generation of these Th populations, with
    dominant effects of IL-4 (Th2) and IL-12 (Th1). Cytokines produced by
    each type of Th cell subpopulation appear to downregulate the activity
    of the others. The choice of effector reaction is determined in part
    by cooperation between the various populations of Th cells: IFN gamma
    from Th1 cells can downregulate Th2 cells, while IL-10 from Th2 cells
    can downregulate Th1 cells (Mossman & Coffman, 1989).

          Finally, it should be noted that the immune system exerts other
    types of responses that do not involve activation of Th cells. These
    include T cell-independent activation of B cells, usually when the
    antigens consist of repeating polysaccharide units (present on
    bacteria such as  Escherichia coli and  Pneumococcus). They also
    include direct activation of T killer cells which bear antigen-
    specific receptors comprising the gamma-delta heterodimer (Bell, 1989;
    De Weger et al., 1989; Raulet, 1989; Haas et al., 1993).  Immunoregulation

          The effector reaction is induced by a finely tuned interplay
    between cells and soluble mediators (Figure 2) (Van Deuren et al.,
    1992). On the one hand, cell-cell contact is required; for instance,
    between the antigen-presenting cell with (processed) antigen on its
    surface and the Th cell. On the other hand, mediators (cytokines or
    interleukins) influence cell function at a distance, after binding to
    specific receptors on the target cell and subsequent signal
    transduction, resulting in cell activation (Foxwell et al., 1992; Taga
    & Kishimoto, 1992). New interleukins and their surface and soluble
    receptors are being identified and characterized rapidly: at least 15
    different interleukins are known at present, in addition to various
    growth factors. As a reflection of their function, these factors are
    designated as chemokines (attracting cells) or cytokines (influencing
    cell function, such as stimulation). Factors synthesized by
    lymphocytes are called lymphokines; those synthesized by monocytes or
    macrophages are called monokines. Among the consequences of cytokine-
    cell interactions are chemotaxis (Miller & Krangel, 1992), production
    of subsequent mediators in the cascade, and down-regulation of
    cellular function (Kimchi, 1992). The role of mediators in cell
    adhesion to vascular endothelium is described below.

          Examples of mediators that influence immune reactions positively
    are IL-1 (Dinarello, 1992), which is secreted by antigen-presenting
    cells and stimulates Th cells; IL-2, which is secreted by Th cells and
    stimulates a variety of T cells to amplify the response; and a number
    of B-cell stimulatory cytokines (Figure 2). Surface receptors for
    these interleukins are present on certain immune cells (and also in
    neuroendocrine tissue, mentioned below) or are expressed during
    activation. For instance, IL-2 receptors and HLA-DR molecules are
    expressed on T cells as part of the activation process, and their
    expression is used to assess the state of cell activation (Shabtai et
    al., 1991).

          In down-regulation, the immune response manifests active
    processes. A number of mediators, such as prostaglandin E2, IL-10, and
    transforming growth factor ß (Brigham, 1989; Fontana et al., 1992;
    Larrick & Wright, 1992), can suppress subsets of T and B lymphocytes.
    After stimulation of Th cells in the initiation of the response,
    precursors of Ts cells are activated which subsequently inhibit Th
    cells from further amplifying the response, in direct cell-cell
    contact or by secretion of soluble inhibitors. The existence of Ts
    cells has been disputed, as part of the function of these cells is
    cytotoxicity, which is exerted by the closely related MHC class I-
    restricted Tc population (Bloom et al., 1992). Immunoregulatory
    circuits have also been documented at the antibody level, where a
    first antibody generates a second one directed to itself. The
    relevance of this antibody-anti-antibody network, the so-called 'anti-
    idiotype' network, remains a subject of speculation.

    FIGURE 2

    FIGURE 2a

    FIGURE 2b

          The immune system is in a continuous state of homoeostatic
    equilibrium. The introduction of an antigen (pathogen) disturbs that
    balance by activating antigen-specific cell clones of T and B
    lymphocyte origin. The system not only allows the proliferation and
    amplification of relevant clones to cope with the antigen, but it also
    searches for (and reaches) a state of newly defined homeostasis.

          Little is known about the phylogenetic development of mechanisms
    for regulating immune responses in vertebrates. Helper, cytotoxic, and
    even suppressor lymphoid functions have been reported in ectothermic
    vertebrates, but the existence of T-cell subpopulations has not been
    demonstrated. T and B cells cooperate in teleost fish and in
    amphibians, and there is indirect evidence that they do so in
    reptiles; however, MHC restriction of these cell interactions has been
    demonstrated only in  Xenopus. The existence of clusters consisting
    of lymphocytes, macrophages, and plasma cells has been documented in
    various lower vertebrate species, which indicates the importance of
    cell-cell interactions in evoking immune responses. Cytokine activity
    resembling that of IL-1 and interferon has been identified in various
    fish species. A T-cell growth factor was first characterized in
     Xenopus. An idiotype-anti-idiotype network has been proposed to
    explain the regulation of antibody production in some bony fish
    (Zapata & Cooper, 1990).  Modifying factors outside the immune system

          Communication with other homeostatic mechanisms in the body is an
    important aspect of immunoregulation; mediators of the response have
    effects not only on the internal regulatory network but also on
    systems outside the immune system. Communication with the clotting and
    kallikrein systems by complement components is one example;
    communication with the central nervous system is another (Ader et al.,
    1990). For instance, IL-1 generated by antigen-presenting cells
    affects the temperature regulatory centre (induction of fever) and the
    sleep regulatory centre (induction of slow-wave sleep) in the
    hypothalamus (Dinarello, 1992).

          One example of interaction between the immune system and the
    central nervous system is the profound influence of stress on immune
    reactivity (Khansari et al., 1990). Both stressful events and the way
    in which individuals cope with stress are involved (Bohus et al.,
    1991), as documented in studies mainly in rats but also in other
    species, including fish (Faisal et al., 1989). In humans, conditions
    of acute psychological stress include bereavement (Bartrop et al.,
    1977), marital disruption (Kiecolt-Glaser et al., 1987), and
    examination periods (Kiecolt-Glaser et al., 1986), and these can be
    associated with a decrease in immune status (Kiecolt-Glaser & Glaser,
    1986) which can result in increased risk for infection, including
    common respiratory tract infections, e.g. influenza (Boyce et al.,
    1977; Clover et al., 1989), infectious mononucleosis (Kasl et al.,
    1979), and herpes virus reactivation (Glaser et al., 1985).

          The hypothalamus-pituitary-adrenal axis is an important pathway
    in the communication between the central nervous system and the immune
    system, resulting in synthesis of glucocorticosteroid hormone by the
    adrenal gland induced by adrenocorticotrophic hormone from the
    pituitary gland (Buckingham et al., 1992). Other mechanisms are those
    mediated by the direct action of neuropeptides, such as opioid
    peptides (van den Bergh et al., 1991), on immune cells; these are
    either stimulatory or down-regulatory, depending in part on
    experimental design and conditions. In addition, almost all lymphoid
    tissues are innervated (Bulloch, 1987; Felten et al., 1987; Kendall &
    Al-Shawaf, 1991), although the role of this neuroregulatory pathway is
    largely unknown (Freier, 1990). The immune system and the
    neuroendocrine system have a number of biologically active mediators
    in common, including cytokines and neuromediators (Fabry et al.,
    1994), and are strongly interrelated (Weigent & Blalock, 1987; Sibinga
    & Goldstein, 1988; Heijnen & Kavelaars, 1991; Heijnen et al., 1991;
    Knight et al., 1992). Drugs that act on the central nervous system,
    like some tranquillizers, antidepressants, benzodiazepines,
    antiepileptics, anaesthetics, and levodopa (an antiparkinson drug),
    may cause immunosuppression (Descotes, 1986). Thus, the principles of
    immunotoxicology may find application in neurotoxicology and endocrine
    toxicology (Snyder, 1989). Some examples of factors that modify the
    immune system, based on studies of the thymus of mice during pregnancy
    and of birds after hatching, are given below. Exogenous conditions may
    have pivotal influences on the structure and function of the immune
    system. In ectothermic vertebrates, these include seasonal changes
    like temperature and photoperiod and are governed by corticosteroid
    and sex hormones. In spring, during the mating period, there is low
    immune reactivity, with thymic involution; in the postmating period
    and the first part of summer, there is maximal development of lymphoid
    tissue and immune reactivity; at the end of summer, activity declines,
    and the lymphoid organs undergo pronounced involution, which persists
    throughout the autumn and winter (Zapata et al., 1992). The lymphoid
    system of ectothermic vertebrates therefore cannot be described in
    morpho-functional terms without taking into account the season in
    which studies were conducted. Seasonal variation in
    immunoresponsiveness is also seen in laboratory animals (Ratajczak et
    al., 1993).  Immunological memory

          A special feature of the immune response is the generation of
    memory after initial contact with an antigen (Gray, 1993). The first
    response includes activation and amplification of antigen-specific T
    or B cells to exert effector reactions; it ends with the return of
    antigen-recognizing cells to the normal resting state (small
    lymphocytes). The second contact with the antigen results in
    recruitment of more antigen-specific cells to give a stronger signal
    and more efficient elimination of the antigen or pathogen. The
    reaction also occurs faster, and there is a stronger binding of
    antibody to antigen. The gradual increase in the binding capacity of

    antibodies during the immune response is known as 'affinity
    maturation'. The memory pool of lymphocytes mediates a faster response
    than the virgin (unprimed) pool.

          The memory effect is most evident in the humoral arm of the
    immune response. The first response gives rise only to IgM class
    antibody, but antibodies of other immunoglobulin classes (especially
    IgG in the internal system) are generated after subsequent contact and
    immunoglobulin class switch. The antibodies thus formed may show
    increased affinity as a result of somatic mutation (Kocks & Rajewski,

          The cellular basis of immunological memory is largely unresolved.
    It appears to be located within the T-cell population, because its
    presence within the B-cell population is apparently of short duration
    and is associated with the presence and stimulatory activity of the
    antigen in germinal centres of secondary lymphoid tissue. The
    generation of memory is the basis for vaccination, performed to
    prevent contracting infectious disease by bringing about contact with
    the pathogen in an attenuated or inactivated, nonpathogenic form.

          It is not known whether immunological memory exists in primitive
    vertebrates. The classical differentiation into primary and secondary
    responses does not exist, as it is mainly immunoglobulin of high
    relative molecular mass that is present, and the repertoire of
    antigen-recognizing specificities is smaller than that in birds and
    mammals. Some fish species may have immunological memory.

    1.2.2  Histophysiology of lymphoid organs  Overview: structure of the immune system

          Components of the immune system are present throughout the body
    (Figure 3). The lymphocyte compartment is lodged in lymphoid organs,
    from which cells can move to sites of infection or inflammation.
    Phagocytic cells of the monocyte-macrophage lineage occur in lymphoid
    organs and also at extranodal sites, such as Kupffer cells in the
    liver, alveolar macrophages in the lung, mesangial macrophages in the
    kidney, and glial cells in the brain (Figure 4). Polymorphonuclear
    leukocytes are present mainly in blood and bone marrow as mature and
    progenitor cells. These cells accumulate at sites of inflammation.

          The lymphoid organs can be classified roughly into two types:
    primary or central (antigen-independent) and secondary or peripheral
    (antigen-dependent). This classification is based on the antigen-
    dependence of cell proliferation and differentiation. It does not hold
    for lower vertebrates, including fish. The bone marrow in higher
    vertebrates is a primary organ, in which are found the pluripotent
    haematopoietic stem cells which differentiate into progenitors of
    myeloid cells (which in turn differentiate into granulocytes,
    monocytes, erythrocytes, and platelets) and lymphoid progenitors

    FIGURE 3

    FIGURE 4

    (Figure 1). The differentiation is not antigen-dependent or antigen-
    driven, but this does not exclude a role for antigens in the process.
    Factors secreted in the periphery during antigen-specific stimulation
    can promote haematopoiesis in the bone marrow (Figure 2) (Fletcher &
    Williams, 1992; Kincade, 1992; Saito 1992; Williams & Quesenberry,
    1992) or inhibit haematopoietic activity (Wright & Pragnell, 1992).
    The bone marrow also functions as a secondary lymphoid organ, because
    terminal antigen-induced lymphoid cell differentiation can occur in
    its microenvironment. For instance, the bone-marrow cells include both
    the memory lymphocyte pool and the major plasma cell population, which
    contributes to the intravascular pool of immunoglobulins. Normally,
    plasma cell differentiation follows antigen presentation at peripheral
    sites, and stimulated cells subsequently migrate to the bone marrow
    for final differentiation. The secondary or peripheral lymphoid organs
    in the body include the lymph nodes, spleen, and lymphoid tissue along
    secretory surfaces like the gastrointestinal and respiratory tracts.

          A second classification is based on the location of lymphoid
    organs, divided into internal organs (some lymph nodes and the spleen,
    in addition to thymus and bone marrow) and external organs (lymphoid
    tissue along secretory surfaces and the lymph nodes that drain the
    mucosa-associated lymphoid tissue (MALT)) (Figure 3). Lymphoid organs
    at these two locations behave somewhat independently in host defence,
    for instance in immunoglobulin synthesis. The main function of the
    external or secretory immune system is to produce (secretory) IgA
    antibody, whereas the internal immune system (mainly bone marrow)
    produces IgG or IgM antibody; the major site of IgE synthesis in the
    body is also along secretory surfaces. The extent of the secretory
    immune system should not be underestimated: About half of the body's
    lymphocytes are located in the secretory immune system, and its
    capacity for immunoglobulin synthesis is about 1.5 times that of the
    internal system. In all vertebrates, intraepithelial lymphocytes and
    nonencapsulated lymphoid infiltrates occur in the intestine. True
    lymphoid organs, such as the tonsils, Peyer's patches, and the
    appendix, have been reported only in higher vertebrates. In birds, the
    caecal tonsil, a blind appendix of the posterior intestine, becomes an
    important peripheral lymphoid organ after involution of the bursa of

          Another organ that contributes to the immune system is the skin.
    It does not contain organized lymphoid tissue, but immune components
    in skin are interconnected with other immune organs, leading to the
    concept of the skin immune system, or skin-associated lymphoid tissue
    (Stingl & Steiner, 1989; Bos, 1990; Nickoloff, 1993; see section

          Immune cells and cellular products are transported between
    lymphoid organs by blood and lymph vessels (Duijvestijn & Hamann,
    1989). For example, Langerhans cells on their way from the skin to a
    lymph node are present in lymph as 'veiled macrophages'. The blood
    circulation contains only a minor part of the body's total pool of
    lymphocytes (estimated at about 1%) and only a selected population,
    i.e. the recirculating lymphocyte pool (Figure 5). Therefore,
    assessment of only the blood lymphoid compartment does not give a
    complete inventory of the body's immune system, as it ignores the
    activities of the non-recirculating cells. In general terms, the blood
    lymphoid compartment does not include cells that are in a state of
    activation, proliferation, or differentiation; such cells are
    typically tissue-bound. This rule is not absolute. For instance, in
    the case of a highly activated B-cell system with hyperplasia of
    germinal centres in lymph nodes, activated B cells may occur in the
    circulation. These cells are normally not part of the recirculating
    pool. With regard to the non-lymphoid cells of the immune system,
    macrophages are tissue-bound (histiocytes), and monocytes are their
    counterpart in blood. Dendritic cells, which are present in very low
    proportions in blood, are the counterparts of Langerhans cells in
    skin, veiled macrophages in lymph, and interdigitating dendritic cells
    in lymphoid tissue. The follicular dendritic cells, i.e. the antigen-
    presenting cells in follicles in lymphoid tissue (see below), do not
    exist in the circulation. Within the myeloid series, the mast cell is
    typically tissue-bound (Figure 6).

          The endothelium lining the blood vasculature has a major role in
    the passage of cells from blood to tissue parenchyma. Adhesion
    molecules on circulating cells and endothelium are involved in this
    process (Patarroyo, 1991; Gahmberg et al., 1992; Gumbiner & Yamada,
    1992). These glycoproteins belong to three families of molecules, the
    immunoglobulin supergene family, the integrins, and the selectins
    (Springer, 1990; Lasky, 1992; Bevilacqua, 1993). They may be present
    on endothelium in the resting state but often require alteration to
    become biologically active in inflammatory processes (e.g. under the
    influence of mediators like IFN gamma). In lymphoid tissue, they are
    called addressins, to indicate their role in the selective homing of
    passenger lymphocytes into internal or external lymphoid tissue.

          The structure and histophysiology of the bone marrow, thymus,
    lymph node, spleen, and MALT are presented in more detail in the
    following sections. The different microenvironments of these organs
    are summarized in Table 3. A detailed description of the histological
    and pathological aspects of rat lymphoid tissue is given by Jones et
    al. (1990).

    FIGURE 5

          Not only conventional histological features but also the
    expression of cell surface markers (immunological phenotype) are
    emphasized. Monoclonal antibodies to marker substances
    (differentiation antigens) on cell populations are used widely in the
    identification of leukocyte populations, especially in cell
    suspensions (flow cytometry) and tissue sections (immuno-
    histochemistry). The wide range of monoclonal antibodies that currently
    exists have been grouped according to the 'cluster of differentiation'
    (CD) nomenclature, in which they are classified according to their
    reactivity to the same cell marker molecule (but not the same epitope
    on those molecules) (Clark & Lanier, 1989; Knapp et al., 1989;
    Schlossman et al., 1994, 1995). The CD nomenclature has been adapted
    for species other than man (Holmes & Morse, 1988; Jefferies, 1988;
    Schuurman et al., 1992a). Some monoclonal antibodies that can be used
    in the identification of leukocytes and stromal cells in tissue sections
    and cell suspension are described in section 4.1.2.

    FIGURE 6

        Table 3.  Microenvironments in lymphoid tissue

    Microenvironment                           Cells present                                   Function

    Bone marrow                                Haematopoietic cells organized as               Differentiation of stem cells into cells
                                               islands within fatty tissue, mature             of the erythroid, myeloid/monocytoid,
                                               leukocytes, plasma cells                        platelet and lymphoid lineage; antibody
                                                                                               synthesis, memory cells

    Cortex                                     Reticular epithelium, immature                  Generation of T-cell competence,
                                               T cells                                         T-cell receptor rearrangement, positive
                                                                                               selection (MHC restriction), negative
                                                                                               selection (autoreactive cells), phenotypic

    Medulla                                    Reticular epithelium, dendritic cells,          Final generation of T-cell competence
                                               T lymphocytes                                   (negative selection), thymic hormone
                                                                                               synthesis, antigen presentation

    Lymph node and spleen
    Paracortex (lymph node),                   Interdigitating cells, Th and Ts cells          Lymphocyte entry through high periarteriolar
    lymphocyte sheath                                                                          lymphoendothelial venules (lymph node) or
                                                                                               central arteriole (spleen), antigen
                                                                                               presentation to Th cells, T-cell proliferation,
                                                                                               differentiation, and regulation (Ts cells)

    Primary follicles, follicular mantle       Dendritic cells (subtype of follicular          Storage of virgin and memory B cells,
    of secondary follicles                     dendritic cells), dendritic macrophages,        recirculating B cells (surface IgM+IgD+)
                                               B cells, small number of T cells

    Table 3 (cont'd)

    Microenvironment                           Cells present                                   Function

    Germinal centre                            Follicular dendritic cells, dendritic           T Cell-dependent B-lymphocyte
                                               macrophages (starry-sky macrophages),           differentiation, antigen presentation in the
                                               B cells (centrocytes, centroblasts),            form of immune complexes (with/without
                                               Th cells                                        complement C3)

    Medulla (lymph node),                      Plasma cells, T effector cells,                 Termination of antigen-specific reaction:
    red pulp (spleen)                          reticular cells, polymorphonuclear              antibody synthesis and immune
                                               granulocytes                                    complex-mediated clearance, Tdth and
                                                                                               Tc cell response

    Marginal zone (spleen)                     Marginal zone macrophages,                      T Cell-independent B-lymphocyte
                                               marginal metallophilic cells,                   proliferation and differentiation, e.g. to
                                               marginal zone B cells                           bacterial polysaccharides, B-cell memory
                                                                                               (surface IgM+IgD- cells)

    Mucosa-associated lymphoid tissue
    Epithelium covering lymphoid               M (microfold) cells                             Transport (uptake) of exogenous
    tissue (e.g. Peyer's patches)                                                              substances

    Follicles and interfollicular              See 'lymph node and spleen'                     For antibody synthesis: precursors of IgA
    areas                                                                                      plasma cells

    Mucosal epithelium                         Epithelial cells, Tc cells, natural             First line of defence, synthesis of secretory
                                               killer cells, gamma-delta T cells               component, transport of IgA (IgM) to

    Lamina propria                             Plasma cells, macrophages                       Synthesis of IgA antibody, phagocytosis
                                                                                               and killing

    MHC, major histocompatibility complex; Th, T helper; Ts, T suppressor; Ig, immunoglobulin; Tdth, delayed-type hypersensitivity T cells;
    Tc, T cytotoxic
          Typing of cells in the T lymphocyte lineage is a good
    illustration of immunological phenotyping. Subsets with other
    functions usually cannot be identified by conventional cytology but
    can be recognized by immunological phenotyping. For example, T cells
    with a helper-inducer function are labelled by antibodies to CD4
    antigens, and cells with a cytotoxic function are labelled by
    antibodies to CD8. The reverse does not hold, however; for example,
    not all CD4+ cells are helper-inducer cells, because T cells in the
    separate subset effecting delayed-type hypersensitivity and some
    macrophage populations are also CD4+*CD8+ T cells are in either
    the cytotoxic or the suppressor subset. Thus, there is no unequivocal
    relationship between CD4 or CD8 expression and cell function, because
    these cell-surface molecules are expressed in relation to the way in
    which antigen is recognized: T cells recognize antigen in the context
    of MHC molecules -- MHC class II molecules for CD4+ cells and MHC
    class I antigens for CD8+ cells (Engleman et al., 1981; Meuer et
    al., 1983). This phenomenon is further discussed in sections

          Monoclonal antibodies have been developed to most subtypes of
    leukocytes, including NK cells. The specificity of antibody 3.2.3,
    anti-NKR-P1, for identifying these cells was described by Chambers et
    al. (1989). Immunological phenotyping of these cells in rat spleen and
    lung is illustrated in Figure 14d.

          NK cells and their activities are an example of the differences
    between cell function, cytology, and immunological phenotype. NK cells
    are characterized by their killer function against selected targets
     in vitro. Cells with cytological features that include cytoplasmic
    granules, called large-granular lymphocytes, presumably have a natural
    killer function, as do cells with an immunophenotype defined by
    certain antibodies. The cells identified in these ways are by no means
    identical and belong to different, overlapping subpopulations.  Bone marrow

          The microenvironment of the bone marrow is found in all of the
    hollow bones of the body and occupies the medullary space of the
    skeleton (Figure 7). Blood enters the marrow from nutrient arteries at
    the place where they bifurcate to form the central artery of the
    medullary canal. Branches of the central artery terminate in
    capillaries within the medullary space or penetrate the endosteum,
    where arterial blood from the nutrient artery mixes with blood from
    muscular arteries in the periosteal capillaries. The blood returns via
    the vascular sinuses to the central sinus and vein. Blood cells in
    various stages of development (Figure 1) intermingle with their
    microenvironment, which is composed of reticular cells (adventitial
    and fibroblastic), adipocytes, endothelial cells, and extracellular

    matrix. These, together with passenger accessory cells (macrophages,
    monocytes, and lymphocytes, especially T cells), support the
    haematopoietic process (Lichtman, 1981; Weiss & Sakai, 1984; Chanarin,
    1985; Weiss & Geduldig, 1991; Mayani et al., 1992).

          In many mammals during ontogenesis, haematopoiesis is first
    located in the yolk sac and fetal liver, until the bone marrow
    develops and becomes functional. In rodents, haematopoiesis also
    occurs in splenic red pulp later in life. This effect is less
    pronounced in other species, but haematopoiesis can occur at other
    sites in the body, e.g. the thymus. In lower vertebrates, a bone
    marrow containing haematopoietic stem cells occurs firstly in
    amphibians. It contains only granulopoietic and lymphoid cells;
    erythropoiesis and thrombopoiesis usually occur in the blood sinusoids
    of the splenic red pulp. In fish and more primitive vertebrates,
    lymphoid tissue is found at many ectopic locations, such as intestine,
    brain, heart, gonads, pro- and mesonephrons, and liver and is
    functionally and structurally similar to the bone marrow of higher
    vertebrates. In birds, the thymus is the main site of erythropoiesis
    during certain periods of life (Kendall & Ward, 1974; Kendall &
    Frazier, 1979).

           Haematopoiesis: The pluripotent haematopoietic stem cells in
    the bone marrow proliferate and differentiate to progenitors of
    myeloid, erythroid, and lymphoid cells. Some further differentiation
    occurs in the marrow, but the final maturation mainly occurs elsewhere
    (Figure 1). The common lymphoid progenitor cell differentiates into a
    T- and a B-lymphoid progenitor. Progenitor T cells then move to the
    thymus for further maturation. B-Lymphoid progenitors mature via the
    pre-B cell stage into virgin B cells, which then leave the
    microenvironment of the bone marrow and lodge in the periphery. In
    birds, this function in B-cell maturation is performed by the bursa of
    Fabricius, an epithelial organ located adjacent to the termination of
    the gastrointestinal tract (B stands for bursa-dependent) (Cooper,
    1982; Du Pasquier, 1989; Zapata & Cooper, 1990). Although the bursa is
    considered essential for the production of B lymphocytes, chickens
    bursectomized early in embryonic life have B cells in their peripheral
    lymphoid organs and can produce antibodies, even if their repertoire
    is very limited. Thus, the bursa seems to be involved in the
    generation of immunoglobulin diversity rather than just in B

          The development of stem cells into more mature cells requires the
    presence of a microenvironment, as described above, and is regulated
    by haematopoietic cytokines (also called haematopoietins) (Fletcher &
    Williams, 1992; Kincade, 1992; Quesniaux, 1992; Saito, 1992; Williams
    & Quesenberry, 1992; Wright & Pragnell, 1992; see also Figure 2).
    Unmyelinated nerve fibres may terminate in the haematopoietic spaces
    (Lichtman, 1981), and neuropeptides may play a role in the regulation
    of haematopoiesis. A microgeographical distribution of the various

    FIGURE 7

    cell lineages in microenvironments has been suggested, such as the
    preferential associations between fibroblasts and granulocytes,
    megakaryocytes, and sinus endothelial and adventitial cells and
    between macrophages and erythroid cells.

          The classical bioassay for haematopoietic stem cell activity is
    measurement of colony-forming units in the spleen of irradiated mice
    after injection of bone-marrow cells. More differentiated progenitors
    can be cultured  in vitro to provide information on the specific
    lineage of the colony-forming cell, to evaluate the progenitor
    activity of macrophages, granulocytes, megakaryocytes, eosinophilic
    granulocytes, and mast cells, as well as that of several cell
    lineages, such as macrophages and granulocytes. Progenitors of
    erythrocytes are assayed as early progenitors or as erythroid burst-
    forming units. Studies of the mechanisms involved have revealed a
    number of factors that promote the differentiation of progenitor
    cells, like granulocyte-macrophage colony-stimulating factor,
    macrophage colony-stimulating factor, granulocyte colony-stimulating
    factor, and c-kit ligand. These mediators are produced by various cell
    types, mainly after activation, and promote haematopoiesis  in vivo
    in conditions of e.g. infection and inflammation. Other soluble
    mediators produced by T cells and by other cells involved in the
    antigen-driven immune response also promote haematopoiesis; these
    include IL-1, IL-3, IL-6, IL-11, interferon and tumour necrosis
    factor. Haematopoiesis in the bone marrow is thus not independent of
    antigenic stimulation and exposure but is not typically antigen-
    driven. For instance, in the case of acute infection, the bone marrow
    produces a large proportion of polymorphonuclear granulocytes within a
    short time, manifested in blood as leukocytosis and a shift in the
    differential count to band-type immature granulocytes. This shift may
    be related to migration of T lymphocytes into the bone marrow
    microenvironment, where they activate haematopoiesis. Antigen-induced
    lymphoid cell differentiation also occurs in the bone marrow; for
    instance, bone marrow cells include the memory lymphocyte pool and the
    major plasma cell population, which contribute to the intravascular
    pool of immunoglobulins.

           Development and aging: The bone marrow is the primary site of
    haematopoiesis throughout life, generating over 95% of the
    haematopoietic activity in adult mammals (Mayani et al., 1992).
    Essentially all of the medullary space in the bones is occupied by
    haematopoietic tissue in mice and rats; in contrast, in adult humans,
    dogs, and rabbits, haematopoietic tissue is restricted to the proximal
    epiphyses of the long bones, the central skeleton, and the skull; most
    bone marrow is gradually replaced by fat cells. The normal mean
    proportions of bone marrow cells and changes with age in rats were
    reported by Valli et al. (1990).  Thymus

          The thymus is a two-lobed organ located in the mediastinum,
    anterior to the major vessels of the heart (Kendall, 1991; Von
    Gaudecker, 1991; Schuurman et al., 1993), although it is located in
    the neck region in the guinea-pig. Its anatomical location complicates
    complete thymectomy  in vivo. The two independent lobes, attached to
    each other only by connective tissue, consist of smaller lobules,
    which have basically the same architecture, with a subcapsular and
    outer cortical area, a cortex, and a medulla (Figure 8). With
    conventional histological stains, the cortex is strongly stained,
    owing to a dense population of small lymphocytes, and the outer cortex
    and medulla show a paler colouring.

          Blood vessels enter the lobules at the cortico-medullary junction
    and extend radially into the cortex. Nerves course along the blood
    vasculature. Fenestrated capillaries are very infrequent in the
    cortex. The thymus is unique among the lymphoid organs in that its
    microenvironment consists of a reticular epithelium (in birds, the
    bursa of Fabricius also has an epithelial framework). Macrophages
    derived from the bone marrow are found in the cortex and medulla as a
    transient population. Dendritic cells in the medulla, resembling
    interdigitating dendritic cells in lymph nodes, have a major function
    in antigen presentation and strongly express MHC class II antigen.

          The thymus appears for the first time in vertebrate phylogeny in
    cartilaginous fish (sharks and rays). More primitive vertebrates lack
    a thymus, although they can manifest cellular immune responses. Except
    in bony fish, the thymus is histologically similar in all vertebrates,
    although it is derived from distinct pharyngeal pouches in different
    species. The classical cortex-medulla demarcation is not present in
    fish thymus, and most thymocytes seem to occupy the central part of
    the organ. In all species, epithelial cells organize a supporting
    network in both cortex and medulla. Hassall's corpuscles, which are
    epithelial aggregates with centrally located cell debris, occur in the
    medulla of the human thymus but are scarce or absent in the rodent
    thymus and in the thymus of ectothermic vertebrates. In the latter,
    epithelial cysts are frequent. The thymus of lower vertebrates, in
    contrast to that of mammals, contains numerous myoid cells; in avian
    thymus, significant erythropoiesis has been documented (Kendall &
    Ward, 1974; Kendall & Frazier, 1979), which may also occur in mammals
    (Kendall, 1980; Kendall & Singh, 1980).

           T-Cell maturation: selection in the thymus: T Cells reside in
    the thymus during their maturation from progenitor cells to
    immunocompetent T cells. This gland has a privileged function in
    promoting the maturation process (Brekelmans & Van Ewijk, 1990;
    Shortman et al., 1990; Van Ewijk, 1991; Boyd et al., 1992). In
    congenitally athymic, 'nude' animals (Schuurman et al., 1992b,c) and
    in thymic aplasia in children with complete Di George's syndrome, the

    FIGURE 8

    absence of a functionally active T-cell system is causally related to
    the aplasia of the organ. The process of T-cell maturation includes a
    number of steps located in different microenvironments (Schuurman et
    al., 1993): The least mature cells, which enter the lobules from the
    bloodstream at the cortico-medullary junction, first move to the outer
    subcapsular cortex, where they appear as large lymphoblasts. They then
    pass through the cortex, where they become small lymphocytes with a
    scanty cytoplasm. Finally, they move to the medulla, where they appear
    as medium-sized lymphocytes. These translocational stages in
    development are monitored on the basis of the immunological phenotype.
    For the CD4-CD8 phenotype, the cells change from CD-CD8- (so-called
    double-negative) at a very immature stage, then change to a CD4-CD8+
    stage and into a CD4+CD8+ (double-positive) phenotype, which is
    found on almost all lymphocytes in the cortex. In the medulla, T cells
    have the phenotype of mature cells, with distinct CD4+CD8- (about
    two-thirds) and CD4-CD8+ (about one-third) populations.

          This phenotypic change is accompanied by a crucial aspect of
    intrathymic T-cell maturation: genesis of the TCR, which consists of
    the alpha-ß heterodimer. Initially, the DNA genomic organization that
    encodes these chains is in germline configuration, with a variety of
    gene segments encoding the variable part of the receptor molecule.
    Before transcription and translation into TCR becomes possible,
    combinations have to be made of the gene segments that encode the
    variable and constant parts of the TCR. This process of gene
    rearrangement requires the thymic microenvironment, and only after it
    is completed can the cell synthesize the receptor. The receptor is
    then expressed on the cell membrane together with the CD3 molecule,
    which may act as the transmembrane signal transducing molecule after
    TCR stimulation; the CD3 molecule is already present in the cytoplasm
    of the cell even before the TCR has been synthesized. T Cells at this
    stage of maturation can be recognized by cytoplasmic staining with CD3

          The TCR gene rearrangement is similar to the rearrangement of
    genes that encode immunoglobulin heavy and light chains, which takes
    place in the bone-marrow microenvironment. Once TCR has been expressed
    at the surface, however, the cell undergoes a process unique to T
    cells, namely, specific selection on the basis of recognition
    specificity (Blackmann et al., 1990; Sprent et al., 1990; Von Boehmer,
    1990). First, the cell is examined for its capacity to recognize an
    antigen in the context of its own MHC (self-restriction); then it is
    allowed to expand (positive selection). Second, the cell is examined
    for its capacity to recognize a self-antigen (autoreactivity). If it
    recognizes a self-antigen, it is blocked from further differentiation
    (negative selection). In this way, the random pool of antigen
    recognition specificities of T cells is adapted to the host's
    situation; the total repertoire of the alpha-ß T-cell population

    (estimated at 1012 different epitopes) changes into the potentially
    available repertoire (estimated at recognition of about 106
    epitopes). Current theories of negative selection state that this step
    is not feasible for all putative autoantigens in the body. Rather, it
    applies to a selection of potentially harmful specificities (in
    particular MHC antigens). If a cell is not selected during positive or
    negative selection, it dies, possibly by suicide or apoptosis
    (McDonald & Lees, 1990). A hallmark of apoptosis is endonuclease-
    induced chromosomal fragmentation into 200 base-pair fragments
    (McConkey et al., 1990). Histologically, apoptosis is recognized by
    the presence of condensed, sometimes fragmented nuclei, which can be
    found in phagocytic macrophages ('tingible body' or 'starry-sky'
    macrophages) (Kendall, 1991).

           Function of the microenvironment: It is generally accepted that
    the epithelial microenvironment of the thymic cortex plays a major
    role in positive selection. This microenvironment expresses MHC class
    I and class II products and shows close interactions with lymphocytes
    morphologically (at the electron microscopic level). This close
    interaction is reflected in the complete inclusion of lymphocytes
    inside the epithelial cytoplasm ('thymic nurse cells') (Van Ewijk,
    1988). Negative selection has been ascribed to either the epithelial
    compartment or the medullary dendritic cells. The different processes
    occurring in early (cortical) and late (medullary) maturation are
    associated with differences in the microenvironment. Epithelial cells
    in the cortex and medulla differ in antigen expression,
    ultrastructural characteristics, and their capacity to synthesize
    thymic hormones such as thymulin, thymic humoral factor, thymosin, and
    thymopoietin. These hormones have a major function late in intrathymic
    T-cell maturation, and the major site of thymic hormone synthesis is
    the medullary epithelium (Dabrowski & Dabrowski-Bernstein, 1990).

          The cortex can be considered a primary lymphoid organ because it
    is an antigen-free microenvironment with a blood-thymus barrier. In
    contrast, antigens can move relatively freely into the medulla and
    encounter antigen-presenting dendritic cells as well as antigen-
    reactive T cells. Thus the medulla has the properties of a secondary
    lymphoid organ (Van Ewijk, 1988; Kendall, 1991).

           Ontogeny, growth, and involution: The thymus in rodents reaches
    full development at around day 17 of gestation, that is about five
    days before birth. In humans, a fully developed thymus is first seen
    at the 16th to 17th week of gestation (Von Gaudecker, 1986), which is
    relatively earlier in the gestation period than in rodents, since the
    human immune system is more mature at birth than that of rodents.
    Nevertheless, a thymus that appears histologically to be fully
    developed may be functionally somewhat immature. For instance, fetal
    thymus from humans (McCune et al., 1988; Namikawa et al., 1990) or
    rats (De Heer et al., 1993) can be transplanted into mice with the
    severe combined immunodeficiency  (scid) mutation and grow for longer

    than tissue obtained postnatally (see also section 4.5.3). After
    birth, when the individual first comes into contact with exogenous
    antigens, the thymus is called upon to provide large numbers of T
    cells to the periphery, and the organ grows in a relatively short time
    -- in humans within three to four weeks, from about 15 to 50 g. In
    rats, the organ reaches it largest relative size about one week after
    birth; the absolute weight is greatest at about six to eight weeks of
    age. After adulthood is reached, the thymus starts to involute
    (Steinmann, 1986; Kuper et al., 1990a; Schuurman et al., 1991a; Kuper
    et al., 1992a), a process that may be related to changes in the
    hormonal status of the individual; circulating thymic hormone is
    reduced to very low levels in adults. The underlying mechanisms are
    not fully understood. The consequences of age-associated involution
    are obvious: emigration of lymphocytes from the thymus decreases
    dramatically, from, for instance, 1.6 × 106/day in one-month-old
    mice to 4 × 104/day in one-year-old mice (Stutman, 1986; Shortman et
    al., 1990). Apparently, the persistent generation of a new antigen-
    recognition repertoire in the T-cell population of adults is not
    needed. Instead, the body can defend itself using the established
    repertoire and extrathymic self-renewal of the T cells. Similar
    processes may occur after artificial involution of the thymus caused
    by toxic compounds or acute stress (including acute disease); this
    aspect is further discussed below.

          It should be emphasized that the basic architecture of the thymus
    is not a fixed histological entity; its features depend on the age and
    the stress hormone status of the individual. A 'normal' architecture
    can be expected only between the late gestational period and young
    adulthood, before the start of age-associated involution. This
    phenomenon has important implications for the selection of rodents
    according to age for studies of immune function and in the
    interpretation of studies of immunotoxicity.

          In mice, severe but reversible changes occur in the thymus during
    pregnancy (Clarke, 1984; Clarke & Kendall, 1989; see also Figure 9).
    The weight of the thymus shows an initial small rise in early
    pregnancy, from about 35 to 40 mg, and dramatically decreases to 15 mg
    or less at day 17 of pregnancy (Clarke, 1984). This involution is
    associated with severe lymphodepletion of the cortex. Cell death is
    seen by the presence of apoptotic figures and phagocytosis in
    macrophages and epithelial cells. Remarkably, the large lymphoblasts
    in the outer cortex remain relatively unattached, and the same applies
    for thymocytes in thymic nurse cells. In birds, changes with breeding
    sessions have been found (Kendall & Ward, 1974; Ward & Kendall, 1975).
    In a study of a wild population of adult red-billed queleas, cyclical
    enlargement and regression of the thymus were documented; at the time
    of mating and laying, most birds, irrespective of sex, showed an
    involuted thymus; on subsequent egg incubation the thymus size
    increased, with a decline in the latter half of the rearing period.  Lymph nodes

          A finely branched lymph vessel system (lymphatics) is involved in
    the return of interstitial fluid in tissue to the blood circulation,
    with lymph nodes spaced at regular intervals (Dunn, 1954; Tilney,
    1971). The major sites of lymph nodes (or groups of lymph nodes) are
    shown in Figure 3; a scheme of the areas drained by distinct lymph
    nodes or lymph node groups is given in Figure 10, a schematic
    presentation of individual lymph node architecture is presented in
    Figure 11, and the histology of a lymph node is shown in Figure 12.
    The main functions of lymph nodes are to filter pathogens from the
    afferent lymph and then to initialize immune reactions. The afferent
    lymphatics penetrate the lymph node capsule and connect with the
    subcapsular sinus, which in turn connects with the cortical and
    medullary sinuses. On the basis of the lymph flow through the node,
    basic units can be recognized, each of which is supplied by its own
    afferent lymph vessel and which comprise part of the paracortex
    (Bélisle & Sainte-Marie, 1981; Sainte-Marie et al., 1990). Afferent
    and efferent blood vessels are connected to the organ at the hilus,
    where the lymph leaves the node via the efferent lymphatic(s). The
    efferent lymphatics drain into other lymph nodes or directly into the
    thoracic duct, which enters the bloodstream, in the rat via the left
    subclavian vein.

          Lymph vessels and lymph nodes occur only in mammals. In some
    birds and monotremes (primitive mammals), primitive lymph nodes
    directly interposed in the lymph circulation have been described.
    Ectothermic vertebrates do not have lymph nodes. Some small lymphoid
    organs such as lymph glands and jugular bodies have been described in
    some species of frogs but not in others. These organs presumably
    filter antigens from both blood and lymph and have been claimed to be
    phylogenetic precursors of mammalian lymph nodes. Likewise, small
    lymphoid aggregates associated with the cardinal veins occur in some

          Lymph nodes are surrounded by a connective tissue capsule. The
    nodes comprise various compartments or microenvironments: (i) the
    outer cortex, with follicles and interfollicular areas; (ii) the inner
    cortex or paracortex; and (iii) the medulla, with medullary cords and
    medullary sinuses. These compartments are easily differentiated into
    sections after conventional histological staining. In the cortex,
    interfollicular areas and the paracortex (T-lymphocyte area) are
    differentiated from follicles by the presence of blood vessels lined
    by high endothelium (high endothelial postcapillary venules, discussed
    below). Follicles (B lymphocyte area) are rounded structures, located

    FIGURE 9

    FIGURE 10

    FIGURE 10a

    FIGURE 10b

    FIGURE 11

    FIGURE 12

    FIGURE 12a

    FIGURE 12b

    mainly immediately underneath the capsule; they present as
    accumulations of small lymphocytes (primary follicle) or a pale-
    stained centre with large lymphoid cells (centrocytes, centroblasts)
    and tingible-body macro-phages surrounded by a mantle with small
    lymphocytes (secondary follicles). The interfollicular areas are
    continuous with the paracortex, and the latter is continuous with the
    medullary cords.

          The arterial blood supply enters the node at the medulla and ends
    in the paracortex as arteriolar capillaries, with branches in the
    follicles. The capillaries feed venules that are lined with high
    endothelial cells. The high endothelial venules run from the
    paracortex into the medullary cords and then leave the node via the
    vein in the hilus. Lymphocytes migrate through the high endothelial
    venules after adhering to the endothelium by specific receptor-ligand
    interactions (Picker & Butcher, 1992). The adhesion molecules on
    lymphocytes and endothelium that are involved in this binding and
    subsequent passage thought the endothelial layer are the addressins,
    reflecting the difference in receptor-ligand interactions that exists
    between lymph nodes of the internal and external lymphoid systems.
    Subsequently, lymphocytes can specifically reach the various nodes,
    using the same route (the blood). After the lymphocytes have migrated
    into the parenchyma, they move into their microenvironment or

           Antigen encounter and immune reactivity: The main route of
    access for antigens and pathogens is the afferent lymph flow; antigens
    can also come into contact with the lymph node tissues via the blood.
    Antigens in the lymph, either free or processed by veiled macrophages,
    enter the node through the subcapsular sinus, which is rich in
    macrophages that can phagocytose free antigen. From there, antigens
    move to the paracortex, where they are presented to CD4+ Th cells by
    the antigen-presenting cells for initiation of the immune response.
    The main antigen-presenting cells in the lymph node are the
    interdigitating dendritic cells, the tissue equivalents of veiled
    macrophages, which can arise from Langerhans cells in the skin (for
    e.g. lymph nodes draining the skin). The antigen-presenting cells
    express MHC class II antigens in high density, enabling the alpha-ß
    TCR of the Th cells to recognize the antigenic determinant complexed
    with the polymorphic ('self') MHC class II molecule. The CD4+
    molecule on the Th cell surface binds to a non-polymorphic determinant
    of MHC class II molecules and strengthens the binding between Th and
    antigen-presenting cells (Janeway, 1992). The cellular interaction
    triggers the synthesis of cytokines like IL-1 and IL-2. This process
    is down-regulated by Ts cells in a way that is not yet completely

          Follicles are involved in antigen-driven B-cell activation,
    somatic mutation, positive and negative selection, and memory and
    plasma cell development (Szakal et al., 1989; Kroese et al., 1990; Liu
    et al., 1992) and are known as primary follicles in the resting state.
    They contain small, IgM+IgD+ virgin B cells in a framework of
    follicular dendritic cells. During stimulation by antigens, the
    follicles change into secondary follicles consisting of a germinal
    centre surrounded by a mantle. Antigen may be transported into the
    follicle by immune (T) cells, but this route is not yet fully
    established. Antigen is presented to B cells in immune complexes, with
    complement split products like C3b, which are trapped in cytoplasmic
    extensions of the follicular dendritic cells. The interaction between
    complement and complement receptors on these cells has a pivotal role
    in the adherence of antigen to them. Fc receptors also play a role,
    but only in rodents. The complement split products in the trapped
    immune complexes have an accessory function in antigen presentation.

          Local CD4+ Th cells assist in B-cell activation. Antigen-driven
    B-cell activation and proliferation in the germinal centre are
    accompanied by an isotype switch of the immunoglobulin class
    synthesized by the B cell. In addition, the affinity of the antibody
    increases as a result of somatic mutation (Kocks & Rajewski, 1989). A
    kind of selection mechanism has been proposed in this antigen-driven
    process, in which cells that produce antibody of higher affinity are
    selected preferentially, and cells that produce antibody of lower
    affinity are not selected and subsequently die, perhaps by apoptosis
    (programmed cell death) (Liu et al., 1989). This selection resembles
    that of developing T lymphocytes in the thymus; it differs from T-cell
    selection by the absence of negative selection and the occurrence of
    somatic mutation. Antigen may remain in the follicular compartment for
    quite some time, thereby causing persistent activation of B cells,
    related to the state of immunological memory within the B-cell
    population. After the antigen disappears, the immunological memory in
    the B cells is short-lived and disappears. B-Cell activation in
    germinal centres leads to activated cells with a specific morphology,
    the so-called centrocytes and centroblasts. Finally, B-cell activation
    leads to the formation of plasma cells, both in the periphery of
    germinal centres but more predominantly in the medullary cords of the
    lymph nodes.

          The main site of effector immune reactions is the medulla. The
    medullary cords house macrophages, granulocytes, activated effector T
    cells, and plasma cells. The effector T cells include CD4+ delayed-
    type hypersensitivity T cells (mediator-producing cells) and CD8+ Tc
    cells. The Tc cells bear an alpha-ß TCR that recognizes antigen in the
    context of the polymorphic determinant of MHC class I molecules. The
    CD8 molecule has an accessory function in this process, since it binds
    a non-polymorphic class I determinant (Janeway, 1992). Antigen
    recognition by Tc cells is thus different from that by Th cells. The

    reaction products of the effector cells, such as lymphokines, and
    effector cells like plasma cell precursors leave the lymph nodes via
    the efferent lymph or blood circulation to go to other sites of the

           Development and aging: Lymph node morphology is dynamic: its
    appearance throughout life is directly related to the type and amount
    of antigenic stimulation. After antigenic contact, the organ increases
    in size within a relatively short time, with high proliferative
    activity of lymphocytes and germinal centre formation, depending on
    the type of reaction and the choice of immunological reaction. In the
    case of B-lymphocyte reactions, hyperplasia of follicles is seen (e.g.
    after bacterial infection); in the case of T-cell reactions, the
    interfollicular areas or paracortex become enlarged (e.g. in viral
    infection). After the reaction is terminated or is transferred to the
    next draining lymph node, it regains its normal small size. Germinal
    centres with an interfollicular microenvironment can develop
    extranodally, especially at sites of chronic inflammation.

          The dynamics of the lymph node can be illustrated by several
    examples. After immunization of the footpad with antigen mixed with an
    adjuvant, such as Freund's complete adjuvant, containing killed
    mycobacteria, the draining popliteal lymph node becomes enlarged (in
    rats, from about 5 mg to more than 100 mg), and granulomatous
    reactions (epitheloid-cell granuloma) can be seen histologically. The
    swelling of lymph nodes is used to assess reactivity towards chemicals
    and in the evaluation of immunomodulatory drugs (Gleichmann et al.,
    1989). In the popliteal lymph node assay, a test substance is injected
    subcutaneously into one footpad and the contralateral side is left
    untreated or injected with vehicle only. The effect of the substance
    is subsequently estimated from the difference in weight between the
    popliteal lymph nodes. Further evidence for immunostimulatory activity
     in vivo is obtained by histological appearance, often manifested as
    follicular hyperplasia.

          Lymph nodes develop relatively late in fetal life: At birth, the
    anlage of unstimulated lymph nodes is present, containing few lymph
    cells. The lymph nodes develop quickly after exposure to many new
    (exogenous) antigens. In adults, they may become relatively quiescent
    and small, with virgin T and B cells and primary follicles. The lymph
    nodes in aged rats are capable of the same degree of activity as those
    in young individuals upon antigenic stimuli. The state and type of
    activation in the various nodes of adult and aged animals differ under
    normal housing conditions and are a reflection of the absence or
    existence of continuous local stimulation with antigens or disease
    processes, like inflammation and the presence of a tumour in the
    drained area (Ward, 1990). The central nodes of the mandibular and
    superficial cervical group, which are continuously exposed to
    (aero)antigens via the oronasopharynx, may contain a considerable

    number of plasma cells and precursors in the medullary cords and
    relatively well-developed germinal centres. Sinal histiocytosis (that
    is, considerable numbers of macrophages in the sinuses) and
    accumulations of pigmented macrophages are often present in the
    mesenteric lymph nodes, which are continuously exposed to antigens via
    the digestive tract. An extensive review of lymph node development and
    aging is given by Losco & Harleman (1992).  Spleen

          The spleen consists of two main compartments: the red and white
    pulp (Van Rooijen et al., 1989; Dijkstra & Sminia, 1990; Laman et al.,
    1992; Van den Eertwegh et al., 1992). A schematic drawing of the
    spleen is presented in Figure 13 and histological views in Figure 14.
    The red pulp consists of blood-filled sinusoids and Bilroth's cords
    containing macrophages, lymphocytes, plasma cells, and NK cells.
    Macrophages perform major functions in clearing blood cells (for
    instance, old red blood cells) and in phagocytosis, especially of non-
    opsonized particles. This high-volume filter function is made possible
    by two factors: the direct contact, unobstructed by blood-vessel
    walls, between phagocytic cells and blood-borne particles; and the
    large blood supply, estimated at about 5% of the total blood volume
    per minute. There are no lymphatics in the spleen.

          The phagocytic function is especially important in the case of
    intravascular pathogenic microorganisms, before antibody formation and
    subsequent opsonization occur (early bacterial septicaemia). The
    mononuclear phagocyte system of the liver (Kupffer cells) plays a
    major role in the removal of opsonized particles. Together with the
    hepatic phagocytic system, splenic macrophages synthesize complement
    components, although this is done mainly by hepatocytes. In rats and
    mice, the red pulp contains nests of (extramedullary) haematopoiesis,
    characterized histologically by megakaryocytes and normoblasts. In the
    case of systemic septicaemia, when pathogenic microorganisms have
    reached the blood either directly or after inadequate filtering
    through lymph nodes, the red pulp increases and contains large
    proportions of (immature) granulocytes. Differentiation between
    septicaemia and extramedullary haematopoiesis is not always easy; the
    decreased or absent white pulp in septicaemia can be helpful in making
    this differentiation.

          Phylogenetically, cartilaginous fish are the first species that
    have a spleen, which consists of lymphoid follicles and a red pulp
    that generally houses developing erythroid cells (Zapata & Cooper,
    1990). The lympho-haematopoietic masses of the intestine that are seen
    in some lower vertebrates (e.g. lampreys) are not primitive spleens
    but rather primitive lymphohaematopoietic organs equivalent to
    mammalian bone marrow. In most bony fishes, the white pulp is poorly
    developed, probably reflecting the existence of other peripheral
    lymphoid organs, e.g. the kidney, which participate actively in the

    FIGURE 13

    FIGURE 14

    FIGURE 14a

    immune response. After antigenic stimulation, the amount of splenic
    lymphoid tissue increases considerably in all lower vertebrates,
    although germinal centres do not occur. At the cellular level,
    however, antigen-presenting cells and cells retaining immune complexes
    on their surface have been described in the spleen of some bony
    fishes, anurous species, and reptiles.

           White pulp: The spleen contains about one-quarter of the body's
    total lymphocyte population; during lymphocyte recirculation, more
    cells pass through the spleen than through all the lymph nodes.
    Lymphocytes in the spleen reside in the white pulp, which consists of
    a central arteriole surrounded by the periarteriolar lymphocyte
    sheath, a T-lymphocyte area. The outer sheath contains B lymphocytes
    and, after antigenic stimulation, plasma cells. Adjacent follicles
    contain B cells. Around the periarteriolar lymphocyte sheath and
    follicles is a corona containing B cells, called the marginal zone;
    this region is easily distinguished, especially in rats. The
    periarteriolar lymphocyte sheath has a microenvironment and a
    passenger leukocyte content similar to that of the lymph node
    paracortex. Some sources claim that the spleen is a rich source of Ts
    cell activity, exceeding that of lymph nodes. The follicles are not
    essentially different in structure and function from those of lymph
    nodes. The spleen performs a major function in humoral immunity by
    synthesizing IgM class antibodies, especially to blood-borne antigens.

          The microenvironment of the marginal zone is unique to the
    spleen. Histologically, the B cells at this site are of medium size;
    on histological staining, they are larger and paler than B cells in
    primary follicles and in the follicular mantle of secondary follicles
    (Figure 14). In addition, they do not show the morphology of the
    centrocytes or centroblasts found in germinal centres, and the
    phenotypic expression (surface IgM+IgD-) indicates that the B
    cells in the marginal zone are a separate population. Special
    macrophage types are present, which are known as marginal zone
    macrophages and marginal metallophilic macrophages. The latter are
    located at the periphery of the white pulp, along the inner border of
    the marginal sinus, and can be stained by silver impregnation.

          The physiological function of the marginal zone has been
    characterized recently. First, the site retains B-lymphocyte memory;
    second, it mediates humoral responses that do not directly involve
    T cells. These T-independent responses are elicited by polysaccharide
    antigens of encapsulated bacteria, which are present in repeating
    units on the microorganism and are presented to the B cells by
    marginal zone macrophages. The response may not be completely T cell-
    independent in all cases, as T cell-derived factors enhance the
    response to some of these antigens. The antibodies generated are
    mainly of the IgM class, as T-cell help is required for an isotype

          In conclusion, the main immunological function of the spleen is
    to defend the body's vascular compartment by generating T cell-
    independent IgM antibody responses to bacterial polysaccharides and by
    exerting an enormous phagocytic power. This function is lost after
    splenectomy, when reduced nonspecific phagocytosis of non-opsonized
    particles, lowered serum IgM levels, and increased susceptibility to
    infections by encapsulated bacteria have been described.  Mucosa-associated lymphoid tissue

          The secretory epithelial surfaces of the body form a major route
    of entry for potentially pathogenic substances. These surfaces include
    the epithelia of the gastrointestinal, upper and lower respiratory,
    and urogenital tracts (Miller & Nicklin, 1987; Sminia et al., 1989).
    The host response at these locations ranges from nonspecific
    constituents, such as a physical or mechanical component (epithelial
    barrier, motility of the gastrointestinal tract, and the mucociliary
    escalator in the respiratory tract), to a chemical component (low
    gastric pH, mucus, lysosomal and digestive enzymes), and antigen-
    specific components of the immune system.

          Nonspecific killer cells are found in significant numbers in the
    lungs and along the epithelium of the gastrointestinal tract, where
    lymphocyte-like cells have been found to kill pathogens, presumably
    without prior sensitization (Hanglow et al., 1990). In mice, these
    cells have been characterized as T cells with a gamma-delta TCR,
    which, in contrast to Tc cells that express alpha-ß TCR, kill targets
    in an MHC-nonrestricted manner (Raulet, 1989). The cells have antigen
    specificity that is encoded at the DNA level by variable gene
    segments, but the repertoire appears to be smaller than that which
    encodes TCR alpha or ß chains. These gamma-delta T killer cells are
    not generated under the strict influence of the thymus, as are their
    alpha-ß T-cell counterparts (Bell, 1989; Haas et al., 1993). Apart
    from their killer activity, these cells may serve as inducing elements
    for the response mediated by alpha-ß TCR-expressing T subsets. In this
    initiating activity, gamma-delta TCR molecules shed from the
    lymphocyte surface may act as antigen-specific factors (De Weger et
    al., 1989).

           Lymphoid tissue: Lymphoid tissue occurs just underneath the
    secretory epithelium, in the duodenum and jejunum as Peyer's patches
    (Figure 15), in the appendix of the large intestine, along the bronchi
    (Sminia et al., 1989), and in the oro- and nasopharyngeal regions
    (Kuper et al., 1992b). These mucosal lymphoid tissues share structural
    and functional characteristics and are strongly interrelated. The
    common designation 'mucosa-associated lymphoid tissue' (MALT) is
    therefore used to refer to bronchus-associated, gut-associated, and
    nasal-associated (nasal cavity and nasopharynx) lymphoid tissue.
    Nasal-associated lymphoid tissue has been identified in horses,

    monkeys, and rats (Figure 16) (Kuper et al., 1990b). In humans and
    domestic animals, the larger lymphoid nodules in the pharyngeal region
    are called tonsils. Together with the intermediate lymphoid tissue,
    the tonsils form Waldeyer's tonsillar ring.

          The organization of MALT is similar to that of lymph nodes, with
    B cell-containing follicles and T cell-containing interfollicular
    areas. Afferent lymph vessels are lacking, because pathogens can enter
    the tissue through the covering epithelial layer. The epithelial cells
    at this location (the 'M' or microfold cells) are often thinner than
    those at other secretory sites, in order to enable efficient passage
    of antigens. Stimulated gut-associated lymphoid tissue and human
    tonsils often have prominent follicles with germinal centres. In
    contrast, germinal centres are scarce in stimulated bronchus-
    associated and nasal-associated lymphoid tissue in rodents, due to the
    fact that immunological reactions occur mainly in the draining
    cervical lymph node. Th medulla-like areas seen in lymph nodes are
    absent in MALT. Lymphocytes and NK cells are found in the lymphoid
    tissue, in interstitial tissue in the lung, and in the lamina propria
    along the gastrointestinal tract.

          The homing specificity of lymphocytes into lymphoid tissue by
    migration through high endothelial venules is described above. The
    specificity of the homing phenomenon to MALT has the advantage that
    the same circulation pathway (i.e. the blood) is used by the secretory
    and internal immune system (with the intrinsic possibility of mutual
    contact). In addition, the antigen message received at one secretory
    site is followed by effects at all secretory surfaces. Thus, after
    antigen presentation in the gastrointestinal tract, effector cells
    (e.g. IgA antibody-synthesizing plasma cells, see below) are found at
    the site of stimulation and at other secretory sites (e.g. the
    respiratory tract). Thus, the major function of MALT is to initiate
    immune responses, which are then passed on to draining lymph nodes,
    such as mesenteric lymph nodes in the gastrointestinal tract.

           The secretory IgA antibody response: The immune response in
    MALT differs from that at other sites of the body in that it is
    devoted to the generation of an IgA antibody response. Thus, MALT
    contains precursors of IgA antibody plasma cells and populations of T
    cells capable of promoting a B-cell immunoglobulin class switch into
    IgA-producing B cells or plasma cells. B-Cell differentiation into
    IgA-producing plasma cells after local antigen presentation is
    accompanied by lymphocyte migration and specific homing. Precursors
    move through draining lymph nodes into the blood and from there to the
    secretory surface, where they lodge as IgA plasma cells in the lamina
    propria. Specific homing mechanisms exist by which these cells are
    able to select the secretory surface of their final mucosal

    FIGURE 15

    FIGURE 16

          In contrast to IgA produced by the bone marrow and circulating in
    blood, IgA synthesized by plasma cells of MALT consists of a dimeric
    immunoglobulin subunit. The two monomers are linked by a polypeptide
    called the J chain (about 15 kDa). These IgA antibodies have their
    main effect outside the body itself, for example in salivary and
    gastrointestinal secretions. The transport from the site of synthesis
    across the epithelial barrier is specifically adapted for dimeric IgA,
    and to a lesser extent for pentameric IgM. Epithelial cells express a
    receptor for these immunoglobulins, called secretory component
    (a polypeptide of about 70 kDa). After binding to this receptor, the
    molecule is transported through the epithelium, possibly through its
    cytoplasm, and excreted on the luminal surface. During this process,
    secretory component attaches to the immunoglobulin molecule (coiled
    around the Fc fragments); the composite molecule, comprising dimeric
    IgA, the J chain, and secretory component, is called secretory IgA. In
    rodents, a similar secretory component-mediated transport occurs in
    the liver (Brown & Kloppel, 1989). Here, a secretory component on the
    hepatocyte surface mediates the passage of dimeric IgA from the
    sinusoids to the bile canaliculi. In this way, dimeric IgA entering
    the liver by the portal vein efficiently recirculates to the bile and
    from there into the gastrointestinal lumen. Secretory IgA is more
    resistant to luminal conditions (especially proteolytic enzymes) than
    dimeric IgA and is thus better able to function there.

          IgA lacks the effector reactivity of IgM and IgG in complement
    activation by the classical cascade, opsonization and phagocytosis, or
    antibody-mediated cellular cytotoxicity. This lack appears to be
    related to the absence of effector systems (complement, phagocytes) in
    secretory fluid. The main function of IgA is to prevent the entry of
    potentially pathogenic substances into the body, by a specific antigen
    exclusion function in which the epithelium is coated with 'antiseptic

           Induction of immunological tolerance: A final feature of MALT
    is its capacity to generate immunological tolerance. After antigenic
    priming at secretory surfaces, subsequent systemic antigenic challenge
    often results in nonresponsiveness (Strobel & Ferguson, 1984;
    Challacombe, 1987; Holt & Sedgwick, 1987; Mowat, 1987). Suppressor
    cells have been found in the spleen and suppressor factors in the
    circulation after local immunization. The induction of tolerance
    pertains primarily to dead microorganisms and inactivated proteins
    which come into contact with the MALT. The mechanism of tolerance
    induction and different responses to live and dead microorganisms is
    not completely defined but is important in tolerance to food antigens
    and the development of food allergies.

          In summary, the host's defence in MALT is different from the
    response of the internal immune system. The responses at this first
    line of defence range from NK cell activity in the epithelium to
    specific IgA antibody-mediated exclusion in the secretory fluid.
    Immune responses to antigens entering the body at secretory sites are
    initiated by lymphocytes in the epithelium, in lymphoid tissue
    immediately underneath the epithelium, and in the lymph nodes that
    drain the site (e.g. the mesenteric node). The liver may also
    contribute to the response, as antigens passing directly into the
    portal vein are efficiently removed and processed by the hepatic
    mononuclear phagocyte system (Kupffer cells). Because MALT can
    function independently of the internal immune system, blood analysis
    alone may not provide complete information on MALT. Instead, analysis
    of secretory fluids, such as saliva (for IgA antibody), bronchoalveolar
    lavage fluid, and jejunal fluid, or direct investigation of the tissue
    itself, are more appropriate approaches.  Skin immune system or skin-associated lymphoid tissue

          As the skin is the largest organ of the body, its principal
    physical function is to act as a barrier to water-soluble compounds,
    to mechanical trauma, and to trauma caused by potentially pathogenic
    microorganisms and the photons of sunlight. The physicochemical
    characteristics of the outermost layer, the corneal or horny substance
    of the epidermis, underlie the resistance to exogenous pathogenic
    substances. The skin also has a host defence function that can be
    designated as immunological. Some studies have suggested that the skin
    might function as a primary organ (Fichtelius et al., 1970; Bos &
    Kapsenberg, 1986), but most of the relevant immune reactions in the
    skin appear to be antigen driven. The components of the skin immune
    system, or skin-associated lymphoid tissue, are the following
    (Streilein, 1983, 1990) (Figure 17): (i) Langerhans cells in the
    epidermis, which are adapted for processing antigen and transporting
    it to the draining lymph node, where they are called interdigitating
    cells and present the antigen to lymphocytes; (ii) epider-motrophic
    recirculating T lymphocyte subpopulations (homing T lymphocytes);

    (iii) keratinocytes, which can synthesize cytokines after activation,
    thereby influencing T-cell differentiation and haematopoiesis; they
    can have an antigen-presenting function, especially after activation
    resulting in MHC class II expression; (iv) Thy-1+ dendritic epidermal
    cells, described in rodent skin epidermis: a special T cell that bears
    the gamma-delta TCR and has an antigen-presenting function; and (v)
    skin-draining lymph nodes comprising high endothelial venules through
    which lymphocytes enter from the blood circulation.

    FIGURE 17

          Immune components exist not only in the epidermis but also in the
    dermis. At this location, T cells and macrophages have preferential
    distributions, especially in the papillary region. T Cells,
    macrophages, mast cells, endothelial cells, and dendritic cells are
    found in the connective tissue of the dermis, as in connective tissue
    at other locations in the body. The reactivity of these cells in the
    dermis may differ, however, from those at other locations. For
    instance, skin mast cells (Van Loveren et al., 1990a) behave
    differently from mast cells at other places. These immunological
    components cannot always be recognized in conventional histological
    preparations of skin. For instance, Langerhans cells and dendritic
    epidermal cells require special immunohistological staining (Figure

          Various inflammatory and immune mediators are also present in
    skin. These include antimicrobial peptides, complement components,
    immunoglobulins, cytokines, fibrinolysins, eicosanoids, and
    neuropeptides. They are partly derived from the blood circulation and
    are partly of local origin (Bos, 1990). Streilein (1990) described the
    function of the skin-associated lymphoid tissue as follows: induction
    of primary immune responses to new cutaneous antigens, expression of
    immunity to previously encountered antigens, and avoidance of
    deleterious immune responses to non-threatening cutaneous antigens.

    1.3  Pathophysiology

    1.3.1  Susceptibility to toxic action

          The dynamic nature of the immune system renders it especially
    vulnerable to toxic influences. The major target sites of the immune
    system for toxicity are presented in Figure 18. The reactions of
    lymphoid cells are associated with gene amplification, transcription,
    and translation, and compounds that affect these processes of cell
    proliferation and differentiation are especially immunotoxic,
    particularly to the rapidly dividing thymocytes and haematopoietic
    cells of the bone marrow. Thus, the disappearance of lymphoid cells
    from bone marrow, blood, and tissue, and thymus weight may be the
    first and most obvious signs of toxicity, as was seen in application
    of the tiered approach to assessing the immunotoxicity of pesticides,
    mentioned previously (Vos & Krajnc, 1983; Vos et al., 1983a). Effects
    on the constituents of the framework (stationary stroma), which
    support and steer the activation, proliferation, and differentiation
    of lymphoid cells, are observed less often (Krajnc-Franken et al.,
    1990; Schuurman et al., 1991b). Such effects result mostly in
    degeneration, ending in atrophy and fibrosis. Alternatively, framework
    cells and passenger leukocytes may persist but are rendered unable to
    function by the toxic insult, and the delicate interactions between
    these cells may be affected. This result of toxicity is not always

    observed by conventional histology; it may be visualized by changes in
    immunolabelling for markers that have functional significance.
    Otherwise, functional assessment  in vivo or  in vitro is required.
    As shown in Figure 18, the effects on specific cells in lymphoid
    tissues are generally reflected in altered histology of lymphoid
    organs, but this is not always the case for effects on responses.

          The skin, respiratory tract, and gastrointestinal tract together
    form an enormous surface that is in close contact with the outside
    world and is potentially exposed to a vast magnitude of microbial
    agents and potential toxicants. The toxic effects on these components
    of the immune system can differ in (histo)pathological manifestation
    from those on internal lymphoid organs. In the human respiratory
    tract, these effects include asthma, fibrosis, and pulmonary
    infections. Examples of inhaled pollutants that may induce these
    effects are oxidant gases and particulates such as silica, asbestos,
    and coal dust. The cellular and biochemical profiles of
    bronchoalveolar lavage constituents after exposure of experimental
    animals and humans by inhalation (Koren et al., 1989) are valuable for
    screening immune-mediated lung injury. The products of pulmonary
    epithelial cells and alveolar macrophages appear to be key factors. A
    number of studies have indicated that lung disease progresses with
    postactivational release of cytokines, such as IL-1, tumour necrosis
    factor, platelet-derived growth factor, and transforming growth
    factors. Alveolar macrophages secrete not only cytokines but also a
    variety of short-lived products that may contribute to altered
    resistance to pulmonary infections and inflammation; these include
    reactive oxygen species, such as superoxide, nitric oxide, and
    hydrogen peroxide, and arachidonic acid metabolites. The overall
    suppression of these humoral systems, in combination with effects on
    e.g. NK cells, may predispose individuals to infectious agents or
    tumour development or may alter the inflammatory and degenerative
    response (Van Loveren et al., 1990b; Khan & Gupta, 1991; Denis, 1992;
    Denis et al., 1993).

          The skin is an important target in immunotoxicology, for instance
    for chemical allergens (Kimber & Cumberbatch, 1992) and ultraviolet B
    (UVB) radiation (Goettsch et al., 1993). The skin can respond to many
    xenobiotics by a specific immune response (contact hypersensitivity)
    or by a nonspecific inflammatory response (contact irritancy); both
    responses are associated with the induction of pro-inflammatory
    cytokines. The cells of the immune system are readily recruited from
    the circulation to the skin in response to dermal stimulation by
    xenobiotics. In addition, various resident immune cells can be
    activated, for instance Langerhans cells during the induction of the

    FIGURE 18

    contact hypersensitivity response. Soluble mediators can be produced
    locally, and antigen-antibody complexes can be formed at the site of
    inflammation. Exogenous factors such as ultraviolet light (Applegate
    et al., 1989) and 7,12-dimethylbenz[ a]anthracene (DMBA) (Halliday et
    al., 1988) can cause the disappearance of Langerhans cells from the
    skin (or the loss of their function), with consequent disturbance or
    dysregulation of the skin's immune function. Keratinocytes, which
    comprise the vast majority of cells in the epidermis, have an
    important role in immune and inflammatory responses, serving as a
    significant source of cytokines, which contribute either
    quantitatively or qualitatively to the nature of the response of the
    skin to exogenous stimulation.

          Reactions to drugs illustrate the skin's susceptibility to toxic
    influences. Cytostatic drugs used in cancer therapy often induce bone-
    marrow depression as a major side-effect, resulting in an increased
    risk for infections, so that blood leukocyte counts, which reflect
    bone-marrow depression, must be monitored during administration.
    Conversely, a number of cytotostatic drugs are immunosuppressive. A
    well-known example is azathioprine (see section A number of
    new immunosuppressive drugs inhibit DNA synthesis, including
    Mizoribine (Bredinin, an imidazole nucleoside), Brequinar sodium (a
    quinoline carboxylic acid derivative), and RS61443 (morpholino-
    ethylester of mycophenolic acid) (Thomson, 1992). Their specificity to
    cells of the immune system may be related to the distinct pathways in
    purine and pyrimidine metabolism that are preferentially used by
    lymphocytes, such as guanine nucleotide synthesis promoted by inosine
    monophosphate dehydrogenase in the case of Mizoribine and RS61443.
    Another example of the particular sensitivity of the immune system to
    toxic damage is its response to irradiation. Ionizing radiation is
    commonly used in cancer therapy. Of the body's constituents, the
    haematopoietic system is particularly sensitive to irradiation; when
    pluripotent stem cells are affected, regenerative activity is lost.
    Other systems destroyed by this treatment, like the intestinal
    epithelium, have an intrinsic self-renewal capacity and do not need
    replacement therapy. The lymphoid constituents of the immune system
    differ in radiosensitivity. The dose of radiation that causes a
    reduction in the cell population by a factor of 1/e (or 0.37), the
    D0, has been estimated at about 0.9 Gy for bone-marrow lymphoid stem
    cells, 0.4-0.9 Gy for pre-B cells, and 0.7 Gy for peripheral
    lymphocytes (Anderson & Williams, 1977; Anderson et al., 1977). The
    thymus is very sensitive to irradiation (Sharp & Watkins, 1981;
    Anderson et al., 1986; Adkins et al., 1988). Cortical lymphocytes
    manifest a D0 value of about 0.6 Gy. The intrathymic T-cell
    precursor has a D0 value of about 1.4 Gy. A refractory population
    (about 20-30%) is present within the medulla, but most cells are
    radiosensitive (D0, about 0.7 Gy). The capacity of thymic stroma to
    support precursor T-cell processing is radioresistant (Huiskamp et
    al., 1988). In addition to cell depletion, peripheral lymphoid tissues

    manifest decreased lymphocyte recirculation at a dose of only 0.5 Gy
    (Anderson et al., 1977), suggesting that high endothelial venules, or
    cell surface molecules involved in lymphocyte migration through these
    venules, are radiosensitive. The sensitivity of lymphocytes to
    ionizing radiation cannot be ascribed solely to their susceptibility
    to death during proliferation. Cells in the resting state disappear
    after irradiation, at a time that does not correspond to their
    physiological half-life. Apparently, the death of lymphocytes occurs
    at phases between cell division. This phenomenon, known as interphase
    death, appears to be a distinct characteristic of lymphocytes and
    sensitizes the immune system to radiation. Thus, only peripheral blood
    lymphocytes need be assessed as dosimeters after accidental
    irradiation (such as at Chernobyl in 1986).

           Toxicity to the thymus: Of the lymphoid cells of the body, the
    lymphocytes of the thymus (thymocytes) are especially susceptible to
    the action of toxic compounds (Schuurman et al., 1992d). Table 4
    presents data on the susceptibility of various thymic components to
    toxic damage, illustrating the particular vulnerability of the
    passenger lymphocyte population. The microenvironment appears to be
    more resistant, mainly on the basis of histopathological assessment.
    Thymocyte depletion, suggestive of toxicity towards this population,
    may actually be an indirect effect, in that the microenvironment is
    damaged and unable to support thymocyte growth. This situation may
    exist in the thymus after exposure to 2,3,7,8-tetrachlorodibenzo-
     para-dioxin (TCDD), which preferentially attacks the thymic
    reticular epithelium, resulting in lymphocyte depletion histologically
    (see section Similarly, the reduction in the cellularity of
    the medulla of the thymus after treatment with cyclosporin A is due to
    the absence of the medullary lymphocyte population, but the basis of
    the reduction is the disappearance of dendritic cells (as seen by
    immunohistochemistry; see section

          The susceptibility of thymocytes to toxicity is also related to
    their fragile composition, especially cortical thymocytes, and to the
    delicate interactions between them and their microenvironment. For
    instance, they are programmed to enter apoptosis when activated during
    the physiological process of selection. A decrease in size or
    involution of the organ may thus be the first manifestation of
    toxicity. It should be noted that stress itself can induce thymic
    involution; furthermore, thymic status is dependent on nutritional
    status and age. The main function of the thymus is to generate the
    T-cell repertoire during fetal and early postnatal life. Its
    susceptibility to toxic compounds and the subsequent effects on the
    cell-mediated immune system are most evident during this period of

        Table 4.  Sensitivity of cell populations in the thymus to toxic chemicals

    Cells                         Location            Compound

      Immature lymphoblasts       Outer cortex        Some organotin compounds,

      Small cells                 Cortex              Glucocorticosteroids, cytostatic drugs
                                                      (e.g. azathioprine)

      Intermediate-sized cells    Medulla             Ammonia caramel (THI)

    Epithelial cells              Cortex              2,3,7,8-Tetrachlorodibenzo-para-dioxin

                                  Medulla             Cyclosporin, FK-506

    Dendritic cells               Medulla             Cyclosporin, FK-506

    Macrophages                   Cortex              Ammonia caramel (THI)

    THI, 2-acetyl-4(5)-tetrahydroxybutylimidazole; caramel colour III
          An ever-increasing number of toxic compounds has been shown to
    affect the immune system. Induced and spontaneous immunopathology in
    rodents have been described in reviews and textbooks (Dean et al.,
    1982, 1985; Irons, 1985; Descotes, 1986; Lebish et al., 1986; Gopinath
    et al., 1987; Luster et al., 1989; Vos & Luster, 1989; Jones et al.,
    1990; Krajnc-Franken et al., 1990; Vos & Krajnc-Franken, 1990;
    Schuurman et al., 1991b; Dean et al., 1994; Frith et al., in press).
    The weight and histology of lymphoid organs are the main parameters in
    evaluating toxicity. The examples given in section 2.2 illustrate the
    various ways in which the immune system is injured.

    1.3.2  Regeneration

          The dynamic nature of the immune system provides it with a strong
    regenerative capacity. In principle, the leukocyte population is
    generated by a single pluripotent progenitor cell in the bone marrow.
    In the thymus, single thymocyte precursors have an enormous capacity
    for expansion (Ezine et al., 1984). Thus, after exposure to xenobiotic
    compounds that destroy the immune system, regeneration can occur
    within a relatively short time: The original architecture is restored

    within three to four weeks after involution caused by radiation or
    treatment with glucocorticosteroid or organotin compounds. The
    restoration process can occur in waves, depending on the sensitivity
    of the precursor T cells in the thymus and bone marrow (Huiskamp et
    al., 1983; Penit & Ezine, 1989). Regeneration does not occur after
    destruction of the white blood cell system, e.g. by sublethal
    irradiation, when the stem cells in the bone marrow are affected. In
    such cases, bone-marrow transplantation is required, and the anlage of
    the lymphoid organs then supports generation of the newly built immune
    system. After bone-marrow transplantation, polymorphonuclear
    granulocytes are the first cells to appear in the circulation, within
    two to three weeks; lymphocytes appear after three to four weeks;
    however, establishment of a fully developed T-cell system takes about
    six months (De Gast et al., 1985). These examples illustrate the
    vulnerability of the bone marrow-derived component to toxic action;
    regeneration requires substitution of this component and not the
    relatively more resistant stromal component in lymphoid tissue.

    1.3.3  Changes in lymphoid organs

          The weight and gross morphology of lymphoid organs are the first
    parameters studied in assessing toxicity, as the response to injury is
    often expressed as changes in tissue or organ weight, size, colour,
    and gross appearance. These observations are combined with leukocyte
    counts, studies of differentiation, and the results of
    histopathological evaluations of lymphoid organs and tissues.
    Conventional histopathology allows evaluation of the effects of
    xenobiotics on the main cell subsets on the basis of their distinct
    morphology, tissue location, and density. In this way, the effects on
    lymphocytes, lymphoblasts, and stromal cells can be evaluated
    (Schuurman et al., 1994; Kuper et al., 1995). The disappearance of
    lymphocytes from blood and tissues and the decrease in size and weight
    are often first seen in the thymus, as this organ is very sensitive to
    toxicity (see above). Evaluations of effects on distinct
    subpopulations must be confirmed by immunohistochemical
    characterization (Schuurman et al., 1992a). The sensitivity of
    histopathology can be increased by quantitative microscopy and
    morphometry and organ cell counts.

          During microscopic examination, important aspects to be
    considered are the cell density and the size of the various
    compartments of the lymphoid organs, as well as qualitative changes
    like germinal centre development; however, microscopic examination of
    lymphoid organs reveals these highly dynamic and complex processes
    only at a static stage. Changes in the number of cells are best
    reported by descriptive terms like 'increases' or 'reductions' in
    cellularity rather than by interpretive terms like 'involution',
    'atrophy', or 'hyperplasia'. The pathology working group of the IPCS-
    European Union international collaborative immunotoxicity study has
    started to develop such a descriptive approach. A window of control

    values and gross and microscopic appearance must be established for
    recognition of deviations from normal; however, normal and control
    values and histological features are influenced by various endogenous
    and exogenous factors, including the age and hormonal (especially sex
    hormone) status of the animal (Kammüller et al., 1989; Goonewardene &
    Murasko, 1992; Kuper et al., 1992a; Losco, 1992; Losco & Harleman,
    1992). The influence of sex hormonal status on the histology and
    histophysiology of lymphoid organs is illustrated by the following
    examples. Considerable changes are seen in the lymphoid organs of mice
    during pregnancy (Clarke, 1984), in birds during hatching (see
    Section 2), and in ectothermic vertebrates during different seasons of
    the year (see section Castration results in an increase in
    thymic size in old animals (Kendall et al., 1990), as has been
    observed in old rats with an involuted thymus after treatment with an
    analogue of luteinizing hormone-releasing hormone (Greenstein et al.,
    1987). Figure 19 shows the wet thymic weight of groups of male rats in
    order to illustrate these phenomena: the mean values were about 500 mg
    in young male rats at 2.5 months of age, about 125 mg in rats at 12-15
    months of age, about 250 mg in aged rats after surgical castration,
    and about 300 mg in aged rats after treatment with the hormone
    (Kendall et al., 1990).

          The neuroendocrine system and the sex steroid hormone balance may
    also underlie the changes in lymphoid organs of ectothermic
    vertebrates with seasonal changes like temperature and photoperiod
    (mentioned above). The thymic status is also dependent on nutritional
    status (Van Logten et al., 1981; Corman, 1985; Mittal et al., 1988;
    Good & Lorenz, 1992), and stressful conditions influence the
    appearance of lymphoid organs, especially the thymus. Finally, housing
    conditions, diet, and microbiological status are important. For
    instance, the presence of gamma-delta T cells in the rodent intestinal
    epithelium is dependent on the presence or absence of microbiological
    stimulation (that is, whether the animals are bred and maintained
    under specified pathogen-free conditions) (Bandeira et al., 1990;
    Dobber et al., 1992; Umesaki et al., 1993). The role of genetic
    factors is reflected in strain differences in lymphoid organ
    histology, e.g. in rats (Losco & Harleman, 1992; Schuurman et al.,
    1992e), seen on examination of data on concurrent and historical

          Genetic factors may also contribute to the individual variability
    in response to a given compound. This variability can be relatively
    high, even among random-bred and inbred rats, as illustrated in the
    following example (Figure 20), derived from a study of the toxicity of
    the immunosuppressive drug azathioprine in random-bred Wistar rats.
    The data on haematological and histopathological parameters were
    subjected to factor analysis in order to facilitate examination of
    individual and group responses to the drug. Factor analysis involves
    clustering of parameters into composite groups, or 'factors', by
    arithmetic manipulation of the data, so that the parameters within a

    cluster are related mathematically. It is up to the investigator to
    decide whether the clustering is meaningful biologically. Figure 20
    shows that not all of the 10 animals that received the highest dose of
    azathioprine had the same response: three were considered to be high
    responders, six low or medium responders, and one a non-responder. The
    high individual variability in response illustrates the significance
    of outliers in studies with a limited number of animals.

          The relevance of changes observed in a study of exposure  in vivo
    must be based on knowledge of the nature of the response. Current
    understanding of the relationship between the structure and function
    of the lymphoid organs and their components often allows only a
    provisional hypothesis to be made about the mechanisms of toxicity.
    Moreover, different mechanisms of tissue injury can yield similar
    histopathological features. Therefore, in-depth, specific histological
    examination of tissue response and immune function are indispensable
    for interpreting changes and for risk assessment. In interpreting
    quantitative changes, like changes in cellularity, it should be noted
    that particular components of the immune system may be decreased in
    number or size (suppressed or involuted) or increased (stimulated or
    expanded), but this does not necessarily reflect the overall effect on
    the immune system or lymphoid organs.

          The presence of tissue damage, protein complex deposits, and
    inflammatory cell infiltrates may indicate the induction of
    autoimmunity or the presence of allergy or hypersensitivity. The site
    at which such responses are seen is often not a lymphoid organ, but
    blood vessels, renal glomeruli, synovial membranes, thyroid, skin,
    liver, or lung, which are well-known sites of autoimmunity and
    hypersensitivity. Non-lymphoid organs should also be examined in order
    to determine whether the effects on the lymphoid system are secondary,
    for instance mediated by acute stress. This aspect is not considered
    in detail in this monograph, which concerns mainly the direct toxic
    action of xenobiotics on the immune system.

    FIGURE 19

    FIGURE 20


    2.1  Description of consequences on human health

    2.1.1  Consequences of immunosuppression

          Since immunosuppressive drugs were introduced clinically to
    prevent allograft rejection more than 25 years ago, the human
    consequences of immunosuppression are well known. Infections and
    cancers are the main consequences of immunosuppressive therapy, as
    exemplified by a number of isolated case reports and epidemiological
    studies (IARC, 1987; Descotes, 1990; IARC, 1990). The cancers are
    often lymphomas and carcinomas, which are likely to be of viral
    origin, especially in immunosuppressed patients. Recurrent respiratory
    viral infections should also be considered as sentinel conditions for
    immunotoxicity, both in individuals and in community-based epidemics,
    including, but not confined to, opportunistic infections. Additional
    evidence that immunosuppression can enhance the risk of cancer is the
    increased incidence of an atypical form of Kaposi's sarcoma and of
    lymphomas frequently located in the brains of patients with AIDS. It
    is important to distinguish between profound immunodepression (mainly
    seen clinically, e.g. after renal transplantation or cytotoxic therapy
    for neoplasia) and the less severe suppression of immune function that
    is more likely to be associated with exposure to an environmental
    immunotoxic agent.  Cancer

          Evidence from three sources, namely cancer patients on
    chemotherapy, organ transplant patients, and patients with autoimmune
    disorders undergoing long-term immunosuppressive therapy, demonstrates
    that immunosuppressed patients are at a higher risk than others of
    developing malignancies (Boyle et al., 1984; Penn, 1988; IARC, 1990;
    Barrett et al., 1993; Bouwes Bavinck et al., 1993; Penn, 1993a,b;
    Descotes & Vial, 1994), although not all immunosuppressive drugs have
    been shown to be carcinogenic, e.g. prednisone and methotrexate (IARC,
    1987). Immunotoxic effects might result in tumour formation through
    reduced immune surveillance, i.e. tumours might escape the guard of
    the immune system. Reduced immune surveillance can thus be regarded as
    tumour promotion.

          The risk for second malignancies after prolonged cancer
    chemotherapy has been shown in numerous case reports and
    epidemiological studies (Henne & Schmähl, 1985; Boivin, 1990; Blatt et
    al., 1992). Acute leukaemia is the most frequently reported second
    cancer (Kyle, 1984); overall, iatrogenic leukaemias account for 10% of
    all leukaemias, with an incidence 5-100 times higher than in the
    general population. Non-Hodgkin's lymphoma develops in 0.5-4.5% of
    patients within 10 years after cytotoxic therapy. The risk for solid

    tumours, e.g. carcinomas of the lung, skin, breast, colon, and
    pancreas, is also increased after cytotoxic therapy but with a
    different trend: the increase in risk is more prolonged and slower
    (Swerdlow et al., 1992).

          Although most cytotoxic drugs are genotoxic, their
    immunosuppressive effects may also account for the increased risk of
    second cancers, as indicated by results obtained in organ transplant
    recipients. The Cincinnati Transplant Tumour Registry collected data
    on more than 3600 cases of cancer in transplant patients up to June
    1988 (Penn, 1988). Large numbers of cancers were also included in the
    Australian and New Zealand Combined Dialysis and Transplant Registry
    (Sheil et al., 1991). Overall, 1-15% of organ transplant patients
    developed cancer within the first five years after transplantation;
    whatever the therapeutic regimen, the incidence of cancer was at least
    three times that of the general population and increased
    logarithmically with the length of follow-up to reach more than 50%
    after 20 years in some series. Cancers of the skin and lips were
    reported in 18% of patients after 10 years of immunosuppressive
    therapy. Squamous-cell carcinoma was the most frequent skin cancer and
    was about 250 times more frequent in transplant patients than in the
    general population (Hartevelt et al., 1990). Lymphomas accounted for
    14-18% of neoplasms in transplant patients, and high-grade non-
    Hodgkin's lymphomas accounted for 95% of these lymphomas. The
    incidence of Kaposi's sarcoma was 400-500 times more frequent in
    transplant patients than in the general population.

          A similar pattern of neoplasias was observed in patients on
    immunosuppressive therapy for autoimmune diseases (Sela & Shoenfeld,
    1988). Non-Hodgkin's lymphomas were 11 times more frequent than in the
    general population, and other cancers, namely leukaemias, primary
    liver cancers, and squamous-cell carcinomas, were also found to be
    more frequent (Penn, 1988; Descotes & Vial, 1994). The respective
    roles of immunosuppressive therapy and of the underlying disease
    remain to be established, however.

          Cancers have been reported to occur after immunosuppressive
    therapy with both cytotoxic and noncytotoxic drugs. Even though the
    respective roles of genotoxicity and immunosuppression are difficult
    to ascertain, cancers have been described in patients on azathioprine
    after organ transplantation (Wessel et al., 1988; Singh et al., 1989)
    or on low-dose methotrexate for autoimmune disorders (Ellman et al.,
    1991; Shiroky et al., 1991; Kingsmore et al., 1992; Kamel et al.,
    1993); immunosuppressive drugs increased the risk of malignancies
    (especially lymphomas) in the treated patients. Interestingly, all of
    the noncytotoxic immunosuppressive drugs were reportedly associated
    with a variety of malignancies presumably related to immuno-
    suppression. Whatever the type of tumour, the time to tumour
    development after treatment with cyclosporin A was shorter (26 months
    on average; 14 months for lymphomas) than after conventional
    immunosuppressive therapy (60 months on average) (Penn, 1988). The

    murine monoclonal antibody OKT3 was also shown to increase the risk
    for lymphoproliferative disorders (Swinnen et al., 1990): Lymphomas
    developed 1-18 months after starting OKT3, and a correlation was found
    between the dose and time to neoplasm development. Lymphoproliferative
    disorders were also shown to occur following treatment with FK 506
    (Reyes et al., 1991). There are insufficient data to conclude a direct
    causal relationship between immunosuppression induced by environmental
    chemicals and the development of cancer; however, there is
    epidemiological evidence that exposure to various potentially
    immunotoxic chemicals (e.g. pesticides, benzene) is associated with
    increased risks for cancers that also occur in immunosuppressed
    patients (e.g. non-Hodgkin's lymphoma and leukaemia).

          No marked difference was found in the relative risks for
    lymphoproliferative disorders associated with the various
    immunosuppressive drugs currently used, suggesting that
    immunosuppression is the causative factor, particularly when account
    is taken of the different carcinogenic potentials of azathioprine and
    cyclosporin A, both clinical reference immunosuppressive agents
    (Ryffel, 1992). Reactivation of latent viruses, e.g. Epstein-Barr
    virus, due to immunosuppression was suggested to be involved. Indeed,
    most lymphoproliferative disorders induced with cyclosporin A or
    methotrexate were B-cell malignant lymphomas associated with this
    viral infection (Starzl et al., 1984; Kamel et al., 1993). Uninhibited
    proliferation of Epstein-Barr virus in B lymphocytes caused by the
    efficient immunosuppression of one or more kinds of controls by
    T lymphocytes is the commonly accepted mechanism. Interestingly,
    Epstein-Barr virus-associated lymphomas in patients on
    immunosuppressive therapy are usually reversible upon cessation of
    treatment (Starzl et al., 1984).  Infectious diseases

          Whatever the primary cause of the immune deficiency, patients
    develop more frequent, more severe, recurring, and often atypical
    infections, depending on the type and severity of the deficiency. The
    complications associated with severe immunosuppression include
    bacterial, viral, fungal, and parasitic infections (Waldman, 1988;
    Barr et al., 1989; Mandell, 1990; Tieben et al., 1994). The pathogens
    most frequently encountered in immunodeficient patients include the
    bacterial agents  Staphylococcus aureus, Streptococci, Escherichia
     coli, Pseudomonas aeruginosa, Listeria monocytogenes, Mycobacterium
     tuberculosis, and atypical mycobacteria. Herpes virus,
    cytomegalovirus, Epstein-Barr virus, and human papillomavirus are the
    leading causes of viral infections in immunosuppressed patients.
    Fungal opportunistic infections include those induced by  Candida,
     Aspergillus, and  Cryptococcus species. The immunotoxicity induced
    by environmental chemicals often results in subtle changes in the
    immune system, which have been suggested to result in increased
    incidences of common infections like influenza and the common cold
    (see section 2.4).

          The respiratory tract is a primary target for infectious
    pathogens, especially in immunosuppressed patients. Pulmonary
    infections and infections of the upper respiratory tract are the most
    common (Frattini & Trulock, 1993). Cytomegalovirus infections, often
    asymptomatic, are particularly frequent in renal transplant patients
    (Rubin, 1990). In addition,  Pneumocystis carinii can cause a
    particular form of pneumonia in immunosuppressed patients. Even though
    respiratory diseases usually predominate, gastrointestinal infectious
    diseases may constitute the leading consequences of immunosuppression
    (Bodey et al., 1986). Infections of the central nervous system and
    isolated fever are also extremely frequent. Interestingly, the type of
    infection that develops in immunosuppressed patients is largely
    dependent on the type of immune defect, as illustrated in Table 5.

          Infectious complications have been commonly described in patients
    treated with various cytotoxic drugs for cancer and with
    immunosuppressants, such as cyclosporin A, for the prevention of
    allograft rejection or the treatment of autoimmune disorders (Kim,
    1989; Descotes & Vial, 1994).
        Table 5.  Pathogens frequently associated with immune defects

    Humoral             Cellular                 Neutrophil          Complement
    immunity            immunity                 functions           system

    Campylobacter       Candida                  Aspergillus         Neisseria
    Echovirus           Coccidioides             Bacteroides         Staphylococci
    Gardia              Cryptococci              Escherichia coli    Streptococci
    Haemophilus         Cytomegalovirus          Klebsiella
    Pneumococci         Herpes virus             Pseudomonas
                        Human papillomavirus
    2.1.2  Consequences of immunostimulation

          The health consequences of immunostimulation are less well
    established than those associated with immunosuppression. A number of
    adverse effects have, however, been reported after treatment with
    immunostimulating drugs, including influenza-like reactions,
    facilitation or exacerbation of underlying diseases, and inhibition of
    hepatic drug metabolism (Descotes, 1992).

          Patients with influenza-like reactions present with mild to
    moderate fever associated with chills, malaise, and hypotension. The
    reaction usually develops within hours after taking an
    immunostimulating drug, and the patient recovers uneventfully within a
    few hours. Such reactions are relatively uncommon with most
    immunomodulating agents but have been shown to limit treatment with
    several recombinant cytokines, e.g. IL-1 and tumour necrosis factor.

          Facilitation and/or exacerbation of underlying diseases have been
    ascribed to most immunostimulating drugs, but the incidence of this
    adverse event differs markedly from one drug to another. Exacerbation
    of chronic infections, psoriasis, and Crohn's disease have been
    reported. More interestingly, several autoimmune diseases are more
    frequent in patients treated with various (recombinant) cytokines,
    e.g. autoimmunity treated with IFN gamma (Jacob et al., 1987),
    thyroiditis with IL-2 (Vial & Descotes, 1993), and lupus erythematosus
    with IFN alpha (Vial & Descotes, 1993). Exacerbation or facilitation
    of allergic reactions to unrelated allergens has also been reported:
    starting an immunostimulating treatment has been associated with
    exacerbation of underlying eczema, asthma, or rhinitis. Allergic
    reactions to radiological contrast media have been shown to be more
    frequent in IL-2-treated patients than controls (Vial & Descotes,

          Oxidative drug metabolism by the hepatic cytochrome P450 system
    has been shown to be inhibited by immunostimulating drugs (Descotes,
    1985) and by administration of bacillus Calmette-Guérin (BCG) vaccine
    or interferon (Vial & Descotes, 1993). Although the mechanism of this
    inhibition is still unknown, activation of macrophages resulting in
    the release of IL-1 and IL-6 has been suggested to be involved.
    Likewise, vaccination has been shown to compromise drug metabolism to
    a sufficient extent that normally therapeutic doses of theophylline
    caused acute toxicity in humans (Renton et al., 1980). Stimulation of
    the immune system has also been shown to alter drug metabolism in
    humans (Renton, 1986). A similar effect of infection has been reported
    in laboratory animals (Selgrade et al., 1984); infection with mouse
    cytomegalovirus before exposure to the insecticide parathion reduced
    the total P450 concentration and dramatically increased the toxicity
    of parathion.

          So far, only a few environmental chemicals have been shown to
    exert immunostimulating properties, e.g. hexachlorobenzene and
    selenium. There have been no reports of clinical reactions to such
    chemicals that are similar to the adverse effects seen with
    immunostimulating drugs.

    2.2  Direct immunotoxicity in laboratory animals

          The following are some illustrative examples of immunotoxic

    2.2.1  Azathioprine and cyclosporin A

          The immunosuppressive effects of azathioprine and cyclosporin A
    are considered because they can shed light on the direct
    immunotoxicity of environmental chemicals.  Azathioprine

          Azathioprine is a thiopurine that is used as cytostatic drug in
    the treatment of leukaemias and as an immunosuppressant in patients
    who have received allogeneic organ transplants or who have autoimmune
    diseases. When used as an immunosuppressant, its main side-effect is
    bone-marrow depression, reflected in blood leukocytopenia; its
    administration must therefore be monitored through blood leukocyte
    counts. Another side-effect, especially after long-term
    administration, is tumour formation (IARC, 1987).

          In rats, azathioprine is cytotoxic for all cell lineages in the
    bone marrow, and strong cellular depletion is observed histologically.
    It decreases the cellularity in thymus, blood, and peripheral lymphoid
    organs, but it is mainly in the thymus that the immature lymphocyte
    population of the cortex is affected. This effect is a general feature
    of most cytostatic drugs. A similar effect is seen in the thymus after
    treatment with glucocorticosteroids, but the molecular mechanism
    resulting in lymphocyte depletion is obviously different: interference
    with DNA synthesis resulting in lymphocyte proliferation in contrast
    to binding to glucocorticosteroid receptors and cell down-modulation.
    Azathioprine affects a number of indicators of immune function, like
    macrophage cytotoxicity (Spreafico et al., 1987), lymphocyte
    proliferation  in vitro after mitogen stimulation (Weissgarten et
    al., 1989) and in the mixed leukocyte reaction (Mellert et al., 1989),
    and cytotoxicity by NK cells (Pedersen & Beyer, 1986; Spreafico et
    al., 1987; Versluis et al., 1989). Both stimulation and suppression of
    these functions have been found in experimental animals, depending on
    the dosage and the time of testing after exposure. These findings are
    in accordance with those in azathioprine-treated patients, who showed
    no change in primary antibody response, a decrease in secondary
    antibody response, and some or no effect on lymphocyte proliferation
     in vitro after mitogen stimulation. The time of testing after the

    start of exposure to azathioprine was a crucial factor in the
    detection of effects. Azathioprine was tested in the IPCS-European
    Union international collaborative immunotoxicity study (see section
    1.1) and showed a significant strain-dependent sensitivity.  Cyclosporin A

          Cyclosporin A is one of the most powerful immunosuppressive drugs
    (Kahan, 1989). It is a neutral lipophilic cyclic peptide consisting of
    11 amino acids (relative molecular mass, 1203 Da) isolated from the
    fungus  Tolypocladium inflatum. Its main use is in bone-marrow
    transplantation to prevent transplant rejection and graft-versus-host
    reactions. It is also used in the therapy of various autoimmune

          A complication of cyclosporin A treatment is nephrotoxicity.
    Another side-effect, especially after long-term administration, is
    tumour formation (IARC, 1987). In its immunosuppressive action,
    cyclosporin A does not affect resting lymphocytes but blocks the
    events occurring after stimulation, particularly the synthesis of
    lymphokines, including IL-1 and IL-2, and IL-2 receptors. The
    synthesis of IL-1 by antigen-presenting cells and of IL-2 by Th cells
    is inhibited, and the synthesis of IFN gamma and tumour necrosis
    factor is blocked. These events occur inside the cell at the
    transcriptional level. Cyclosporin A binds to an intracellular
    receptor, cyclophilin, forming a complex with calcineurin; this
    complex in turn interferes with the activation of genes, resulting in
    inhibition of lymphokine gene transcription (Baumann et al., 1992;
    Sigal & Dumont, 1992).

          An interesting feature of cyclosporin A is its specific action on
    the thymus and the induction of autoimmune phenomena. Rats treated
    with total body irradiation and syngeneic or autologous bone-marrow
    transplantation, followed by treatment with cyclosporin A at a dose of
    about 10 mg/kg body weight per day subcutaneously for four weeks,
    developed signs of acute graft-versus-host reactions, with lymphocytic
    infiltration at multiple epithelial sites (Glazier et al., 1983). A
    similar pseudo-graft-versus-host reaction has also been evoked in
    mice. It is associated with thymic changes, because it can be
    transferred in whole thymus or thymocytes (Sakaguchi & Sakaguchi,
    1988). Histologically, the medullary area is diminished (Beschorner et
    al., 1987a; Schuurman et al., 1990; see also Figure 21). The medullary
    stroma shows a decrease in MHC class II expression, indicating a loss
    of dendritic cells, which has been confirmed by electron microscopy
    (De Waal et al., 1992a). As these cells normally contribute to the
    negative selection process, their depletion (or reduced MHC class II
    expression) may be related to an absence of negative selection. The
    autoreactive T cells may even attack the medullary epithelium.

    FIGURE 21

    FIGURE 21a

          The effect of cyclosporin A on thymic functions, i.e. the
    induction of 'leakiness', with export of T cells that have not been
    negatively selected, has not yet been studied for other drugs, but may
    not be specific to cyclosporin A. It represents a distinct mechanism
    of autoimmunity induced by the action of toxic compounds on the immune
    system, mediated via thymic selection. Although the medullary area is
    reduced in young rats after treatment with cyclosporin A, this is not
    the case in one-year-old rats, which presumably have a lesser output
    of mature T cells because of thymic involution (Beschorner et al.,

          The effect of cyclosporin A in inducing syngeneic graft-versus-
    host disease in rodents has an application in clinical medicine:
    Patients treated for cancer with high-dose chemotherapy and/or total
    body irradiation, followed by autologous bone-marrow transplantation,
    develop a recurrence of the original tumour at a higher incidence than
    patients who receive an allogeneic bone-marrow transplant. This
    difference has been ascribed to the addition of a graft-versus-tumour
    effect to the graft-versus-host reaction. Trials have now been
    initiated to induce a graft-versus-host reaction with cyclosporin A
    treatment after autologous bone-marrow transplantation, in order to
    reduce tumour recurrence. The initial results are promising (Hess et
    al., 1992; Yeager et al., 1993; Kennedy et al., 1994).

          Interestingly, two other immunosuppressive drugs, FK-506 and
    rapamycin, which also interfere with gene activation in T lymphocytes,
    do not bind to cyclophilin but to another intracellular receptor, the
    FK-binding protein. The effect of FK-506 on the thymus is similar to
    that of cyclosporin A, i.e. a decrease in the medulla (Pugh-Humphreys
    et al., 1990), and rapamycin causes severe acute involution with
    disappearance of lymphocytes from the cortex (Zheng et al., 1991).
    These findings indicate that the two compounds have different
    molecular mechanisms of action on the thymus from those of cyclosporin
    A, which have not yet been elucidated.

    2.2.2  Halogenated hydrocarbons  2,3,7,8-Tetrachlorodibenzo-para-dioxin

          The halogenated hydrocarbon most closely studied for its
    immunotoxic effects is TCDD. It has a variety of toxic effects, with a
    remarkable interspecies variation; however, it causes atrophy of the
    thymus and immunotoxicity in all species investigated (Vos & Luster,
    1989; Holsapple et al., 1991; Neubert, 1992; Kerkvliet & Burleson,
    1994). Atrophy of the thymus is reflected histologically by lymphocyte
    depletion of the cortex (Figure 22). Functionally, cell-mediated
    immunity appears to be suppressed in a dose-dependent fashion, as
    manifested in delayed-type hypersensitivity responses, rejection of
    allogeneic skin transplants, graft-versus-host reactivity, and
    lymphocyte proliferation  in vitro after mitogen stimulation. This

    immune suppression is age-related: more severe immunotoxic effects are
    observed after perinatal administration than after administration in
    adulthood (Vos & Moore, 1974; Thomas & Hinsdill, 1979). TCDD can also
    impair antibody-mediated immunity after primary or secondary
    immunization. A sensitive parameter of the immunotoxicity of TCDD and
    TCDD congeners in mice is suppression of the T cell-dependent antibody
    response to sheep red blood cells in mice (Vecchi et al., 1980; Davis
    & Safe, 1988; Kerkvliet et al., 1990). No effects have been observed
    on classical macrophage functions.

          In mice, susceptibility to TCDD is genetically determined and is
    segregated at the locus that encodes a cytosolic protein which
    mediates aryl hydrocarbon hydroxylase activity (Poland & Knutson,
    1982). This Ah (aromatic hydrocarbon) receptor has a high affinity for
    TCDD and is strongly active in mouse and rat thymus (Gasiewicz &
    Rucci, 1984), particularly in epithelial cells (Greenlee et al., 1985;
    Cook et al., 1987). Ah receptor-dependent immunotoxicity has been
    demonstrated in mice for thymic atrophy and the antibody response to
    sheep red blood cells (Tucker et al., 1986; Kerkvliet & Burleson,
    1994); however, the importance of Ah receptor-mediated events in
    chronic, low-level TCDD immunotoxicity is controversial (Morris et
    al., 1992).

          Immunosuppression in adult mice manifests almost exclusively as
    suppressed antibody responses and does not appear to be related to
    thymic atrophy in experiments in thymectomized (Tucker et al., 1986)
    and nude (Kerkvliet & Brauner, 1987) mice. Both T and B lymphocytes
    involved in antibody responses can, however, be affected by TCDD. For
    example, exposure to TCDD  in vivo alters regulatory lymphocyte
    function (Kerkvliet & Brauner, 1987) and antigen-specific T lymphocyte
    activation (Lundberg et al., 1992). TCDD also inhibits T-independent
    antigen responses (Vecchi et al., 1983) and T-dependent responses when
    only B cells are treated (Dooley & Holsapple, 1988). Studies of the
    effects of TCDD on enriched B-cell populations  in vitro have shown
    that it selectively inhibits late stages of the cell cycle and the
    development of B cells into plasma cells after antigen-specific
    activation (Luster et al., 1988). The molecular events responsible for
    TCDD immunosuppression have not been examined in detail. While early
    events in B-cell maturation, such as inositol phosphate accumulation,
    are not affected (Luster et al. 1988), activation of protein kinase
    (Kramer et al., 1987) and tyrosine kinase (Clark et al., 1991) have
    been observed.

          A consequence of TCDD-induced immunosuppression is impaired
    resistance to infection by bacterial, viral, and protozoan
    microorganisms (Vos et al., 1991). In various mouse strains with
    different treatment schedules, TCDD suppressed resistance to models 
    of infectious diseases with  Salmonella bern, S. typhimurium,
     Streptococcus pneumoniae, herpes II,  Plasmodium yoelli and influenza
    viruses. Various effects have been reported on resistance to 

    FIGURE 22

    FIGURE 22a

     L. monocytogenes. TCDD had no effect on the mortality of mice
    infected with  Herpes suis (pseudorabies), whereas the mortality of
    mice infected with influenza virus was enhanced by a single oral dose
    of TCDD as low as 10 ng/kg body weight (Burleson et al., in press).

          Many studies have been performed to investigate the mechanisms of
    TCDD-induced thymic atrophy, and a number have presented evidence that
    the effect may occur through an action on epithelial cells:

    1.    The enhanced lymphoproliferation of thymocytes after coculture
          with cultured mouse and human epithelial cells was reduced when
          the epithelial cells were pretreated with TCDD (Greenlee et al.,
          1985; Cook et al., 1987).

    2.    In mouse radiation chimaeras, TCDD-induced suppression of Tc
          lymphocytes is determined by the host (epithelium) and not the
          donor (bone marrow, subsequently thymocytes) (Nagarkatti et al.,

    3.    Histological and electron microscopy studies of TCDD-exposed rats
          reveal formation of epithelial aggregates and a more
          differentiated state of cortical epithelium, indicating that TCDD
          acts on the thymic epithelium (De Waal et al., 1992b, 1993).

          A direct action of TCDD on rat thymocytes has also been
    documented  in vitro as cell death due to apoptosis (McConkey et al.,
    1988), but this effect requires higher concentrations than those that
    affect epithelial cell function  in vitro. In bone marrow, TCDD
    affects myelopoiesis (Luster et al., 1985a) but may be more selective
    for prothymocytes (Fine et al., 1989, 1990; Holladay et al., 1991;
    Blaylock et al., 1992), thus indirectly affecting thymic function.  Polychlorinated biphenyls

          Polychlorinated biphenyls (PCBs) are important environmental
    chemicals shown in numerous studies to have immunotoxic properties.
    PCB mixtures alter several morphological and functional aspects of the
    immune system in rodents, guinea-pigs, rabbits, and chickens (Vos &
    Luster, 1989). The first suggestion that PCBs might affect the immune
    system came from observations on the weight and histology of lymphoid
    organs. Oral exposure of chickens to PCBs resulted in small spleens
    (Flick et al., 1965) and atrophy of lymphoid tissue (Vos & Koeman,
    1970). Similar effects were noted in rabbits and guinea-pigs
    (Figure 23). Dermal application of PCBs to rabbits caused lymphopenia,
    atrophy of the thymic cortex, and a reduced number of germinal centres
    in spleen and lymph nodes (Vos & Beems, 1971). Oral exposure of guinea-
    pigs significantly reduced the number of circulating lymphocytes
    and the relative thymus weight (Vos & Van Driel-Grootenhuis, 1972).

    FIGURE 23

          Functional tests have been focused on humoral immune responses.
    Exposure of guinea-pigs, rabbits, mice, and rats to PCBs at different
    regimens reduced antibody production to foreign antigens, including
    tetanus toxoid, pseudorabies virus, sheep red blood cells, and keyhole
    limpet haemocyanin (Vos & Van Driel-Grootenhuis, 1972; Koller &
    Thigpen, 1973; Loose et al., 1977; Wierda et al., 1981; Exon, 1985;
    Kunita et al., 1985). These data are in line with the observations of
    Loose et al. (1977) and Thomas & Hinsdill (1978) that exposure to PCBs
    lowered circulating immunoglobulin levels in mice. No reduction was
    reported in antibody responses to bovine serum albumin (Talcott &
    Koller, 1983).

          The response to sheep red blood cells in the plaque-forming cell
    assay has been used to establish dose-response relationships for
    several potentially immunotoxic Aroclors in mice given a single
    intraperitoneal injection of PCB mixtures (Davis & Safe, 1989). These
    studies indicate that the higher chlorinated PCB mixtures are more
    immunotoxic than the lower chlorinated Aroclors (Allen & Abrahamson,
    1973; Loose et al., 1978; Tryphonas, in press). Data on the effects of
    PCBs on total serum immunoglobulin levels have not been reported in
    non-immunized animals.

          While the suppressive effects of PCBs on humoral immunity are
    well documented, the effects on cell-mediated immune parameters are
    less clear. The delayed-type hypersensitivity reaction to tuberculin
    was suppressed in guinea-pigs (Vos & Van Driel-Grootenhuis, 1972) but
    not in rabbits treated with PCBs (Street & Sharma, 1975). Decreased
    delayed-type hypersensitivity reactions were reported in mice by Smith
    et al. (1978) but not by others (Talcott & Koller, 1983). Kerkvliet &
    Baecher-Steppan (1988) reported that 3,4,5,3',4',5'-hexachlorobiphenyl
    reduced Tc lymphocyte activity in the spleens of mice. In contrast,
    the graft-versus-host reaction was increased following PCB treatment
    (Carter & Clancy, 1980). Studies on the mitogen-induced responses of
    splenic mononuclear leukocytes from PCB-treated mice  in vitro
    resulted in either enhanced or unaltered responses (Bonnyns &
    Bastomsky, 1976; Wierda et al., 1981; Davis & Safe, 1989; Smialowicz
    et al., 1989).

          Functional impairment of the non-specific resistance of macro-
    phages has been reported, including reduced phagocytic activity and
    clearance of pathogenic bacteria by the spleens and livers of PCB-
    exposed animals (Smith et al., 1978) and decreased NK cell activity
    (Talcott et al., 1985; Smialowicz et al., 1989). Exposure of mice to
    PCBs also enhanced their sensitivity to endotoxin shock (Loose et al.,
    1978; Thomas & Hinsdill, 1978).

          PCB treatment was shown to protect mice and rats against
    Ehrlich's tumour (Keck, 1981) and Walker 256 tumours (Kerkvliet &
    Kimeldorf, 1977), shown as reduced tumour growth and metastasis after
    transplantation; in other studies, however, no influence of PCB on
    tumour-cell implants was reported (Koller, 1977; Loose et al., 1981).
    PCBs also affect the resistance of animals to infectious diseases.
    Thus, ducklings exposed to low levels of PCBs were more susceptible to
    challenge with duck hepatitis virus (Friend & Trainer, 1970), and mice
    were more susceptible to challenge with Moloney leukaemia virus
    (Koller, 1977),  Plasmodium berghei (Loose et al., 1978),
     S. typhimurium (Loose et al., 1978),  L. monocytogenes (Thomas &
    Hinsdill, 1978), and  Herpes simplex and  Ectromelia viruses
    (Imanishi et al., 1980).

          The immunotoxic effects of PCBs have also been investigated in a
    number of studies with non-human primates. Decreased titres of anti-
    sheep red blood cells have been observed in PCB-exposed rhesus (Thomas
    & Hinsdill, 1978) and cynomolgus monkeys (Hori et al., 1982; Truelove
    et al., 1982; Kunita et al., 1985). Immunotoxic effects were also
    reported in adult female rhesus monkeys and their infants (exposed
     in utero and through lactation) after low-level exposure (Tryphonas
    et al., 1989, 1991a,b). In this study, five groups of female rhesus
    monkeys were administered PCB (Aroclor 1254) at 0, 5.0, 20.0, 40.0, or
    80.0 µg/kg body weight per day orally. Immunological effects were
    reported after both 23 and 55 months of exposure and comprised
    significantly decreased IgM and IgG responses to sheep red blood cells
    at the lowest dose. Alterations in T-cell subsets were reported in the
    group receiving the high dose in comparison with the controls, which
    were characterized by an increase in Ts/Tc (CD8) cells and a reduction
    in the relative numbers of Th/inducer cells (CD4) and in the CD4:CD8
    ratio. No effects were seen on total lymphocytes or on B cells or on
    total serum IgG, IgM, and IgA levels. A further study indicated that
    Aroclor 1254 had no effect on B lymphocytes, since antibody responses
    to T-independent pneumoccocal antigen were not significantly affected.
    A trend for reduced incorporation of 3H-thymidine by mitogen-induced
    lymphocyte proliferation was noted only for the T mitogens
    phytohaemagglutinin and concanavalin A and not for the B pokeweed
    mitogen. A significant augmentation of NK cell activity was noted at
    the highest dose. Total serum complement activity (CH50) was also
    increased. The serum levels of corticosteroids (hydrocortisone), which
    were measured throughout the study, were not affected by treatment
    (Loo et al., 1989), clearly indicating that the changes in several of
    the immune parameters were direct effects of Aroclor 1254 on the
    immune system.  Hexachlorobenzene

          Hexachlorobenzene (HCB) is a highly persistent chemical which was
    used in the past as a fungicide. Emissions to the environment now
    occur owing to its use as a chemical intermediate and its presence as
    a by-product in several chemical processes. It is an immunotoxic
    compound (Vos, 1986), with different effects in rats and mice. In
    rats, the main changes seen after subacute exposure are increased
    weights of spleen and lymph nodes; the serum levels of IgM are also
    increased. Histologically, the spleen shows hyperplasia of follicles
    and the marginal zone (Figure 24); the lymph nodes have more follicles
    with germinal centres and greater proportions of high endothelial
    venules, indicating activation (Figures 25 and 26). High endothelial-
    type venules are also induced in the lung (Figure 27), and macrophages
    accumulate in lung alveoli (Kitchin et al., 1982; Vos, 1986; see also
    Figure 28).

          Functional assessment showed an increase in cell-mediated
    immunity (delayed-type hypersensitivity) and an even greater increase
    in antibody-mediated immunity (primary and secondary antibody response
    to tetanus toxoid). Macrophage functions were unaltered. Stimulation
    of immune reactivity occurs at dietary levels as low as 4 mg/kg after
    combined pre- and postnatal exposure for six weeks, whereas the
    conventional parameters of hepatotoxicity are not altered at this dose
    (Vos et al., 1979a; Vos, 1986). The developing immune system of the
    rat therefore seems to be particularly vulnerable to the immunotoxic
    action of HCB. Reduced NK cell activity has also been found in the
    lung after oral exposure to 150-450 mg/kg HCB in the diet (Van Loveren
    et al., 1990c).

          Studies on the mechanism of action of HCB indicate a role for
    T cells: congenitally athymic  rnu/rnu rats, which lack T cells, do
    not manifest the hyperplasia of B lymphocytes in splenic follicles and
    the marginal zone after administration of the compound; but
    endothelial cell proliferation and macrophage accumulation in the lung
    are apparently T cell-independent, as these effects were seen in
    athymic animals (Vos et al., 1990b). In contrast to the
    immunostimulatory effect in rats, HCB suppresses cell-mediated and
    antibody-mediated immunity in mice, as well as their resistance to
    protozoan infections ( Leishmania and  Plasmodium berghei) and to
    inoculated tumours (Loose et al., 1977, 1978, 1981). The
    susceptibility of mice to HCB is also higher after pre-or perinatal
    administration (Barnett et al., 1987). Recent studies indicate that
    the immunostimulatory effect of HCB in rats may be related to

    FIGURE 24

    FIGURE 25

    FIGURE 26

    FIGURE 27

    FIGURE 27a

    FIGURE 28

    1.  Exposure to HCB of Lewis rats, which develop autoimmune disease
    after sensitization with complete Freund's adjuvant (adjuvant
    arthritis) or with guinea-pig myelin (experimental allergic
    encephalomyelitis), had clear effects (Van Loveren et al., 1990c):
    Whereas the allergic encephalomyelitis response was severely enhanced,
    the arthritic lesions were strongly suppressed.

    2.  Wistar rats treated with HCB produce antibodies to autoantigens;
    thus, IgM, but not IgG, levels against single-stranded DNA, native
    DNA, rat IgG (representing rheumatoid factor), and bromelain-treated
    mouse erythrocytes (indicating that phosphatidylcholine is a major
    autoantigen) were elevated. It has been suggested that HCB activates a
    B-cell subset committed to the production of these autoantibodies and
    associated with various systemic autoimmune diseases (Schielen et al.,

    2.2.3  Pesticides

          A large number of studies have focused on the immunotoxicity of
    pesticides. Because of the chemical heterogeneity of these compounds
    as a class, the reported effects vary widely (Barnett & Rodgers,
    1994).  Organochlorine pesticides

          The evidence for the immunotoxicity of organochlorine pesticides
    as a class is inconclusive.

          DDT: Wistar rats treated with 40 mg/kg body weight per day DDT
    orally for 60 days showed increased anti-bovine serum albumin titres
    (Lukic et al., 1973). Studies by Vos et al. (Vos & Krajnc, 1983, Vos
    et al., 1983a), however, showed no changes in thymus or spleen
    weights, leukocyte counts, or total serum IgG and IgM levels at doses
    up to 800 mg/kg body weight per day.

           Chlordane: Prenatal exposure of mice to chlordane was reported
    to reduce contact and delayed hypersensitivity responses, suggesting
    an effect on T-cell responses. Attempts to elucidate the mechanism
    have, however, been unsuccessful. Johnson et al. (1986) observed
    increased lymphocyte proliferation only at a dose of 8 mg/kg body
    weight in B6C3F1 mice and concluded that chlordane has no significant
    immunotoxicity in this model.

           Chlordecone: Chlordecone reduced thymus and spleen weights by
    40% in Fischer rats at at a dose of 10 mg/kg body weight per day but
    had no significant effect at or below 5 mg/kg body weight per day.
    T-Lymphocyte proliferation was unaffected at all doses (Smialowicz et
    al., 1985a).

           Lindane: Lindane had various effects on the anti-sheep red
    blood cell response, depending on the immunization protocol. Specific
    IgM levels were unchanged by parenteral immunization after four weeks'
    treatment with 150 mg/kg in the diet, but specific IgG2b levels were
    raised after intragastric immunization (André et al., 1983); however,
    the duration of  Giardia muris infection was significantly prolonged.
    Five weeks' oral treatment with up to 12 mg/kg of diet decreased the
    antibody titre to TY3 vaccine in rabbits in a dose-dependent manner
    (Desi et al., 1978).

           Toxaphene: Toxaphene given at 100 or 200 mg/kg of diet
    decreased anti-bovine serum albumin antibody titres in Swiss mice
    treated for eight weeks and in the offspring of dams given the same
    diet. Macrophage activity was also reduced in these offspring, but
    there were no changes in the delayed-type hypersensitivity response to
    purified protein derivative (Allen et al., 1983).

           Endosulfan: Endosulfan had no immunotoxic effect in Wistar rats
    (Vos & Krajnc, 1983; Vos et al., 1983a).  Organophosphorus compounds

          Single doses of the insecticides parathion, malathion, and
    dichlorvos cause significant reductions in anti-sheep red blood cell
    plaque-forming cell responses (Casale et al., 1983, 1984). The
    relevance of these findings is questionable, however, as they occurred
    only if cholinergic or parathion symptoms were also induced.
    Administration of multiple doses of malathion resulted in conflicting
    findings: C57Bl6 mice given four doses of 240 mg/kg body weight over
    eight days had unchanged plaque-forming cell responses to sheep red
    blood cells. In contrast, rabbits treated with 5-10 mg/kg body weight
    per day over 5-6 weeks had reduced antibody titres after vaccination
    with  S. typhimurium. Parathion also failed to suppress anti-sheep
    red blood cell plaque-forming cell formation when given as four doses
    of 4 mg/kg body weight (Casale et al., 1983). Parathion-methyl given
    to rabbits for four weeks did not affect immune responses (Desi et
    al., 1978). In mice, however, both cellular and humoral responses were
    reported to be suppressed by subacute administration of parathion
    (Wiltrout et al., 1978).

          The immunotoxicity of MPT-IP (the industrial compound for the
    production of Wofatox EC50, containing 60% parathion-methyl) was
    studied in mice given single oral doses of 8.9 mg/kg body weight or
    repeated doses of 0.890 or 0.445 mg/kg body weight for four weeks.
    Depending on the day of administration, the single dose increased the
    IgM plaque-forming cell content of the spleen and the serum anti-sheep
    red blood cell antibody titre. In the subacute system, the smaller
    dose (0.445 mg/kg) increased the splenic plaque-forming cell content
    and serum antibody titre (Institoris et al., 1992).

          Dimethoate was administered by gavage to three generations of
    Wistar rats at doses of 14.1, 9.39, and 7.04 mg/kg body weight
    (equivalent to 1/50, 1/75, and 1/100 of the LD50), and parathion-
    methyl was administered at doses of 0.436, 0.291, a,d 0.218 mg/kg body
    weight. The highest dose of dimethoate significantly decreased the
    plaque-forming capacity of spleen cells in the first generation and
    increased thymic weight in the second and third generations. All three
    doses of parathion-methyl decreased the number of red blood cells and
    the haematocrit value, and the two highest doses decreased the
    leukocyte count. The nucleated cell content of the bone marrow was
    increased in the second and third generations, and decreased relative
    thymic weight was seen at all three doses in the third generation
    (Institoris et al., 1995). In a similar experiment, dichlorvos was
    administered at doses of 1.85, 1.24, or 0.972 mg/kg body weight. A
    significant decrease in leukocyte count, lowered spleen cellularity,
    and decreased plaque-forming capacity were seen with the highest dose
    in the second generation. In the third generation, there was a dose-
    dependent decrease in femoral bone-marrow cellularity (Institoris et
    al., in press).  In vitro, 250 µmol/litre of paraoxon, a parathion
    metabolite, suppressed mitogenic lymphocyte proliferation in spleen
    cells from Sprague-Dawley rats (Pruett & Chambers, 1988).

          Reduction of antibody titre against Ty3 vaccine was observed by
    the end of six weeks' oral treatment of rabbits with 5-100 mg/kg body
    weight of malathion or with 1.25 or 2.5 mg/kg body weight of
    dichlorphos (Desi et al., 1978). In the same system, a dose-dependent
    decrease was observed in the tuberculin skin reaction after
    administration of 0.31, 0.62, or 1.25 mg/kg body weight of
    dichlorphos. The cholinesterase activity of red blood cells was
    decreased only by the two higher doses.

          Convincing evidence for immunotoxicity has been obtained only for
     O,O,S-trimethylphosphorothiate ( O,O,S-TMP), a contaminant of
    various commercial organophosphorus formulations, such as malathion,
    fenitrothion, and acephate. This compound was shown to suppress
    humoral and cellular immunity in mice exposed to 10 mg/kg body weight
    orally (Devens et al., 1985). Several organophosphorus derivatives can
    alter some immune functions  in vitro, including mitogen-induced
    lymphocyte proliferation (Pruett & Chambers, 1988), T-lymphocyte
    cytotoxicity, and production of hydrogen peroxide by macrophages
    (Pruett, 1992), at concentrations that can theoretically be attained
     in vivo.

          Several mechanisms have been proposed to explain organo-
    phosphorus-induced immunosuppression (Pruett, 1992). A direct
    cholinergic mechanism is unlikely to be involved, as the addition of
    various cholinergic agonists does not suppress immune responses  in
     vitro. In addition,  O,O,S-trimethylphosphorodithioate, a structural
    analogue of  O,O,S-TMP, modulates cholinesterase activity but does
    not alter immune competence. An indirect mechanism involving

    stress caused by neurotoxicity has also been proposed. Finally, a
    direct action on cells of the immune system, and particularly
    macrophages, has been suggested to be involved. Mice treated with
     O,O,S-TMP, which is not neurotoxic, generate a population of
    macrophages, contraindicating lymphocyte proliferation. Antigen
    processing and presentation by these highly activated (inflammatory)
    macrophages are severely impaired; however, the changes in macrophage
    function are not correlated with suppression of humoral or cellular
    immunity. While there is no direct evidence that B and T lymphocytes
    are the predominant targets of organophosphorus compounds, their
    mechanisms of action on macrophages are largely unknown.  Pyrethroids

          Dose-dependent decreases in the serum anti- S. typhimurium
    antibody titre and in the tuberculin skin reaction were observed in
    rabbits fed 25, 12.5, or 6.25 mg/kg body weight of technical-grade
    cypermethrin (93.5%) for seven weeks (Desi et al., 1985). Single oral
    doses (23.5, 20.7, or 18.7 mg/kg body weight) of supermethrin, the
    active substance of the pyrethroid pesticide Neramethrin EC 50,
    decreased the number of IgM plaque-forming cells in the spleens of
    mice but had no effect on the delayed-type hypersensitivity reaction.
    Repeated doses of 2.97, 1.49, and 0.743 mg/kg body weight caused only
    slight changes in the leukocyte count and in the nucleated cell
    content of femoral bone marrow (Siroki et al., 1994).  Carbamates

          Carbaryl induced marked increases in serum IgG1 and IgG2, but not
    IgA, IgG3, or IgM, levels of mice exposed to 150 mg/kg of diet for one
    month (André et al., 1983). Rabbits given carbaryl at 4-150 mg/kg of
    diet for four weeks had no changes in anti-sheep red blood cell
    haemolysin or haemagglutinin titres or in the delayed-type
    hypersensitivity response to tuberculin, whereas oral treatment with
    carbofuran at 0.5-20 mg/kg of diet for four weeks induced a 60-75%
    decrease in the delayed-type hypersensitivity response (Street &
    Sharma, 1975). Aldicarb induced no changes in a large battery of
    assays for immune function and host resistance in B6C3F1 mice exposed
    to 0.1-1000 mg/litre of drinking-water daily for 34 days (Thomas et
    al., 1987).  Dinocap

          Dinocap is a dinitrophenol compound used as a fungicide. Female
    C57Bl/6J mice were given doses of 12.5-50 mg/kg body weight per day by
    gavage for 7 or 12 days. All mice given the highest dose died after
    four days. Mice given 25 mg/kg for 12 days had decreased thymus
    weights and cellularity and increased spleen weights but no changes in
    body weight, leukocyte count, lymphoproliferative response to B- or 
    T-cell mitogens, mixed lymphocyte reaction, or NK cell activity of
    spleen cells; lymphoproliferative responses to concanavalin A and

    phytohaemagglutinin in thymocytes were reduced. In mice exposed for
    seven days to 25 mg/kg body weight per day, the cytotoxic T lymphocyte
    response to P815 mastocytoma cells was enhanced, and there was a
    significant reduction in the IgM and IgC plaque-forming cell response
    to sheep red blood cells.  In vitro in murine thymocytes, a
    concentration of 10 µg/ml dinocap for 72h suppressed the proliferative
    response to concanavalin A and phytohaemagglutinin; exposure for as
    little as 30 min suppressed the mitogen-stimulated response with no
    direct cytotoxicity (Smialowicz et al., 1992a).

    2.2.4  Polycyclic aromatic hydrocarbons

          A major concern for human health is the carcinogenic potential of
    most polycyclic aromatic hydrocarbons (PAHs). Interestingly, those
    which are carcinogenic also have potent immunosuppressive properties,
    whereas those which are not carcinogenic lack marked immunotoxic
    effects (Ward et al., 1985; White, 1986). Suppression of humoral
    immunity has been observed frequently after exposure to a number of
    PAHs, including benzo[ a]pyrene, DMBA, and 3-methylcholanthrene (Ward
    et al., 1985). Structure-activity studies by White et al. (1985), in
    which the antibody-forming cell response was used to evaluate 10 PAHs
    in B6C3F1 and DBA/2 mice, demonstrated a wide spectrum of activity:
    compounds like benzo[ e]pyrene and perylene were not immunotoxic,
    whereas dibenz[ a,h)anthracene and DMBA were potent immunosuppressors
    of the plaque-forming cell response. Interestingly, the DBA/2 mice
    were more susceptible to the immunosuppressive effects than the B6C3F1

          PAHs also suppress cell-mediated immunity. T-Lymphocyte
    cytotoxicity and mixed lymphocyte responsiveness were found to be
    impaired by most PAHs. Differences between PAHs are seen, however, in
    that benzo[ a]pyrene may be less suppressive of cell-mediated
    immunity than DMBA, accounting for the greater host susceptibility to
     L. monocytogenes and PYB6 sarcoma challenges in DMBA- than in
    benzo[ a]pyrene-treated rodents (Ward et al., 1985). Thurmond et al.
    (1987) evaluated immunosuppression in B6C3F1 ( Ah-responsive) and
    DBA/2 ( Ah-nonresponsive) mice and in  Ah-congenic C57Bl/6J
    (responsive B6-AhbAhd and nonresponsive B6-AhdAhd) mice after
    exposure to DMBA in a battery of immunological assays, including
    evaluation of organ weights, plaque-forming cell response, mitogen
    responses, and mixed lymphocyte responses. The authors concluded that
    the immunosuppressive action of DMBA was independent of the  Ah locus
    and associated induction of cytochrome P1-450 metabolizing enzymes.

          The mechanisms of PAH-mediated immunosuppression remain to be
    elucidated. PAHs may exert their immunotoxic effects as the parent
    compound or as metabolites.  In vitro many of the metabolites of
    benzo[ a]pyrene and DMBA are immunosuppressive, the diol metabolites
    being the most potent (Kawabata & White, 1987; Ladics et al., 1991).

    Several possible mechanisms of action have been proposed, including
    altered interleukin levels (Lyte & Bick, 1986; Pallardy et al., 1989),
    a direct effect on transmembrane signalling (Pallardy et al., 1992),
    and alterations in intracellular calcium mobilization (Burchiel et
    al., 1991; Davis & Burchiel, 1992).

          Earlier studies suggested that Th cells or faulty antigen
    recognition by T cells were possible mechanisms of DMBA-induced
    immunosuppression (House et al., 1987, 1989). Myers et al. (1987) also
    reported that benzo[ a]pyrene alters macrophage antigen presentation.
    Studies by Ladics et al. (1992) demonstrated that the only splenic
    cell type capable of metabolizing benzo[ a]pyrene was the macrophage
    and that the predominant immunosuppressive metabolite formed was the
    benzo[ a]pyrene-7,8 diol epoxide, which is also believed to be the
    ultimate carcinogenic metabolite of benzo[ a]pyrene.

    2.2.5  Solvents  Benzene

          Exposure to benzene is associated with myelotoxicity, and a
    strong correlation was noted between lymphocytopenia and abnormal
    immunological parameters. The myelotoxicity may be due, in part, to
    altered differentiation of marrow lymphoid cells, as suggested by the
    finding that acute exposure of IgM+ cell-depleted marrow cultures to
    hydroquinone, an oxidative metabolite of benzene, blocked the final
    maturation stages of B-cell differentiation (King et al., 1987). In
    addition, it was shown that the hydroquinone metabolite inhibits
    lectin-stimulated lymphocyte agglutination and mitogenesis by reacting
    with intracellular sulfhydryl groups (Pfeifer & Irons, 1981).

          Immunosuppression associated with exposure to benzene was found
    in rabbits to be an impaired antibody response together with an
    increased susceptibility to tuberculosis and pneumonia. Similarly,
    C57Bl/6 mice exposed to benzene had a lower antibody response and
    reduced mitogen-induced lymphocyte proliferation (Wierda et al.,
    1981). Chronic inhalation of concentrations as low as 30 ppm impaired
    resistance to  L. monocytogenes (Rosenthal & Snyder, 1985).
    Similarly, increased susceptibility to PYB6 tumour cell challenge was
    seen at concentrations that also impaired Tc lymphocyte function.

          The mechanism of benzene-induced immunosuppression is unclear.
    Cellular depletion may be the major effect, although B- and T-cell
    dysfunction may also be involved. The antiproliferative effects of
    benzene may be related to its ability to alter cytoskeletal
    development through inhibition of microtubule assembly. Polyhydroxy
    metabolites of benzene ( para-benzoquinone and hydroquinone) have
    been shown to bind to sulfhydryl groups on the proteins necessary for
    the integrity and polymerization of microtubules. This effect may
    alter cell membrane fluidity and may explain the sublethal effect of
    benzene on lymphocyte function.  Other solvents

          Hexanediol (1.2 mg/kg per day for seven days) decreased thymus
    and spleen weights, antibody production, and delayed-type
    hypersensitivity in mice (Kannan et al., 1985). Humoral immunity was
    suppressed to a greater extent in female than in male mice after a
    four-month exposure to trichloroethylene in the drinking-water at
    doses of 0.1, 1.0, 2.5, or 5.0 mg/ml; cell-mediated immunity and bone-
    marrow stem-cell colonization were inhibited only in females (Sanders
    et al., 1982). The immunotoxicity of glycol ethers and some of their
    metabolites has been studied in rats by measuring the plaque-forming
    cell response to trinitrophenyl lipopolysaccaride. The glycol ethers
    2-methoxyethanol and 2-methoxyethylacetate were immunosuppressive, as
    was the principal metabolite of the latter, 2-methoxyacetic acid. The
    glycol ethers 2-(2-methoxyethoxy)ethanol, bis(2-methoxyethyl) ether,
    2-ethoxyethanol and its principal metabolite 2-ethoxyacetic acid,
    2-ethoxyethyl acetate, and 2-butoxyethanol were not immunosuppressive
    (Smialowicz et al., 1991, 1992b, 1993)

          Dichloroethylene did not induce immunotoxic changes in mice given
    up to 2 mg/litre per day for 90 days (Shopp et al., 1985). Similarly
    negative findings were obtained with trichloroethane (Sanders et al.,

    2.2.6  Metals

          Heavy metals have been shown to alter immune responsiveness in
    laboratory animals (Koller, 1980). Alterations in B lymphocyte
    function have been observed most frequently after exposure to lead and
    cadmium, but T-cell and macrophage changes have also been described.
    In addition, exposure to metals is correlated better with impaired
    resistance to experimental infections than with changes in B-
    or T-cell functions. Interestingly, immunostimulation has been
    shown to occur at levels of exposure lower than those associated with
    immunosuppression. Metals have also been shown to induce immuno-
    potentiation, at lower doses than those that cause immunosuppression.  Cadmium

          Conflicting results have been obtained with regard to the effect
    of cadmium on humoral immunity in animals (Descotes et al., 1990).
    Cell-mediated immunity, however, is consistently depressed after both
    short- and long-term exposure, and phagocytosis and NK cell activity
    are found to be depressed. Susceptibility to  L. monocytogenes, Herpes
     simplex 1 and 2, and influenza virus was increased in B6C3F1 mice
    exposed for long periods (Thomas et al., 1985a).  Lead

          Experimental studies suggest that lead has immunosuppressive
    effects in rodents (Lawrence, 1985; Descotes et al., 1990; Koller,
    1990). Early studies demonstrated that lead can suppress the humoral
    immune response of mice exposed as adults (Koller & Kovacic, 1974) and
    of rats exposed pre- and postnatally (Luster et al., 1978). In
    contrast, no change in humoral immunity was found in mice exposed to
    0.08-10 mmol/litre in drinking-water (Lawrence, 1981) or after a
    10-week oral treatment with 13, 130, or 1300 mg/kg of diet (as lead
    acetate) (Koller & Roan, 1980). Delayed-type hypersensitivity was
    found to be depressed by lead acetate and lead chloride but not in
    mice treated with lead oxide, nitrate, or carbonate. The most
    consistent finding in experimental studies of the effects of lead on
    host resistance, however, is increased susceptibility to infectious
    agents (McCabe, 1994).

          With respect to nonspecific host defence mechanisms, mice treated
    with lead at doses of 5, 10, or 20 µg/kg body weight given intra-
    peritoneally once or at doses of 25, 50, or 100 µg/kg body weight
    given orally once showed increased clearance of colloidal carbon
    (Schlick & Friedberg, 1981). Furthermore, treatment of mice with 130
    or 1300 ppm of lead orally for 10 weeks impaired the phagocytosis of
    sheep red blood cells (Koller & Roan, 1977). Lead also has consistent
    overall effects on host resistance to infection. Thus, treatment
    resulted in significantly decreased resistance of mice to  Klebsiella
     pneumoniae (Hemphill et al., 1971) and  S. typhimurium, and decreased
    resistance of rats to a bacterial endotoxin and to a challenge with
     E. coli, S. epidermidis, or  S. enteritidis. The increased
    susceptibility of rodents to Gram-negative bacteria after exposure
    to lead is likely to be due to hypersensitivity to an endotoxin of
    bacterial origin (Cook et al., 1974, 1975)

          Organolead compounds, such as tetrethyllead, can also be
    immunotoxic (Luster et al., 1992).  Mercury

          Mercuric salts have been shown repeatedly to depress both humoral
    and cellular immunity and nonspecific host defences in animals. For
    instance, B6C3F1 mice given mercuric chloride orally for seven weeks
    had decreased thymus and spleen weights, an impaired plaque-forming
    cell response, and inhibition of lymphocyte proliferation at a daily
    dose of 75 mg/litre of drinking-water (Dieter et al., 1983).
    Methylmercury was reported to decrease humoral immunity in mice
    treated orally for three weeks with 0.5, 2, or 10 mg/litre drinking-
    water (Blackley et al., 1980).  Organotins

          Several organotins have been shown to be markedly immunotoxic and
    are considered as prototype immunotoxicants (Penninks et al., 1990),
    even though no human data are available.

          Di- n-octyltin dichloride at 50 or 150 mg/kg of diet for six
    weeks induced a dramatic, dose-related decrease in the weight of the
    thymus in rats, associated with a less severe decrease in spleen and
    lymph node weights (Seinen & Willems, 1976). The numbers of cells in
    the thymus and spleen, but not the bone marrow, were decreased.
    Histologically, lymphocyte depletion was seen in the thymus and in
    thymus-dependent areas of the spleen. Interestingly, thymic atrophy
    recovered quickly after cessation of exposure (Seinen et al., 1977).
    It was later shown to be associated with a 25% decrease in peripheral
    blood lymphocytes, with a preferential loss of Th lymphocytes. As
    expected, T-cell functions, such as the delayed-type hypersensitivity
    response and T-lymphocyte proliferation, were depressed. Inhibition of
    humoral immunity was also seen, with reduced numbers of plaque-forming
    cells and decreased circulating antibody titres. NK cell activity was
    not affected, whereas susceptibility to  L. monocytogenes infection
    was markedly increased.

          Immune function is not impaired in guinea-pigs or mice fed
    di- n-octyltin dichloride, which correlates with the absence of
    thymic atrophy (Seinen & Penninks, 1979). Mice treated intravenously
    or intraperitoneally develop thymic atrophy, however, suggesting
    interspecies variability in the disposition of dialkyltins after oral
    intake, although other, poorly understood mechanisms may account for
    this variability (Penninks et al., 1990). No interspecies differences
    in lymphocyte functions were noted after exposure  in vitro.

          Generally similar findings were made with the trialkylorganotin,
    tri- n-butyltin oxide (TBTO). As trisubstituted organotins are
    rapidly metabolized to disubstituted derivatives, the latter are
    considered to be involved in the reported thymic effects (Snoeij et
    al., 1988). In a short-term study in rats, pronounced effects were
    found on the lymphoid organs: thymus (Figure 29), spleen, and lymph
    nodes. These effects were most pronounced in thymus-dependent areas
    (Figure 30) (Krajnc et al., 1984). Interestingly, thymus atrophy also
    occurred in fish, as guppies exposed to organotin compounds showed
    severe thymic atrophy (Figure 31). In function tests (Vos et al.,
    1984), rats that were exposed to TBTO for six weeks after weaning had
    suppressed delayed-type hypersensitivity responses to ovalbumin and
    tuberculin and suppressed IgG responses to sheep erythrocytes.  In
     vitro mitogen responses to concanavalin A in thymus and spleen and
    NK cell activity in both the spleen and the lungs were decreased (Van
    Loveren et al., 1990b). Exposure to TBTO at 20 or 80 mg/kg of diet for
    six weeks led to decreased resistance to infection with
     L. monocytogenes or  Trichinella spiralis. The latter effect was

    evidenced by increased numbers of adult worms in the gut as a result
    of impaired worm expulsion, increased numbers of muscle larvae in the
    striated tissue, decreased inflammatory responses around these larvae,
    and decreased antibody responses to  T. spiralis, especially in the
    IgE class (Vos et al., 1984). After long-term exposure (15-17 months)
    to 5 or 50 mg/kg of diet, delayed-type hypersensitivity was not
    suppressed, but assays for NK cell activity and resistance to
    infection indicated suppression.

          As the immune responsiveness of older animals can be expected to
    be less strong than that of younger rats, the effects of exposure to
    immunotoxic chemicals may become evident less easily; however, tests
    for function still indicated immunotoxicity. In experiments in which
    exposure to TBTO was begun only at 12 months of age, both infection
    models showed immunotoxicity to TBTO. Very few studies have focused on
    the immunotoxic effects of chemicals on the gut immune system, but the
    studies of TBTO showed both a decreased capacity of the host to expel
    adult  T. spiralis worms from the gut and increased production of
    serum IgA specific for this parasite (Vos et al., 1990a).

          The mechanism of the immunotoxicity of organotin compounds has
    been investigated extensively (Penninks et al., 1990). A direct
    influence on the synthesis of thymic hormones is uncertain, as
    conflicting results have been obtained in different experiments.
    Interference with the influx of prothymocytes can be ruled out, as
    thymic atrophy develops too rapidly. Interestingly, organotins reduced
    the proliferative activity of thymocytes and the number of
    proliferating thymoblasts within 24h after exposure was begun, at a
    time when thymic atrophy was not evident. This selective effect on
    thymoblasts and the physiological destruction of most cortical
    thymocytes would result in marked depletion and, finally, in thymic
    atrophy.  Gallium arsenide

          Gallium arsenide is an intermetallic compound used widely in the
    electronics industry, primarily in the manufacture of transistors and
    light-emitting diodes. A single intratracheal instillation of 50, 100,
    or 200 mg/kg body weight into female B6C3F1 mice resulted in a dose-
    related decrease in the IgM and IgG antibody response to sheep
    erythrocytes. Similarly, cell-mediated immunity, as evaluated by the
    delayed-type hypersensitivity reaction to keyhole limpet haemocyanin
    and the mixed lymphocyte response, was also decreased in a dose-
    dependent way. Increases were observed in complement C3 levels,
    mitogen response to lipopolysaccharide, and NK cell activity. No
    effects were observed on response to T-cell mitogens, total complement
    CH50 activity, or host resistance to  Plasmodium yoelii or
     Streptococcus pneumoniae; however, a significant decrease in host
    resistance was observed to  L. monocytogenes and B16F10 tumour
    challenge (Sikorski et al., 1989).

    FIGURE 29

    FIGURE 30

    FIGURE 31  Beryllium

          Beryllium induces a variety of diseases, including granulomatous
    lung (chronic beryllium disease) and skin conditions. These
    granulomatous reactions involve a lymphocyte response to beryllium
    salts. The major lymphocyte population consists of Th cells (CD4). The
    T-cell response to beryllium is IL2-dependent (Saltini et al., 1989).
    The antigen has not been identified, but may be a beryllium-protein
    complex. There appears to be a genetic predisposition, as the majority
    of patients with beryllium lung disease share a particular HLA-Dp
    allele (HLA-DpB1) (Richeldi et al., 1992). The development and
    maintenance of lung and skin granulomas depend on the presence of
    antigen, antigen-presenting cells, and memory T lymphocytes and the
    release of proinflammatory cytokines by macrophages and lymphocytes
    (Boros, 1988; Kunkel et al., 1989).

    2.2.7  Air pollutants

          Pollutants characteristic of occupational and urban environments
    may cause or aggravate pulmonary diseases. Pulmonary defence
    mechanisms to pathogens comprise mechanical defences, nonspecific
    defences (ingestion by phagocytic cells, lysis of virus-infected
    cells), and specific immunity. A number of studies in experimental
    animals have shown that exposure to air pollutants, including ozone,
    nitrogen dioxide, sulfur dioxide, some volatile organic compounds, and
    metal particulates, adversely affects pulmonary defences, and
    primarily nonspecific defences important in clearing certain Gram-
    positive bacteria from the lung (Graham & Gardner, 1985; Jakab &
    Hmieleski, 1988; Selgrade & Gilmour, 1994).

          In dogs, exposure to ozone at 3 ppm for 2 h per day for three
    days markedly increased the number of epithelial neutrophils, whereas
    the number of circulating neutrophils was decreased (O'Byrne et al.,
    1984). A significant decrease in absolute thymocyte numbers was also
    observed in mice continuously exposed to 0.7ppm ozone for three to
    seven days (Li & Richters, 1991). Decreased spleen and thymus weights
    were reported in mice exposed to ozone alone or in combination with
    nitrogen dioxide (Fujimaki, 1989; Goodman et al., 1989). The numbers
    of neutrophils and alveolar macrophages in bronchoalveolar lavage
    fluid were found to be increased in rats, and T-lymphocyte
    infiltrations were seen in ozone-induced lesions of mice.
    Accumulations of macrophages are located mainly at the bronchoalveolar
    junction and in alveoli (Figures 32 and 33).

    FIGURE 32

    FIGURE 33

          Modulation of nonspecific defence mechanisms by ozone has also
    been described (Goldstein et al., 1971; Holt & Keast, 1977; Van
    Loveren et al., 1988a, 1990b). Thus, phagocytic activity in alveolar
    macrophages is suppressed, but this depends on the concentration and
    duration of exposure; enhanced phagocytic activity was also observed.
    Alterations in the macrophage production of arachidonic acid
    metabolites, resulting in increased prostaglandin 2 production, have
    been suggested to be involved (Gilmour et al., 1993). NK cell activity
    is either unaffected or stimulated by low ozone concentrations,
    whereas high concentrations decreased both the number and the activity
    of splenic and pulmonary NK cells (Burleson et al., 1989; Van Loveren
    et al., 1990b). Ozone also affects T cells (Dziedzic & White, 1986;
    Van Loveren et al., 1988a; Bleavins & Dziedzic, 1990; Dziedzic et al.,
    1990). Ozone-induced systemic dysfunction has been reported in animals
    and probably contributes to impaired host defences (Aranyi et al.,
    1983). Humoral immunity, e.g. circulating antibody titres to a variety
    of antigens and the plaque-forming cell response to sheep
    erythrocytes, is depressed after exposure to ozone; cellular immunity
    is also inhibited. The numbers of all major T lymphocyte subsets,
    mitogen-induced T lymphocyte proliferation, and delayed-type
    hypersensitivity responses were all shown to be decreased. Numerous
    studies with infectivity models show that exposure to ozone has an
    adverse influence on the host defences to respiratory infections (Van
    Loveren et al., 1994), and most of the studies demonstrate that the
    primary targets are the alveolar macrophages (Selgrade & Gilmour,

          Although the influence of other air pollutants such as nitrogen
    dioxide and sulfuric acid on host defences has been the subject of
    fewer studies, the available data suggest that they have similar
    adverse effects (Graham & Gardner, 1985). In view of the numerous
    possible targets of air pollutants on respiratory defences and because
    of the intricate mechanisms involved, infectivity models in animals
    are particularly relevant for ascertaining the likely consequences of
    air pollution for exposed human populations.

    2.2.8  Mycotoxins

          Mycotoxins are structurally diverse secondary metabolites of
    fungi that grow on feed. Mycotoxin-induced immunosuppression may be
    manifested as depressed T- or B-lymphocyte function, decreased
    antibody production, or impaired macrophage activity. Immuno-
    stimulation may also be observed with the tricothecenes under
    some experimental conditions. Similar effects have been found on the
    proliferative responses of human and rodent lymphocytes  in vitro
    (Lang et al., 1993). Most of the data have been obtained  in vivo or
     in vitro in animal systems, and there is only limited evidence that
    mycotoxins are immunosuppressive in humans (Lea et al., 1989).

          Dietary exposure to various mycotoxins resulted in decreased
    antibody production, T-lymphocyte proliferative response, delayed-type
    hypersensitivity, and NK cell activity (Pestka & Bondy, 1990).
    Interestingly, dietary intake was associated with increased
    susceptibility to experimental infections.

          Aflatoxin is markedly immunosuppressive in cattle and poultry
    (see below). Thymic atrophy, suppression of mitogen-induced T- and
    B-lymphocyte proliferation, and decreased antibody responses to
    various microbial antigens and sheep erythrocytes have been observed
    (Corrier, 1992). Cell-mediated immune responses appear to be affected
    at lower concentrations than antibody responses. The mechanism of
    action seems to be related to impaired protein synthesis.

          Ochratoxin, a mycotoxin produced by several species of
     Aspergillus and  Penicillium, causes depletion of lymphoid cells in
    the spleen and lymph nodes of dogs, swine, and mice (Corrier, 1992).
    The dose, the route of administration, and the animal species appear
    to be critical factors, however; for instance, administration of 13 mg
    of ochratoxin to mice in six intraperitoneal injections did not impair
    T-lymphocyte proliferation (Luster et al., 1987), whereas
    intraperitoneal injections of 5 mg/kg body weight for 50 days did
    (Prior & Sisodia, 1982). Ochratoxin also impairs NK cell activity and
    increases tumour cell growth in mice (Luster et al., 1987).

          The trichothecenes, including T-2 toxin and deoxynivalenol
    (vomitoxin), are a structurally related group of mycotoxins produced
    by  Fusarium. T-2 toxin has been studied extensively and has been
    shown to induce lymphoid depletion in the thymus, spleen, and lymph
    nodes of numerous laboratory animals (Pestka & Bondy, 1990; Corrier,
    1992). In addition, mitogen-induced T- or B-lymphocyte proliferation,
    antibody production, and macrophage activation have been found to be
    depressed after exposure to either T-2 toxin or vomitoxin. The
    impaired immune responsiveness is associated with increased
    susceptibility to a variety of experimental infections. As the
    tricothecenes are currently considered to be the most potent small-
    molecule inhibitors of protein synthesis in eukaryotic cells, the
    immunosuppression associated with exposure to these mycotoxins is
    likely to be directly or indirectly linked to inhibition of protein

    2.2.9  Particles  Asbestos

          Exposure to asbestos is associated with the development of
    inflammatory, fibrotic, and malignant (i.e. pleural mesothelioma and
    bronchogenic carcinoma) diseases in humans. Although the pathogenesis
    of asbestos-induced lung diseases is complex, a number of observations

    indicate that immune processes influence the development and
    resolution of both the inflammatory response and fibrotic lesions. For
    example, exposure to asbestos is associated with alterations in
    cellular and humoral-mediated immunity, including reduction of
    lymphocyte mitogenesis, delayed hypersensitivity responses, and
    primary antibody responses (Hartmann et al., 1984; Hartmann, 1985;
    Bissonette et al., 1989; Miller, 1992). In addition, immunodeficient
    mice resolve asbestos-induced inflammatory and fibrotic responses only
    with difficulty (Corsini et al., 1994), suggesting that immune
    mediators with anti-inflammatory or anti-fibrotic activity (e.g. IL-4
    or INF gamma) are involved. Furthermore, it is well established that
    alveolar macrophages and type II epithelial cells secrete inflammatory
    cytokines, chemokines, and growth factors in response to asbestos
    (Driscoll et al., 1990; Rosenthal et al., 1994), and these mediators
    are directly involved in the inflammatory responses (e.g. inflammatory
    cell recruitment) and fibrogenesis (e.g. fibroblast proliferation).  Silica

          Experimental animals have been used extensively to define the
    pathogenesis of silicosis (Uber & McReynolds, 1982). Several immune
    changes have been demonstrated in guinea-pigs, including depression of
    humoral and cellular immunity and increased susceptibility to
    infectious agents. Similarly, mice exposed to silica showed decreased
    lipopolysaccharide-induced proliferation of B lymphocytes and
    depressed plaque-forming cell responses (Scheuchenzuber et al., 1985).
    Antibody responses to T-independent antigens, however, were less
    markedly depressed than responses to T-dependent antigens, suggesting
    an additional effect on T-cell control of humoral immunity. The
    effects of silica on cellular immunity depend on the dose and route of
    entry of antigens. The concanavalin A-induced proliferation response
    of spleen T lymphocytes was increased, whereas that of mesenteric
    lymph node T lymphocytes was depressed. The aberrations of humoral and
    cellular immunity induced by silica are thus complex, and it remains
    to be established how these immune changes correlate with the
    induction of lung fibrosis or autoantibodies, the major adverse
    consequences of exposure to silica. In addition, silica is markedly
    toxic to macrophages and activates alveolar macrophages, granulocytes,
    and monocytes (Gusev et al., 1993). Infectivity models consistently
    show an increased susceptibility of silica-exposed rodents to
    infectious pathogens.

    2.2.10  Substances of abuse

          The immunotoxic consequences of exposure to substances of abuse
    are difficult to ascertain in most instances as confounding factors,
    such as intercurrent infections secondary to intravenous injection,
    may contribute to the observed changes. Recent research has provided
    evidence, however, that substances of abuse can directly affect the
    immune system (Descotes, 1986; Watson, 1990; Friedman et al., 1991a;
    Watson, 1993).

          In rodent lymphocytes  in vitro, D9-tetrahydrocannabinol
    depressed the proliferative responses of T lymphocytes in a dose-
    dependent manner (Friedman et al., 1991b). Further to the early
    findings that opiates adversely affect immune competence (Cantacuzene,
    1898), an increasing body of evidence shows that exogenous opioids
    have a variety of effects on cells of the immune system (Rouveix,
    1993). At pharmacological concentrations, opiates suppress antibody
    production, lymphocyte proliferation, and delayed-type
    hypersensitivity and decrease NK cell activity in various animal
    models. In addition, phagocytosis is impaired. Opioid peptides can,
    however, also have a stimulatory effect on the immune system,
    depending on the experimental conditions. ß-Endorphin affects cytokine
    production in rat and mouse T-cell cultures  in vitro; e.g. it
    stimulates the synthesis of IL-2, IL-4, and INF gamma, thereby
    inducing MHC class II expression on B cells (van den Bergh et al.,
    1993a,b, 1994).

          In general, short-term exposure of mice, rats, and guinea-pigs to
    mainstream tobacco smoke either produces no significant immuno-
    modulatory effect or a slight immunostimulation, which returns
    to normal shortly after cessation of exposure (Johnson et al., 1990).
    In contrast, subchronic or chronic (more than one year) exposure is
    generally immunosuppressive: cellular immunity, e.g. mitogen-induced
    lymphocyte proliferative response, and NK cell activity are impaired
    after long-term exposure to tobacco smoke. The humoral immune response
    is also depressed, as shown by decreased antibody titres, and animals
    exposed to cigarette smoke for extended periods are more susceptible
    to tumour and infectious challenge than naive animals.

    2.2.11  Ultraviolet radiation

          The earliest indication that ultraviolet radiation (UVR) affects
    the immune system came from studies of host resistance to UVR-induced
    tumours in mice (Kripke, 1974). Subsequent studies showed that low
    doses of UVR suppress contact hypersensitivity responses to chemical
    sensitizers (Toews et al., 1980) and that systemic immunosuppression
    (depressed contact hypersensitivity in unirradiated skin) occurs after
    exposure to higher doses (Jessup et al., 1978). Irradiated mice were
    also found to be less resistant to infection (Giannini, 1990). Other
    studies (Noonan & De Fabo, 1990) have determined that systemic
    suppression of immunoreactivity is not a function of the dose of UVR
    but rather of the interval between irradiation and immunization of the
    mice. Thus, induction of contact hypersensitivity responses in mice
    exposed to low doses of UVR was not affected when the animals were
    immunized through unirradiated skin immediately after exposure to UVR;
    however, sensitization was suppressed if three days were allowed to
    elapse between irradiation and immunization. It has also been shown
    that the dose of UVR required to induce 50% suppression of the immune
    response depends on the strain of mouse and the type of antigen used
    (Noonan & De Fabo, 1990; Noonan & Hoffman, 1994).

          The mechanism of UVR-induced suppression of cellular immunity has
    not been elucidated, nor has a single initial event been identified
    that leads to suppression of immunoreactivity. Currently, induction of
    pyrimidine dimers in DNA (Kripke et al., 1992) and isomerization of
    urocanic acid (Noonan & De Fabo, 1992) are the leading contenders.
    Increased suppressor cell activity (Brodie & Halliday, 1991) and
    efferent lymphatic blockade, which inhibits lymphocyte homing, may be
    responsible for the UVR-associated accumulation of lymphocytes in
    lymph nodes in UVR-exposed areas (Spangrude et al., 1983) and have
    been proposed as possible causes of immunosuppression. Exposure to UVR
    has also been shown to alter the pattern of cytokine production by
    T cells, from a response dominated by Th1 (i.e. favouring delayed
    hypersensitivity responses) to one dominated by Th2 (i.e. favouring
    antibody production) (Araneo et al., 1989; Simon et al., 1990).
    Exposure to UVR has been reported to affect Langerhans cells directly,
    such that their interaction with T cells induces specific antigen
    tolerance in the Th1 subpopulation (Simon et al., 1990) and
    preferential activation of the Th2 population (Simon et al., 1991).
    This may be the reason that mice exposed to UVR are more susceptible
    to infection with the protozoan  Leishmania major (Giannini, 1992),
    since resistance to infection with this intracellular parasite is
    dependent on the magnitude of the Th1 response of the host (Reed &
    Scott, 1993). In addition, reduced resistance to  T. spiralis was
    found in rats exposed to UVR on days 5-10 of infection (Goettsch et
    al., 1993). Altered cytokine production profiles may also be
    responsible for increased sensitivity to  Mycobacterium lepraemurium,
    an intracellular pathogen that induces a chronic and eventually fatal
    infection in susceptible mice. In a comparison of susceptible (BALB/c)
    and resistant (C57Bl/6J) mice, Brett & Butler (1986) determined that
    resistance to infection is correlated with the ability of mouse
    lymphocytes to elaborate cytokines that activate macrophages, rather
    than with the actual development of a delayed hypersensitivity
    response to bacterial antigens. Jeevan & Kripke (1990) and Jeevan et
    al. (1992) reported that irradiation of BALB/c mice resulted in
    decreased resistance to infection, as measured by bacterial counts and
    length of survival after infection. Elevated bacterial counts were
    seen in animals exposed to doses of UVR that did not suppress the
    delayed hypersensitivity response to bacterial antigens, suggesting
    that the underlying mechanism of UVR-induced suppression of resistance
    to infection is independent of suppressed delayed hypersensitivity.

    2.2.12  Food additives

          There is little information about the effects of food additives
    on the immune system. An early study showed that the preservative
    methylparaben and the antioxidants butylated hydroxyanisole, butylated
    hydroxytoluene, and propylgallate suppress the in-vitro T-dependent
    antibody response, whereas vanillin and vanillic acid stimulate it
    (Archer et al., 1978). The significance of these findings  in vivo
    has yet to be established.

          The immunotoxicity of 'caramel colour', which covers a large
    number of complex products used as food colorants, has been
    investigated. One of the compounds in this group, 2-acetyl-4(5)-
    tetrahydroxybutylimidazole (THI) (caramel colour III), has been found
    to be immunotoxic in rodents (Kroplien et al., 1985). THI induces a
    rapid reduction in the number of B and T cells in blood, spleen, and
    lymph nodes and morphological changes in the thymus of rats, with an
    increased number of mature medullary thymocytes and a decreased number
    of cortical macrophages. THI might reduce the migration of mature
    thymocytes into the periphery, as a decrease in the number of recent
    ER4+ thymic emigrants was found in the spleens of exposed rats
    (Houben et al., 1992). Functional studies indicate changes in Th cell
    function, an increased capacity to clear the Gram-positive bacterium
     L. monocytogenes, and modulation of the activity of adherent splenic
    cells (Houben et al., 1993). It has been hypothesized that THI exerts
    an antivitamin B6 action by competing with pyridoxal 5'-phosphate for
    binding to the cofactor site of one or more pyridoxal 5'-phosphate-
    dependent enzymes.

    2.3  Immunotoxicity of environmental chemicals in wildlife and
         domesticated species

          Most of the concern about chemical contamination of wildlife
    populations has been focused on the aquatic ecosystem, and a growing
    body of literature has appeared on the effects of pollution on the
    health status of aquatic life. These studies deal mainly with the
    occurrence of tumours and infectious diseases in fish and marine
    mammals. These are multifactorial diseases in which perturbations of
    the immune system may play a part.

    2.3.1  Fish and other marine species  Fish

          Fish diseases are being monitored on a routine basis at various
    sites in North America and Europe. In Europe, most of the programmes
    are carried out under the auspices of the International Council for
    Exploration of the Sea. National and local studies have been directed
    to estuarine, marine, and brackish waters suspected of being polluted,
    such as in the vicinity of industrial areas and after major oil
    spills. The common diseases that are discussed in relation to
    pollution are certain skin diseases, such as lymphocystis, papillomas,
    fin rotting, and skin ulcers (Vethaak & ap Rheinallt, 1992), as they
    are easily identified grossly and are therefore potentially useful for
    biomonitoring. Since most diseases of fish have a viral or bacterial
    etiology, and elevated incidences have been correlated with chemical
    pollution, immunotoxicity may play a role. A causal relationship
    between chemical pollution and a disease state induced by
    immunosuppression cannot be finally established, however, since many
    confounding factors exist in the natural environment. Liver neoplasia

    and precursor lesions have been used to biomonitor environmental
    pollution in flatfish (Malins et al., 1988; Vethaak & ap Rheinallt,
    1992; Vethaak, 1993); however, the role of the immune system is not

          Effects observed in field studies are modified or confounded by
    numerous factors, in particular for feral fish, for which there are
    deficient case histories and limited knowledge of their migratory
    patterns and biology (Vethaak, 1993). Extensive epidemiological
    surveys are required that include specific parameters that have been
    validated in experiments under (more) controlled conditions (in
    mesocosms or the laboratory). Changes in disease patterns may suggest
    immune alterations, but this should be demonstrated. Since most
    diseases have a complex etiology, it will be difficult to establish
    the role of immunotoxic effects under field conditions. Circumstantial
    evidence can be obtained in these instances, although in the case of
    feral animals mesocosm or laboratory experiments must carried out in
    order to reach a final conclusion (Secombes et al., 1992; Vethaak,

          The etiological components and their role in the pathogenesis of
    many diseases in fish in the field are, as yet, poorly understood, and
    laboratory experiments are often indispensable for background
    knowledge. Even when such scientific deficiencies are resolved,
    laboratory studies will still be needed, since function tests under
    controlled conditions yield the most reliable and sensitive methods of
    assessing immunological stress and must often accompany field studies,
    as mentioned above. Findings from laboratory situations do not
    necessarily imply effects in the field, however; in particular, when
    results from the laboratory are extrapolated to field situations,
    there is often a discrepancy between the levels of exposure.  Marine mammals

          Marine mammals are of special interest to the discipline of
    immunotoxicology. As the highest predators in highly contaminated
    marine environments, these animals are exposed to a large number of
    environmental chemicals, some of which have been identified as
    potentially immunotoxic. Persistent lipophilic halogenated compounds
    such as PCBs, polychlorinted dibenzodioxins, polychlorinated
    dibenzofurans, and pesticides accumulate in the marine food chain and
    are thus biomagnified in marine mammals. The concentrations of PCBs in
    the blubber layer of marine mammals are higher than in any other
    wildlife species measured (Table 6). In times of food shortage and
    other stressful circumstances, these lipids are mobilized, thereby
    releasing their toxic burden.

        Table 6.  Concentrations of polychlorinated biphenyls (PCBs) in herring and the blubber
              layer of marine mammals

    Species                       Source              Total PCBs          Reference
                                                      (µg/g lipid)

    Herring                       Atlantic Ocean      0.0003-0.001        De Swart et al.
                                  (United Kingdom)                        (1994)

    Herring                       Baltic Sea          0.0035-0.0045       De Swart et
                                  (Sweden)                                al. (1993)

    Weddell seal                  Weddell Sea         0.07-0.09           Luckas et al.
    (Leptonychotes wedelli)       (Antarctic)                             (1990)

    Harbour seal                  Atlantic Ocean      1-13                Luckas et al.
    (Phoca vitulina)              (Iceland)                               (1990)

    Harbour seal                  Baltic Sea          21-140              Luckas et al.
    (Phoca vitulina)              (Sweden)                                (1990)

    Beluga whale                  St Lawrence         15-700              Muir et. al.
    (Delphinapterus leucas)       River (Canada)                          (1990);
                                                                          Martineau et al.

    Striped dolphin               Mediterranean       100-2600            Kannan et al.
    (Stenella coeruleoalba)       Sea (Spain)                             (1993)
          Because of the high level of exposure of marine mammals, they may
    be expected to be the first wild animals to suffer from
    immunosuppression due to chronic exposure to environmental
    contaminants. Toxicological research over the last 20 years has
    identified environmental chemicals as the source of many disorders in
    marine mammals. In both field studies and controlled experiments, PCBs
    have been linked to reproductive problems. As early as 1976, a high
    incidence of premature parturition was seen in California sea-lions
     (Zalophus californianus), which was caused by a viral infection and
    was suggested to be linked to higher levels of pollutants in animals
    that aborted as compared with mothers that gave birth to healthy pups
    (Gilmartin et al., 1976). Pathological changes in the uteri of seals
    in the highly polluted Baltic Sea, in some cases leading to sterility,
    could be correlated with increased levels of PCBs (Helle et al.,
    1976). In addition, several studies have linked skeletal deformities

    in grey seals  (Halichoerus grypus) and harbour seals  (Phoca
     vitulina) in the Baltic Sea to high levels of organochlorines in
    their environment (Bergman et al., 1992; Mortensen et al., 1992). In
    porpoises  (Phocoenoides dalli) living in the north-western part of
    the North Pacific, a negative correlation was found between serum
    testosterone levels and DDE concentrations in blubber (Subramanian et
    al., 1987). In an experimental situation, two groups of harbour seals
    were fed fish containing different levels of pollutants. Seals that
    were fed fish from the heavily polluted western part of the Dutch
    Wadden Sea had significantly lower pup production than seals feeding
    on less polluted fish (Reijnders, 1986). In the same study, it was
    shown that the seals fed polluted fish also had significantly reduced
    levels of vitamin A and thyroid hormone in their serum (Brouwer et
    al., 1989). No parameters of immune function were studied.

          Such correlative observations in the natural environment, in
    combination with the results of semi-field studies, suggest that the
    current levels of contaminants may be adversely affecting certain
    marine mammal populations. The occurrence of a large number of
    epizootics among seals and dolphins inhabiting polluted coastal areas
    -- among bottlenose dolphins  (Tursiops truncatus) on the Atlantic
    coast of the United States in 1987 and in the Gulf of Mexico in 1990,
    striped dolphins  (Stenella coeruleoalba) on the coast of France in
    1989 and in the Mediterranean Sea in 1990-92, Baikal seals  (Phoca
     sibirica) in Lake Baikal in 1987, and harbour seals in north-west
    Europe in 1988 -- led to both public and scientific discussions about
    the possible contribution of environmental pollutants to these disease
    outbreaks. Owing to the complexities of the relationships between
    toxicants and the immune system and the difficulties in obtaining
    samples fit for use in immunotoxicological studies, it has been
    impossible thus far to conclusively demonstrate immunosuppression in
    free-ranging marine mammals.

          Another immunotoxicological experiment was carried out in which
    two groups of juvenile harbour seals  (Phoca vitulina) were fed fish
    from the Baltic Sea or from the relatively unpolluted Atlantic Ocean.
    The diets were analysed for concentrations of potential immunotoxic
    chemicals: the estimated daily intakes of TCDD-like organochlorines
    were 270 and 35 ng/day for the two groups, respectively. Immunological
    function in the two groups was examined by measuring mitogen- and
    antigen-induced proliferative responses of lymphocytes, NK cell
    activity, serum antibody levels after immunization with primary
    antigens, and delayed-type hypersensitivity reactions. These
    techniques had to be validated for application to seals, as virtually
    nothing was known about the cellular immune system of marine mammals
    (De Swart et al., 1993, 1994). Seals fed herring from the contaminated
    Baltic Sea had significantly depressed immune function, as measured by
    decreased NK cell activity (Ross et al., in press) and T-cell mitogen-
    induced lymphocyte proliferation (De Swart et al., 1994), and

    significantly lower delayed-type hypersensitivity and antibody
    responses to immunization with ovalbumin (Ross et al., 1995). The
    functional changes were accompanied by increased numbers of
    neutrophils in the peripheral blood (De Swart et al., 1994). Since NK
    cells are an important line of defence against viruses, and
    lymphocytes (especially T cells) play a major role in the clearance of
    viral infections, functional suppression of these leukocytes may
    contribute to the severity of epizootic episodes among marine mammals.

          These experiments not only provide the first demonstration of
    pollution-induced impairment of immune function in marine mammals but
    indicate that mammals in general can undergo such impairment as a
    consequence of chronic exposure to the levels of pollution found in
    their natural habitats. It is still difficult, however, to link the
    disease outbreaks among marine mammals directly to pollution-induced
    impairment of immune function.

    2.3.2  Cattle and swine

          The effects of antimicrobial, corticosteroid, and hormonal
    compounds have been investigated in cattle, mainly as lymphocyte
    proliferative responses and neutrophil functions  in vitro. The
    results are in keeping with those obtained in humans (Black et al.,

          Few studies have dealt specifically with the direct immunotoxic
    effects of pesticides and environmental pollutants. No statistical
    difference was found between control and polybrominated biphenyl-
    exposed animals in the numbers of circulating total, T, and B
    lymphocytes, serum immunoglobulin levels, mitogen-induced
    proliferative responses of lymphocytes, antibody response to keyhole
    limpet mitogen, or cell-mediated response to purified protein
    derivative (Kateley & Bazzell, 1978). In contrast, peripheral blood
    lymphocytes from sows fed polybrominated biphenyls for 12weeks had
    significantly decreased responses to mitogens (Howard et al., 1980).

          The mycotoxin tricothecene produced by  Fusarium and several
    other fungi was shown to reduce lymphoid tissue cellularity and serum
    IgA, IgG, and IgM concentrations and to impair neutrophil migration,
    chemotaxis, and phagocytosis in cattle exposed to high doses (Buening
    et al., 1982; Mann et al., 1983). Similarly, aflatoxin was reported to
    suppress the mitogen-induced proliferative response of bovine
    lymphocytes (Paul et al., 1977). Other mycotoxins, e.g. ochratoxin A
    and zearalenone, have been suggested to be immunosuppressive in cattle
    (Black et al., 1992).

    2.3.3  Chickens

          Chickens have been used in a number of immunological studies, as
    the unique bursa of Fabricius, the avian organ for B-cell development,
    underlies the need for a separate avian model in immunotoxicology.
    Exposure of adolescent chickens to cyclophosphamide decreased the
    levels of antibodies to various antigens without decreasing graft-
    versus-host reactivity (Lerman & Weidanz, 1970). Nutritional
    deficiencies in selenium or vitamin E have also been shown to impair
    the humoral immune responses of adolescent chickens (Marsh et al.,
    1981). Exposure to cyclophosphamide  in ovo results in decreased
    antibody forming capacity, decreased responsiveness to
    phytohaemagglutinin and concanavalin A, and decreased weights and
    altered morphology of the thymus, spleen, and bursa of Fabricius
    (Eskola & Toivanen, 1974). Peritoneal macrophages were not affected
    after exposure to cyclophosphamide  in ovo, as judged by their
    number, superoxide anion production, and surface expression of Ia
    antigen and transferrin receptor (Golemboski et al., 1992). Dietert et
    al. (1985) showed that exposure to aflatoxin B1  in ovo did not alter
    humoral immunity; however, two parameters of cell-mediated, graft-
    versus-host, and cutaneous basophil hypersensitivity reactions were
    depressed. Methyl methanesulfonate decreased the bactericidal action
    of peritoneal macrophages for  E. coli after exposure  in vitro
    (Qureshi et al., 1989). TCDD impairs B-cell development in the bursa
    of Fabricius in chicken embryos (Nikolaidos et al., 1990).

    2.4  Immunotoxicity of environmental chemicals in humans

          Although limited, various lines of evidence derived from case
    reports, clinical studies, and well-designed longitudinal studies
    imply that environmental agents can affect the human immune system.
    While these data raise concern about potential health effects, they
    rarely refer to clinical disease, for a number of reasons. For
    example, there may be sufficient redundancy or reserve in the immune
    system that 'moderate' levels of immunosuppression do not result in
    disease. Alternatively, the clinical changes most likely to be
    associated with moderate immunosuppression, e.g. increased severity or
    frequency of pulmonary infections, do not occur. Well-designed
    clinical studies with adequate populations and appropriate monitoring,
    follow-up, and documentation of exposure have rarely been conducted.
    Examples of published reports that attribute immune changes in human
    populations exposed to environmental agents are summarized below. As
    the reader will note, these studies range from poorly defined to
    relatively large longitudinal studies.

    2.4.1  Case reports

          In an unsubstantiated study, a cluster of cases of Hodgkin's
    disease reported in a small town in Michigan (United States) was
    ascribed to chronic immune stimulation by mitogenic substances in the
    environment (Schwartz et al., 1978). Immunological studies of family

    members revealed a large number of individuals with altered ratios of
    T-lymphocyte subpopulations, autoantibodies, infections, recurrent
    rashes, and NK cell function. A report of a four-year study of workers
    engaged in the manufacture of benzidine, a human bladder carcinogen,
    suggested that individuals with depressed cell-mediated immunity (as
    judged by skin testing) had precancerous conditions and subsequent
    neoplasms (Gorodilova & Mandrik, 1978); no cases of neoplastic disease
    were registered in workers with normal immunological responses.

    2.4.2  Air pollutants

          The association betweeen changes in immunological parameters and
    host resistance and inhalation of particulate materials and oxidant
    gases is well established (Folinsbee, 1992). For example, decreases in
    delayed-type hypersensitivity response, circulating T-cell numbers,
    and T-cell proliferation have been observed with, and sometimes
    preceding, asbestos-related diseases, i.e. fibrosis, asbestosis, and
    mesothelioma (Kagan et al., 1977a; Gaumer et al., 1981; Lew et al.,
    1986; Tsang et al., 1988). B-Cell responses are increased, however, as
    evidenced by increased serum and secretory (primarily IgA)
    immunoglobulins (Kagan et al., 1977b). Kagan et al. (1979) also
    reported an association between exposure to asbestos and B-cell
    lymphoproliferative disorders. Several studies have shown altered NK
    cell activity after exposure to asbestos (Kubota et al., 1985; Tsang
    et al., 1988). In studies of NK cell responses in asbestos workers,
    Lew et al. (1986) found that immune changes may occur independently of
    any early neoplastic process. Similarly, there have been multiple
    observations of abnormal antibody production, decreased cell-mediated
    immune responses, and decreased resistance to disease in people
    occupationally exposed to silica (Uber & McReynolds, 1982).

          Oxidant gases have been associated with an increased prevalence
    of respiratory infections, particularly bacterial, and potential
    immune effects, but the data are less convincing than those from
    studies of rodents. The association between air pollution in
    industrialized areas and altered health status has been well
    established in epidemiological studies (French et al., 1973). A number
    of studies have linked exposure to air pollutants (ozone, nitrogen
    dioxide, sulfur dioxide, environmental tobacco smoke) with an
    increased incidence, severity, or duration of symptoms associated with
    respiratory infections (Lunn et al., 1967; French et al., 1973;
    Durham, 1974; Harrington & Krupnick, 1985; Neas et al., 1991; Schwartz
    et al., 1991; Schwartz, 1992; US Environmental Protection Agency,
    1990), although several studies of nitrogen dioxide failed to show
    such an association (Speizer et al., 1980; Ware et al., 1984; Samet et
    al., 1993). In Ontario, Canada, increased air pollution from sulfur
    dioxide and ozone during the summer was directly related to hospital
    admissions for acute respiratory symptoms (Bates & Sizto, 1983).
    Goings et al. (1989) subjected young adult volunteers to 1, 2, or
    3 ppm nitrogen dioxide for 2h per day on two consecutive days and then
    administered influenza virus intranasally. Although no statistical

    differences were observed, the frequencies of infections in exposed
    volunteers (91%) were higher than the 56-73% in healthy individuals,
    suggesting that nitrogen dioxide may play a role in increasing
    susceptibility to infection. In assessments of air pollution at home,
    young children in households with gas stoves had a higher incidence of
    respiratory disease and decreased pulmonary function than children in
    households with electric stoves. This difference was related to
    increased levels of nitrogen dioxide in homes with gas stoves, which
    reached peak values of > 1100 mg/m3 (Melia et al., 1977; Speizer et
    al., 1980). In contrast, Samet (1994) found no association between
    indoor levels of nitrogen dioxide and respiratory infections in
    children. A study of schoolchildren in Chattanooga (United States)
    showed an increased incidence of respiratory illness associated with
    atmospheric nitrogen dioxide levels (Shy et al., 1970).

          Examination of hospital admissions in Massachusetts (United
    States) in 1980 and 1982 revealed a positive association between 1-h
    maximum ozone levels in the summer months and daily admissions for
    pneumonia and influenza (Ozkaynak et al., 1990). An effect of
    atmospheric pollution, including oxidant gases, was also seen on the
    number of influenza cases in Sofia, Bulgaria (Kalpazanov et al.,
    1976); however, no demonstrable adverse effect on the course of a
    rhinoviral infection was seen in young adult male volunteers after
    exposure to moderate levels of ozone (0.3ppm for 6 h/day over a period
    of five days) (Henderson et al., 1988), and children living in areas
    with high ozone concentrations had lowered CD4:CD8 ratios of
    peripheral lymphocytes but no higher incidence of chest colds (Zwick
    et al., 1991). The phagocytic activity of alveolar macrophages
    (obtained by bronchoalveolar lavage) and other functions were impaired
    in human volunteers exposed to ozone (Devlin et al., 1991). The
    sensitivity of human and mouse macrophages to the effects of ozone is
    similar (Selgrade et al., 1995).

          Not all epidemiological studies have showed a correlation between
    air pollution and respiratory disease. Some of the discrepancies from
    experimental studies may be due to the parameters used to assess
    enhancement of infection. In experimental studies, increases in viral
    titres in the respiratory tract tend to be taken as an indication that
    the exposure enhances infection, whereas epidemiological studies rely
    on symptoms, many of which could be related to enhanced inflammatory
    or even allergic responses to the virus in the absence of viral

          Controlled studies (in an environmental chamber) showed that
    acute exposure to ozone causes an inflammatory response in the lower
    airways of human subjects (Koren et al., 1989; Devlin et al., 1991).
    The inflammatory response was manifested by increases in various
    inflammatory indicators including polymorphonuclear neutrophils
    (Figure 34), proteins, fibronectin, IL-6, and tryptase (Koren et al.,
    1989, 1994).

    FIGURE 34

          The proinflammatory effects of ozone raise the possibility that
    it can increase the sensitivity of people with atopic asthma to
    challenge with a specific allergen. Several studies have investigated
    the effect of exposure to pollutants on subsequent reactivity to
    antigen in atopic human volunteers. Molfino et al. (1991) reported
    that exposure to 0.12 ppm ozone significantly increased bronchial
    responsiveness to antigen in some individuals. Although they
    acknowledged weaknesses in the design of the experiment and
    recommended that the findings be confirmed, their observations are in
    accordance with those of the majority of epidemiological studies. In
    contrast, Bascom et al. (1990) found no alteration in the acute
    response to nasal antigen challenge in allergic patients pre-exposed
    to ozone in comparison with exposure to air; however, they did report
    increased upper respiratory tract inflammation after exposure to ozone
    in these patients in the absence of antigen challenge. Similarly, the
    bronchial response to inhaled grass pollen was unaffected by prior
    exposure to 0.1 ppm nitrogen dioxide (Orehek et al., 1981). The topic
    of sensitization and the role of ozone in exacerbating asthma has been
    reviewed (Koren & Blomberg, in press).

    2.4.3  Pesticides

          Pesticides can alter the human immune system. For example, women
    chronically exposed to low levels of aldicarb-contaminated groundwater
    had altered numbers of T cells and decreased CD4:CD8 ratios (Fiore et
    al., 1986). While this finding was not confirmed in studies in animals
    (see section, follow-up studies by Mirkin et al. (1990)
    confirmed the immune changes in those individuals still available for
    study, although the population was considerably smaller.

    2.4.4  Halogenated aromatic hydrocarbons

          A number of chemical accidents have resulted in human exposure to
    halogenated aromatic hydrocarbons. A feed supplement for lactating
    cows, inadvertently contaminated with polybrominated biphenyls, was
    used in more than 500 dairy herds and poultry farms in Michigan
    (United States) in 1973. Diary farm residents had reduced proportions
    of circulating T Iymphocytes and reducedlymphoproliferative
    responsiveness  in vitro (Bekesi et al., 1978); these changes
    persisted during follow-up (Bekesi et al., 1987). Silva et al. (1979),
    however, were unable to detect any immune abnormalities in a similarly
    exposed cohort.

          In Taiwan, more than 2000 people were exposed in 1979 to rice oil
    contaminated with PCBs and polychlorinated dibenzofurans. The clinical
    features in many of the exposed individuals included chloracne,
    pigmentation of skin, liver disease, and respiratory infections. When
    the immune status of the exposed individuals was examined one year
    after exposure, decreased concentrations of serum IgM and IgA, but not
    IgG, were reported, in addition to decreased numbers of circulating Th
    cells. The proportions of Ts/Tc cells and B lymphocytes were within
    the control values. Suppression of delayed-type hypersensitivity to
    recall antigens (streptokinase, streptodornase, tuberculin), enhanced
    mitogen-induced lymphocyte proliferation, and increases in
    sinopulmonary infections have been reported in this population (Chang
    et al., 1981, 1982; Lu & Wu, 1985). Many of the effects were
    transient, since two years after exposure most of the clinical
    abnormalities and laboratory parameters had returned to normal. A
    similar incident occurred in Japan in 1978, resulting in the 'yusho'
    syndrome. The immune system was assumed to be affected because of an
    increased frequency of respiratory infections and lowered serum IgM
    and IgA concentrations (Shigematsu et al., 1978). Another incident of
    intoxication with PCBs and polychlorinated dibenzofurans occurred
    after exposure to contaminated soot of fires in electrical equipment
    (Elo et al., 1985). The exposed people had serum PCB concentrations up
    to 30 mg/litre. The number of blood T cells was lower five weeks after
    exposure but had returned to normal values seven weeks later. Lowered
    CD4:CD8 ratios and lymphocyte proliferation after mitogen stimulation
    were also seen. Nine of the 15 most heavily exposed persons suffered
    from at least one infection of the upper respiratory tract. No overt
    long-term effects or chloracne were observed.

          The existing data also suggest that neonates are particularly
    sensitive to the immunotoxic effects of PCBs. Thus, higher incidences
    of colds and gastrointestinal (vomiting, abdominal pain) and
    dermatological (eczema, itchy skin) manifestations were observed in
    breast-fed infants of women occupationally exposed to the PCBs
    Kanechlor 500 and 300 than in infants born to unexposed women. The
    incidence of these symptoms increased with increasing length of
    breast-feeding (Hara, 1985). Epidemiological studies of women who
    consumed contaminated fish from the Great Lakes indicated that the
    maternal serum PCB level during pregnancy was positively associated
    with the number and type of infectious illnesses suffered by their
    breast-fed infants, especially during the first four months of life.
    The incidence of infections in the infant was correlated strongly with
    the highest rate of maternal fish consumption (Swain, 1991).

          A number of studies have been conducted of the immune status of
    people exposed to TCDD. In 1976, an accident occurred at a chemical
    plant in Seveso, Italy, in which high concentrations of TCDD were
    released into the local environment. An evaluation of 44 exposed
    children (20 with chloracne) showed no overt changes in immune status
    (Reggiani, 1980), although the adequacy of the control populations
    used has been questioned. In a study of residents of an area of
    Missouri (United States) with long-term exposure (average, three
    years) to low levels of TCDD in contaminated soil, no clinical
    symptoms were recorded, although a number of individuals showed
    changes in cell-mediated immunity, manifested as altered delayed-type
    hypersensitivity (Hoffman et al., 1986). In the follow-up
    investigation, however, the skin anergy was not confirmed (Evans et
    al., 1988). The serum concentration of the thymic hormone thymosin-a1,
    which has been related to the toxic action of this compound on the
    thymus, was reduced (Stehr-Green et al., 1989; Hoffman, 1992).
    Jennings et al. (1988) found an increased frequency of circulating
    antinuclear antibodies and immune complexes in TCDD-exposed workers.

    2.4.5  Metals

          Exposure to metals may also affect the immune system. Workers
    with elevated blood lead levels (30-90 µg/100 ml) had increased
    suppressor cell activity (Cohen et al., 1989), lowered lymphocyte
    proliferation after mitogen stimulation  in vitro (Jaremin, 1983),
    decreased IgA concentrations in saliva, and lowered complement C3
    levels (Ewers et al., 1982). These individuals also had an enhanced
    prevalence of respiratory infection (Ewers et al., 1982). The
    immunotoxic effects of lead may be dose-dependent, since neither
    humoral nor cellular parameters were affected after long-term, low-
    level exposure (Reigart & Graber, 1976; Kimber et al., 1986). In
    unrelated studies, the cationic heavy metal mercury was associated
    with immune complex disease in humans (Makker & Aikawa, 1979).

          In contrast to the database on the immunotoxic effects of
    cadmium, lead, and mercury in experimental animals  in vivo and the
    results of mechanistic studies  in vitro, the data on the effects of
    heavy metals on the human immune system are scanty and refer mainly to
    occupational exposure. These studies nevertheless provide evidence
    that at least mercury and lead affect the immune system (Moszczynski
    et al., 1990a; Bernier et al., in press).

          Significantly decreased levels of serum IgG and IgA, but not IgM,
    IgD, or IgE, were reported in workers occupationally exposed to
    metallic mercury vapours for 20 years in comparison with unexposed
    controls (Moszczynski et al., 1990b). These workers had blood mercury
    levels of < 50 µg/litre. Similarly, significantly decreased IgG and
    IgA levels were observed in workers with urinary mercury levels of
    0.029-0.545 mg/litre (Bencko et al., 1990). Studies of a small number
    of people exposed to mercury in dental amalgams have shown increased
    levels of IgE (Anneroth et al., 1992), an increased incidence of
    asthma (Drouet et al., 1990), and development of contact dermatitis
    (Gonçalo et al., 1992). Total lymphocyte, CD4, and CD8 levels were
    higher in exposed people than in controls (Eedy et al., 1990).

          Epidemiological data indicate that the main effects of
    occupational exposure to lead are on cellular aspects of the immune
    system and that humoral parameters remain relatively insensitive to
    such exposure. Thus, serum IgG, IgM, and IgA levels remained within
    the normal range in workers exposed for 4-30years, with a mean blood
    lead level of 38.4 µg/dl, in comparison with a mean control level of
    11.8 µg/dl (Kimber et al., 1986). Similarly, no effects were noted on
    serum IgG or IgA levels in a cohort exposed to lead oxides at a
    reported concentration within the plant of 266 µg/m3, who had an
    estimated blood lead level of 64 µg/dl; however, the response of
    lymphocytes from the exposed group to stimulation with
    phytohaemagglutinin and concanavalin A  in vitro was significantly
    lower than that of controls (Alomran & Shleamoon, 1988). Decreased
    serum Ig levels were reported in occupationally exposed workers with a
    mean blood lead level of 46.9 µg/dl, but the duration of exposure was
    not reported (Castillo-Mendez et al., 1991). In another study, no
    significant effects were noted on serum immunoglobulin levels after
    exposure to lead, but the levels of secretory IgA, which plays a major
    role in the defence against respiratory and gastrointestinal
    infections, was significantly decreased in workers with blood lead
    levels of 21-90 µg/dl. The incidence of influenza infections per year
    was significantly higher in these workers than in the control group
    (Ewers et al., 1982).

          Studies on the effects of lead on lymphocyte levels in
    occupationally exposed workers have produced inconclusive results. One
    study found an increase in absolute B lymphocyte counts and CD8 cells
    (Coscia et al., 1987), while a decrease in total T lymphocytes and the
    CD4 subset was reported in another set of workers (Fischbein et al.,

    2.4.6  Solvents

          Certain organic solvents may induce immune changes in humans.
    Benzene-induced pancytopenia with associated bone-marrow hypoplasia, a
    classical sign of chronic exposure to benzene, results in an
    immunodeficiency state due to the reduced numbers of immunocompetent
    cells (Goldstein, 1977). Alterations in the numbers of certain
    lymphocyte subsets, e.g. CD3 and CD4 lymphocytes, have also been
    reported in workers exposed to solvents (Denkhaus et al., 1986),
    suggesting that the effects may be somewhat specific.

    2.4.7  Ultraviolet radiation

          Numerous reports have shown that UVR inhibits contact
    hypersensitivity of the skin to sensitizers such as
    dinitrochlorobenzene (DNCB) (O'Dell et al., 1980; Halprin et al.,
    1981; Hersey et al., 1983a,b; Kalimo et al., 1983; Sjovall et al.,
    1985; Friedmann et al., 1989; Yoshikawa et al., 1990; Vermeer et al.,
    1991; Cooper et al., 1992). The dose required to produce
    immunosuppressive effects in humans is similar to that in C57Bl/6
    mice, the strain phenotypically most sensitive to UVR-induced immune
    suppression (Noonan & Hoffman, 1994).

          Human buttock skin was exposed to four daily doses of
    144 mJ/cm2 UVR, and the irradiated site was sensitized with DNCB
    immediately after the last exposure; the inner surface of the forearm
    was challenged 30days later with DNCB and contact hypersensitivity
    assessed. Forty percent of the volunteers failed to develop contact
    hypersensitivity and were designated sensitive, suggesting that, as in
    mice, susceptibility to UVR is genetically controlled. The sensitive
    phenotype also appeared to be a risk factor for the development of
    skin cancer (Yoshikawa et al., 1990). Suppression of contact
    hypersensitivity is seen in a similar percentage of black-skinned
    individuals, indicating that melanin cannot protect against this
    phenomenon (Vermeer et al., 1991). In another study, human buttock
    skin was exposed to 0.75 or two minimal erythemal doses (MED) of UVB
    (1 MED = 29.1-32.5mJ/cm2, depending on the individual) for four days
    and sensitized through irradiated skin immediately after the last
    exposure to DNCB; subjects were challenged with diluted DNCB at a
    distal site three weeks later. Some subjects were also exposed to four
    MED (moderate sunburn) and sensitized three days later with DNCB.
    Analysis of overall individual responses revealed decreased
    frequencies of fully successful immunizations in all UVB-exposed
    groups. The rate of immunological tolerance to DNCB (lasting up to
    four months) in the groups that were initially sensitized on skin that
    had received erythemagenic doses of UVB was 31%, whereas it was 7% in
    unirradiated controls (Cooper et al., 1992).

          A dose-response relationship was established in the studies of
    Cooper et al. (1992) in a comparison of subjects with types I-III skin
    (fair to moderately fair) who received various doses or schedules of
    UVR from a bank of FS20 fluorescent sun lamps (rich in UVB) with
    respect to their ability to mount an immune response to DNCB. A linear
    inhibition of immune responsiveness was seen, the first detectable
    decrease occurring at 0.75 of the individual's MED, reaching complete
    inhibition of responsiveness for 95% of subjects when two MED were
    administered every day for four days before immunization. Similar
    inhibition occurred when DNCB was administered through skin that had
    been exposed to a single dose of fourMED three days earlier. A dose-
    response curve for fair-skinned subjects was constructed by plotting
    the dose in total MED administered against the degree of immune
    response to DNCB (Figure 35). The 50% immune suppressive dose was
    calculated to be about 100mJ/cm2 of UVB.

          Unresponsiveness to a contact sensitizer applied to UV-irradiated
    skin can thus be induced in a proportion of individuals after exposure
    to moderate levels of UVR, and at least some individuals become
    immunologically tolerant in a manner similar to experimental animals.
    Taken together, these data suggest that the systemic immunosuppression
    induced in mice by UVR also occurs in humans, possibly through a
    similar mechanism. UVR appears to alter antigen presentation and the
    expression of Langerhans (CDla+DR+) cells, which is followed by an
    influx of CDla+DR+ monocytes that preferentially activate CD4+
    (suppressor-inducer) cells, which induce maturation of CD8+ Tc
    Iymphocytes (Cooper et al., 1986; Baadsgaard et al., 1990). The UVR
    wavelengths responsible for induction of CDla-DR+ cells are
    predominantly within the UVB band and to a lesser extent in the C band
    (Baadsgaard et al., 1987, 1989).

          UVR from solarium lamps also suppressed NK cell activity in the
    blood of subjects exposed for 1h per day for 12 days and tested one
    and seven days after exposure; the activity returned to normal by 21
    days after exposure (Hersey et al., 1983a). The effects of UVR on NK
    cell activity are attributed to A radiation (Hersey et al., 1983b).

          The depressed immune function observed in irradiated rodents
    reflects anecdotal observations in humans, i.e. that exposure to
    sunlight exacerbates certain infectious diseases, particularly those
    involving the skin. For example, it was noted at the turn of the
    century that smallpox lesions were worsened by exposure to sunlight
    (Finsen, 1901), and herpetic lesions and viral warts may be
    reactivated or exacerbated by sunlight (Giannini, 1990). It has also
    been hypothesized that sunlight affects susceptibility to infection
    with the bacteria that cause leprosy (Patki, 1991). Lesions of  Herpes
     simplex virus type I and type II can be reactivated by exposure to
    UVR (Spruanoe, 1985; Klein, 1986). Using the criteria established by

    FIGURE 35

    Yoshida & Streilin (1990) for the UVB-sensitive phenotype, Taylor et
    al. (1994) reported that 66% of individuals who have a history of
    herpes lip lesions provoked by exposure to sunlight were sensitive to
    UVB, in comparison with 40-45% of the general population and 92% of
    skin cancer patients. Exposure of immunosuppressed patients to
    sunlight can increase the incidence of viral warts caused by
    papillomavirus (Boyle et al., 1984; Dyall-Smith & Varigos, 1985). It
    is also known that UVR exacerbates the clinical course of systemic
    lupus erythematosus, an autoimmune disease (Epstein et al., 1965). The
    effects of UVR on the risk of infectious disease have been reviewed by
    Koren et al. (1994). In contrast, certain infectious diseases appear
    to be cured by sunlight; the most notable are erysipelas (Giannini,
    1990), a skin disease caused by  Streptococcus, and skin lesions
    caused by Herpes zoster virus.

    2.4.8  Others

          A large number of therapeutic drugs and drugs of abuse may also
    alter human immune function in humans. These include:


    Therapeutic agents
    Alkylating agents
       Nitrogen mustards: cyclophosphamide, L-phenylalanine mustard,
       Alkyl sulfonates: busulfan
       Nitrosoureas: carmustine (BCNU), lomustine (CCNU)
       Triazenes: dimethyltriazenoimidazolecarboxamide (DTIC)

    Anti-inflammatory agents
       Aspirin, indomethacin, penicillamine, gold salts
       Adrenocorticosteroids: prednisone

       Purine antagonists: 6-mercaptopurine, azathioprine, 6-thioguanine
       Pyrimidine antagonists: 5-fluorouracil, cytosine arabinoside,
       Folic acid antagonists: methotrexate (amethopterine)

    Natural products
       Vinca alkaloids: vinblastine, vincristine, procarbazine
       Antibiotics: actinomycin D, adriablastine, bleomycin, daunomycin,
            puromycin, mitomycin C, mithramycin
       Antifungal agents: griseofulvin
       Enzymes: L-asparaginase
       Cyclosporin A

    Estrogens: diethylstilbestrol, ethinylestradiol

    Substances of abuse

    Adapted from Dean & Murray (1990)


    3.1  General testing of the toxicity of chemicals

          The fact that substances used in various aspects of modern life
    can be simultaneously beneficial and harmful to human life creates a
    legislative and regulatory dilemma. In order to balance the desire to
    use the many new substances that enter the market every year and the
    economic benefit that is associated with their use on the one hand
    with the health and safety of the population on the other is an
    important challenge to governmental authorities. Legislative and
    regulatory efforts to minimize and control the risk of adverse effects
    on human health has resulted in a system for assessing and classifying
    the potential risk of exposure to chemicals. Potential adverse effects
    can be assessed in studies with experimental animals. In conducting
    such studies, attention must be paid to ethical and regulatory
    requirements for animal welfare and to good laboratory practice.

          In assessing and evaluating the toxic characteristics of a
    chemical, its oral toxicity may be determined once initial information
    has been obtained by acute testing. Toxicity is routinely tested
    according to guidelines, one of which is guideline No. 407 of the
    Organisation for Economic Co-operation and Development (OECD) for
    testing of chemicals, the 'Repeated Dose Oral Toxicity - Rodent:
    28-day or 14-day Study' (Organisation for Economic Co-operation and
    Development, 1981). This guideline has undergone three revisions, the
    most recent of which (January 1994) includes parameters of
    immunotoxicological relevance (see Table 7). Depending on the amount
    of a chemical to be produced and the expected exposure to the
    chemical, testing according to this guideline may in many countries
    provide the only information on its safety, including potential
    toxicity to the immune system. The information yielded by this type of
    testing is therefore decisive in determining how chemicals are used in
    society. Subsequent guidelines have been defined for use in follow-up
    studies if more exposure is expected or if there is a suspicion of
    toxicity on the basis of structural analogy with other known
    compounds. These include 90-day studies of oral toxicity, long-term
    studies, and studies of reproductive effects. Although such guidelines
    include more parameters of the immune system than OECD test guideline
    No. 407, detection of potential immunotoxicity may still not be
    adequately addressed. In practice, the best procedure is to carry out
    appropriate tests on a case-by-case basis, at increasing levels of
    complexity, when concerns are raised in more general toxicological

        Table 7.  Parameters of OECD Test Guideline 407 that relate to the immune system

    Current Guideline 407         Proposal for updating              Proposal for updating              Proposal for updating
    (adopted 21 May 1981)         Guideline 406                      Guideline 407 (revision of         Guideline 407 (revision of
                                  (February 1991)                    January 1993)                      January 1994)

    Total and differential        Total and differential             Total and differential             Total and differential
     leukocyte count               leukocyte count                    leukocyte count                   leukocyte count

                                  Weight of spleen and thymus        Weight of spleen and thymus        Weight of spleen and thymus

    Histopathology of spleen      Histopathology of spleen,          Histopathology of spleen,          Histopathology of spleen, thymus,
                                  thymus, lymph node, and            thymus, lymph nodes (one           lymph nodes (one relevant to the
                                  bone marrow                        relevant to route of               route of administration and a
                                                                     administration and a distant       distant one to cover systemic
                                                                     one to cover systemic effects),    effects), small intestine (including
                                                                     and bone marrow                    Peyer's patches), and bone marrow

    Histopathology of target      Histopathology of target           Histopathology of target           Histopathology of target
     organs                       organs                             organs                             organs

    OECD, Organisation for Economic Co-operation and Development
              An insight into the type of information that the OECD test
    guideline No. 407 yields is given below. In this test, the substance
    is administered orally in daily graduated doses to groups of
    experimental animals, one dose per group for 28 or 14 days. The
    preferred rodent species for this test is the rat, although others may
    be used. At least three doses and a control should be used. The
    highest dose should result in toxic effects but not produce an
    incidence of fatalities which would prevent a meaningful evaluation;
    the lowest dose should not produce any evidence of toxicity and should
    exceed a usable estimate of human exposure, when available. Ideally,
    the intermediate dose level(s) should produce minimal observable toxic

          In compliance with the guideline, the following examinations are
    carried out:

    (a)   haematology, including haematocrit, haemoglobin concentration,
          erythrocyte count, total and differential leukocyte count, and a
          measure of clotting potential such as clotting time, prothrombin
          time, thromboplastin time, or platelet count;

    (b)   clinical biochemistry of blood, including blood parameters of
          liver and kidney function. The selection of specific tests is
          influenced by observations on the mode of action of the
          substance. Suggested determinations are: calcium, phosphorus,
          chloride, sodium, potassium, fasting glucose, serum alanine
          aminotransferase, serum aspartate aminotransferase, ornithine
          decarboxylase, gamma-glutamyl transpeptidase, urea nitrogen,
          albumin, blood creatinine, total bilirubin, and total serum
          protein. Other determinations that may be necessary for an
          adequate toxicological evaluation include analyses of lipids,
          hormones, acid-base balance, methaemoglobin, and cholinesterase
          activity. Additional clinical biochemistry may be used when
          necessary, to extend the investigation of any observed effects.

    (c)   pathology, including gross necropsy, with examination of the
          external surface of the body, all orifices, and the cranial,
          thoracic, and abdominal cavities and their contents. The liver,
          kidneys, adrenal glands, and testes are weighed wet as soon as
          possible after dissection to avoid drying. Liver, kidney, spleen,
          adrenal glands, heart, and target organs showing gross lesions or
          changes in size are preserved in a suitable medium for possible
          future histopathological examination. Histopathological
          examination is performed on the preserved organs or tissues of
          the group given the high dose and the control group. These
          examinations may be extended to animals in other dosage groups,
          if considered necessary to further investigate changes observed
          in the high-dose group.

          A properly conducted 28- or 14-day study will provide information
    on the effects of repeated doses and can indicate the need for
    further, longer-term studies. It can also provide information on the
    selection of doses for longer-term studies.

          It is clear that the 1994 guideline is not suitable for adequate
    assessment of the potential adverse effects of exposure to a test
    chemical on the immune system, since the immunological parameters are
    restricted to total and differential leukocyte counts and the
    histopathology of the spleen. An evaluation of this test (Van Loveren
    & Vos, 1992) indicated that over 50% of the immunotoxic chemicals in a
    series of about 20 chemicals would not have been identified as such if
    the tests had strictly adhered to the guideline. In fact, it is even
    doubtful if chemicals indicated as immunotoxic only on the basis of
    guideline No. 407 would in practice have been picked up: For instance,
    in a toxicological experiment, a small but significant change in the
    percentage of basophilic leukocytes would by itself probably not be
    considered to be biologically relevant in the absence of any other
    parameter to suggest that an effect on the immune system might have
    been present.

          These data indicate that extension of OECD test guideline No. 407
    is necessary in order adequately to assess potential immunotoxicity.
    It is recommended that additional immunological parameters be included
    in this guideline in order to increase its power (Vos & Van Loveren,
    1987; Basketter et al., 1994).

          Guidelines also exist for follow-up studies if greater exposure
    is expected or if there is a suspicion of toxicity on the basis of
    structural analogy with other compounds. In these studies, potential
    toxicity to the immune system is generally addressed somewhat more
    extensively than in guideline No. 407. For instance, in a 90-day study
    of oral toxicity, the OECD guidelines prescribe that histopathological
    examination be done on the thymus, a representative lymph node, and
    the sternum with bone marrow, in addition to the spleen. Even with
    these additional parameters, it is highly questionable whether
    potential immunotoxicity is adequately assessed. For this purpose, a
    variety of tests is available, which are described in section 4.
    Depending on what is already known about the toxicity of the test
    compound, different panels of tests (also referred to as tiers) are
    selected for immunotoxicological evaluation. Usually, if little or no
    information is available, a dose range including high doses is used;
    lower, overtly nontoxic doses are chosen if some knowledge is
    available about the physical and chemical properties, toxicokinetics,
    structure-activity relationships, and intended use.

    3.2  Organization of tests in tiers

          Immunotoxicity can be assessed in a tiered approach (Luster et
    al., 1988; Van Loveren & Vos, 1989). Generally, the objective of the
    first tier is to identify potentially hazardous compounds (hazard
    identification). If potential immunotoxicity is identified, a second
    tier of tests is carried out to confirm and further characterize the

          Various approaches have been suggested for evaluating the
    potential immunotoxicity of compounds. Most are similar in design, in
    that the first tier is usually a screen for immunotoxicity and the
    second tier consists of a more specific confirmatory set of studies or
    in-depth mechanistic studies. Since the use of the tiers is usually
    tailored to the goals or objectives of the organization that proposes
    them, they differ in respect of the specific assays recommended and
    the organization of the assays into tiers.

    3.2.1  United States National Toxicology Program panel

          The tiers and assays originally adopted by the NTP, based on the
    proposed guidelines for immunotoxicity evaluation in mice reported by
    Luster et al. (1988), are shown in Table 8.

        Table 8.  Panel adopted by the US National Toxicology Program for detecting immune
              alterations after exposure of rodentsa to chemicals or drugs
    Parameter                     Procedures

    Screen (Tier I)

    Immunopathology               Haematology: complete and differential blood count
                                  Weights: body, spleen, thymus, kidney, liver
                                  Cellularity: spleen
                                  Histology: spleen, thymus, lymph node

    Humoral immunity              Enumerate IgM antibody plaque-forming cells to
                                    T-dependent antigen (sheep red blood cells)
                                    Lipopolysaccharide mitogen response

    Cell-medicated                Lymphocyte blastogenesis to mitogens
    immunity                        (concanavalin A)
                                  Mixed leukocyte response to allogeneic leukocytes

    Nonspecific immunity          Natural killer cell activity

    Comprehensive (Tier II)

    Immunopathology               Quantification of splenic B and T cell numbers

    Humoral-mediated              Enumeration of IgG antibody response to sheep red
    immunity                        blood cells

    Cell-medicated                Cytotoxic T lymphocyte cytolysis; delayed
    immunity                        hypersensitivity response

    Nonspecific immunity          Macrophage function: quantification of resident
                                    peritoneal cells, and phagocytic ability (basal and
                                    activated by macrophage activating factor)

    Host resistance               Syngeneic tumour cells
    challenge models              PYB6 sarcoma (tumour incidence)
    (end-points)b                 B16F10 melanoma (lung burden)
                                  Bacterial models: Listeria monocytogenes (mortality);
                                    Streptococcus species (mortality)
                                  Viral models: influenza (mortality)
                                  Parasite models: Plasmodium yoelii (parasitaemia)

    a  The testing panel was developed using B6C3F1 female mice.
    b  For any particular chemical tested, only two or three host resistance models
       are selected for examination.
          In this approach, the tier 1 assay is limited; it includes assays
    for both cell-mediated and humoral-mediated immunity and for innate
    (nonspecific) immunity with the inclusion of NK cell assays. It also
    includes immunohistopathology, which is part of the standard protocol
    for studies of subchronic toxicity and carcinogenicity conducted by
    the NTP. Tier II represents a more extensive evaluation and includes
    additional assays for assessing effects on cell-mediated, humoral, and
    innate immunity, in addition to host resistance. In this approach,
    animals are usually evaluated at only one time, so that the
    possibility for recovery or reversibility of immunological changes is
    not evaluated. A 14-day exposure period is employed routinely;
    however, 30- and 90-day exposures have been used, depending on the
    pharmacokinetic properties of the chemical being tested. The dose used
    in this tier system tends to be lower than those in several of the
    other approaches followed. In the NTP approach, dose levels are
    selected whenever possible that have no effect on body weight or other
    toxicological end-points. The approach has therefore focused on
    compounds for which the immune system is the most sensitive target.
    This is in marked contrast to other approaches, in which the highest
    dose is usually the maximum tolerated dose.

          The assays that make up the NTP tier approach have undergone
    various revisions, partly on the basis of an immunotoxicological
    review of compounds evaluated in this tier structure (Luster et al.,
    1992). The mitogen assays were first moved from tier I to tier II and
    have now been dropped altogether: They were found to be insensitive
    and to add little when run in conjunction with other assays that have
    a proliferative component, such as the mixed leukocyte response and
    the plaque assay. Furthermore, the only macrophage phagocytic assay
    routinely carried out in immunotoxicological studies conducted for the
    NTP is evaluation of the functional activity of the mononuclear
    phagocyte system, which is an in-vivo assay for phagocytosis.

          Studies by Luster et al. (1992) show that the potential
    immunotoxicity of a compound can be reasonably predicted with a few
    selected assays. As additional data become available, further changes
    to the NTP tiers will most likely be forthcoming.

    3.2.2  Dutch National Institute of Public Health and Environmental
           Protection panel

          The tier approach for immunotoxicological evaluation followed at
    the National Institute of Public Health and Environmental Protection
    (RIVM) in the Netherlands (Vos & Van Loveren, 1987) is shown in
    Table 9. This approach is based essentially on OECD test guideline
    No. 407, which suggests that the maximum tolerated dose be used as the
    high dose in the study. As a result, significantly higher doses are
    used than in the NTP approach in evaluating compounds for
    immunotoxicity. Additionally, the standard exposure period is 28 days,
    and the animal species routinely used is the rat instead of the mouse.

    This type of testing can therefore be performed in the context of
    studies in rats to determine the toxicological profile of a compound.
    At least three doses should be used, the highest having a toxic effect
    (but not mortality) and the lowest producing no evidence of toxicity.
    Moreover, immunotoxicity tests carried out in the context of such
    testing should not in any way influence the toxicity of the chemical
    (e.g. immunization or challenge with an infectious agent). In the NTP
    panel, the highest dose to which mice are exposed is chosen so that no
    overt toxicity, i.e. changes in body weight or gross pathological
    effects, is observed. As tests for immunotoxicity must be fairly
    sensitive in order to preclude false negatives, the NTP tier I
    includes functional assays. With a broader dose range that includes
    overt toxicity, potential immunotoxicity is more likely to be
    observed, without the inclusion of functional tests. If functional
    assays are to be included in the first tier, those tests that require
    sensitization of animals would require inclusion of satellite groups.
    In OECD test guideline No. 407 for testing chemicals, none of the
    other systems is approached functionally.

    It has been suggested that the NK cell assay be added to tier 1 (Van
    Loveren & Vos, 1992). Since the assay does not require animals to be
    sensitized or challenged, the same animals can be used without
    affecting other toxicological parameters, and thus an additional
    satellite group of animals is unnecessary.

    3.2.3  United States Environmental Protection Agency, Office of
           Pesticides panel

          The United States Environmental Protection Agency has proposed a
    tiered approach to the evaluation of biochemical pest control agents,
    which fall under the subdivision M guidelines for pesticides (Sjoblad,
    1988). The proposed tiers are shown below. Tier 1 of this approach
    includes functional assays for evaluating humoral immunity, cell-
    mediated immunity, and innate immunity. Thus, while Tier 1 is
    considered by the Agency to be an immunotoxicity screen, it is much
    more encompassing than the first tier of the other approaches. By
    providing options in the selection of assays for tier 1, the approach
    can easily accommodate both the rat and the mouse as the laboratory
    animal species used. In this approach, the tier 2 studies provide
    information sufficient for risk evaluation, including information on
    the time course of recovery from immunotoxic effects and host
    resistance to infectious agents and tumour models. Additional
    functional tests would be required if a dysfunction were observed in
    tier 1 tests or if data from other sources indicated the compound
    could produce an adverse effect on the immune response.

        Table 9.  Methods for detecting immunotoxic alterations in the rat evaluated by the
              Dutch National Institute of Public Health and Environmental Protection,
              Bilthoven, Netherlands
    Parameters               Procedures
    Tier 1

    Nonfunctional            Routine haematology, including differential cell counts
                             Serum IgM, IgG, IgA, IgE determination; lymphoid organ
                               weights (spleen, thymus, local and distant lymph nodes)
                             Histopathology of lymphoid tissues, including mucosa-
                               associated lymphoid tissue
                             Bone-marrow cellularity
                             Analysis of lymphocyte subpopulations in spleen by flow

    Tier 2

    Cell-medicated           Sensitization to T-cell dependent antigens (e.g. ovalbumin,
    immunity                   tuberculin, Listeria), and skin test challenge
                             Lymphoproliferative response to specific antigens (Listeria)
                             Mitogen responses (concanavalin A, phytohaemagglutinin)

    Humoral                  Serum titration of IgM, IgG, IgA, IgE responses to
    immunity                   T-dependent antigens (ovalbumin, tetanus toxoid,
                               Trichinella spiralis, sheep red blood cells) by ELISA
                             Serum titration of T-cell-independent IgM response to
                               lipopolysaccharide by ELISA
                             Mitogen response to lipopolysaccharide

    Macrophaand              Phagocytosis and killing of Listeria by adherent spleen
    function                   and peritoneal cells  in vitro
                             Cytolysis of YAC-1 lymphoma cells by adherent spleen
                               and peritoneal cells

    Natural killer           Cytolysis of YAC-1 lymphoma cells by non-adherent
    function                   spleen and peritoneal cells.

    Host resistance          Trichinella spiralis challenge (muscle larvae counts and
                               worm expulsion)
                             Listeria challenge (spleen and lung clearance)
                             Cytomegalovirus challenge (clearance from salivary gland)
                             Endotoxin hypersensitivity;
                             Autoimmune models (adjuvant arthritis, experimental
                               allergic encephalomyelitis)

    Ig, immunoglobulin; ELISA, enzyme-linked immunosorbent assay

    Subdivision M guidelines: proposed revised requirements by the US
    Environmental Protection Agency for testing the immunotoxicity of
    biochemical pest control agents

    AI.    Tier 1

    A.    Spleen, thymus, and bone-marrow cellularity

    B.    Humoral immunity (one of the following)

          1.   Primary and secondary IgG and IgM responses to antigen; or,
          2.   Antibody plaque-forming cell assay

    C.    Specific cell-mediated immunity (one of the following)

          1.   One-way mixed lymphocyte reaction assay; or,
          2.   Effect of agent on normal delayed-type hypersensitivity
               response; or,
          3.   Effect of agent on generation of cytotoxic T-lymphocyte

    D.    Nonspecific cell-mediated immunity

          1.   Natural killer cell activity and
          2.   Macrophage function

    II.   Tier 2

    A.    Required if:

          1.   Dysfunction is observed in tier 1 tests
          2.   Tier 1 test results cannot be definitively interpreted
          3.   Data from other sources indicate immunotoxicity

    B.    General testing features:

          1.   Evaluate time course for recovery from immunotoxic effects.
          2.   Determine whether observed effects impair host resistance to
               infectious agents or to tumour cell challenge.
          3.   Perform additional specific, but appropriate, testing
               essential for evaluation of potential risks.
          This Agency has also suggested that immunotoxicological screening
    be conducted in evaluating conventional chemical pesticides
    (subdivision F guidelines; see below); however, unlike those of
    subdivision M, these guidelines are not designed as a tiered testing
    scheme. If the immunotoxicity screen listed in subheading I were added

    to subchronic and/or chronic studies in subdivision F, it would be a
    more effective screen for immunotoxicity than is currently available.
    If this proposed screen indicates that the immune system is a
    sensitive target, the Agency considers that it may be necessary to
    evaluate the risk for immunotoxic effects as under subheading
    II. Currently, these suggestions have not been promulgated as official
    guidelines or regulations.

        Evaluations suggested by the US Environmental Protection Agency as
    appropriate additions to Subdivision F guidelines for immunotoxicity
    screening (subheading I) and possible additional data appropriate for
    risk evaluation of chemical pesticides (subheading II)

    I.    Immunotoxicity screen
          A.   Serum immunoglobulin levels (e.g. IgG, IgM, and IgA)
          B.   Spleen, thymus, and lymph node weights
          C.   Spleen, thymus, and bone-marrow cellularity and cell
          D.   Special histopathology (e.g. enzyme histochemistry,
          E.   More complete evaluation of 'premature' mortality of test
               animals, as possibly related to immunosuppressive effects

    II.   Immunotoxicity risk evaluation
          A.   Host resistance to challenge with infectious agent and/or
               tumour cells
          B.   Specific cell-mediated immune responses (e.g. mixed
               leukocyte response, delayed-type hypersensitivity
               response,cytotoxic T lymphocyte assays)a
          C.   Nonspecific cell-mediated immune responses (i.e. natural
               killer cell activity, macrophage function)a
          D.   Time course for recovery from adverse immunological effects

    a  Measures of specific and nonspecific cell-mediated immune
       responses that also may be considered useful in an immunotoxicity
    3.2.4  United States Food and Drug Administration, Center for Food
           Safety and Applied Nutrition panel

          The United States Food and Drug Administration is considering
    testing guidelines for evaluating the immunotoxic potential of direct
    food additives (Hinton, 1992). The multifaceted approach is included
    in the draft revision of the  Toxicological Principles for the Safety
     Assessment of Direct Food Additives and Color Additives Used in Food
    is (US Food and Drug Administration, 1993). The testing requirements
    are based on the 'concern level' of the substance, assigned on the
    basis of the available toxicological information or the substance's

    structural similarity to known toxicants and on estimated human
    exposure from its proposed use. A compound with high toxic potential
    and high exposure would be assigned a high initial 'concern level'
    (3), and one with low toxic potential and low exposure would be
    assigned a low initial level (1).

          In general, substances will be evaluated for immunotoxic
    potential on a case-by-case basis. Two types of immunotoxicity tests
    and procedures are defined in this approach: Type 1 tests are those
    that do not involve perturbation of the test animal (i.e.
    sensitization or challenge). These are further divided into 'basic'
    tests, which include haematology and serum chemistry, routine
    histopathological examination, and determination of organ and body
    weights, and 'expanded' tests, which are logical extensions of the
    'basic' tests and include those that can be performed retrospectively.
    Type 2 tests include injection of or exposure to antigens, infectious
    agents, vaccines, or tumour cells. In general, type 2 tests require a
    satellite group of animals for immunological evaluation. The sets of
    'basic' and 'expanded' type 1 tests are defined as level-I
    immunotoxicity tests, and the sets of type 2 tests are defined as
    level-II tests. Some level-I tests can be used to screen for
    immunotoxic effects, while others focus on the mechanism of action or
    the cell types affected by the test substance. Level-II tests are
    conducted to define the immunotoxic effects of food and colour
    additives more specifically. The recommended testing scheme is shown

          The functional tests generally require sensitization of exposed
    rats and controls and subsequent analysis of the responses to the
    sensitizing antigens. For this reason, functional tests are not
    readily conducted in the first tier of immunotoxicity testing. As
    guidelines for routine toxicology experiments preclude compromising
    the experiment by any agent other than the test chemical, the second
    tier of immunotoxicity testing, with immune function tests, requires a
    separate set of experiments. The antigens that are used to sensitize
    the exposed and control rats may be relatively simple antigens, such
    as ovalbumin or tetanus toxoid, or more complex antigens, such as
    sheep red blood cells, bacteria, or parasites. The responses can occur
    in various arms of the immune system, which consequently must be
    measured with different assays. For instance, humoral responses can be
    measured by determination of specific antibodies in serum; the
    appropriate tests for cellular responses are proliferative responses
    of lymphocytes to the specific antigens  ex vivo/in vitro or delayed-
    type hypersensitivity responses to injection with antigen  in vivo.

    Recommendations of the United States Food and Drug Administration for
    testing the immunotoxicity of direct food additives

    Basic testing (rat model)
          Complete blood count, differential white blood cell count;
          Total serum protein, albumin:globulin ratio;
          Histopathology, gross and microscopic (spleen, thymus, lymph
            nodes, Peyer's patches, and bone marrow);
          Lymphoid organ and body weights

    Retrospective level-I testing (possible in a standard toxicology
          Electrophoretic analysis of serum proteinsa (when positive or
           marginal effect is noted in basic testing);
          Immunostaining of spleen and lymph nodes for B and T cellsa
            (quantification of total immunoglobulins);
          Serum autoantibody screen and deposition of immunoglobulins
            (micrometry for semiquantification of the proliferative

    Enhanced level-I testing (possible for more complete screening in the
    standard toxicology study core group, with a satellite animal group,
    or in a follow-up study)
          Cellularity of spleen (lymph nodes and thymus when indicated);
          Quantification of total B and T cells (blood and/or spleen);
          Mitogen stimulation assays for B and T cells (spleen);
          Natural killer cell functional analysis (spleen);
          Macrophage quantification and functional analysis (spleen);
          Interleukin-2 functional analysis (spleen);
          When indicated or for more complete analysis, other end-points
            such as total haemolytic complement activity assay in serum

    Level-II testing with a satellite group or follow-up study for
    screening of functional immune effects
          Kinetic evaluation of humoral response to T-dependent antigen
            (primary and secondary responses with sheep red blood cells,
            tetanus toxoid, or other);
          Kinetic evaluation of primary humoral response to a
            T-independent antigen such as pneumococcal polysaccharides,
            trinitrophenyl-lipopolysaccharide, or other recognized
          Delayed-type hypersensitivity response to known sensitizer of
            known T effector cell;
          Reversibility evaluation

    Recommendations (cont'd)

    Enhanced level-II testing with a satellite group or follow-up study
    for evaluation of potential immunotoxic risk
          Tumour challenge (MADB106 or other in rat);
          PYB6 sarcoma (in mouse);
          Infectivity challenge (Trichinella, Candida or other in rat;
            Listeria or other in mouse) a Recommended for inclusion in
            basic testing

    a  Recommended for inclusion in basic testing

          Not all functional assays require prior sensitization of the test
    animals, e.g. proliferative responses of lymphocytes  ex vivo/in vitro
    to mitogens which are either specific for T cells, giving information
    on cellular immunity, or for B cells, providing data on humoral
    immunity. The phagocytic and lytic activity of macrophages and the
    nonspecific cytotoxic activity of NK cells can also be measured
     ex vivo/in vitro, without prior sensitization of the test animals.
    Both types of activity are examples of nonspecific defence mechanisms,
    directed to bacteria and certain tumour cells and to tumour cells and
    virally infected cells, respectively. Since measurement of these types
    of activity does not require prior sensitization of the host, such
    functional tests can be considered for inclusion in the first tier of
    testing for immunotoxicity in routine toxicology.

    3.3  Considerations in evaluating systemic and local immunotoxicity

    3.3.1  Species selection

          Selection of the most appropriate animal model for
    immunotoxicology studies has been a matter of great concern. Ideally,
    toxicity testing should be performed with a species that responds to a
    test chemical in a pharmacologically and toxicological manner similar
    to that anticipated in humans, i.e. the test animals and humans
    metabolize the chemical similarly and have identical target organs and
    toxic responses. Toxicological studies are often conducted in several
    animal species, since it is assumed that the more species that show a
    specific toxic response, the more likely it is that the response will
    occur in humans. Data from studies in rodents on target organ toxicity
    at immunosuppressive doses for most immunosuppressive therapeutic
    agents have generally been predictive of later clinical observations.
    Exceptions to the predictive value of rodent toxicological data are
    infrequent but occurred in studies of glucocorticoids, which are
    lympholytic in rodents but not in primates (Haynes & Murad, 1985).
    Although certain compounds exhibit different pharmacokinetic
    properties in rodents and in humans, rodents still appear to be the

    most appropriate animal model for examining the non-species-specific
    immunotoxicity of compounds, because of established toxicological
    knowledge, including similarities of toxicological profiles, and the
    relative ease of generating data on host resistance and immune
    function in rodents. Comparative toxicological studies should be
    continued and expanded, however, as novel recombinant biological
    compounds and natural products that enter safety testing will probably
    have species-specific host interactions and toxicological profiles.

          The quantitative and possibly the qualitative susceptibility of
    an individual animal to the immunotoxicity of a selected agent can be
    influenced by its genetic characteristics, indicating not only a need
    to consider species but also strain. Rao et al. (1988) described two
    approaches for selecting appropriate genotypes for toxicity studies.
    The first is to select genotypes that are representative of an animal
    species, which by virtue of similar metabolic profiles may also
    exhibit a sensitivity similar to that of man, such as random-bred
    mice. A second approach is to attempt to identify genotypes that are
    uniquely suitable for evaluating a specific class of chemicals, such
    as the use of  Ah-responsive rodent strains in studies with
    polyhalogenated aromatic hydrocarbons. In many cases, however, this
    approach requires considerable knowledge of the mechanisms of toxicity
    of the compound, which may not be available. One compromise has been
    to use Fl hybrids which have the stability, phenotypic uniformity, and
    known genetic traits of an inbred animal, yet have the vigour
    associated with heterozygosity. The description of the genetic
    relationships between inbred mouse strains on the basis of the
    distribution of alleles at 16 loci (Taylor, 1972) has made possible
    rational selection of appropriate Fl hybrids, such as the B6C3Fl

    3.3.2  Systemic immunosuppression

          Because of this complexity, the initial strategies devised by
    immunologists working in toxicology and safety assessment were to
    select and apply a tiered panel of assays in order to identify
    immunosuppressive or, in rare instances, immunostimulatory agents in
    laboratory animals (US National Research Council, 1992). Although the
    configuration of these testing panels varies according to the
    laboratory conducting the test and the animal species used, they
    include measurements of one or more of the following: (i) altered
    lymphoid organ weights and histology, including immunohistology; (ii)
    quantitative changes in the cellularity of lymphoid tissue, peripheral
    blood leukocytes, and/or bone marrow; (iii) impairment of cell
    function at the effector or regulatory level; and/or (iv) increased
    susceptibility to infectious agents or transplantable tumours.

          A variety of factors must be considered in evaluating the
    potential of an environmental agent or drug to adversely influence the
    immune system. These include appropriate selection of animal models
    and exposure variables, consideration of general toxicological
    parameters and mechanisms of action, as well as an understanding of
    the biological relevance of the end-points to be measured. Treatment
    conditions should be based on the potential route, level, and duration
    of human exposure, the biophysical properties of the agent, and any
    available information on the mechanism of action. Moreover,
    toxicokinetic parameters, such as bioavailability, distribution
    volume, clearance, and half-life, should be measured. Doses should be
    selected that will allow establishment of a clear dose-response curve
    and a no-observed-effect level NOEL). Although, for reasons explained
    earlier, it is beneficial to include a dose that induces overt
    toxicity, any immune change observed at that dose should not
    necessarily be considered to be biologically significant, since severe
    stress and malnutrition are known to impair immune responsiveness.
    Many laboratories routinely use three doses but generally conduct
    studies to define the range of doses before a full-scale
    immunotoxicological evaluation. If studies are being designed
    specifically to establish reference doses for toxic chemicals,
    additional exposure levels are advisable. In addition, inclusion of a
    'positive control' group, treated with an agent that shares some of
    the characteristics of the test compound, may be advantageous when
    experimental and fiscal constraints permit.

          The selection of the exposure route should reflect the most
    probable route of human exposure, which is most often oral,
    respiratory, or dermal. If it is necessary to deliver an accurate
    dose, a parenteral exposure route may be required; however, this may
    significantly change the metabolism or distribution of the agent from
    that which would occur following natural exposure.

    3.3.3  Local suppression

          Local immune suppression has received less attention than
    systemic immune suppression, and this is noteworthy, since the surface
    that is exposed to the environment, i.e. the skin, the respiratory
    tract, and the gastrointestinal tract, are the major ports of entry of
    antigens and pathogens. While a variety of validated methods are
    currently available to detect chemical skin sensitizers in humans and
    experimental animals, there is no standard method to assess the
    potential of chemicals to induce local immunosuppression in the skin.
    Furthermore, although increasing evidence suggests that the
    consequence of skin immunosuppression would be an increase in
    neoplastic and infectious diseases of the skin, definitive data are
    still lacking. In contrast, considerable efforts are being deployed to
    develop sensitive models for monitoring skin irritants. For example,
    human keratinocyte cultures and keratinocyte-fibroblast co-cultures

    have been examined for end-points ranging from changes in cell
    viability to production and loss of various bioactive products. Few
    test systems are available for the gut and the respiratory tract.


          This section comprises general descriptions of methods used for
    evaluating immunotoxicity.

    4.1  Nonfunctional tests

    4.1.1  Organ weights

          It is routine practice in toxicology to weigh organs that are
    potentially affected by the compound that is being investigated. The
    immunological organs that are suitable for weighing in screening for
    potential immunotoxicity are: the thymus, which plays a decisive role
    in the development of the immune system and which is affected by many
    immunotoxicants; the spleen, which is the repository for many
    recirculating lymphocytes; and the lymph nodes, which are important
    for the induction of immune processes. Determination of the weight of
    draining lymph nodes (depending on the route of exposure, i.e.
    mesenteric nodes for oral exposure and bronchial nodes for inhalation)
    in addition to distant lymph nodes (such as popliteal lymph nodes for
    determining systemic effects) is the best. Mesenteric nodes, in
    particular, occur in a string within non-lymphoid fatty tissue, and
    care must be taken to remove this non-lymphoid tissue so that the
    weight can be adequately determined. The cellularity of these organs
    is another indication of the effects of chemicals on the immune
    system. Furthermore, cell suspensions can be prepared from lymphoid
    organs in order to assess the distribution of subpopulations of
    lymphoid cells and to test their functionality within the organs.
    Under OECD guideline No. 407, histopathological examination of
    lymphoid organs and tissues is crucial for detecting the effects of
    chemicals on the immune system. Therefore, upon termination of
    exposure to a compound in a toxicological experiment, organs such as
    the spleen should first be weighed; subsequently, they are divided
    into parts which are also weighed, and one or more parts are used for
    histopathological examination and the remainder to prepare cell
    suspensions that can be evaluated for distribution of lymphocyte
    subpopulations or can be assessed functionally.

    4.1.2  Pathology

          The histopathology of the thymus, spleen, and draining and
    distant lymph nodes, of the mucosal immune system (Peyer's patches in
    the gut or bronchus and nose-associated lymphoid tissue in the
    respiratory tract), and of the skin immune system should be evaluated,
    depending on the route of exposure. The first level of evaluation
    should be of haematoxylin-eosin stained, paraffin-embedded slides. A
    more sophisticated level of evaluation is immunoperoxidase staining of
    special cell types.

          Many monoclonal antibodies are available for mice, rats, and
    humans to detect differentiation antigens, cell adhesion molecules,
    and activation markers on haematolymphoid and stromal cells involved
    in immune responses. A list of some monoclonal antibodies that can be
    used in the identification of leukocytes and stromal cells in (frozen)
    sections of lymphoid tissue is presented in Table 10; a selection of
    these is reviewed below.

          For a further description of these markers, and the cells that
    express them, reference may be made to the introductory section and to
    descriptions in the literature (Brideau et al., 1980; Bazin et al.,
    1984; Dallman et al., 1984; Dijkstra et al., 1985; Joling et al.,
    1985; Vaessen et al., 1985; Joling, 1987; Hünig et al., 1989; Kampinga
    et al., 1989; Portoles et al., 1989; Schuurman et al., 1991a).

          These markers are usually stained in frozen tissue sections of
    6-8 µm, fixed in acetone. A three-step immunoperoxidase procedure is
    most suitable: the first step includes the monoclonal antibody
    specific for the determinants to be studied (see above), the second
    step, rabbit anti-mouse immunoglobulin, and the third step, swine
    anti-rabbit immunoglobulin, the latter two antibodies conjugated to
    horseradish peroxidase. The peroxidase activity can be developed by
    3,3-diaminobenzidine tetrahydrochloride with hydrogen peroxide as
    substrate, and the sections can be counterstained with Mayer's
    haematoxylin to facilitate evaluation. Negative controls are prepared
    by omitting the antibody in the first step or replacing it with an
    irrelevant one. Under these conditions, only the peroxidase activity
    of polymorphonuclear cells, when present, is visualized, and no
    immunolabelling is found.

          In general, histopathological evaluation provides a semi-
    quantitative estimation of effects. The experienced pathologist can do
    this adequately in studies carried out 'blind', especially if the
    effects are clear. For more subtle effects, morphometric analysis is a
    valuable addition, especially when supported by software for assessing
    the values of parameters such as size, surface, and intensity of
    staining. The compartments of the immune system, i.e. specific T and B
    lymphocyte areas in spleen and lymph nodes and cortical and medullary
    areas of immature and differentiated thymocytes within the thymus, and
    the numbers of specialized cells per surface unit are parameters that
    are well suited for morphometric analysis.

    4.1.3  Basal immunoglobulin level

          Serum immunoglobulin levels are often altered after exposure of
    rats to immunotoxic chemicals (Vos, 1980; Vos et al., 1982, 1984,
    1990a; Van Loveren et al., 1993a). This is not surprising, as the
    total levels measured are a function of the humoral aspects of the
    immune system, which react to the antigens that the host encounters.
    For this reason, measurement of antibody levels is potentially
    valuable in screening for immunotoxicity. Since the amount of antibody

        Table 10.  Some monoclonal antibodies to leukocytes and stromal cells used in immunohistochemical studies of tissue sections and flow
               cytofluorography on cell suspensions

    CD        Relative       Mouse               Rat            Human          Reactivity
              mol. mass

    T Cells

    CD1       gp43,45,       Ly-38                              OKT6,          Lymphocytes in thymic cortex, Langerhans cells in skin,
              49,12                                             a-Leu-6        interdigitating cells

    CD2       gp50           Ly-37,              MRC OX-34,     a-Leu-5,       All T cells in thymus and peripheral lymphoid organs, subset
                             NSM46.7,            MRC OX-54,     OKT11          of macrophages (rat). Sheep erythrocyte receptor, leukocyte
                             RM2-5               MRC OX-55                     function antigen:-2 (LFA-2). Ligand f or LFA-3 (CD58)

    CD3       gp19-29        CD3-1,KT3,          IF4, G4.18     a-Leu-4,       T Cells in thymic medulla and peripheral lymphoid organs
                             145-2C11                           OKT3           (T-cell receptor-associated, cytoplasmic in precursor T cells
                                                                               in thymus)

    CD4       gp65           Ly-4, L3T4,         MRC OX-35,     a-Leu-3,       Lymphocytes in thymic cortex, about two-thirds of T cells in
                             YTS 177.9           MRC OX-38,     OKT4           peripheral lymphoid organs, subset macrophages, microglia
                                                 (ER2), W3/25                  T helper/inducer and delayed-hypersensitivity phenotype.
                                                                               MHC class II binding, receptor for human immunodeficiency

    CD5       gp65-62        Ly-1,Lyt-1          MRC OX-19,     a-Leu-1        Lymphocytes in thymic cortex (faint). All T cells in thymic
                                                 HIS47                         medulla and peripheral lymphoid tissue, subset of B cells

    CD6       gp120                                             Tü 33          T Cells in thymic medulla and peripheral lymphoid organs

    CD        Relative       Mouse               Rat            Human          Reactivity
              mol. mass

    CD7       gp41                                              WT1,B-F12,     Prethymic T-cell precursors, all T cells in thymus and fewer
                                                                a-Leu-9        in peripheral lymphoid organs

    Table 10 (cont'd)

    CD        Relative       Mouse               Rat            Human          Reactivity
              mol. mass

    T Cells (contd)

    CD8       gp32-33        Ly-2,3,Lyt-2,3,     MRC OX-8       a-Leu-2,       Lymphocytes in thymic cortex, about one-third of T cells in
                             YTS 105.8                          OKT8           peripheral lymphoid organs, splenic sinusoids (T cytotoxic/
                                                                               suppressor phenotype, NK cells). MHC class I binding

    CD24      p45,55,65      J11d,M1/69          SRT1           BA-1           B Cells in germinal centres and corona, myeloid cells,
                                                                               thymic cortex cells (rodents). Heat-stable antigen (HSA)

    CD43      gp115          Ly-48               W3/13,HIS17    DFT-1,         (Pro)thymocytes, T cells, plasma cells, cells in bone
                                                                WR-14          marrow, polymorphonuclear granulocytes, brain cells.
                                                                               Leukosialin, sialophorin

    CDw       p25-30         Thy-1               (ER4),         5F10           Thymocytes, T lymphocytes, connective tissue structures,
    90                                           MRC OX-7,                     epithelial cells, fibroblasts, neurons, subset of bone-marrow
                                                 HIS51                         cells, plasma cells, stem cells (T-activation molecule)

                             Thy-2                                             Thymocytes

              p40-55         H57-597             R73,HIS42      WT31,          T-Cell receptor a-b chain. Mature T cells in thymic medulla
                                                                TalphaF1,      and peripheral lymphoid tissue

              p40-55         GL3,GL4,            V65            CgammaM1,      T-Cell receptor gamma-delta chain
                             UC7-13D5                           11F2,
                                                                TCR delta1,

              p41-55                             MRC OX-44                     Prothymocytes, lymphocytes in thymic medulla, T and B


    Table 10 (cont'd)

    CD        Relative       Mouse               Rat            Human          Reactivity
              mol. mass

    T Cells (contd)

              p41,47                             MRC OX-2                      Thymocytes, dendritic cells, B cells, brain cells
                                                 ER3,ER7,                      Subset of thymocytes and peripheral T cells, subset of
                                                 ER9,ER10                      myeloid cells

                                                 HIS44                         Most lymphocytes in thymic cortex, small subset of
                                                                               medullary lymphocytes, erythroid cells, cells in germinal

                                                 HIS45                         Some lymphocytes in thymic cortex, most medullary
                                                                               thymocytes and peripheral T cells, subset of B cells.
                                                                               Quiescent cell antigen (QCA-1)

    MHC class I              (Various antibodies to polymorphic and            All nucleated cells, including leukocytes and stromal
                             non-polymorphic epitopes)                         cells; for T cells absent on thymic cortex cells (human)

    B Cells

    CD9       gp24                                              BA-2           Germinal centres (faint); some cells in thymic cortex. Late
                                                                               pre-B cells

    CD10      gp100                                             BA-3,          Germinal centres (faint); some cells in thymic cortex
                                                                W8E7           Common acute lymphoblastic leukaemia antigen (CALLA)

    CD19      gp95                                              a-Leu-12,      B Cells in germinal centres and mantles, follicular dendritic
                                                                B4, FMC63      cells

    CD20      p35            Ly-44                              B1, a-Leu-16   B Cells in germinal centres and mantles, follicular dendritic

    Table 10 (cont'd)

    CD        Relative       Mouse               Rat            Human          Reactivity
              mol. mass

    B Cells (contd)

    CD21      gp140                                             B2,BL13,       B Cells in germinal centres and mantles (faint), follicular
                                                                HB-5           dendritic cells (C3d receptor, CR2, receptor for Epstein-Barr

    CD22      gp135          Lyb-8.2,                           a-Leu-14,To    B Cells in germinal centres and mantles, cytoplasmic in
                             Cy34.1                             To 15,RFB4,    precursor B cells

    CD23      p45            Ly-42                              a-Leu-20,      Some B cells in marginal centres and mantles, activated B
                                                                Tü 1           cells, subset of follicular dendritic cells (IgE Fc receptor)

    CD24      p45,55,65      J11d,M1/69          SRT1           BA-1           B Cells in germinal centres and corona, myeloid cells, thymic
                                                                               cortex cells (rodents). Heat-stable antigen (HSA)

    CD37      gp40-45                                           BL14           B Cells in germinal centres and mantles

    CD38      gp45                                              a-Leu-17,      Lymphocytes in thymic cortex, cells in germinal centres,
                                                                OKT10          plasma cells (immature lymphoid cells, plasma cells)

    CDw75     p53?                                              LN1, OKB4      B Cells in germinal centre, in corona (faint), macrophages,

    CD79a     p33,40                                            mb-1           B Cells, Ig alpha chain

    CD79b     p33,40                                            B29            B Cells, Igß chain

                             p200                (HIS14)                       All B cells, including TdT+ precursors

                             p200                (HIS22)                       All B cells in corona, pre-B cells

    Table 10 (cont'd)

    CD        Relative       Mouse               Rat            Human          Reactivity
              mol. mass

    B Cells (contd)

    MHC class II             (Various antibodies to polymorphic and            B Lymphocytes, activated T cells, monocytes/macrophages,
                             non-polymorphic epitopes)                         interdigitating cells, Langerhans cells, epithelia, endothelia

                                                                               B Cells (surface); in germinal center IgM+IgG+IgA+ and in
                                                                               anti-immunoglobulin corona IgM+IgD+, plasma cells

    Monocytes/macrophages, myeloid cells

    CD13      p130-150       ER-BMDM-1                          a-Leu-M7,      Monocytes, granulocytes, dendritic reticulum cells
                                                                My7            (aminopeptidase N)

    CD14      p55                                ED9            UCH-M1, B-A8,  Monocytes, some granulocytes and macrophages

    CD15      p170-190                                          a-Leu-M1       Granulocytes, some monocytes (lacto-N-fucose pentaosyl)

    CD16      p50-70                                            a-Leu-11       NK cells, subset of T cells, neutrophilic granulocytes,
                                                                               activated macrophages. IgG-FcRIII, low affinity, complexed IgG

    CD33      p67                                               a-Leu-M9,      (Precursor) granulocytes, macrophages, Langerhans cells.
                                                                My9            Myelin-associated protein

    CD68      p110                                              Ki-M6,Ki-M7    Macrophages (specific)

              p160           F4/80                                             Monocytes-macrophages

              p32            Mac-2                                             Thioglycollate-elicited peritoneal macrophages

              p92-110        Mac-3                                             Peritoneal macrophages

    Table 10 (cont'd)

    CD        Relative       Mouse               Rat            Human          Reactivity
              mol. mass

    Monocytes/macrophages, myeloid cells (contd)

                             4F7                                               Dendritic cells in skin, bone marrow

                                                 ED1                           Monocytes/macrophages

                                                 ED2,HIS36                     Subset of macrophages (F4/80-like)

                                                 ED3                           Subset of macrophages, restricted, negative in thymus
                                                 MRC OX-41                     Granulocytes, macrophages, dendritic cells

                                                 MRC OX-62                     Dendritic cells (integrin-like)

                                                 (IF119)                       Dendritic cells

                                                 HIS48                         Granulocytes

                                                                Mac-387        Macrophages

    Natural killer cells

    CD16      p50-70                                            a-Leu-11       NK cells, subset of T cells, neutrophilic granulocytes, activated
                                                                               macrophages. IgG-FcRIII, low affinity, complexed IgG

    CD56      p220/135                                          a-Leu-19,      NK cells, monocytes, neuroectodermal cells NKH-1, isoform of
                                                                B-A19          neural cell adhesion molecule (NCAM)

    CD57      p110                                              a-Leu-7,       NK cells, subset of T cells, some B cells, some epithelial cells,
                                                                VC1.1          monocytes, neuroendocrine cells, NKH-1

                             a-asialo-GM1                                      NK cells, stromal components

    Table 10 (cont'd)

    CD        Relative       Mouse               Rat            Human          Reactivity
              mol. mass

    Natural killer cells (contd)

                             NK-1.1,2B4,         3.2.3                         NK cells (NKR-P1 gene family)
                             3A4, 5E6

    Follicular dendritic cells

                                                 ED5,ED6,       Ki-M4,DRC-1    Follicular dendritic cells
                                                 MRC OX-2

    Epithelial cells (thymus)

                             (Various anti-keratin antibodies)                 Epithelium

                             (ER-TR4),4F1        HIS38          TE-3,(MR3,     Thymic cortex epithelium

                             (ER-TR5),IVC4       (HIS39)        TE-4,(MR19),   Thymic subcapsular or medullary epithelium

    Complement receptors

    CD11b     p160           Ly-40,              MRC OX-41,     Mac-1,         Granulocytes, macrophages, CD5+ B cells, C3b1R, CR3
                             M1/70               MRC OX-42,     a-Leu-15

    CD21      gp140                                             B2,BL13        B Cells in germinal centres and mantles (faint), follicular
                                                                               dendritic cells (C3d receptor, CR2, receptor for Epstein-Barr

    CD35      p220                                              To 5           Follicular dendritic cells, macrophages, B cells in corona
                                                                               (faint), renal glomerular epithelium. C3bR, CR1

    Table 10 (cont'd)

    CD        Relative       Mouse               Rat            Human          Reactivity
              mol. mass

    IgG-Fc receptors

    CD16      p50-70                                            a-Leu-11       NK cells, subset of T cells, neutrophilic granulocytes, activated
                                                                               macrophages; IgG-FcRIII, low affinity, complexed IgG

    CD32      gp140          Ly-17                              3E1,CIKM5      B Cells, myeloid cells, macrophages; IgGFcRII, low affinity,
                                                                               complexed IgG

    CD64      p75                                               32.2           Monocytes; IgG-FcRI, high affinity, monomeric IgG

    ß1-Integrin (CD29-CD49) family

    CD29      p130           9EG7                               B-D15          Ubiquitous, not on erythrocytes; ß1 chain of all CD49 antigens

    CD49a     p200                                              TS2/7          Activated T cells, monocytes, smooth muscle cells. Very late
                                                                               antigen-1 (VLA-1), ligand of collagen, laminin

    CD49b     p155                                              31H4,AK7,      T Cells, B cells, thrombocytes, fibroblasts, endothelium.
                                                                P1E6           Very late antigen-2 (VLA-2), ligand of collagen I, II, III,
                                                                               and IV, laminin

    CD49c     p145                                              11G5,P1B5      B Cells, renal glomeruli, basal membranes. Very late antigen-3
                                                                               (VLA-3), ligand of collagen, laminin, fibronectin, and invasin

    CD49d     p150           R1-2,               P12520,        HP2/1,44H6,    Thymocytes, lymphocytes, monocytes, NK cells, eosinophilic
                             MFR4.B              MR?4           L25.3          granulocytes, erythroblasts. Very late antigen-4 (VLA-4),
                                                                               ligand of VCAM-1, fibronectin

    CD49e     p160           MFR5,                              SAM-1          Monocytes, leukocytes, œmemoryœ T cells, fibroblasts,
                             P12750                                            thrombocytes and muscle cells. Very late antigen-5 (VLA-5),
                                                                               ligand of fibronectin

    Table 10 (cont'd)

    CD        Relative       Mouse               Rat            Human          Reactivity
              mol. mass

    ß1-Integrin (CD29-CD49) family (contd)

    CD49f     p150           GoH3                               Go-H3,4F10     T Cells, thymocytes, monocytes, thrombocytes. Very late
                                                                               antigen-6 (VLA-6), ligand of laminin and invasin

    ß2-Integrin (CD11-CD18) family

    CD11a     p180           Ly-15,              WT.1           YTH-81.5,      T and B cells, NK cells, erythroid and myeloid stem cells.
                             2D7,                               B-B15,         Leukocyte function-associated antigen-1 (LFA-1) involved in cell
                             M17/4                              G-25.2         adhesion, ligand for intercellular adhesion molecule (ICAM)-1
                                                                               (CD54), ICAM-2 (CD102), ICAM-3 (CD50)

    CD11b     p160           Ly-40,              MRC OX-41,     Mac-1,         Granulocytes, macrophages, CD5+ B cells. C3b1R, CR3
                             M1/70               MRC OX-42,     a-Leu-15

    CD11c     p150                                              a-Leu-M5,      Monocytes, macrophages, granulocytes (faint), activated
                                                                S-HCL-3        lymphocytes. CR4

    CD18      p95            YTS213.1,           WT.3           BL5            All lymphocytes. ß-Chain of CD11 antigens

                             p160-95                            ED7,ED8        CD11-CD18 molecule

    Table 10 (cont'd)

    CD        Relative       Mouse               Rat            Human          Reactivity
              mol. mass


    Terminal deoxynucleotidyl transferase (TdT)                                Immature (lymphoid) cells in bone marrow and thymic cortex
                                                                               (nuclear staining)

    CD25      p55            Ly-43, AMT13,       MRC OX-39      Tac,a-IL2-R    Activated lymphocytes at scattered locations in thymus and
                             7D4,3C7                                           T-cell areas in peripheral lymphoid organs. Interleukin-2
                                                                               receptor alpha chain

    CD122     p75            5H4,                               CF1,Mik-ß2,    NK cells, T cells, B cells, monocytes. Interleukin-2 receptor
                             TM-ß1                              Mik-ß3         ß chain

    CD26      p120           H194-112            MRC OX-61      134-2C2        (Activated) T cells. Dipeptidyl peptidase IV, in mouse T-cell
                                                                               activation molecule (THAM)

    CD30      p105                                              Ki-1,Ber-H2    Sporadic cells in thymic (cortex) and T cell areas in
                                                                               peripheral lymphoid organs, some plasma cells. Activated
                                                                               lymphocytes,Hodgkin cells

    CD34                                                        MY 10,8G12     Haematopoietic progenitor cells, capillary endothelium
                                                                QBEND/10       Human progenitor cell antigen (HPCA)

    CD44      p65-85         Ly-24               MRC OX-49,     a-Leu-44,      Prothymocytes, T cells, small B cells. Lymphocyte homing
                             IM7                 MRC OX-50      F10-44-2       receptor. Phagocytic glycoprotein-1 (PgP-1), HCAM

    CD45      p180-210       Ly-5                MRC OX-1,      T29/33         All leukocytes. Common leukocyte antigen

    Table 10 (cont'd)

    CD        Relative       Mouse               Rat            Human          Reactivity
              mol. mass

    Others (contd)

    CD45R     p190-220       B220                MRC OX-22,     a-Leu-18,      All B cells, subset of T cells. Common leukocyte antigen.
                             MRC OX-32,          MB1,MT2                       HIS24 restricted to strains of the RT7.2 allotype and labels
                             HIS24                                             all peripheral B cells except cells in marginal zone, pre-B

    CD45RA    p205-220       14.8                MRC OX-33      HI100          B Cells, T cytotoxic-suppressor cells (faint), subset of
                                                                               thymocytes.In humans, also CD4+ subset (naive-virgin

    CD45RO    p190-220                                          UCH-L1         T Cells in immature and memory stage. Common leukocyte

    CD54      p90            KAT-1               1A29           84H10,B-C14    Endothelial cells, many activated cell types. Intercellular
                             3E2                                HA58           adhesion molecule-1 (ICAM-1)

    CD71      p95            YTA74.4,C2          MRC OX-26      B3/25          Proliferating cells in germinal centres, some cells in thymus
                                                                               and T-cell areas in peripheral lymphoid organs, stromal
                                                                               cells. Transferrin receptor

                                                                Ki-67          Proliferating cells in germinal centres, some cells in thymus
                                                                               and T-cell areas in peripheral lymphoid organs. Proliferation
                                                                               antigen present in late G1, S, G2, and M phases
                             PCNA                PCNA           PCNA           Proliferating cells in germinal centres, some cells in thymus
                                                                               and T-cell areas in peripheral lymphoid organs

                             MEL14                                             Recirculating T and B cells. Lymphocyte homing receptor

    MHC, major histocompatibility complex; NK, natural killer; Ig, immunoglobulin
    CD nomenclature from: Clark & Lanier (1989); Knapp et al. (1989); Schlossman et al. (1994, 1995)
    Antibodies within parentheses are not commercially available.
        in serum is a function of the antibody's half-life, the longer the
    study the more likely it is that an effect will be observed.
    Immunoglobulin levels can be influenced by the cleanness of the
    facility: studies conducted in facilities with excellent husbandry
    will have lower basal levels than those conducted in 'dirty'
    facilities where animals are constantly exposed to foreign antigens,
    including pathogens.

          Measurement of basal immunoglobulin levels is useful only after
    subchronic or chronic exposure, i.e. with sufficient time for normal
    metabolic elimination. Basal levels of immunoglobulin decrease only
    when synthesis is reduced or prevented such that metabolized
    immunoglobulins are not replaced. The parameter therefore yields
    little information about possible mechanisms of immunotoxicity, and
    should rather be regarded as a screening parameter; this is in fact
    true for most non-functional tests. The IgM and G classes have usually
    been measured; however, since the two other classes (A and E) are
    biologically very important (for instance in mucosal immunity and
    allergic manifestations), they should also be measured.

          Total IgM and IgG concentrations in serum can be analysed by
    means of a 'sandwich' enzyme linked immunosorbent assay (ELISA), as
    described by Vos et al. (1982). Total IgA and IgE concentrations can
    be analysed in an essentially similar way, except that the microtitre
    plates are coated with monoclonal anti-rat IgA (Van Loveren et al.,
    1988b) or monoclonal anti-rat IgE antibodies (MARE-1), respectively,
    and immunoglobulins bound to these antibodies in serum samples are
    detected by sheep anti-rat IgA or monoclonal anti-kappa chains of rat
    immunoglobulins (MARK-1), conjugated with peroxidase.

          Data from the ELISA are usually reported as percentages of
    control values, and a titration curve based on pooled sera is
    prepared; optimal dilutions of exposed and unexposed groups are then
    plotted from this curve. The deviation of the dilution of the test
    groups from the control groups is expressed, with the dilution in the
    control group set at 100%. While studies in rats indicate that
    measurement of basal immunoglobulin levels is useful in predicting the
    immunotoxic effects of compounds, studies conducted in mice at the NTP
    do not, and measurement of basal immunoglobulins is not included in
    either tier of their testing panel (Luster et al., 1988). There are
    several possible reasons for the difference in the usefulness of basal
    immunoglobulin levels in rats and mice. First, in the studies of Vos
    and colleagues, cited above, the exposure period was routinely longer
    than the 14-day studies conducted within the NTP; since serum antibody
    level is a function of the antibody half-life, longer studies are more
    likely to detect an effect. Furthermore, the doses used at the NTP
    were often lower, by design, than those used in rats. A final possible
    explanation, which remains to be confirmed, is that immunoglobulin
    synthesis in rats is more sensitive than that in mice.

    4.1.4  Bone marrow

          Bone marrow is an important haematopoietic organ and a source of
    precursors for lymphocytes and other leukocytes. Changes in the bone
    marrow are therefore likely to result in alterations of
    immunocompetent cell populations, which may be long lasting or
    permanent and thus serve as an indicator of potential immunotoxicity.
    In a study to validate immunotoxicological parameters, bone-marrow
    cellularity was shown to be an indicator of the immunotoxicity of
    cyclosporin A, used as the model compound (Van Loveren et al., 1993a).
    Determination of cellularity in stained slides of bone marrow,
    evaluation of smears, and actual counts of the numbers of cells within
    bone marrow are practical. For this purpose, both ends of a femur are
    cut off, and bone-marrow cells are collected by flushing balanced salt
    solution through the femur with a 21-gauge needle. The concentration
    of nucleated cells is determined in a Coulter counter; a differential
    count of cells can be done visually in May-Grunvald Giemsa-stained
    cytospin preparations.

    4.1.5  Enumeration of leukocytes in bronchoalveolar lavage fluid,
           peritoneal cavity, and skin

          Mononuclear phagocytes in the alveoli of the lung play an
    important role in clearing inhaled particles, including
    microorganisms, from the lung. The numbers of cells and alterations in
    their function can be end-points of the toxicity of inhaled chemicals.
    In order to study these parameters, methods for harvesting the cells
    from the lungs should be easy to perform, guarantee the sterility of
    the cell harvest, and be standardized. Methods involve use of either a
    syringe (Blusse Van Oud Alblas & Van Furth, 1979) or a complex system
    of syringes, tubes, and valves (Moolenbeek, 1982). These methods often
    result in contamination of the harvested cell population; moreover,
    they are laborious and cannot easily be standardized since the syringe
    is operated manually. In a more recently developed method (Van
    Soolingen et al., 1990), an excised lung is placed in a pressure
    chamber and connected to a cannula through which lavage fluid can be
    introduced into the lung and transferred from the lung into a test
    tube. This procedure is repeated several times to obtain an optimal

          Enumeration of mononuclear cells in the peritoneal cavity can
    also best be performed by harvesting these cells by lavage. Because
    of the architecture of this organ, three or four cycles of
    intraperitoneal injections of lavage fluid, followed by gentle
    massaging of the abdomen, and aspiration of the fluid with the syringe
    that was also used for injection suffice.

          Langerhans cells in the skin can be enumerated with
    histopathological techniques. Frozen tissue sections are used, stained
    with immunoperoxidase techniques including markers for MHC class II
    antigens or specific markers, as indicated above. Morphometric
    analysis may provide a quantitative basis for this type of evaluation.

    4.1.6  Flow cytometric analysis

          Evaluation of phenotypic markers has proved to be one of the most
    sensitive indicators of immunotoxic compounds. The availability of
    fluorescent activated cell sorter (FACS) analysis units and
    fluorescent cell counter units in immunotoxicology laboratories has
    made analysis of cell populations routine. Determination of the
    phenotype of lymphoid cells is a non-functional assay, although it has
    often been inappropriately grouped with functional tests. The presence
    or absence of a particular marker on the surface of a cell does not
    reveal the functional capability of the cell. The usefulness of
    surface marker analysis for predicting potential immunotoxicity has
    been demonstrated. In studies conducted by Luster et al. (1992), a 91%
    concordance was found for correct identification of immunotoxic
    compounds on the basis of studies of surface markers alone.

          As indicated above (section 4.1.2), numerous markers are
    expressed on the cells of the immune system. Essentially, the same
    reagents as used on tissue sections are applied on cells that have
    been isolated from tissues, body fluids, or lavage fluids in
    suspension (see above). Furthermore, both polyclonal and monoclonal
    antibodies are available for detecting these surface markers. While
    many of the markers have been used in immunological investigations,
    very few have been evaluated with a large number of immunosuppressive
    compounds. The markers that have been routinely used in studies of
    immunotoxicity conducted for the NTP in mice and the cell types they
    identify are shown in Table 11. The CD4:CD8 ratio in spleen has been
    shown to concord best with the immunotoxicity of these surface markers
    (Luster et al., 1992).

    Table 11.  Phenotypic markers on lymphocyte subpopulations used in
               studies of immunotoxicity by the United States National
               Toxicology Program

    Surface marker                Cell type

    sIg+                          Pan B cells
    Thy 1.2+ or CD3+              Pan T cells
    CD4+CD8-                      T Helper/delayed-type
                                          hypersensitivity cells
    CD8+CD4-                      T Suppressor-cytotoxic cells
    CD8+CD4+                      Immature T cells

          The identification of phenotypic markers in rats has not
    developed as rapidly as in mice; however, antibodies to rat cell
    surface markers are now becoming available commercially and are being
    used in immunotoxicological assessments (Smialowicz et al., 1990). The
    monoclonal antibodies currently used for this purpose are: OX4 or
    MARK-1 for B cells, W3/13 or OX 19 for T cells, R79 for the T cell
    receptor, W3/25 for CD4 cells, and OX 8 for CD8 cells.

          In enumerating the cell types in lymphoid tissue, both
    percentages and absolute cell numbers should be reported. Of the two,
    absolute cell numbers are by far the most meaningful. Compounds that
    affect all populations equally and thus do not change the relative
    percentages of the various cell types may be missed if only
    percentages are evaluated. In addition, significant differences in the
    magnitude of an effect on one or more of the populations can be
    observed when the data are evaluated as absolute numbers and not as
    percentages. As indicated above, the absolute changes more closely
    reflect the events occurring in the animal and should thus be given
    priority in interpreting data.

          FACS analysis is also being used to determine the activation
    state of various cell types, on the basis of changes in detectable
    activation markers. Some of the activation markers that have been
    studied are F4/80 (Austyn & Gordon, 1981), Mac-1 (Springer et al.,
    1979), Mac-2 (Ho & Springer, 1984), transferrin receptor (Neckers &
    Cossman, 1983), and IL-2 receptor (Cantrell et al., 1988). While
    activation markers are of value in studying the mechanism of action of
    compounds, their usefulness as predictors of immunotoxicity has yet to
    be firmly established.

    4.2  Functional tests

    4.2.1  Macrophage activity

          Phagocytic activity is the first line of defence against many
    pathogens. Macrophages can phagocytose many particles, including
    bacteria, and can lyse and inactivate them. Alterations in phagocytic
    activity are therefore important potentially adverse effects of
    chemicals on the immune system. The capacity to ingest particles
     in vitro can be measured, and activity  in vivo can be measured by
    determining the clearance of bacteria, such as  L. monocytogenes.
    This test is dealt with in section

          Several assays have been developed for evaluating various types
    of phagocytosis in mice and can also be used in rats, with slight
    modifications. Innate and non-immune-mediated phagocytosis by
    macrophages can be evaluated by determining the uptake of fluorescent
    latex covaspheres (Duke et al., 1985). Macrophages and peritoneal
    exudate cells are placed on a tissue culture slide and incubated with
    the covaspheres for 24h on a rocking platform. The slides are then

    fixed with methanol. The slide chambers are evaluated under a
    fluorescent microscope, and macrophages with more than five latex
    covaspheres are counted as positive for phagocytosis. The results are
    expressed as percentage of phagocytosis, which is calculated as the
    ratio of macrophages positive for phagocytosis to total macrophages
    counted. In order to distinguish phagocytosed latex covaspheres from
    those that are merely associated with the macrophage surface, the
    cells are exposed for 30-60s to methylene chloride vapour. By
    immersing the slides in this manner, the covaspheres that have not
    been phagocytosed are dissolved, while those inside the macrophage
    remain intact (Burleson et al., 1987). Phagocytosed fluorescent latex
    particles can easily be quantified under the fluorescence microscope.
    While this assay is straightforward, it is labour intensive, and
    reading the slides, shifting back and forth from the fluorescent
    field, and counting the macrophages is time-consuming.

          A radioisotopic procedure, the chicken erythrocyte assay, can be
    used to evaluate both adherence to and phagocytosis of particles by
    macrophages. The phagocytic capacity is measured as an immunologically
    mediated (Fc receptor) response. Macrophages are added to each well of
    a 24-well tissue dish and allowed to adhere for a 2-3-h incubation
    period. Nonadherent cells are washed, and chicken erythrocytes
    labelled with 51Cr are added to each well; then a subagglutinating
    dilution of antisera to chicken erythrocytes is added to each well and
    the plate incubated for 1h. The plates are then washed to remove
    unbound erythrocytes; an ammonium chloride solution is added to lyse
    adhered erythrocytes, and the supernatant is collected and counted to
    determine adherence of the erythrocytes to the macrophages. Next, both
    the macrophages and the phagocytosed chicken erythrocytes are lysed by
    addition of 0.1 N sodium hydroxide, and the solution is counted to
    determine the amount of phagocytosis. Three to six wells in each group
    do not receive 51Cr and are used to evaluate the DNA content
    (Labarca & Paigen, 1980). The data are expressed as adherence counts
    per minute, phagocytosed counts per minute, and specific activity for
    adherence and phagocytosis. Specific activity is determined by
    dividing the number of adhered or phagocytosed counts per minute by
    the DNA content per well. The data must be expressed in terms of
    specific activity, since compounds that affect the macrophages'
    ability to adhere to the 24-well culture dish will significantly alter
    the results obtained.

          While both the nonspecific and immune-mediated phagocytosis
    assays are useful for understanding the potential mechanisms of action
    of compounds, changes in phagocytic activity in these in-vitro assays
    have not been found to be predictive of immunotoxicity. For example, a
    single intratracheal exposure to gallium arsenide resulted in
    increased adherence and phagocytosis by chicken erythrocytes but
    decreased phagocytosis of latex covaspheres (Sikorski et al., 1989).

          The phagocytosis assay that is most predictive of altered
    macrophage function is evaluation of the functional ability of the
    mononuclear phagocyte system. This is a holistic assay for measuring
    the capacity of the fixed macrophages of the mononuclear phagocyte
    system, where macrophages provide the first line of defence against
    both pathogenic and non-pathogenic blood-borne particles. The fixed
    macrophages of the mononuclear phagocyte system line the liver
    endothelium (Kupffer cells), the spleen, the lymph nodes (reticular
    cells), the lung (interstitial macrophages), and other organs such as
    the thymus and bone marrow. When the assay is conducted in mice, the
    animals are injected intravenously with 51Cr-labelled sheep
    erythrocytes, and a 5-µl blood sample is taken from the clipped tail
    at 3-min intervals over a 15-min period. A final 30-min blood sample
    is taken, and 1h after injection the animals are sacrificed and the
    liver, spleen, lungs, thymus, and kidneys are removed, weighed, and
    counted in a gamma counter. The 60-min time interval after injection
    of sheep erythrocytes was selected as the time of sacrifice since it
    represents the plateau for particle uptake by the selected organs
    (White et al., 1985). Blood clearance of the radiolabelled cells is
    expressed as vascular half-life and as a phagocytic index, which is
    determined by the slope of the clearance curve. Organ distribution is
    expressed as percent organ uptake and counts per minute per milligram
    of tissue (specific activity). The assay can detect both stimulation
    and inhibition of the mononuclear phagocyte system. Bick et al. (1984)
    reported marked stimulation of the mononuclear phagocyte system after
    treatment with diethylstilbestrol; more recently, morphine sulfate was
    shown to decrease vascular clearance and hepatic and splenic
    phagocytosis significantly (LeVier et al., 1993).

    4.2.2  Natural killer activity

          NK activity against neoplastic and virus-infected targets has
    been clearly demonstrated  in vitro and is thought to play an
    important role  in vivo in providing surveillance against neoplastic
    cells and as a first line of defence against viruses (Herberman &
    Ortaldo, 1981). In humans, rats, and mice, most cells with NK activity
    can be identified by morphological (although the definition is not
    morphological) and functional characteristics (Timonen et al., 1981).
    Most of the cells that show NK activity are nonadherent, non-
    phagocytic lymphocytes and are morphologically associated with large
    granular lymphocytes (Timonen et al., 1982). Although cells with NK
    activity do not strictly belong to the T-cell lineage, they can
    express T cell-associated markers and express surface receptors, such
    as those for the Fc portion of IgG and the ganglioside asialo GM1.
    Some of these markers are also expressed by monocytes, macrophages,
    and polymorphonuclear leukocytes (Herberman & Ortaldo, 1981). Within
    4h, the cells can show nonantigen-specific cytotoxic activity
     in vitro and  in vivo against certain (NK-sensitive) tumour cell
    lines and virus-infected cells.

          The cells have enhanced cytolytic function after activation with
    a variety of stimuli, including viral infection (Stein-Streinlein et
    al., 1983), BCG (Tracey et al., 1977), IL-2 (Henney et al., 1981;
    Domzig et al., 1983; Lanier et al., 1985; Malkovsky et al., 1987),
    interferon, and interferon inducers (polyI:C) (Tracey et al., 1977;
    Oehler & Herberman, 1978; Djeu et al., 1979a,b). NK activity  in vitro
    can be stimulated with IL-2 and interferon (Tracey et al., 1977; Djeu
    et al., 1979b). Anti-asialo GM1 antibody can strongly inhibit
    cytotoxic NK activity both  in vitro and  in vivo (Kasai et al.,
    1980, 1981; Yosioka et al., 1986). This antibody binds to the cell
    surface glycolipid GM1 and suppresses the lytic activity of effector
    cells. Large granular lymphocytes are found in several lymphoid
    organs. Many cells with high NK activity are found in spleen and
    peripheral blood (Rolstad et al., 1986); lymph nodes have less NK
    activity, and thymus and bone marrow show only marginal activity. NK
    activity can also be demonstrated in the bronchus-associated lymphoid
    tissue in the lungs. Moreover, large granular lymphocytes can migrate
    from the circulation into the extravascular tissue and can even be in
    contact with the lumen of the alveoli (Timonen et al., 1982; Reynolds
    et al., 1984; Rolstad et al., 1986; Prichard et al., 1987). The
    presence of large granular lymphocytes associated with NK activity in
    the lungs is probably of great importance, because the lungs
    constitute a major site for neoplastic disease (metastatic spread) and
    viral infections. NK cells may also operate in certain types of
    bacterial infections. In experimental animals, suppression of NK cell
    activity increased the numbers of metastases after transplantation of

          The clinical significance of altered NK cell activity in humans
    has not clearly been established. Asymptomatic individuals with low NK
    cell responses may be at some risk for developing upper respiratory
    infections and for increased morbidity (Levy et al., 1991); and
    extreme susceptibility to severe and repeated herpes virus infection
    was reported in an individual without NK cells (Biron et al., 1989).
    It is obvious therefore that exposure to toxic substances that alter
    NK activity can have biological consequences, and testing this
    activity is important in assessing potential immunotoxicity.

          The procedure for determining NK activity is as follows: cell
    populations that exert NK activity (usually enriched peripheral blood
    mononuclear cells or spleen cells) are cultured with NK-sensitive
    target cells. A cell type frequently used for this purpose is the YAC
    lymphoma cell line, which has been applied to mice, rats, humans, and
    even seals. YAC lymphoma target cells are radiolabelled with 51 Cr,
    and lysis of the cells, resulting in release of chromium, within 4h is
    used to estimate the cytolytic activity of the NK cells within the
    cell population. This assay has been used to demonstrate the effects
    of numerous compounds on NK activity in rats (e.g. TBTO, ozone, and
    HCB: Vos et al., 1984; Van Loveren et al., 1990c), mice (Luster et
    al., 1992), and harbour seals (Ross et al., in press).

    4.2.3  Antigen-specific antibody responses

          Most antibody responses require not only B cells, which, after
    maturation into plasma cells, produce antibodies, but also the help of
    T lymphocytes. A variety of T cell-dependent antigens can be used for
    this purpose, and an excellent one is tetanus toxoid. A typical
    immunization schedule in rats comprises intravenous immunization on
    day 0 followed by a booster on day 10. Primary and secondary IgG and
    IgM responses can then be measured in serum, taken on day 10 (just
    before the booster) and day 21, respectively. The primary IgM response
    is the immunoglobulin response that is least under the control of T
    cells. As tetanus toxoid is also used for human immunization, the
    responses to this antigen may be useful in extrapolating experimental
    data to humans. The responses can be determined in an ELISA (Vos et
    al., 1979b).

          Another widely used T cell-dependent antigen is ovalbumin. This
    antigen can be and has been used to induce all classes of antibody
    responses, i.e. IgM, IgG, IgA, and IgE, that can be measured with the
    ELISA (Vos et al., 1980; Van Loveren et al., 1988b). The classical
    assay of specific IgE responses is the passive cutaneous anaphylaxis
    reaction. Serial dilutions are injected into the skin of rats,
    sensitizing local mast cells; the specific antigen is then injected
    intravenously, simultaneously with Evans blue. Mast cell products are
    released where IgE meets the antigen, and IgE is cross-linked on the
    membranes of the mast cells, leading to extravasation of Evans blue.
    The titre can be determined from the magnitude of the reaction at each
    dilution of IgE. ELISA techniques and the specific reagents to detect
    IgE in an ELISA that are now available make this test preferable.

          Ovalbumin induces not only humoral responses but also delayed-
    type hypersensitivity. Sensitization to ovalbumin in Freund's complete
    adjuvant enhances responses and makes it possibile to assay both
    responses in one animal. Delayed-type hypersensitivity can also be
    directed to purified protein derivative, with responses induced by the
    adjuvant (Vos et al., 1980). At least in mice, however, immunization
    in complete adjuvant skews responses in the direction of Th1
    responses, i.e. delayed hypersensitivity, and hence suppresses Th2-,
    IgE-, and IgA-dependent immune responses.

          A few antigens can induce humoral immune responses without
    involvement of T lymphocytes. One example is trinitrophenol-Ficoll
    (lipopolysaccharide). Sensitization of animals to this antigen yields
    immunoglobulin responses that can be measured in an ELISA. This is a
    useful test for use in mechanistic studies to separate the effects of
    compounds on B and T cells.

    4.2.4  Antibody responses to sheep red blood cells  Spleen immunoglobulin M and immunoglobulin G plaque-forming
             cell assay to the T-dependent antigen, sheep red blood cells

          A widely used particulate T cell-dependent antigen is sheep red
    blood cells. Antibody titres induced after sensitization can be
    assayed with various techniques; one that is widely used is the
    plaque-forming cell assay, or antibody-forming cell response. This
    assay is relatively simple and can be conducted with inexpensive
    equipment found in most laboratories, but the optimal concentration of
    sheep red blood cells must be injected. As the antigenicity of red
    blood cells varies significantly from sheep to sheep, time must be
    invested to obtain cells from a sheep that repeatedly gives a high
    response (>= 1500 plaque-forming cells/106 spleen cells). The
    number of cells administered (about 2 × 108) should also be
    optimized for both mice and rats in the laboratory conducting the
    assay. The intravenous route is that preferred for sensitization;
    intraperitoneal injections can be used but significantly increase the
    potential for nonresponding animals as a result of a poor injection.
    Animals are sacrificed on day 4 after injection, and spleen cells are
    prepared by mincing the spleen between two frosted microscope slides,
    teasing it apart with forceps, or passing it through a small mesh
    screen; all of these methods are satisfactory, and that used to
    prepare single splenocyte cultures varies from laboratory to
    laboratory. An aliquot of cells is added to sheep erythrocytes and
    guinea-pig complement; these are placed in a microscope slide chamber
    when the Cunningham assay method is used (Cunningham & Szenberg,
    1968), or, in the Jerne method, cells and guinea-pig complement are
    added to a test tube containing warm agar and after thorough mixing
    the test tube mixture is plated in a petri dish and covered with a
    microscope cover slip (Jerne et al., 1974). In either case, the
    preparations are then incubated at 37°C for 3-4h to allow plaques to
    develop. The plaques are counted under a Bellco plaque viewer. A
    plaque results from the lysis of sheep erythrocytes and is elicited as
    a result of the interaction of complement and antibodies directed
    against sheep erythrocytes, which are produced in response to the
    intravenous sensitization. As each plaque is generated from a single
    IgM antibody-producing plasma cell, the number of IgM plaque-forming
    cells present in the whole spleen can be calculated. The data are
    expressed as specific activity (IgM plaque-forming cells/106 spleen
    cells) and IgM plaque-forming cells per spleen.

          By incorporating rabbit anti-mouse or anti-rat IgG antibody into
    the preparation of spleen cells, complement, and sheep red blood
    cells, the number of IgG antibody-forming cells present in the spleen
    can also be determined. This number is calculated by subtracting the
    number of IgM plaque-forming cells from the total number of both IgM
    and IgG plaque-forming cells. The optimal IgG primary response is
    observed five days after sensitization (Sikorski et al., 1989).

          The T-dependent IgM response to sheep red blood cells is one of
    the most sensitive immunotoxicological assays currently in use. Luster
    et al. (1992) reported that the individual concordance of the plaque-
    forming cell assay for predicting immunotoxicity was the highest of
    all the functional assays (78%). Furthermore, use of this assay in
    combination with either NK cell activity or surface marker analysis
    resulted in pairwise concordances for predictability of more than 90%.

          While the plaque-forming cell assay has been shown to be
    sensitive and predictive, the procedure does have its limitations. As
    indicated earlier, the effect of the test compound on the immune
    system is evaluated only in spleen cells, and effects on other
    antibody-producing organs and tissues are not determined. The assay is
    somewhat laborious, and it is preferable that several people
    participate, to help in removing spleens, preparing cell preparations,
    counting cells, and adding preparations to either microscope slide
    chambers or agar dishes. An additional drawback is that the assay must
    be conducted on the same day as the animals are sacrificed. This is in
    marked contrast to the ELISA, in which sera can be frozen and
    evaluated at a later date. While the slides and petri dishes can be
    placed in a cold room or refrigerator and counted the next day, this
    procedure is not recommended, as they tend to dry out to some extent,
    making viewing and discerning plaques more difficult.  Enzyme-linked immunosorbent assay of anti-sheep red blood
             cell antibodies of classes M, G, and A in rats

          An alternative to the plaque-forming cell assay is ELISA of anti-
    sheep red blood cell antibody titres in serum. Antigen preparations
    made from ghosts of sheep erythrocytes by extraction with potassium
    chloride are used to coat the bottoms of the wells of 96-well
    microtitre plates. Serum samples from rats immunized with sheep
    erythrocytes are titrated onto these plates using specific polyclonal
    antibodies to rat IgM or IgG, to which peroxidase is conjugated. IgA
    has also been assayed, using monoclonal anti-rat IgA antibodies and
    polyclonal rat anti-mouse IgG conjugated with peroxidase. The ELISA of
    serum titres of IgM, IgG, and IgA to sheep erythrocytes is an easy,
    reliable method that can be used to detect the effects of chemicals on
    the immune system of the rat (Van Loveren et al., 1991; Ladics et al.,

          The assay measures titres of specific antibodies, in contrast to
    the plaque-forming cell assay which determines the number of cells
    that are actually responsible for production. The ELISA assesses the
    production of antibodies, either per cell or in terms of the total
    capacity of the host to produce these antibodies  in vivo. In
    interpreting the effects of exposure to chemicals, account must be
    taken of the fact that the cells used in the assay are derived from
    specialized parts of the body, such as the spleen, and alterations in
    the numbers of antibody-producing cells in such an organ in rats
    immunized with sheep red blood cells cannot give information on other,

    inaccessible pools of antibody-producing cells. In the ELISA,
    alterations in titres due to exposure to chemicals indicate changes in
    the immune potential of the exposed animals. In screening for the
    effects of chemicals on the immune system, therefore, ELISAs may be
    preferable, but for studies on specific immunosuppressive mechanisms,
    the plaque-forming cell assay, although labour- and time-intensive, is
    a powerful tool for obtaining information complementary to the data
    provided by the ELISA. Unfortunately, it is not always possible to
    perform the two assays with material from the same animal. The peak
    response in the plaque-forming cell assay in both rats (Fischer 344)
    and mice (B6C3F1) occurs on day 4 after sensitization, while the peak
    response in the ELISA occurs on day 6 for rats and day 4-5 for mice
    (Temple et al., 1993). In order to detect the effects of chemicals on
    the immune response to sheep red blood cells, it is preferable to
    choose the optimal conditions, or to follow the kinetics, of the

    4.2.5  Responsiveness to B-cell mitogens

          Responsiveness to lipopolysaccharide is another estimate of
    humoral immune response, as solely B cells respond to this mitogen.
    Although the responses of rats to this mitogen are less pronounced
    than those of mice, good results can be obtained, and the
    immunosuppressive effects of chemicals can be detected (Vos et al.,

          An alternative B-cell mitogen is  S. typhimurium mitogen (STM),
    a water-soluble, proteinaceous extract derived from the cell walls of
     S. typhimurium; it is a more potent mitogen for rat B lymphocytes
    than lipopolysaccharide (Minchin et al., 1990). In both mice and rats,
    the polyclonal activation of B lymphocytes is a multistep process. In
    mice, mitogens alone can provide all the signals necessary for
    proliferation and differentiation; in the rat, STM stimulation induces
    B lymphocytes to proliferate without differentiating. The addition of
    lymphokines to STM-stimulated B cells also failed to stimulate them to
    differentiate (Stunz & Feldbush, 1986). Nevertheless, this mitogen is
    useful for evaluating effects on the proliferative ability of rat B
    lymphocytes. Smialowicz et al. (1991) showed a decrease in the STM
    response in Fischer 344 rats after oral exposure to 2-methoxyethanol.

          Unlike the bell-shaped mitogen dose-response curves observed with
    T-cell mitogens, the proliferative response of B lymphocytes to both
    lipopolysaccharide and STM rises quickly at low concentrations of the
    mitogens and plateaus at higher concentrations. As a result, a single
    concentration on the plateau phase of the mitogen response curve is
    sufficient to evaluate the effects of a test compound on B-cell
    mitogen-driven proliferation. One of the reasons that the mitogen
    assays appear to be insensitive is that the cells must remain in
    culture for several days in order to obtain a peak response. As a
    result, they may recover from the immunomodulatory effects of the test
    compounds during this in-vitro phase. This is a common problem with

    many ex-vivo/in-vitro assays, including the cytotoxic T lymphocyte and
    mixed leukocyte response assays; because of the short, 4-h period of
    the NK cell assay, this is less of a concern.

    4.2.6  Responsiveness to T-cell mitogens

          The proliferative ability of T lymphocytes after stimulation with
    mitogens can be measured by the uptake of 3H-thymidine in a manner
    similar to that used to measure B-cell proliferation (Anderson et al.,
    1972). Concanavalin A and phytohaemagglutinin are T-cell mitogens in
    both rats and mice; pokeweed mitogen stimulates the proliferation of
    both T and B cells and thus lacks specificity. Although both
    concanavalin A and phytohaemagglutinin stimulate T lymphocytes,
    T cells responsive to concanavalin A have been reported to be less
    mature than those responsive to phytohaemagglutinin (Stobo & Paul,
    1973). Multiple concentrations of these mitogens should be used to
    ensure that a peak response is obtained: both produce a bell-shaped
    dose-response curve, and too high a concentration can result in a
    suboptimal response.

          Historically, mitogens have been included in the battery of tests
    for evaluating potential immunotoxicity, because the assay is one that
    can also be carried out in humans. Human studies, however, are
    conducted on peripheral blood, while most studies of rodent lymphocyte
    transformation are conducted using spleen or lymph node cells. Thus,
    the argument that the assay has clinical relevance is not well
    founded. Furthermore, as the response of lymphocytes is extremely
    robust, the assay lacks sensitivity. After a significant number of
    compounds were evaluated for potential immunotoxicity in mitogen
    assays, use of this assay was shifted from the tier 1 screen
    originally described by Luster et al. (1988) to the tier 2
    comprehensive evaluation. Use of the mitogen assay has now been
    removed completely from studies conducted for the NTP, since other
    assays in which cellular proliferation is required (e.g. plaque-
    forming cell assay, mixed leukocyte reaction) were considered to be
    more sensitive, and the data obtained from the mitogen assays add
    little if any to an evaluation of the potential immunotoxicity of test

    4.2.7  Mixed lymphocyte reaction

          In the mixed lymphocyte reaction (also known as mixed lymphocyte
    culture), suspensions of responder T lymphocytes from spleen or lymph
    nodes are co-cultured with allogeneic stimulator cells. The foreign
    histocompatibility antigen (MHC class I or class II molecules)
    expressed on the allogeneic stimulator cells serves as the activating
    stimulus for inbred populations. In noninbred populations, a pool of
    allogeneic cells can be used as stimulators. The assay analyses the
    ability of T cells to recognize allogenic cells as 'non-self' as a
    result of the presence of different MHC class II antigens on their

    surface. In response to the class II antigens, the spleen or node
    cells proliferate. Because a sufficiently large number of T cells in
    the mixed lymphocyte population respond to the stimulator population,
    the responder T cells need not be primed. Proliferation of the
    responder cells is one of the parameters for T-cell responsiveness to
    cellular antigens. If the allogeneic stimulator cell suspension
    contains T cells, their uptake of 3H-thymidine must be prevented by
    gamma-irradiation or mitomycin C, in order to preclude background
    thymidine uptake.

    4.2.8  Cytotoxic T lymphocyte assay

          The Tc lymphocyte assay is a continuation of the mixed lymphocyte
    reaction response in which the T lymphocytes further differentiate
    into cytotoxic effector cells under the influence of various
    cytokines. In mice, the assay is usually conducted using P815
    mastocytoma cells as the sensitizer and target cell (Murray et al.,
    1985). Mice are exposed  in vivo to the test agent, and spleen cells
    are then removed and placed in culture flasks with the P815
    mastocytoma cells. After a five-day co-culture period, the spleen
    cells are harvested and added to fresh P815 mastocytoma cells which
    have been radiolabelled with 51Cr as sodium chromate. After a 4-h
    incubation, the percentage cytotoxicity is determined by measuring the
    specific release of 51Cr into the supernatant. The five days of
    culture are necessary for the T lymphocytes to differentiate into
    cytotoxic effector cells. Unfortunately, this extended period in
    culture may give the spleen cells sufficient time to recover from any
    adverse effects of the test compound, although such effects may have
    been present at the time the spleen cells were removed from the
    animal. This inherent limitation of the assay detracts from its
    usefulness in assessing the immunotoxicity of test compounds.

          A holistic Tc lymphocyte assay has been described, in which the
    animal is sensitized after injection of the irradiated target cells
    (Devens et al., 1985). Inhibiting the ability of the sensitizing cells
    to proliferate either through irradiation or mitomycin C treatment
    before injection prevents development of Tc lymphocytes in the animal.
    Smialowicz et al. (1989) developed an assay in rats in which effector
    cells are generated in culture by incubating cells with lymph node
    cells from Wistar/Furth rats, and 51Cr-labelled W/Fu-G1 tumour cells
    are used as the target cells. The assay requires four days in culture
    and can be run simultaneously with the rat mixed lymphocyte reaction,
    thus providing information on the test compound's ability to affect
    proliferation and differentiation into effector cells.

    4.2.9  Delayed-type hypersensitivity responses

          Delayed-type hypersensitivity responsiveness is a reflection of
    the capacity of the cellular immune system to execute immune responses
    and especially those dependent on IL-2 and INF gamma, which include
    attraction and activation of nonspecific mononuclear leukocytes

    (macrophages-monocytes). Many systems can be used, depending on the
    antigen. One is sensitization to BCG, followed by challenge with
    purified protein derivative, to which sensitivity is induced. Another
    example is ovalbumin, to which sensitization is most efficient if the
    ovalbumin is emulsified in complete Freund's adjuvant. In this system,
    delayed hypersensitivity can be measured to both purified protein
    derivative and ovalbumin (Vos et al., 1980). Another antigen is
     L. monocytogenes: This system is particularly interesting since it
    can be used in the context of experiments in which host resistance to
    this pathogen is also measured (Van Loveren et al., 1988a).

          Delayed hypersensitivity responses can be measured after
    sensitization to  Listeria by subcutaneous injection of the test
    antigen into the ears. Prior to and 24 and/or 48h after challenge, the
    increment in ear thickness can be measured with a micrometer by a
    person unaware of the experimental group. The background ear swelling
    responses of similar, unimmunized control animals are subtracted from
    the swelling responses found in immunized animals.

          Several delayed-type hypersensitivity assays have been developed
    and used for evaluating immunotoxicity in the mouse. Most have
    involved measuring swelling in either the footpad or the ear after
    sensitization and challenge with a protein antigen. Studies by
    LaGrange et al. (1974) demonstrated that sheep erythrocytes could
    elicit a delayed-type hypersensitivity response after a single
    injection into the foot pad; however, more sheep erythrocytes were
    needed to elicit the delayed-type hypersensitivity response than to
    produce the optimal humoral immune response. Foot pad swelling can be
    measured with a micrometer, as described for rats or by a more
    objective, isotopic procedure, as described by Paranjpe & Boone (1972)
    and Munson et al. (1982). The delayed-type hypersensitivity response
    to sheep erythrocytes was previously considered to be a good assay for
    detecting effects on cell-mediated immunity; however, the lack of
    persistence of the response (LaGrange et al., 1974; Askenase et al.,
    1977) and the possible contribution of antibody to the response raised
    concern about the specificity of the assay when sheep erythrocytes are
    used as the eliciting antigen. Benzo[ a]pyrene, a compound that
    selectively affects humoral but not cell-mediated immunity in adult
    mice, appears to decrease cell-mediated immunity when measured in the
    sheep erythrocyte assay but has no effect on delayed-type
    hypersensitivity when evaluated in the keyhole limpet haemocyanin
    assay. The effect in the sheep erythrocyte assay is observed at doses
    of benzo[ a]pyrene that decrease antibody production, suggesting a
    significant antibody component of the swelling observed (White, 1992).

          Keyhole limpet haemocyanin is another protein antigen used in
    evaluating delayed-type hypersensitivity responses. Holsapple et al.
    (1984) characterized the response to this antigen in the mouse,
    showing that it produced the classical delayed-type hypersensitivity
    response both with and without adjuvant. Two immunizations with

    keyhole limpet haemocyanin were required, however, to produce a
    response equivalent to one obtained with complete Freund's adjuvant.
    In these studies, animals were sensitized with subcutaneous injections
    of keyhole limpet haemocyanin in the shoulder area, with seven days
    between the sensitizations. They were then challenged with the same
    antigen injected intradermally into the central portion of the pinna
    of one of the ears. Increases in ear thickness were evaluated by both
    micrometer readings and radioisotopically. The unchallenged ear was
    used as an individual control for each animal, and a group of
    unsensitized but challenged animals was used to control for
    nonspecific and background effects. The results indicated that,
    whenever possible, the use of adjuvant in delayed-type
    hypersensitivity studies should be avoided. Despite the fact that
    complete Freund's adjuvant boosted the responses to keyhole limpet
    haemocyanin, it partially masked the dexamethasone-induced suppression
    of the response. In some cases, however, delayed-type hypersensitivity
    responses are difficult to induce without adjuvant.

          The studies currently conducted in mice and rats with this assay
    are holistic assays for evaluating cell-mediated immunity. Since
    sensitization and challenge occur in the intact animal, all components
    of the immune system are present to respond in a physiologically
    relevant manner. This type of assay is much more valuable for
    evaluating the effects of compounds on cell-mediated immunity than are
    in-vitro assays such as the mixed leukocyte response or Tc cell assay.
    Luster et al. (1992) reported that the delayed-type hypersensitivity
    response assay in mice was highly predictive (100% concordance) of
    immunotoxicity when used in combination with the NK cell assay and the
    plaque-forming cell assay.

    4.2.10  Host resistance models  Listeria monocytogenes

          Relevant mechanisms of defence against  L. monocytogenes include
    phagocytosis by macrophages and T cell-dependent lymphokine production
    which enhances phagocytosis (Mackaness, 1969; McGregor et al., 1973;
    Takeya et al., 1977; Pennington, 1985; Van Loveren et al., 1987).
    Humoral immunity is not relevant in protection against infection in
    this model. Clearance of  Listeria after infection by, for instance,
    the intravenous or the intratracheal route can be assessed at various
    times after infection by determining the numbers of colony forming
    units in the spleen or lungs, respectively. This can be done by
    classical methods (Reynolds & Thomson, 1973) that involve the
    following steps: Serial dilutions of homogenates of the organs,
    prepared in mortars with sterile sea sand, are plated onto sheep blood
    agar plates; after a 24-h incubation at 37°C, the colonies are counted
    to determine the number of viable bacteria in the organ. Differences
    in the numbers of bacteria retrieved from the organs are an indication
    of the clearance of the bacteria, i.e. the rate at which the host
    disposes of the bacteria after infection.

          Histopathology after a  Listeria infection can also be valuable.
    For instance, exposure to ozone before an intratracheal infection with
     Listeria affects pathological lesions due to the infection (Van
    Loveren et al., 1988a): Pulmonary infection with  Listeria induces
    histopathological lesions characterized by foci of inflammatory cells,
    such as lymphoid and histiocytic cells, accompanied by local cell
    degeneration and influx of granulocytes. If rats are exposed to ozone
    for one week before infection, the lesions are much more severe than
    in unexposed animals and persist at times when either ozone-associated
    or infection-associated effects alone would have resolved. The quality
    of the lesions is also influenced by prior exposure to ozone: mature
    granulomas were found in  Listeria-infected rats that were also
    exposed to ozone.

          When mice are challenged with  Listeria, mortality is the usual
    end-point monitored; however, clearance and organ bacterial colony
    counts can also be determined.  L. monocytogenes is a Gram-positive
    bacterium. The resistance of mice to the organism is genetically
    regulated, and the susceptibility of the B6C3F1 strain, the strain
    designated by the NTP for immunotoxicity studies, comes from the C3H
    parent, since the C57Bl/6 mouse is resistant (Kongshavn et al., 1980).
     Listeria can easily be stored at -70°C at a stock concentration of
    approximately 108 colony forming units per ml. In studies in mice,
    three challenge levels are routinely selected to produce 20, 50, and
    80% mortality in the vehicle control animals. Mortality is recorded
    daily for 14 days. Treatment groups consisting of 12 mice per group
    have been found to be useful for obtaining statistically meaningful
    data on host resistance. This assay is extremely reproducible when the
    organism is administered intravenously.

          The  Listeria assay can detect both protection from and
    increased susceptibility to chemicals and drugs. Morahan et al. (1979)
    used the model to demonstrate a dose-dependent decrease in host
    resistance after exposure to delta-9-tetrahydrocannabinol, the major
    psychoactive constituent of marijuana. The  Listeria host resistance
    assay is that most often used in immunotoxicological assessment of
    compounds, and numerous examples of its use can be found in the
    literature. It is one of the primary models used by the NTP for
    evaluating immunosuppression. Since  Listeria is a human pathogen,
    appropriate precautions are needed in conducting the assay.  Streptococcus infectivity models

          Two species of  Streptococcus have been used widely in bacterial
    host resistance models for immunotoxicological assessment.
     S. pneumoniae has been used primarily for evaluating systemic
    immunity.  S. zooepidemicus has also been used to evaluate systemic
    immunity but is used extensively to evaluate the effects of drugs and
    chemicals on the local immunity of the pulmonary system.

           S. pneumoniae is a Gram-positive coccus to which host
    resistance is multifaceted (Winkelstein, 1981). The first line of
    defence against this organism is the complement system. Activation of
    the complement system can result in direct lysis of certain strains of
     S. pneumoniae; however, owing to the nature of their cell wall, some
    strains are resistant to lysis by complement. Complement can still
    participate directly in the removal of these bacteria as a result of
    deposition of complement component C3 on their surface, which
    facilitates phagocytosis by polymorphonuclear leukocytes and
    macrophages. In the later stages of the infection, antigen-specific
    antibody plays a major role in controlling the infection. Thus,
    compounds that affect complement, polymorphonuclear leukocytes, B-cell
    maturation and proliferation, or the production of antibody can be
    evaluated in this system.  S. pneumoniae is an excellent model for
    evaluating immunotoxicity, since it elicits multiple immune components
    which participate in host resistance, each of which can be a potential
    target for an adverse effect of a xenobiotic. To date, this model has
    had limited success in rats.

          Preparation of  S. pneumoniaefor challenge is slightly more
    complicated than the procedures used for  Listeria; however, the
    potential of the model for detecting immunotoxic compounds makes the
    additional steps worthwhile. Stock preparations of  S. pneumoniae
    (ATCC 6314) are easily maintained at -70°C in defibrinated rabbit
    blood, and aliquots of the stock preparation can be removed and grown
    in culture at various dilutions to obtain the desired challenge
    concentration. An alternative approach is to grow the organism in
    culture and to monitor the bacterial concentrations by measuring the
    turbidity of the culture. A 5-µl aliquot of the stock preparation is
    used to inoculate 50ml of brain-heart infusion broth, which is
    incubated at 37°C, and the turbidity of the overnight culture is
    determined with an Abbott Biochromatic analyser system or another
    instrument that can sensitively measure changes in culture turbidity.
    The overnight culture is diluted with fresh brain-heart infusion broth
    to yield an absorbence difference of 0.020-0.025. The turbidity of the
    subculture is monitored periodically, and when the optimal density
    reaches an absorbence difference of 0.080, the subculture is rapidly
    cooled in an ice bath and diluted to the desired inoculum level. The
    turbidity of each inoculum is checked in the analyser, and adjustments
    are made to obtain the preselected differences in absorbence.
    Routinely, one day after the last exposure, female mice are challenged
    intraperitoneally with 0.2ml of the  S. pneumoniae inoculum. If the
    inoculum is administered intravenously, extremely high challenge
    levels must be used, which may reflect the efficiency of the
    mononuclear phagocyte system to clear and kill the organism. Three
    innocula, each at a different concentration, are prepared to give a
    range of lethality (e.g. 20, 50, and 80%), and a sample of each is
    serially diluted and placed on blood agar plates to determine the
    number of colony-forming units administered to the animals. Owing to
    the rapid onset of infection, mortality is recorded twice daily for
    seven days. In studies by White et al. (1986), when female B6C3F1 mice

    were exposed daily for 14 days to 1,2,3,6,7,8-hexachlorodibenzo-
     para-dioxin or TCDD by gavage, they were found to have decreased
    host resistance to  S. pneumoniae, which is consistent with the
    decrease in complement activity caused by these compounds.

          Another species of  Streptococcus that has been used as a host
    resistance model is  S. zooepidemicus, a group C streptococcus
    (Fugmann et al., 1983). Exposure to  N-nitrosodimethylamine was shown
    to decrease host resistance to this strain significantly. Infection
    with  S. zooepidemicus may be dependent on an antibody-mediated
    response, since the time to death after challenge is considerably
    longer than with  S. pneumoniae. Numerous studies have demonstrated
    that aerosolized  S. zooepidemicus is one of the most sensitive
    indicators of the toxicity of air pollution: Mice exposed for short
    periods to single or mixed pollutants before infection with an aerosol
    of  S. zooepidemicus and then assessed for mortality over 20 days had
    increased mortality with increasing concentrations of ozone (Coffin &
    Gardner, 1972; Ehrlich et al., 1977), nitrogen dioxide (Ehrlich &
    Henry, 1968; Sherwood et al., 1981), sulfur dioxide (Selgrade et al.,
    1989), metal particulates (Gardner et al., 1977; Adkins et al., 1979,
    1980; Aranyi et al., 1985), phosgene (Selgrade et al., 1989), and
    other volatile organic compounds (Aranyi et al., 1986). With many of
    these compounds, enhanced susceptibility to infection has been
    demonstrated at concentrations at or below the United States national
    ambient air quality standards or threshold limit values. With all of
    these compounds, enhanced mortality has been associated with failure
    to clear bacteria from the lung and suppression of alveolar macrophage
    phagocytic function. This model has recently been adapted to rats. In
    this species, both ozone (Gilmour & Selgrade, 1993) and phosgene (Yang
    et al., 1995) delayed clearance of bacteria from the lungs and
    enhanced the inflammatory response (polymorphonuclear leukocytes in
    lavage fluid) at concentrations that do not themselves produce
    inflammation; however, mortality does not occur in this species. While
    the bacteria have generally been administered as aerosols, Sherwood et
    al. (1988) showed that similar results could be obtained when they
    were administered intratracheally or intranasally. Since some strains
    of  Streptococcus are pathogenic to humans, appropriate precautions
    must be taken when using this host resistance model.  Viral infection model with mouse and rat cytomegalovirus

          Cytomegalovirus infections are widely distributed in humans, with
    about 60-90% of the population infected. Human cytomegalovirus
    infections occur in several forms, the most serious being congenital
    and perinatal infection and infection of immunosuppressed individuals.
    Less serious forms include post-perfusion syndrome and some cases of
    infectious mononucleosis; however, the vast majority of postnatal
    infections in immunocompetent individuals are clinically asymptomatic.
    More severe disease may occur in immunodeficient hosts, such as
    transplant patients (Naraqi et al., 1977; Pass et al., 1978; Marker et

    al., 1981; Rubin et al., 1981). Primarily on the basis of
    morphological considerations, cytomegaloviruses are classified as
    members of the family Herpesviridae. Because these viruses have a
    relatively protracted replication cycle, a slowly developing
    cytopathology characterized by cytomegaly, and a relatively restricted
    host range, they are grouped into the beta Herpesviridae subfamily
    (Roizman et al., 1981; Roizman, 1982). The roles of several arms of
    the immune system in resistance to cytomegalovirus have been studied
    extensively in mice. The role of humoral immunity is not well
    understood. It was suggested initially that neutralizing antibodies do
    not play a pivotal role in recovery from cytomegalovirus infection in
    mice (Osborn et al., 1968; Tonari & Minamishima, 1983); however, the
    role of antibodies in neutralization of murine cytomegalovirus and in
    antibody-dependent cell-mediated cytotoxicity is now recognized
    (Manischewitz & Quinnan, 1980; Quinnan et al., 1980; Farrell &
    Shellam, 1991). Cytomegalovirus-specific Tc cells can be detected in
    cytomegalovirus-infected mice (Ho, 1980; Quinnan et al., 1980). NK
    cell activity appeared to be the most effective, especially during the
    initial stages of infection (Bancroft et al., 1981; Selgrade et al.,
    1982; Bukowski et al., 1984). Enhanced susceptibility to infection has
    been demonstrated in mice when macrophage function was blocked by
    silica, and transfer of syngeneic adult macrophages to suckling mice
    significantly increased their resistance to mouse cytomegalovirus
    infection (Selgrade & Osborn, 1974). An inverse correlation is seen
    between the virulence of mouse cytomegalovirus and its infectivity for
    peritoneal macrophages (Inada & Mims, 1985), suggesting that
    attenuated virus may be controlled, in part, by macrophages. Since the
    rat virus acts very much like the attenuated mouse virus, macrophages
    may be even more important in rats. Macrophages may facilitate the
    generation of latent infection (Booss, 1980; Yamaguchi et al., 1988).

          Enhanced susceptibility to mouse cytomegalovirus has been
    demonstrated after treatment of mice with cyclophosphamide,
    cyclosporin A, nickel chloride, or DMBA. Treatment with benzo[ a]-
    pyrene or TCDD did not affect susceptibility to this infection
    (Selgrade et al., 1982). Enhanced susceptibility was correlated with
    chemical suppression of virus-augmented NK cell activity during the
    first week of infection. In rats, exposure to immunotoxic agents such
    as organotin compounds led to altered resistance to rat
    cytomegalovirus (Garssen et al., 1995).

          Experimentally, rodents can be inoculated intraperitoneally with
    a species-specific cytomegalovirus, and the concentration of the virus
    in tissue can be determined in a plaque-forming assay, which is a
    modification of the method described by Bruggeman et al. (1983, 1985).
    Rat embryo-cell monolayers are prepared in 24-well plates. Different
    organs (salivary gland, lung, kidney, liver, spleen), obtained at
    various times after infection, are homogenized in a tissue grinder and
    stored as 10% weight/volume samples at -135°C until use. The confluent
    monolayers are then infected with 10-fold serial dilutions of the
    organ suspensions. After centrifugation, the suspension is removed,

    and 1 or 0.6% agarose is added. After incubation at 37°C in 5% carbon
    dioxide for seven days, the cells are fixed in 3.7% formaldehyde
    solution, the agarose layer is removed, and the monolayer is stained
    with 1% aqueous methylene blue. Plaques are counted under a
    stereoscopic microscope.

          In PVG rats, cytomegalovirus is detectable eight days after
    infection, although the virus load is much higher on days 15-20. The
    viral load in the salivary gland is higher than that in other organs,
    i.e. spleen, lung, kidney, and liver. In contrast, in Lewis rats and
    BN rats the viral load in e.g. the kidney was higher than that in the
    salivary gland during the first week after infection (Bruning, 1985),
    perhaps due to a strain difference (Bruggeman et al., 1983, 1985).
    Total body irradiation of PVG rats with 60Co one day before
    infection with cytomegalovirus increased the viral load in the
    salivary gland, lung, kidney, spleen, and liver over that in
    unirradiated PVG rats; histological analysis also indicated a higher
    viral load in the salivary gland of infected rats. The mucosal
    epithelium of the salivary gland contains enlarged cells with nuclear
    inclusion bodies; these could be detected in irradiated, infected rats
    only if the salivary gland was dissected and fixed 15 days after
    infection. These results are in agreement with those of Bruggeman et
    al. (1983), who found that gamma irradiation also induced higher viral
    loads in the salivary gland of BN rats. Taken together these results
    indicate a role for cellular immunity in resistance to this virus in
    rats.  Influenza virus model

          Influenza virus A2/Taiwan H2N2 has been used as a viral
    challenge in evaluating alterations in host resistance of mice after
    exposure to various compounds. Compounds that decrease host resistance
    to the virus are  N-nitrosodimethylamine (Thomas et al., 1985b) and
    TCDD (House et al., 1990a). Compounds that do not to alter host
    resistance to this pathogen include ozone (Selgrade et al., 1988),
    benzo[ a]pyrene, benzo[ e]pyrene (Munson & White, 1990), methyl
    isocyanate (Luster et al., 1986), and Pyrexol (House et al., 1990b).
    Mortality is the end-point routinely measured in evaluating decreased
    host resistance to influenza virus, which is usually instilled
    intranasally (Fenters et al., 1979). Host resistance to this virus has
    been reported to be mediated by cell-mediated immunity (Ada et al.,
    1981), interferon (Hoshino et al., 1983), and antibody (Vireligier,
    1975). This model had been suggested for use in evaluating compounds
    that affect humoral immunity; however, its inability to detect such
    compounds indicates that it is not suitable. A possible explanation
    for the discrepancy is that administration of the virus by intranasal
    instillation may invoke local immune mechanisms in the lung and may
    not adequately reflect systemic immunocompetence. In several cases,
    enhanced mortality has been demonstrated in the absence of effects on

    viral titres in the lung (Selgrade et al., 1988; Burleson et al., in
    press), indicating that enhanced mortality does not always reflect
    effects on virus-specific immune defences.

          Influenza virus has been used in evaluating immunotoxicity in
    rats, after adaptation. Studies by Ehrlich & Burleson (1991) showed
    that rats exposed to phosgene had significantly decreased host
    resistance. TCDD was shown to affect the resistance of rats to the
    adapted influenza virus RAIV (Yang et al., 1994).

          As influenza virus is a human pathogen, appropriate precautions
    must be taken.  Parasitic infection model with Trichinella spiralis

          Resistance to infection with the helminth  T. spiralis has been
    evaluated in both mice and rats after exposure to a variety of
    chemicals. In humans, as in other carnivores, infection occurs by
    eating meat containing infectious larvae. The life cycle of the worm
    is as follows: Infectious larvae excyst in the acid-pepsin environment
    of the stomach, rapidly migrate to the jejunum, and penetrate host
    intestinal epithelial cells. Sexually mature parasites are present
    within three to four days after infection. The viviparous females
    produce larvae that migrate via the lymphatic and blood vessels to
    host muscle, where they encyst and are encapsulated within a host-
    derived structure. Encapsulated muscle larvae can survive for years
    within this structure.

          An intense inflammatory response, comprised mainly of mast cells
    and eosinophils, accompanies intracellular infection in the intestine.
    T Cell-dependent immunity plays a crucial role in this inflammatory
    response (Manson-Smith et al., 1979; Vos et al., 1983b; Wakelin,
    1993), which is responsible for the expulsion of adult parasites.
    Antibodies damage the reproductive structures of the female parasite
    (Love et al., 1976), have a major role in the rapid elimination of
    subsequent infections in rats (Appleton & McGregor, 1984), and
    sensitize migrating newborn larvae for destruction by granulocytes
    (Ruitenberg et al., 1983).

          The number of encysted muscle larvae is typically much higher in
    immunosuppressed animals than in immunocompetent animals, due perhaps
    to delayed expulsion of adult worms from the intestine, decreased host
    control of parasite fecundity, decreased destruction of migrating
    larvae, or a combination of resistance defects. These end-points of
    host resistance to  T. spiralis infection and class-specific antibody
    titres can be measured by standard techniques (Van Loveren et al.,
    1994). Histological evaluation of the inflammatory infiltrate
    surrounding encysted muscle larvae has also been described (Van
    Loveren et al., 1993b).

          It should be noted that direct effects of the chemical under
    study can affect the outcome of infection. For example Bolas-Fernandez
    et al. (1988) determined that cyclosporin A delays expulsion of adult
    parasites from the intestines of rats but does not increase the number
    of larvae encysted in host muscle. This was determined to be a direct
    effect of cyclosporin A on the fecundity of female parasites rather
    than on immunity to infection. Animals are infected by oral gavage
    with known numbers of larvae, isolated from infected donor muscle.
    Because infection is spread only by consumption of infected meat or
    freshly isolated larvae, there is little danger of the infection
    spreading to other animals housed in the same room.  T. spiralis is a
    human pathogen and must be handled as such; normal laboratory
    practices are sufficient to prevent accidental infection.

           T. spiralis infection has been used as a host resistance model
    in both rats and mice. In general, chemicals that suppress T-cell
    function suppress resistance to  T. spiralis infection. Thus, TBTO
    (Vos et al., 1990b), diethylstilbestrol (Luebke et al., 1984), TCDD
    (Luebke et al., 1994, 1995), and the virustatic agent acyclovir
    (Stahlmann et al., 1992) had deleterious effects on resistance.  Plasmodium model

          Two strains of  Plasmodium have been used to evaluate the
    potential immunotoxicity of compounds.  P. yoelii (17XNL) is a
    nonlethal strain that produces a self-limiting parasitaemia in mice.
    Resistance to this organism is multifaceted and includes specific
    antibody, macrophage involvement, and T cell-mediated functions
    (Luster et al., 1986). In this assay, animals are injected with 106
    parasitized erythrocytes, and the degree of parasitaemia is monitored
    over the course of the infection by taking blood samples. In control
    animals, the peak response usually occurs 10-14 days after injection.
    The degree of parasitaemia can be evaluated by a variety of methods,
    e.g. manually, by counting parasitized erythrocytes in blood smears
    (Luebke et al., 1991). Host resistance to  P. yoelii has been used to
    assess the immunotoxicity of benzidine (Luster et al., 1985b),
    diphenylhydantoin (Tucker et al., 1985), TCDD (Tucker et al., 1986),
    pyran copolymer (Krishna et al., 1989), gallium arsenide (Sikorski et
    al., 1989), and 2'-deoxycoformycin (Luebke et al., 1991).

           P. berghei is lethal to mice and certain strains of rats and
    has been used in assessing immunotoxicity (Loose et al., 1978). Host
    resistance depends on specific antibody production and ingestion and
    destruction of antibody-coated  Plasmodium by phagocytic cells such
    as macrophages. T Lymphocytes may also be involved in host resistance
    to the organism (Bradley & Morahan, 1982). Mortality has been
    evaluated after injection of 106  Plasmodium-infected erythrocytes.
    Compounds that have been evaluated for immunotoxicity in this model
    system include 4,4'-thiobis(6- tert-butyl- meta-cresol) (Holsapple
    et al., 1988), dietary fish-oil supplement (Blok et al., 1992), and

    styrene (Dogra et al., 1992). Neither  P. yoelii nor  P. berghei is
    infectious in humans; infection of animals can occur only through
    parenteral injection of contaminated blood.  B16F10 Melanoma model

          The B16F10 tumour cell line is a malignant melanoma that is
    syngeneic with the C57Bl/6 mouse, which is one of the parents of the
    B6C3F1 mouse. This tumour line was selected for its propensity to
    metastasize to the lung. The assay is an outgrowth of the work of
    Fidler and colleagues (Fidler, 1973; Fidler et al., 1978). NK cells
    and macrophages have been proposed to be involved in host resistance
    to this metastasizing tumour; however, T lymphocytes have also been
    shown to play a role (Parhar & Lala, 1987). This host resistance assay
    is referred to as an artificial metastasis model, since the tumour
    cells are administered by intravenous injection, usually into the tail
    vein, and lodge in the lung, which is the first capillary bed they
    encounter. The B16F10 tumour cells can be stored frozen and can easily
    be grown in culture before use. Routinely, 1-5 × 105 cells are
    injected intravenously into sentinel mice (i.e. untreated mice
    injected with the highest challenge level of tumour cells), and the
    tumour burden is monitored in order to select the optimal day of

          Two parameters are routinely used to assess tumour burden. One is
    DNA synthesis in the lungs of mice bearing tumours. Since background
    DNA synthesis in the lungs of mice without tumours is extremely low,
    any detectable rate is a result of the presence of a tumour. In order
    to measure synthesis, mice are pulsed intraperitoneally one day before
    sacrifice with 0.2 ml of 10-6 mol/litre of 5-fluorodeoxyuridine,
    followed 30 min later by 2 µCi of 125I-iododeoxyuridine administered
    by the intravenous route. After sacrifice, the lungs are removed,
    placed in Bouin's fixative solution, and counted with a gamma counter.
    A second indicator of tumour burden is visual enumeration of tumour
    nodules after fixation in Bouin's solution. The visibility of the
    black nodules of the melanin-producing B16F10 tumour cells on the
    yellow background of the fixed lung tissue allows enumeration of up to
    200-250 nodules on the surface of the lungs. A good correlation has
    been shown between number of tumour nodules and radioactivity present
    in the lungs (White, 1992). Thus, if the tumour nodules become too
    numerous to count, the results of the study can still be determined
    from the radioassay. This system has been useful in demonstrating
    decreased host resistance after systemic exposure to the tumour
    promoter phorbol myristate acetate (Murray et al., 1985),
    intratracheal exposure to gallium arsenide (Sikorski et al., 1989),
    and exposure to nickel chloride (Smialowicz et al., 1985b); it has
    also been used to show enhanced host resistance after exposure to
    manganese chloride (Smialowicz et al., 1984) and 4,4'-thiobis(6- tert-
    butyl- meta-cresol) (Holsapple et al., 1988).  PYB6 Carcinoma model

          The PYB6 tumour cell line is a fibrosarcoma originally induced
    with a polyoma virus in C57Bl/6 mice. Host resistance to the tumour
    includes NK cell activity and T cell-mediated killing (Urban et al.,
    1982). While PYB6 cells can easily be grown in culture, they should be
    passed through an animal before use in challenge studies for
    immunotoxicity (Luster et al., 1988). In studies with the PYB6 line,
    mice are injected in the thigh with 1-5 × 103 viable tumour cells
    and are then palpated weekly to detect the development of tumours at
    the injection site. The end-points evaluated include the incidence of
    tumours and time to tumour appearance; tumour size can also be
    measured. This assay has been useful in detecting decreased host
    resistance to many compounds, including Aroclor 1254 (Lubet et al.,
    1986), DMBA (Dean et al., 1986), and benzene (Rosenthal & Snyder,
    1987).  MADB106 Adenocarcinoma model

          A tumour model used to evaluate host resistance in rats is the
    MADB106 rat mammary adenocarcinoma, which is syngeneic with the
    Fischer 344 rat. NK cells appear to play a major role in host defence
    to this tumour (Barlozzari et al., 1985). In this model, survival time
    after injection of the cells is the usual end-point monitored.
    Compounds that decrease host resistance to the tumour can decrease
    both the percentage survival and the survival time of treated animals.
    Control rats begin to succumb to the adenocarcinoma two to three weeks
    after an intravenous injection of 2 × 106 tumour cells. Smialowicz
    et al. (1985b) showed a significant decrease in the survival of
    animals treated with a single intramuscular dose of nickel chloride,
    which was correlated with a decrease in NK cell activity.

    4.2.11  Autoimmune models

          Autoimmune models can also be used to investigate whether a
    compound exacerbates induced or genetically predisposed auto-immunity.
    These models are used mainly to elucidate the pathogenesis of
    autoimmunity and the effect of immunosuppression in
    immunopharmacology. Few studies have been reported, although the
    relevance of the model for extrapolation to humans may be good. A
    number of autoimmune models are available in rats and mice. Autoimmune
    phenomena can be either induced or occur spontaneously. In induced
    models, an autoantigen is isolated from a target organ obtained from
    another species (generally bovine), and the animal is immunized with
    this purified antigen in adjuvant. Examples in the rat (Calder &
    Lightman, 1992) are experimental encephalomyelitis elicited by bovine
    spinal cord antigen (Stanley & Pender, 1991), experimental uveitis
    elicited by bovine retinal S-antigen (Fox et al., 1987), and adjuvant
    arthritis elicited by  Mycobacterium containing H37RA adjuvant
    (adjuvant arthritis) or collagen (Holmdahl et al., 1990; Klareskog &
    Olsson, 1990; Wooley, 1991). Autoimmune phenomena and associated organ

    pathology normally emerge in almost all immunized animals within two
    to three weeks. Depending on the effector reaction and the
    reversibility of the damage, the disease either stops when the damage
    is complete (e.g. uveitis, resulting in blindness of the animal) or
    the autoimmune reaction is transient and animals recover (adjuvant
    arthritis). In some models, animals subsequently experience a relapse
    around day 30 (experimental encephalomyelitis). The induction and
    development of autoimmunity in these animals are mediated by T cells
    that show the cytokine expression pattern (IL-2, INF gamma) of the Th1
    subset. The effector phase of disease symptoms is also mainly a T
    cell-mediated process, to which CD4+ cells, CD8+ cells, and
    macrophages contribute. Lewis (RT11) rats are particularly
    susceptible. These autoimmune models can be induced in other species,
    such as mice (Baker et al., 1990) and rhesus monkeys (Rose et al.,
    1991). They are generally accepted as models of human (organ-specific)
    autoimmune diseases, e.g. experimental allergic encephalomyelitis as a
    model of multiple sclerosis, experimental allergic uveitis as a model
    of idiopathic posterior uveitis, and adjuvant arthritis as a model of
    rheumatoid arthritis.

          Autoimmunity can also be induced by metals (Druet et al., 1989;
    Bigazzi, 1992). A well-known example is glomerulopathy induced by
    mercuric chloride in BN (RT1n) rats. The process is initiated by T
    cells with a cytokine synthesis pattern (IL-4) of the Th2 subset. The
    relative incidence of these cells is much higher in BB rats than in
    other strains. After the cells of the Th2 subset have been stimulated,
    there is polyclonal stimulation of B lymphocytes, leading to synthesis
    of antibodies (including pathogenic antibodies) to the glomerular
    basement membrane. These antibodies subsequently mediate autoimmune
    destruction of renal glomeruli. This model is the best studied model
    of 'drug'-induced autoimmunity. Mercuric chloride elicits
    glomerulonephritis in other rat strains, in which glomerular
    destruction is not due to anti-glomerular autoantibodies but is
    mediated by immune complexes deposited in the glomerulus (Druet et
    al., 1989).

          In spontaneous models, predisposition to the development of
    autoimmune phenomena and disease is determined by the genetic
    composition of the animal strain. Well-known examples are BB rats
    (Like et al., 1982; Guberski, 1994) and NOD mice (Lampeter et al.,
    1989; Leiter, 1993), which develop autoimmune pancreatitis and
    subsequently diabetes. Within the pancreas, the islets of Langerhans
    are infiltrated by T lymphocytes and macrophages; subsequent
    destruction of the islets results in diabetes. These spontaneous
    models are considered to be animal models of human diabetes (Dotta &
    Eisenbarth, 1989; Lampeter et al., 1989; Riley, 1989). Other examples
    are the systemic autoimmunity that emerges in certain mouse strains
    (Guttierez-Ramos et al., 1990) like (NZB × NZW)F1 mice (Theofilopoulos
    & Dixon, 1985) and the mixed lymphocyte reaction in  lpr (Matsuzawa
    et al., 1990) and  gld mice (Roths et al., 1984). The spontaneous
    pathology in these animals resembles various disease manifestations in

    human systemic lupus erythematosus and is similarly mediated by immune
    complexes that deposit in tissue. In (NZB × NZW)F1 mice, mainly lupus
    nephritis is induced by immune complexes; in the mixed lymphocyte
    reaction in  lpr mice, both joint manifestations and
    glomerulonephritis are seen. The genes associated with autoimmune and
    immune complex disease are not known. In comparison with induced
    models, these models have the advantage of spontaneous, gradual
    development of autoimmune disease symptoms; however, this is a
    disadvantage in experimental design, as not all animals develop
    disease, and emerging disease develops at different times or ages.

          In general, treatment of animals with immunosuppressive drugs
    that interfere with signal transduction (cyclosporin, FK-506,
    rapamycin) or cell proliferation (cytostatics like azathioprine,
    mizoribine, and brequinar), and anti-inflammatory agents
    (corticosteroids) inhibit the development of symptoms in these models.
    Exposure to immunotoxic chemicals may also lead to alterations in the
    course of disease emergence. For instance, HCB, which leads to
    immunoenhancement in rats, markedly enhanced the severity of allergic
    encephalomyelitis (Van Loveren at al., 1990c). In contrast, arthritic
    lesions were strongly suppressed in HCB-exposed Lewis rats, indicating
    that HCB has biologically significant immunotoxic effects. Although
    the contrasting effects in the two autoimmune models are not yet
    understood, and clear dose-effect relationships have yet to be
    established, this type of information should be obtained for risk

    4.3  Assessment of immunotoxicity in non-rodent species

          While most immunotoxicological evaluations are conducted in mice
    or rats, use of other species is increasing.

    4.3.1  Non-human primates

          Various non-human primates, including  Macaca mulatta (rhesus
    macaques),  M. nemestrina (pig-tailed macaques),  Cercocebus atys
    (sooty mangabeys),  M. fasicularis (cynomolgus monkeys), and
    marmosets have been used in immunotoxicological studies. Many of the
    assays carried out in mice or rats can be adapted for use with non-
    human primates. Strategies and methods used in studies of humans have
    also been introduced in studies on non-human primates. Monoclonal
    antibodies generated to human leukocyte subsets can be used in
    phenotyping blood mononuclear cells of e.g. marmoset monkeys
     (Callithrix jacchus) (Neubert et al., 1990, 1991), although the
    possibility of such use differs, depending on the evolutionary
    distance of the non-human primate from humans. While most of the
    assays conducted in non-human primates involve serum or peripheral
    blood, some assays, such as those used to measure delayed-type
    hypersensitivity, are holistic, in that the animals are sensitized
     in vivo and then evaluated  in vivo at the challenge site (Bugelski
    et al., 1990; Bleavins & Alvey, 1991).

          The effects of chronic exposure to the PCB Arochlor 1254 on the
    immune response of rhesus monkeys have been evaluated by Tryphonas et
    al. (1989). In these studies, the lymphocyte response to concanavalin
    A and phytohaemagglutinin was evaluated, as were total serum
    immunoglobulin levels, antibodies to sheep red blood cells, and
    numbers of T and B cells in peripheral blood. In later studies
    (Tryphonas et al., 1991a,b), one-way mixed lymphocyte cultures,
    antibodies to pneumococcal antigens, phagocytic mononuclear cell
    function, NK function, haemolytic complement activity, and production
    of IL-1, tumour necrosis factor, thymosins, and interferon were
    evaluated in monkeys exposed to Arocolor 1254. Ahmed-Ansari et al.
    (1989) evaluated phenotypic markers and function in three species of
    non-human primate. The functional assays included NK cell activity,
    lymphocyte transformation, and antigen presentation. Extensive studies
    included the evaluation of more than 20 phenotypic markers or
    combinations of markers for each of the three monkey species. Use of
    the monkey as a test species is likely to increase as more and more
    biotechnology and recombinant products are produced.

    4.3.2  Dogs

          While dogs are not the species of choice for immunotoxicological
    studies, they are used predominantly in assessing toxicological
    safety, and virtually all of the assays used for assessing immunotoxic
    potential have been adapted for use in dogs. These include evaluation
    of basal levels of IgA, IgG, and IgM (Glickman et al., 1988),
    allergen-specific serum IgE (Kleinbeck et al., 1989), mononuclear
    phagocyte function (Thiem et al., 1988), NK cell activity (Raskin et
    al., 1989), Tc cell activity (Holmes et al., 1989), and mitogen-and
    cell-mediated immune responses (Nimmo Wilkie et al., 1992).

    4.3.3  Non-mammalian species

          Non-mammalian species are also used extensively for evaluating
    the potential adverse effects of compounds and agents on the immune
    system.  Fish

          Because of their environment, fish are an excellent model for
    studying the effects of water-and sediment-borne pollutants. There are
    several other good reasons for studying immunotoxicity in fish: many
    of their diseases are related to environmental quality, various
    environmental pollutants have immunotoxic potential, and many of the
    diseases have an immune component. Moreover, there is concern about
    the health status of aquatic ecosystems in relation to pollution, and
    fish will be useful target species for developing biomarkers (see
    box). Fish are easy to obtain, there is an extensive body of
    knowledge, and their economic interest (aquaculture) facilitates the
    finding of research resources. At present, immunotoxicology in fish is

    not as sophisticated as that in mammals. Screening and functional
    tests are being developed in the laboratory but cannot yet be applied
    in the field.

          A wide range of species is used for field and laboratory studies.
    The choice of species depends on its biology (migratory or local,
    marine or freshwater; sediment-dwelling or pelagic) and on experience
    in the laboratory. A lack of consistency, e.g. becuse of a limited
    number of species (as in mammalian immunotoxicology) makes this field
    of research diffuse and extended and may result in limited progress;
    nevertheless, a variable or consistent effect over a variety of
    species is certainly a valuable observation. Some species seem to be
    preferred, such as trout, salmon, and carp, which are practical, owing
    to their size, for sampling blood and tissues for laboratory studies.
    Smaller species, such as guppies  (Poecilia reticulata) and medaka
     (Oryzias latipes), have secured a niche in aquatic toxicology owing
    to the ease of husbandry and relatively low cost; moreover, because of
    their small size, whole animals can be used for histopathological
    examination (Wester & Canton, 1991), but their application in
    immunotoxicology may be limited because of difficulty in obtaining
    adequate blood and tissue samples. For studies of saltwater species,
    bottom-dwelling flatfish are commonly used in field studies and, to a
    certain extent, in studies of mesocosms and in the laboratory. In
    Europe, the flounder  (Platichthys flesus) and dab  (Limanda limanda)
    are popular target species since they are susceptible to certain
    recognizable diseases and are commonly available.

          A compehensive variety of parameters is listed by Anderson (1990)
    and Weeks et al. (1992). A modified set based on those lists and
    assays used in rodent immunotoxicology is presented in the box above
    and classified as tier 1 (screening tests) and tier 2 (functional
    assays). Parameters commonly mentioned in the literature are discussed

           Blood cell counts and differential counts: As leukocytes play a
    major role in specific and nonspecific humoral and cellular immune
    responses, this parameter is used as a measure of the status of the
    defence system, in particular in tier 1 testing. It is relatively easy
    to test blood samples drawn from live animals, but many environmental
    factors unrelated to defence may modify leukocyte status (Anderson,
    1990). The use of monoclonal antibodies directed against individual
    cell types may improve their identification (Bly et al., 1990; Van
    Diepen et al., 1991). Another possible parameter is the haematocrit;
    however, it has no known specificity for any immune function, although
    it may be considered as a general indicator of stress.


    Candidate biomarkers for immunotoxicity in fish

    Tier 1: Screening tests

    *     Conventional haematology, including total and differential blood
          cell counts, surface markers (flow cytometry), and macrophage
          density and morphology: easy, nonspecific

    *     Serum immunoglobulin concentrations in naive (unstimulated) fish:
          easy, limited specificity

    *     Lymphoid organ weight (mainly spleen, occasionally thymus):

    *     Histopathology of the thymus, spleen, and kidney: possible, can
          be specific

    Tier 2: Functional assays

    *     Humoral immune response (agglutination, enzyme-linked
          immunosorbent assay): possible, can be specific

    *     Cellular immune response (allograft rejection in scale, skin, or
          eye): possible, can be specific

    *     Macrophage functions (phagocytosis, bacterial killing, migration,
          chemiluminescence): limited specificity

    *     Host resistance (bacterial infections): possible, can be
          specific, relevant
           Nonspecific defence: Other indicators of nonspecific defence
    have been proposed as indicators of immunological stress. These
    include acute-phase proteins (Fletcher, 1986), the levels of which
    appear to be stress hormone-dependent; and lysozyme and ceruloplasmin
    activity, which are reduced in carp exposed to trichlorphon  in vivo
    (Siwicki et al., 1990).

           Morphology: The spleen is easy to excise and weigh in animals
    of adequate size and could thus serve as a biomarker, although it is
    not commonly reported in the literature. One reason may be that a
    major and variable portion of the spleen consists of storage blood or
    erythropoietic tissue (Fänge & Nilsson, 1985); the lymphoid tissue is
    poorly developed and is mainly associated with melanomacrophage
    centres (Zapata, 1982, 1983; Fänge & Nilsson, 1985; Van Muiswinkel et
    al., 1991); and, after immunization, only a small proportion of the
    plaque-forming cells is found in the spleen, in contrast to the kidney

    pronephros (Van Muiswinkel et al., 1991). The role of the spleen in
    most fish species thus seems to be limited, although melanomacrophage
    centres are abundant.

          Experimental immunotoxicology in mammals has demonstrated that
    the weight of the thymus, a primary and exclusively immunological
    organ, is a sensitive indicator of thymic effects. This parameter is
    not commonly used in fish. One reason is that the thymus has a complex
    location in some species, which makes clean dissection nearly
    impossible. Other reasons are inconsistencies in thymic morphology,
    histopathology, and morphometry (Ghoneum et al., 1986; Wester &
    Canton, 1987). The latter paper described studies in which guppies
    exposed to TBTO showed dose-dependent atrophy of the thymus, as seen
    in rats (Krajnc et al., 1984). Both species also showed a concomitant
    increase in 'neutrophils', which suggests functional compensation.
    This response could not be reproduced in medaka (Wester et al., 1990),
    stickleback, or flounder (P. Wester, unpublished results), probably
    indicating species specificity. Thymic lymphocyte function can also be
    tested  in vitro.

           Macrophage function tests: Macrophages are an important cell
    population for both specific (antigen processing and presentation) and
    nonspecific (phagocytosis and destruction) defence. They are
    considered to be a relatively primitive defence mechanism and are
    therefore of major importance to lower animals (Ratcliffe & Rowly,
    1981). Much effort has been devoted to establishing macrophage
    parameters as biomarkers for immune effects in fish; a possible reason
    for this preference is the fact that these cells are fairly easy to
    obtain, e.g. by peritoneal washing or removal of kidney pronephros,
    and many function tests do not require sophisticated techniques or
    species-specific reagents or markers (Mathews et al., 1990). The tests
    include determination of chemotaxis, phagocytosis, pinocytosis, and
    chemiluminescence. Zelikoff et al. (1991) studied the applicability of
    trout peritoneal macrophages for immunotoxicology, stressing the need
    for systematic baseline information. In addition to the tests listed
    above, they studied the morphology and spread of resident and
    stimulated peritoneal macrophages and concluded that these cells share
    many morphological and functional properties with their mammalian
    counterparts and may thus be useful indicators in immunotoxicology.
    Many case studies in fish species have been published, demonstrating
    the sensitivity of one or more parameters to chemicals, including PAHs
    (Weeks & Warinner, 1986; Zelikoff et al., 1991) and pentachlorophenol,
     in vitro (Anderson & Brubacher, 1993).

           Melanomacrophage centres: Melanomacrophage centres, or
    macrophage aggregates, are widely distributed throughout the fish
    body, in particular in spleen, liver, and kidney. They are composed of
    clusters of swollen, rounded cells (macrophages) that stain pale-tan
    to black. This parameter must be determined histopathologically. Their
    occurrence and morphology have been described (Agius, 1985), but their

    function is not yet fully understood. The presence of pigments
    (haemosiderin, lipofuscin, and ceroid) indicates storage of effete
    biological material (erythrocytes, biomembranes) (Wolke, 1992). The
    melanin present may be a generator of the bactericidal hydrogen
    peroxide (Roberts, 1975), and the presence of antigens indicates a
    role in immune reactions, e.g. antigen presentation. An increase in
    melanomacrophage centres can be found with age and after stress
    (Blazer et al., 1987), as confirmed in field studies (Vethaak &
    Wester, 1993). Moreover, a large number of relatively small, pale
    centres was seen in animals caught in late winter, when conditions are
    more stressful, including spawning with associated migration and
    starvation (Vethaak & Wester, 1993). The presumption that the small
    size and pale appearance are indicators of recent development is
    supported by the observation that in liver tumours composed of
    relatively young, fast-growing tissue melanomacrophage centres are
    usually absent or definitely smaller. As a consequence, when these
    centres are used as general parameters of stress, the study groups
    must be matched for age.

          Because they are characteristic for fish and because of the
    multiplicity of their functions, these structures deserve special
    attention in the context of defining biomarkers for immunotoxicity. In
    addition, they are easy to monitor, since they do not require special
    preparation other than routine histological procedures, including
    morphometry. Since melanomacrophage centres can be considered
    primitive analogues of the mammalian lymph follicle (Payne & Fancey,
    1989), it has been suggested that their presence indicates immune
    capacity or function, although their role in this context has not yet
    been established and the implications of a change in this parameter
    for the integrity of the defence systems remains unclear. The density
    of melanomacrophage centres in liver or spleen has been successfully
    correlated with environmental sediment (Payne & Fancey, 1989) and
    along a gradient of pollution in the North Sea (Bucke et al., 1992).
    Other studies have reported an increase in melanomacrophage centre
    density after contact with chemical contaminants (Blazer et al., 1987;
    Secombes et al., 1992), which may indicate accumulation of cytotoxic
    waste or immune stimulation.

          At present, macrophage function and melanomacrophage centres are
    the most widely used and promising indicators of the effects of
    environmental stress (Blazer et al., 1987). Their relationship to
    other components of the immune system remains to be clarified,
    however, in tests with immunotoxicants.

           Humoral immune response: Determination of circulating
    immunoglobulin levels in serum is useful for testing the net result on
    an immunological pathway  in vivo. The response can be measured in
    'naive' animals (total immunoglobulin) or after exposure to an
    antigen, e.g. to verify the efficacy of vaccination in aquaculture.
    Sheep red blood cells can be used as a standard antigen, and the
    immune response can be measured by agglutination tests. ELISA tests,

    which are sensitive and specific, can also be used (Arkoosh &
    Kaattari, 1990). A related test is the haemolytic plaque assay which
    identifies antibody-producing cells (splenic lymphocytes) (Anderson,
    1990), but which has been used to only a limited extent in fish.

           Specific lymphocyte stimulation tests: Functional tests widely
    used in mammalian immunotoxicology, in which lymphocytes are
    stimulated  in vitro by exposure to mitogens such as
    lipopolysaccharide, phytohaemagglutinin, and concanavalin A, can also
    be used in fish. Proliferation is monitored by measuring the
    incorporation of 3H-thymidine into DNA. The test is not antigen-
    specific but provides information on the capacity of the entire B
    (lipopolysaccharide) or T (phytohaemagglutinin, concanavalin A) cell
    population. It is used to only a limited extent in fish
    immunotoxicology, although Faisal & Hugget (1993) gave an elegant
    demonstration of significant suppression of this parameter in spot
     (Leiostomus xanthurus) under field conditions; this was shown to be
    related to site and pollution in controlled laboratory experiments.

           Specific cellular immune responses: Tests described in the
    literature to measure cellular immune responses are scale or skin
    allograft rejection, a relatively simple test (Zeeman & Brindley,
    1981), and eye allograft rejection (Khangarot & Tripathi, 1991);
    delayed rejection was seen in carp after exposure to copper. These
    tests are applied to only a limited extent.

          The tests described above are mainly tier 2 tests. The tests most
    often used in immunotoxicology, however, are those for host resistance
    (challenge by infections or tumours). The results of such tests are
    rarely reported in the literature and have not been validated. For
    ultimate proof of immunotoxicity, all phases of a test (maintainence,
    exposure, and infection) must be conducted under strictly controlled
    laboratory conditions. When suitable (often species-specific)
    pathogens are standardized, such tests are valuable and necessary for
    estimating the practical consequences of suspected immunotoxicity.
    Although the incentive for undertaking immunotoxicological studies in
    fish is usually epidemiological observation of a suspected toxic
    component, ultimate challenge experiments must be carried out before a
    final conclusion about immunotoxic mechanisms can be drawn.

          More emphasis has been given to the development of biomarkers
    than to their application in the field, for several reasons, including
    the lack of specificity and the lack of association between effects at
    the level of the biomarker and the population (Mayer et al., 1992).
    Some comments on and some needs in this field are as follows:

    *     Immunological biomarkers in fish have great potential: many have
          not yet been fully explored, probably owing to practical
          limitations of lack of specificity and predictivity.

    *     The number of animal species should be limited in order to
          concentrate research, which often requires species-specific
          knowledge and reagents. Standardization could be achieved by
          choosing well-defined inbred strains of fish (e.g. carp or

    *     A tiered approach is highly recommended for obtaining knowledge
          on the specificity of the biomarker.

    *     More knowledge is needed on the epidemiology, mechanisms, and
          etiology of diseases in fish, and particularly the predictive
          value of immune parameters and the influence of hormesis.

    *     In terms of relevance for the organism, a test that monitors the
          net result of a cascade of reactions (e.g. specific antibody
          production, host resistance) is more predictive than a single,
          nonspecific cell parameter (e.g. macrophage activity  in

    *     In identifying potential biomarkers for immunotoxicity, evidence
          should be available that the levels tested in the laboratory are
          relevant for field conditions and that the effect is directly
          related to the immune system.  Chickens

          Another non-mammalian species that has been studied extensively
    with regard to the structure and function of its immune response is
    the chicken. It is therefore not surprising that the chicken has
    emerged as the predominant avian model for assessing compounds for
    potential immunotoxicity. Humoral responses to different antigens have
    been assessed routinely (Lerman & Weidanz, 1970; Marsh et al., 1981).
    The weights of the thymus, spleen, and bursa of Fabricius have been
    used, in combination with decreased antibody responses  in vivo and
    lymphocyte responses to phytohaemagglutinin and concanavalin A
     in vitro (Eskola & Toivanen, 1974). Graft-versus-host and
    cutaneous basophil hypersensitivity have also been used to detect
    immunosuppression in chickens (Dietert et al., 1985). The availability
    of chicken cell lines (Sung et al., 1992) will facilitate studies of
    the mechanisms of action of compounds on the immune responses of this
    avian model.

    4.4  Approaches to assessing immunosuppression in vitro

          The complexity of the immune system and the requirement of many
    agents for metabolism and distribution to produce an immunotoxic
    response has resulted in the almost exclusive use of animal models

     in vivo for immunotoxicity assessment. Culture systems have been
    used extensively, however, to study the mechanisms by which agents
    produce immunosuppression.

          Since most of the assays used for assessing immunotoxicity are
    ex-vivo/in-vitro tests, they are easily adapted to completely in-vitro
    assays for assessing immunosuppression. The direct addition of
    compounds in various assays, including those involving NK cells,
    lymphocyte proliferation, mixed leukocytes, and Tc lymphocytes, has
    been used to determine the mechanisms by which compounds alter the
    immune response at the cellular and subcellular level. Similarly, the
    action of benzene and its metabolites on bone marrow has been studied
    extensively  in vitro (Gaido & Wierda, 1987), and the effects of TCDD
    on thymocytes have been well studied in thymic epithelium co-cultures
    (Greenlee et al, 1985). One of the most useful in-vitro assays for
    studying immunosuppression is the T-dependent antibody response to
    sheep erythrocytes. This assay, also known as the Mishell-Dutton assay
    (Mishell & Dutton, 1967), has been used extensively in studying the
    cellular target of immunotoxicants. It is the in-vitro counterpart of
    the in-vivo plaque-forming cell assay, but sensitization with sheep
    erythrocytes takes place in splenic cell culture and the plaque
    response is measured on day 5 after addition of the erythrocytes. The
    Mishell-Dutton assay has been used to study the structure - activity
    relationships of various immunosuppressive compounds (Kawabata &
    White, 1987; Davis & Safe, 1991). Since T cells, B cells, and
    macrophages are needed for the response and an adverse effect on any
    of these cell types can produce immunomodulation, it has proved
    to be a sensitive assay for evaluating compounds  in vitro for
    immunosuppressive activity. Furthermore, since the various cell types
    that participate in the response can easily be separated, individually
    treated, and then reconstituted in the culture system, it is an
    excellent assay for determining which cell type is adversely affected
    by the compound. Using this approach, White & Munson (1986)
    demonstrated that asbestos suppresses the response by affecting
    macrophages; Shopp & Munson (1985) showed that the primary action of
    phorbol ester on the antibody response occurs through an effect on B
    cells; and Johnson et al. (1987) found that  N-nitrosodimethylamine
    affects primarily B cells.

          As indicated above, one of the limitations of in-vitro systems is
    that exogenous metabolic activation systems are often required. While
    lymphocytes can metabolize some compounds, such as benzo[ a]pyrene,
    to active metabolites (Ladics et al., 1992), other potent
    immunosuppressive compounds such as cyclophosphamide require a
    metabolic activation system. Such preparations usually consist of a
    9000 x g supernatant of liver (S9). Using S9 preparations, Tucker &
    Munson (1981) showed that cyclophosphamide could be activated to an
    immunosuppressive form  in vitro. Similarly, naphthalene could be
    metabolized to an immunosuppressive metabolite (Kawabata & White,
    1990). An alternative approach to the S9 activation system is a

    hepatocyte co-culture system, which has been shown to be capable of
    activating several parent compounds to their immunosuppressive
    metabolites (Yang et al., 1986).

          Predictive in-vitro systems based on immune cells of human origin
    are particularly attractive, given the uncertainties of extrapolating
    the results of experimental studies to humans and the accessibility of
    immune cells in human peripheral blood. Although many of the immune
    cells obtained from human blood are immature forms, the large numbers
    and diverse populations (i.e. polymorphonuclear leukocytes, monocytes,
    NK cells, T cells, and B cells) that can be obtained provide an
    attractive alternative or adjunct to conventional studies in
    experimental animals. As a consequence, a number of studies have been
    conducted to compare the functional response of human and rodent
    lymphocytes to putative immunosuppressive agents  in vitro (Cornacoff
    et al., 1988; Luo et al., 1992; Wood et al., 1992; Lang et al., 1993).
    Although these studies were hampered by the lack of assays to assess
    primary antigen-specific immune responses in human lymphocytes, a
    relatively good interspecies correlation has been observed in the
    limited responses available. Furthermore, several of these assays have
    been successfully modified to include co-culture with primary
    hepatocytes (Kim et al., 1987) to allow for chemical metabolism.

    4.5  Future directions

    4.5.1  Molecular approaches in immunotoxicology

          A promising avenue for early detection of immunotoxicity may be
    measurement of the expression of various interleukins. Cytokines are
    involved both in regulation of the immune system and in pathological
    phenomena, hence alterations in their pattern of expression may be
    early indicators of immunotoxicity. Such testing can be done at the
    level of mRNA expression, on mRNA extracted from lymphoid tissue taken
    from exposed animals, or in tissue sections, so that the alterations
    can be evaluated in the context of morphological indications of the
    toxic effects. The signal of the cytokine that is being tested must
    therefore be strong enough to be picked up in material from exposed
    animals whose immune system has not received other stimuli, i.e.
    sensitization or infection. This may not be true for all cytokines;
    ex-vivo stimulation of cells that are part of the immune system may be
    necessary, although the tests then become more laborious and must in
    fact be considered functional assays, like tests for mitogen

          Very sensitive analysis can be done with the semiquantitative
    polymerase chain reaction, which is a powerful technique for
    elucidating early kinetic changes of cytokine expression, before
    translation and secretion (Saiki et al., 1985). In addition, since
    immunosuppressive agents can enhance or inhibit the ultimate
    production and secretion of cytokines at various stages such as
    transcription, the splicing of mRNA, translation of mRNA into

    polypeptides on ribosomes, post-translational processing, and
    secretion, potential molecular targets can be dissected by such
    techniques. Several other molecular approaches may be used, including
    northern blotting, dot-slot blotting, in-situ hybridization, and
    antisense oligonucleotides for inhibiting the translation of specific

    4.5.2  Transgenic mice

          The development of molecular genetic techniques has allowed not
    only the isolation and analysis of specific genes but also the
    manipulation of embryonic genes. Transgenic technology can be used in
    immunology to generate mice that lack virtually any genetic control
    mechanism or specific cell subpopulations. As a consequence, complex
    systemic responses can be dissected into individual components, and
    the mechanism by which immunosuppressive agents exert their affects
    can be better understood. Two strategies are used to induce genetic
    aberrations in transgenic mice (Bernstein & Breitman, 1989). One
    involves the introduction of genes that produce toxins, such as
    diphtheria toxin or the A subunit of ricin, into targeted cell
    subpopulations. The second strategy involves the thymidine kinase
     (tk) gene from  Herpes simplex virus: When certain nucleotide
    analogues are administered and are metabolized exclusively by viral
    thymidine kinase, the metabolites are lethal only to cell
    subpopulations that express the  tk gene. Both approaches are
    inducible systems for killing cells  in vivo. Although gene ablation
    techniques can be used to generate mutant animals that lack specific
    cells  in vivo, a small proportion of cells appeared to escape from
    targeted cell death in virtually every study using bacterial toxins or
    viral  tk genes. While this may cause problems in determining the
    qualitative roles of ablated cell populations, these techniques hold
    promise for understanding the selective toxicity of drugs and
    environmental agents on the immune system.

          Other promising avenues are the use of animals transgenic with
    respect to certain specificities of the TCR. If a gene that encodes
    for a certain antigen specificity is introduced into the genome, that
    specificity may be the only one that is expressed by the T cells. The
    effects of immunotoxicants that affect the (positive and/or negative)
    selection process that takes place in the thymus could be studied
    elegantly with such models, when either undesired specificities (which
    should be negatively selected) or desired specificities (which should
    be positively selected) are introduced.

    4.5.3  Severe combined immunodeficient mice

          Another approach that may warrant further exploration is the use
    of severe combined immunodeficient CB-17  scid/scid (SCID) mice
    grafted with human immune cells. Xenogeneic lymphoid cells and/or
    tissues can be successfully transferred to SCID mice (McCune et al.,
    1988; Namikawa et al., 1990; Barry et al., 1991; Greiner et al., 1991;

    Surhe & Sprent, 1991). SCID mice have been grafted with human fetal
    lymphoid tissue in order to study human haematopoiesis (McCune et al.,
    1988) or with human peripheral blood lymphocytes to allow production
    of human immunoglobulins, including secondary antibody responses
    (Mosier, 1990). SCID mice have also been used to study autoimmunity
    and potential antiviral therapeutics. While these animal models still
    have limitations (Pollock et al., 1994), they may ultimately provide
    predictive models for examining potential immunosuppressive agents.

          In particular, SCID mice co-implanted with human fetal thymus and
    liver tissue fragments (SCID/hu mice) offer the possibility of
    studying the human thymus  in vivo in an isolated xenogeneic
    environment (McCune et al., 1988; Namikawa et al., 1990) and the
    effects of immunotoxicants on these grafts. This system is
    particularly interesting with regard to those immunotoxicants for
    which the thymus is one locus of action. The placement of human fetal
    thymus under the SCID mouse renal capsule, followed by an intravenous
    injection of fetal liver cells (McCune et al., 1988), and
    co-implantation of human fetal liver and fetal thymus under the renal
    capsule of SCID mice (Namikawa et al., 1990) have resulted in
    reconstitution of SCID/hu mice; the fetal thymic implants increased in
    size, and were found to be vascularized. The architecture and
    antigenic distribution of these thymic grafts were virtually
    indistinguishable from those of normal, age-matched human thymus.
    Human stem cells were found to home to and differentiate within the
    grafted human thymus, and phenotypically mature and functional human
    T cells were found in the peripheral circulation of these mice (McCune
    et al., 1988; Krowka et al., 1991; Vandekerckhove et al., 1991). As
    such, the SCID/hu model can be helpful in immunotoxicological research
    on the human thymus. When data obtained in experimental animals are
    extrapolated to the human situation, a 'control' model, between the
    SCID/hu mouse model and the intact laboratory animal (rat), is
    desirable in order to test for possible differences in thymic
    behaviour, because of its location under the kidney capsule: Thymic
    blood flow and therefore the toxicokinetic behaviour of the thymus may
    differ. For this reason the SCID/ra model was developed, by implanting
    rat fetal thymus and liver tissue fragments under the SCID mouse renal
    capsule. The outcomes of exposure of rats and SCID/ra mice can be
    compared and the influence of thymus location and mouse metabolism on
    extrapolation from SCID/hu to humans can then be determined.

          Implantation of fetal rat thymus and liver tissue yields thymic
    grafts that are virtually indistinguishable from normal, age-matched
    rat thymus (De Heer et al., 1993). After implantation of rat fetal
    thymus and liver tissue, the thymic grafts increase considerably in
    size. Histologically, the SCID/ra thymic graft bears a close
    resemblance to normal rat thymus, and the (immuno)histology of the
    SCID/hu and SCID/ra mouse thymic grafts is comparable. Differences are
    found, however, in peripheral reconstitutions of SCID/hu and SCID/ra
    mice: Whereas large numbers of circulating donor rat T cells are found

    in the blood and peripheral lymphoid organs of SCID/ra mice, only a
    small number of donor T cells are found in the SCID/hu. This implies
    that the data for extrapolating immunotoxic data from rats to humans
    must be confined to thymic effects. With this restriction in mind, the
    outcome of experiments with SCID/hu and SCID/ra mice can be used to
    compare the sensitivity of the human and rat thymus and can thus yield
    important information for the process of human risk assessment.

    4.6  Biomarkers in epidemiological studies and monitoring

          There is a difference between assays of the immune system and
    biomarkers. Many validated tests can indicate alterations to the
    immune system, including its function, so that most assays can be
    helpful for hazard identification. Not every validated assay of the
    immune system is a biomarker, however. The IPCS (1994a) definition of
    a biomarker is one that indicates exposure (and is specific for
    exposure), indicates susceptibility to adverse effects, and/or is
    predictive of disease associated with exposure. Biomarkers should be
    used to characterize risk due to exposure, on the basis of
    identification of the hazard.

          Within this strict definition, it is clear that not many
    biomarkers are available for immunotoxicity (as is true for other
    systems), especially for assessing immunotoxicity or individual
    susceptibility to immunotoxicity. Some assays may be useful in
    epidemiological studies. In any event, more epidemiological studies
    are needed to obtain a better view of the usefulness of biomarkers for
    detecting immunotoxic events and hence the possible health risks that
    may be associated with exposure to immunotoxicants.

    4.7  Quality assurance for immunotoxicology studies

          In many countries, studies to support the safety of a compound or
    drug must be conducted in accordance with the requirements for 'good
    laboratory practice' of the agency that is evaluating the material.
    Immunotoxicological studies conducted to support the safety of a new
    drug or chemical should follow at least the 'spirit' of good
    laboratory practice. The OECD has published their principles of good
    laboratory practice, with supporting publications on their
    application, and these have been adopted into legislation in a number
    of countries. IPCS (1992) has published a monograph,  Quality
     Management for Chemical Safety Testing, covering the important
    aspects of good laboratory practice in a nonregulatory context and
    quality control of chemical analyses.

          In the United States, good laboratory practice for conducting
    nonclinical laboratory studies for submission to the Food and Drug
    Administration has been detailed. The standards for studies on
    pesticides submitted to the Environmental Protection Agency were also
    published, as were the procedures to be followed in conducting studies

    submitted on compounds covered by the Toxic Substance Control Act.
    Each of these sets of standards is periodically updated by the
    respective agencies, and studies must be conducted in accordance with
    the most recent updates. While there are some differences in the
    wording of the standards, they are generally similar.

          Good laboratory practice includes written protocols for
    evaluating potential immunotoxicants and the establishment of standard
    operating procedures for assays. Each laboratory must run the assays
    frequently enough to establish historical control values, and
    the results of any study conducted to evaluate a compound for
    immunotoxicity must be judged in the context of the historical control
    values for the laboratory and appropriate controls. Incorporation of
    positive control compounds in the study design provides additional
    confidence that the assays are being conducted correctly, particularly
    when the tested compound shows no effect.

          The selection of assays to be used in evaluating compounds for
    immunotoxicity remains a subject of active discussion. Other parts of
    this document address this issue in detail. Regardless of which assays
    are used, however, they must be standardized and be recognized as
    validated and meaningful. Significant advances have been made in the
    standardization and harmonization of assay procedures for assessing
    immunotoxicity, mainly as a result of the willingness of leading
    laboratories in the field to share their standard operating procedures
    openly with other laboratories. Published papers and books on
    immunotoxicity test methods also contribute to the standardization
    process. As a result of studies by Luster et al. (1988), the assays
    used by the NTP for evaluating potential immunotoxicants have been
    accepted as validated assays in mice.

    4.8  Validation

          An important requirement of tests for evaluating immunotoxicity
    is that they be validated. While there is no agreed definition of
    validation, tests must meet certain requirements. In toxicology,
    validation is the process by which the reliability and relevance of a
    test to identify human health risk is established (Balls et al.,
    1990). A flow diagram of a proposed validation process and its end-
    point, the acceptance of a method by regulatory authorities for
    submission of toxicological data, is presented in Figure 36.

          Four parameters must be considered in determining the validity of
    a testing method: specificity, sensitivity, accuracy, and precision.
    Specificity is based on the rate of false-positive results generated.
    Sensitivity is determined by the ability to identify true-positive
    results. These two parameters determine the level of predictability or
    relevance. In order to determine specificity and sensitivity, the
    method is evaluated with a set of compounds of known positive and
    negative immunosuppressiveness. This approach was used by the NTP in

    FIGURE 36

    evaluating the predictability of various assays (Luster et al., 1988).
    In a subsequent study, the potential immunotoxicity (defined as a
    dose-related effect on any of two immunotoxicological parameters with
    no effect on body weight) was determined for 51 chemicals in mice,
    using a variety of general and functional immunological parameters
    (Luster et al., 1992).

          Accuracy is determined by the ability to measure the intended
    end-point truly. Precision is the ability to reproduce results from
    experiment to experiment or between laboratories. In the NTP studies
    in mice (Luster et al., 1988), four laboratories participated in the
    inter-laboratory validation process. A number of international studies
    are in progress on the precision of several assays, using the rat as a
    model for immunotoxicological evaluations, in an attempt to bring the
    level of acceptance of immunotoxicological studies in rats to the
    level that has been achieved for mice. Most of these studies are
    multinational and represent an interaction of industry, government,
    and academia to achieve this common goal.

          A comparative study in Fischer 344 rats with cyclosporin A (White
    et al., 1994) encompasses nine laboratories in Canada, Europe, and the
    United States. The primary focus of the study is on the use of
    functional assays for detecting immunosuppression; lymphoid organs and
    tissue are weighed and examined histopathologically in several of the
    laboratories. The functional assays used in this protocol include the
    plaque-forming cell assay or ELISA to sheep erythrocytes, splenocyte
    proliferative assays to concanavalin A and STM, the NK cell assay, and
    the mixed leukocyte response. Splenocyte surface markers were also
    analysed. The study design was similar to that used by the NTP, with a
    14-day exposure and administration by oral gavage. The preliminary
    results demonstrated excellent reproducibility of the results for the
    plaque-forming cell assay, splenocyte proliferative assays to
    concanavalin A and STM, and the mixed leukocyte response. Differences
    were observed between the laboratories in the results of the test for
    NK cell activity.

          The IPCS-European Union international collaborative
    immunotoxicity study in rats is also in progress. The study involves
    20 laboratories in Canada, Europe, Japan, and the United States; its
    design is based on the OECD test guideline No. 407 for a 28-day
    toxicity study. The study focuses primarily on the ability to detect
    immunotoxic compounds on the basis of organ weights, pathological
    findings, and 'enhanced pathology', which includes additional
    evaluation of lymphoid tissues not currently required by test
    guideline No. 407. Functional assays were also conducted; the core
    assays included the plaque-forming cell assay or ELISA to sheep
    erythrocytes, splenocyte proliferative assays (concanavalin A and
    STM), and the NK cell assay. The study was conducted in two phases. In
    the first phase, azathioprine was used as the test compound, various
    strains of rat were used, and each laboratory established its own

    doses on the basis of a predetermined maximum tolerated dose. In the
    second phase, cyclosporin A was the test compound, only three strains
    of rat were used, and a more structured protocol was followed. A
    report on phase I of the study has been drafted, and the data from
    phase II are currently being analysed. All of the laboratories found
    that azathioprine is immunosuppressive, even though several strains of
    rats were used, different dose levels were administered, and no
    standard protocol was followed. The preliminary results with
    cyclosporin A show good agreement between the laboratories for the
    plaque-forming cell assay but some differences for the splenocyte
    proliferative assays (concanavalin A and STM) and the NK cell assay.

          A third interlaboratory study in rats has been organized by the
    German Bundesgesundheitsamt, in Berlin, with German and French
    participants. The design is also based on OECD test guideline No. 407
    for a 28-day repeated-dose study in Wistar rats. Cyclosporin A was
    selected as the test substance. The live phase of this study has been
    completed, the data are being analysed, and the final report is being

          Information obtained from studies of predictability, accuracy,
    and precision, such as those described above, must undergo peer review
    before publication. A major goal of the validation process is to
    determine which methods should be recommended in the testing
    guidelines of regulatory agencies. Figure 36 shows each input and
    output of information and the action steps. The process of developing
    and obtaining acceptance of testing guidelines is based on three major
    inputs: (1) publication of reports in peer-reviewed journals;
    (2) guidelines for deciding whether a method is valid; and
    (3) implementation of test methods that are interpretable by
    scientists involved in assessing biologically relevant risks and the
    results of which can be incorporated into quantitative dose-response
    analyses. The proposed process is built around the generation of these
    major inputs. Two of the issues that will arise in the development of
    guidelines are: 'How many and what type of compounds should be
    included in the validation process?' and 'Should the compounds be
    shown to be immunosuppressive in both humans and mice?'


    5.1  Introduction: immunocompetence and immunosuppression

          An immune response in the fully mature, immunologically competent
    individual provides protection against a myriad of infectious agents
    and environmental hazards. The immune system acts as a self-restoring
    (homeostatic) system which can quickly return to normal levels of
    function after periods of marked stimulation and response. This self-
    regulation allows the individual to recover from or circumvent the
    toxic effects of many potentially damaging environmental hazards.
    There are many well-known clinical conditions of inherited deficiency
    in immunological function; some result in specific defects in antibody
    formation, others consist of T-cell and/or metabolic defects, while
    others include impairment of both B- and T-cell function. These
    conditions, known as primary immunodeficiency disorders, are due to
    definable, inheritable genetic defects. Clinical studies of these
    disorders have demonstrated the importance of the immune system to
    host defence and individual survival, showing that individuals with
    partial or absolute defects in T-cell function rarely survive beyond
    infancy or early childhood. In contrast, individuals with defects in
    B-cell function, resulting in a deficiency in antibody formation, may
    suffer from a variety of chronic, recurrent infectious diseases and
    diminished health but can survive with appropriate therapy when the
    underlying disorder is recognized. Study of these genetic
    immunodeficiency states has also provided considerable information
    about the functions of human B and T cells which would otherwise not
    have been determined.

          Impairment of the function of a key component of the immune
    system results in a diminished immune response (immunosuppression) or
    immunodeficiency. Acquired immunodeficiency states were recognized
    only sporadically until the late 1970s, when a syndrome appeared that
    spread rapidly through certain groups and produced a generalized type
    of immunosuppression known as acquired immunodeficiency syndrome
    (AIDS). AIDS was found to be due to retroviruses that infect and
    destroy Th (CD4+) cells in humans (Fauci et al., 1991). CD+
    lymphocytes have been identified in experimental studies as the key
    cells in the recognition and secondary processing of antigens. Thus,
    progression of AIDS is associated with progressive loss of Th cells
    and increased frequencies of infection by bacterial, fungal, viral,
    and parasitic agents and of certain types of neoplasms.

    5.2  Considerations in assessing human immune status related to

          The assessment of immunotoxicity in humans exposed to potentially
    immunotoxic compounds is much more complicated than in experimental
    animals. Issues such as logistics, appropriate controls, magnitude and
    pattern of exposure, and confounding parameters such as medication,
    drug abuse, and illness must be considered. Other considerations that

    should be taken into account in comparing human immune status with
    that in laboratory animals in relation to immunotoxicity are as

          (1) The human population is heterogeneous and genetically
    disparate; it can be considered as 'wildlife'. Inbred laboratory
    animals are, by definition, genetically identical; outbred laboratory
    animals typically have a larger genetic variability than inbred
    animals but a variability that is much smaller than that in wildlife
    populations. Genetic constitution, which accounts for the variability,
    has consequences for the antigen recognition capacity of the immune
    system, especially for the T-lymphocyte population. Antigen
    recognition by T cells is restricted to the MHC haplotype of the
    individual and therefore differs between (allogeneic) individuals.
    Inbred, and most outbred, laboratory animals are much more alike in
    antigen recognition capacity than wildlife populations. For instance,
    the repertoire of certain inbred mouse strains lacks part of the
    spectrum of T-cell specificity, as seen by the absence of T cells that
    express distinct 'variable gene families' in the repertoire of TCR
    specificities. Such 'gaps' in the repertoire have thus far not been
    detected in the outbred human population by similar methods of
    detection (Hu et al., 1993), perhaps because each individual in an
    outbred population expresses the MHC products of both parents and can
    in principle multiply the repertoire of MHC-restricted reactions by a
    factor of 2 (including MHC I and MHC II).

          Interindividual variability has obvious consequences for
    immunotoxicity, in which the response to the chemical or drug
    underlies the mechanism of toxicity. Its effect on direct toxicity is
    presumably less, but the manifestation of toxicity is often reduced
    immune reactivity (e.g. increased incidence of infections) and hence
    determined by individual reactions to antigens.

          (2) The human population, like populations of 'wildlife' animals,
    is continuously exposed to environmental stimuli. It is well known
    that it is not necessary that each member of a population be protected
    ('immune') but that a certain proportion of protected individuals must
    be reached in order to achieve 'herd immunity'. That is to say, the
    whole population is protected when a certain percentage of individuals
    is immune. In contrast, when this percentage falls below the required
    incidence (which differs for different infectious agents), the
    population as a whole loses its protected state, and an infectious
    epidemic can result. This situation easily arises in small groups in a
    country where vaccination is minimal. Well-known examples are the
    outbreaks of poliomyelitis in the Netherlands and the hepatitis A
    virus outbreaks in China. This phenomenon should be taken into account
    in epidemiological studies and associated laboratory investigations
    (e.g. antibody levels to microorganisms) in assessing immunotoxicity
    in human populations.

          (3) Most of the human population is continuously exposed to
    environmental stimuli and maintains its ability to respond to foreign
    material from the pool of immunological memory. From the first
    postnatal period through to adulthood, the T-cell repertoire is
    generated by the thymus; later, this generation of cells is reduced to
    a low level. Strict MHC restriction implies that the T-cell population
    cannot easily change specificity, e.g. by somatic mutation of
    the genes that encode the TCR. There is some evidence from
    immunophenotyping, both in mice and humans, that the T-cell population
    shifts gradually during life, from naive (committed) T cells to memory
    T cells. Within the B-cell population, the situation is different.
    Here, the repertoire changes continuously, due to somatic mutation
    presumably associated with 'affinity maturation' in lymph nodes. This
    phenomenon may result in the emergence of B cells with a strong
    affinity for stimuli and the disappearance of low-affinity cells. It
    is not known whether neoantigens or pathogens are recognized by
    'affinity matured' or memory B cells or by naive B cells. In the
    absence of information on this aspect, it can be suggested that most
    of the immune capacity of adults is deployed for memory reactions,
    whereas in young people the contribution from the naive pool is
    higher. This is reflected in infectious epidemics, when microorganisms
    like influenza change to phenotypes that cannot be recognized by the
    memory pool, an aspect to be kept in mind when choosing immune tests
    to be used in evaluating immunotoxicity. Primary responses, like those
    to keyhole limpet haemocyanin antigen, are considered to be more
    sensitive than secondary responses, like those to tetanus toxoid.
    Another example of this effect is the composition of the recall
    antigens used in testing delayed-type hypersensitivity (Borleffs et
    al., 1993). Both primary and secondary antibody responses, however,
    are valuable for evaluating the intrinsic naive and memory immune
    capacity of individuals, although secondary responses are less
    sensitive to immunological insults.

          (4) For a number of infectious microorganisms, the immune
    response does not result in complete elimination of the invader but
    rather in its 'silent' integration into the genome. Certain viruses,
    like herpes viruses, cytomegalovirus, and Epstein-Barr virus, are
    dealt with in this way. An individual is considered to be a carrier of
    the virus (postinfection status) on the basis of the presence of
    antibodies. In other words, individual postinfection is a continuous
    defence against these viruses, often with sufficient capacity to keep
    the virus in a silent form. In diminished immune capacity, this
    natural protection can be lost, and infections can re-occur after
    viral reactivation. Primary infection and reactivation therefore have
    different pathogeneses, although the subsequent disease may be
    characterized by the same symptoms. This situation is well known
    clinically, when high doses of immunosuppressive drugs are given for
    long periods. The relevance of this observation in immunotoxicity
    testing must be established.

          (5) Ex-vivo diagnosis in humans is often restricted to
    haematological investigations, so that only information on the
    circulatory pool of cells and plasma factors is obtained. For example,
    the distribution of immunoglobulins differs in the intravascular and
    extravascular spaces. Only about 1% of the total lymphocyte pool is
    present in blood (1010 cells out of the total of about 1012), and
    this population represents only the recirculating pool of cells and
    not the tissue-bound cells that participate actively in immunological
    responses. Investigations of peripheral blood cells can be somewhat
    misleading: for instance, patients infected with the human
    immunodeficiency virus (HIV)-1 may show severe depression of CD+
    cells in blood but less reduction in CD cells in lymphoid tissue
    (Schuurman et al., 1985).

          It is considerably more complex to establish immune changes in
    humans than in animals, since noninvasive tests are limited, the
    levels of exposure to an agent (i.e. dose) are difficult to establish,
    and the responses in the population are extremely heterogeneous. With
    respect to the latter, the variation in immune responses (genetic or
    environmental) can exceed a coefficient of variation greater than
    20-30%. Because many of the immune changes in humans that follow
    exposure to chemicals may be sporadic and subtle, recently exposed
    populations must be studied and sensitive tests for assessing the
    immune system be performed. Since many of the immune tests used in
    humans have a certain degree of overlap (redundancy), it is also
    important that a positive diagnosis not be based on a change in one
    test but on a profile (pattern) of changes, similar to that observed
    in primary or secondary immunodeficiency diseases. For example, low
    CD:CD8 ratios are often accompanied by changes in skin reactions to
    recall antigens. The Clinical Immunology Subcommittee of WHO and the
    International Union of Immunological Societies published methods for
    examining changes in the human immune system and described their
    pitfalls (Bentwich et al., 1982, 1988); however, most of the tests
    were selected on the basis of observations in patients with primary
    immunodeficiency diseases. Such individuals suffer from severe
    recurring infections, and their degree of immunosuppression is
    probably considerably greater than that induced by chemicals. Thus,
    some of the methods may be of limited value for examining potential
    chemical-induced immunosuppression, and further evaluation of methods
    is needed.

          In view of the difficulties in identifying chemical-induced
    immunosuppression in humans, establishment of exposure levels (e.g. in
    blood or tissue) would not only be useful but would in many instances
    be essential for determining a cause-effect relationship. Clinical
    disease may not necessarily have to be present in order for
    immunosuppression to be meaningful, for several reasons. Firstly,
    there are uncertainties about the extent of the reserve capacity of
    the immune system and whether the relationship between immune function
    and clinical disease shows a linear or a threshold response. In a

    linear relationship, even minor changes in immune function would be
    related to an increase in disease, if the population examined is large
    enough. While the relationship at the low end of the dose-response
    curve is unclear, at the high end of the curve (i.e. severe
    immunosuppression), clinical disease is readily apparent. This is
    exemplified by increased incidences of the opportunistic infections
    that occur in AIDS patients. Secondly, clinical disease may be
    difficult to establish, as neoplastic diseases may involve a
    10-20-year latency before tumour appearance, and increased infection
    rates are difficult to ascertain in epidemiological surveys (e.g.
    increased numbers of cases of severe common cold).

    5.3  Confounding variables

          The normal population has a wide range of immunological
    responses, with no apparent health impact. In addition to the
    underlying population variability, certain host characteristics and
    common exposures may be associated with significant, predictable
    alterations in immunological parameters. If not recognized and
    effectively addressed in study design or statistical analysis, these
    confounding factors may severely alter the results of population

          Examples of factors associated with measurable alterations in
    immunological parameters are age, race, sex, pregnancy status, acute
    stress and the ability to cope with stress, coexistent disease or
    infection, nutritional status, lifestyle, tobacco smoking, and some
    medications. The effect of acute stress on the immune system is
    mentioned in section Protein calorie restriction and
    deficiencies of trace elements such as zinc have also been associated
    with immune deficiency (Chandra, 1992; Good & Lorenz, 1992; Chandra,
    1993). Periodic influences, ranging from daily to seasonal, also
    exist; some are relatively minor, but others are of a magnitude that
    may rival the expected effect of a low-level exposure to a toxic
    agent. They are therefore of primary concern in large epidemiological
    studies. For example, African Americans have, on average, serum IgG
    levels that are 15-20% higher, neutrophil counts that are 10-15%
    lower, and a proportion of circulating B cells that is significantly
    higher than those of Caucasians, with no discernible health
    implications. Cigarette smoking is associated with a significant
    decrease in IgG level and an increase in leukocyte count, independent
    of ethnic differences. Therefore, it is imperative that population
    norms and reference ranges be supplemented by detailed analysis of
    potential confounding factors. Study designs should include
    considerations of matching, stratification, and subgroup analysis to
    control for these potential effects. As new immunological assays are
    developed, normative data will be required, particularly for ethnic
    minorities, children, the aged, and certain groups potentially at high
    risk, such as pregnant and lactating women.

          Certain endocrine diseases and conditions may be associated with
    significant alterations in immune function (e.g. adrenal dysfunction).
    Some medications, such as corticosteroids, phenytoin, and nonsteroidal
    anti-inflammatory agents, may depress a variety of immune functions.
    Questionnaires and population surveys should allow the collection of
    sufficient information to make it possible to consider these factors
    in data analysis and interpretation.

          An increasingly important consideration in any analysis of immune
    function, particularly in relation to immune deficiency, is the
    potential presence of HIV infection, which causes widespread
    alterations in virtually all elements of the immune system. Even a
    small proportion of unrecognized HIV-infected individuals in a study
    population may significantly affect the results and the interpretation
    of data. When immunological studies indicate decreased immune
    parameters consistent with HIV infection, testing for the virus should
    be considered; otherwise, interpretation of the results of
    immunological tests of immune dysfunction, particularly among
    populations with potentially high rates of HIV infection, may be
    severely limited.

          In assessing human immunotoxicity, it is useful to establish the
    presence of infectious, allergic, or autoimmune diseases in order to
    ensure completeness and to rule out additional confounding variables.
    A clue to the type of immunological defect is often provided by the
    kind of infection observed. For example, patients with impaired
    humoral immunity have an increased incidence of recurrent infections
    with encapsulated bacterial pathogens (e.g.  Pneumococcus and
     Haemophilus influenzae), which can induce chronic sinopulmonary
    infection, bacteraemia, and meningitis. In contrast, if cellular
    immunity is intact, the patients will have less severe infections with
    fungal and viral agents. Abnormalities of T cells and impairment of
    cell-mediated immunity predispose individuals to infections with a
    wide variety of agents, including viruses that cause disseminated
    infections (e.g.  Herpes simplex virus, varicella-zoster virus, and
    cytomegalovirus), fungi that cause mucocutaneous candidiasis, and
    parasitic organisms including the protozoan  Pneumocystis carinii.

    5.4  Considerations in the design of epidemiological studies

          An important factor in assessing the usefulness of an
    epidemiological study for risk assessment is its design. The commonest
    design used in immunotoxicology is the cross-sectional study, in which
    exposure and disease status (in this case, changes in immunological
    function) are measured at one time or over a short period. The immune
    function of 'exposed' subjects is compared with that of a comparable
    group of 'unexposed' individuals. Important considerations in using
    the data provided by such studies in risk assessment have been
    discussed (E. Ward, unpublished manuscript):

    (1)   What is the relationship between changes in immune function and
          human health risk?

    (2)   Are the selection procedures for study subjects adequately

    (3)   Is there evidence that the exposed group was actually exposed to
          the substance of interest?

    (4)   Has the possibility that other exposures, of either the entire
          population or individuals, been accounted for?

    (5)   Are the 'exposed' and 'unexposed' populations comparable with
          respect to factors other than the exposure of interest?

    (6)   Have major individual aspects (such as illness and use of
          medications) that may influence the outcome of tests for immune
          function been accounted for?

    (7)   Has inter- or intralaboratory variability been controlled for?
          Was the laboratory that ran the tests for immune function able to
          distinguish between samples from 'exposed' and 'unexposed'

    5.5  Proposed testing regimen

          Biological research involving human subjects must be conducted in
    accordance with ethical standards and involve scientific procedures
    designed to ensure the safety of the subjects (Council for
    International Organizations of Medical Sciences, 1993). Below are
    shown a testing scheme proposed by WHO for preliminary evaluation of
    individuals exposed to immunotoxicants, an approach developed by a
    working group organized by the United States Centers for Disease
    Control and Agency for Toxic Substances and Disease Registry, and that
    proposed by a panel of the United States National Academy of Science
    (US National Research Council, 1992). The three approaches have many

    5.6  Assays for assessing immune status

          A plethora of tests has been developed to assess immunity in
    humans (Bentwich et al., 1982, 1988), which are described in
    laboratory manuals (Lawlor & Fischer, 1988; Miller et al., 1991;
    Coligan et al., 1994). Many of these tests are now commercially
    available in kits. A systematic approach to the evaluation of immune
    function, which is based on simple screening procedures, followed by
    appropriate specialized tests of immune function, usually permits the
    definition of an immune alteration. The tests should include
    evaluation of the B-cell system, of the T-cell system, and of
    nonspecific resistance (polymorphonuclear leukocytes, monocytes and
    macrophages, NK cells, the complement system). Although some exogenous


    Assays suggested by WHO for assessing immunotoxicity in all persons
    exposed to immunotoxicants

    1.    Complete blood count with differential counts

    2.    Antibody-mediated immunity (one or more of following):
          *    Primary antibody response to protein antigen (e.g. epitope-
               labelled influenza vaccine)
          *    Immunoglobulin concentrations in serum (IgM, IgG, IgA, IgE)
          *    Secondary antibody response to protein antigen (diphtheria,
               tetanus, or poliomyelitis)
          *    Natural immunity to blood-group antigens (e.g. anti-A and

    3.    Phenotypic analysis of lymphocytes by flow cytometry
          *    Surface analysis of CD3, CD4, CD8, CD20

    4.    Cellular immunity
          *    Delayed-type hypersensitivity skin testing using Multitest
          *    Primary delayed-type hypersensitivity reaction to protein
               (keyhole limpet haemocyanin)
          *    Proliferation to recall antigens

    5.    Autoantibodies and inflammation
          *    C-Reactive protein
          *    Autoantibody titres to nuclei, DNA, mitochondria and IgE
               (rheumatoid factor)
          *    IgE to allergens

    6.    Measure of nonspecific immunity
          *    Numbers of natural killer cells (CD56 or CD60) or cytolytic
               activity against K562
          *    Phagocytosis (nitroblue tetrazolium or chemiluminescence)

    7.    Clinical chemistry


    Screening panel recommended for human studies by the United States Centers for
    Disease Control and Agency for Toxic Substances and Disease Registry

    *     Complete blood count with differential counts
          -    absolute lymphocyte count
          -    granulocyte count
          -    platelet count
          -    absolute eosinophil count
          -    examination of peripheral smear

    *     Immunoglobulins
          -    IgG
          -    IgA (optional)
          -    IgM (optional)

    *     C-Reactive protein

    *     Autoantibody screening panel
          -    Antinuclear antibody
          -    Rheumatoid factor
          -    Anti-thyroglobulin antibody

    *     Peripheral blood leukocyte surface markers
          -    CD2/CD3
          -    CD4/CD8
          -    CD8/CD3
          -    CD19/CD20

    *     Clinical chemistry in serum
          -    Blood urea nitrogen
          -    Cholesterol
          -    Creatinine
          -    Total bilirubin
          -    Alkaline transaminase
          -    Alkaline phosphatase
          -    Total protein (albumin:globulin ratio)

    Tests recommended by the panel of the United States National Academy of Sciences
    for studies of persons exposed to immunotoxicants

    Tier 1 (all persons exposed to immunotoxicants) 

    I.     Humoral immunity
           *    Immunoglobulin concentrations in serum (IgM, IgG, IgA, IgE)
                and immunofixation electrophoresis
           *    Natural immunity: Antibody levels to ubiquitous antigens
                (e.g. anti-A and anti-B group substances in individuals of
                non-AB blood type)
           *    Secondary antibody responses to proteins (e.g. diphtheria,
                tetanus, poliomyelitis) and polysaccharides (e.g.
                pneumococcal, meningococcal)

           Note: In immunization studies, live microorganisms should not be
           given to persons suspected of being severely immunocompromised.

    II.    Lymphocytes
           *    Enumeration of B and T cells in blood
           *    Surface analysis of CD3, CD4, CD8, CD20
           *    Secondary delayed-type hypersensitivity reaction (e.g.
                candida, diphtheria, tetanus)
           *    Alternative: Multiple antigen skin test kit 

    III.   Autoantibody titres (to red blood cells, nuclei, DNA,
           mitochondria, IgE (rheumatoid factor)

    Tier 2 (all persons with abnormal Tier 1 test results and
    a fraction of the total exposed population to be determined
    by a statistician)

    I.     Humoral immunity
           *    Primary antibody response to protein and polysaccharide

           Note: A panel of antigens should be developed that can be used
           in sequential studies on a given individual, since a particular
           antigen can be used only once to assess a primary response.

    II.    Cellular immunity
           *    Proliferative response to mitogens (phytohaemagglutinin,
                concanavalin A) and possible antigens such as tetanus;
                primary delayed-type hypersensitivity reaction to keyhole
                limpet haemocyanin

           Note: Here, too, a panel of standard antigens is needed for
           sequential testing; these could be the same as those used to
           assess primary antibody responses.

    Tests recommended by the panel of the United States National Academy of Sciences
    for studies of persons exposed to immunotoxicants

    III.   Natural killer cells, monocytes, and other T- and B-cell markers
           *    CD5, CD11, CD16, CD19, CD23, CD64; class II MHC on T cells
                by two-colour flow cytometry for co-expression of class II
                and a T-cell marker such as CD3

    IV.    Serum levels of cytokines (e.g. IL-1, IL-2, IL-6) and of shed or
           secreted cellular activation markers and receptors (e.g. CD25).

    V.     Class I and II MHC antigen typing

    Tier 3 (to be considered for persons with abnormalities
    in Tier 2 tests or for a random fraction of the entire
    population in Tier 2)

    *      If a proportion of CD16 cells of Tier 2, III, is abnormal:
           nonspecific killing of a tumour cell line to test for natural
           killer function
    *      If primary delayed-type hypersensitivity reaction in Tier 2, II,
           is abnormal:cell proliferation in response to phorbol ester and
           calcium ionophore,anti-CD3 antibody, and a staphylococcal
           enterotoxin B (experimental)
    *      Generation of secondary cell-mediated immune reactions
           (proliferation and MHC-restricted cytotoxicity)  in vivo,  e.g.
           with influenza virus (experimental)
    *      Immunoglobulin subclass levels in serum (IgA1, IgA2, IgG1-4)
    *      Antiviral titres (e.g. influenza, parainfluenza,
           cytomegalovirus, human immunodeficiency virus) in serum (no
           deliberate immunization)
    agents can alter several elements of the human immune system, others
    have a primary effect on a single element. For example, low doses of
    cyclosporin A selectively affect T cells by acting on the production
    of IL2 and IL2 receptors. Conversely, the anticonvulsive drug
    phenytoin acts primarily on the humoral immune system, leading to a
    selective deficiency of IgA.

           A number of immune function assays recommended for inclusion in
    a simple screening panel for assessing human immune function after
    potential exposure to xenobiotics believed to affect the immune system
    are described below. It should be noted that there are many indicators
    of altered immune function in humans which may not be specific markers
    for exposure, immune disease or susceptibility (IPCS, 1994a;
    IPCS/Department of Health, 1995).

    5.6.1  Total blood count and differential

           A complete blood count, with differential absolute counts of
    lymphocytes, granulocytes, eosinophils, and platelets, are basic
    components of immunotoxicology. These tests are useful in defining the
    general health status of a population, since they are relatively well
    standardized over most age, sex, and race groups. Such counts are also
    essential for interpreting the results of ex-vivo/in-vitro functional
    tests, described below, since functional tests reflect a combination
    of numbers of cell types and activity per cell.

           The absolute lymphocyte count is critical: Higher absolute
    counts are found in children than in adults, but lymphocyte counts
    consistently below 1500/mm3 are indicative of lymphocytopenia, and a
    higher count signals a defect in the T-cell system or effects on the
    bone marrow. Lymphocytopenia can occur not only in primary immune
    deficiency but also as a result of viral infections, malnutrition,
    stress, autoimmune diseases, and haematopoietic malignancies.
    Examination of bone marrow may be indicated to exclude some other
    factors once lymphocytopenia has been confirmed. Additional assessment
    of cell mediated immunity and direct measurement of T-cell parameters,
    such as lymphocyte phenotypic markers, may also be indicated.

           Review of peripheral smears for morphological abnormalities of
    the white and red cells adds useful information for interpreting raw
    cell counts, such as the atypical lymphocytosis of many acute viral
    infections. The absolute eosinophil count can be very helpful in
    delineating allergic disorders, vascular collagen diseases, and
    parasitic manifestations.

    5.6.2  Tests of the antibody-mediated immune system

           Evaluation of antibody-mediated immunity involves measurement of
    serum concentrations of immunoglobulins and assessment of antibody
    formation after immunization or measurement of 'natural antibodies'.  Immunoglobulin concentration

           Several methods are available for measuring the concentrations
    of the five major classes of immunoglobulin, IgM, IgG, IgA, IgD, and
    IgE, in serum, including single radial diffusion, double diffusion in
    agar gel, immunoelectrodiffusion, radioimmunoassay, ELISA, and
    automated laser nephelometry. Single radial diffusion is widely used.
    Gel diffusion methods are very sensitive to differences in diffusion
    constants and thus to differences in molecular size.

           The serum concentration of each of the major immunoglobulins
    should be determined, with the exception of IgD (which occurs
    predominantly on the cell surface). The determinations must be well
    standardized because antisera vary in quality. Since serum
    immunoglobulin concentrations vary with age and environment,
    appropriate norms must be used. Patients can manifest a deficiency in
    all immunoglobulin classes (common variable hypogammaglobulinaemia),
    or they may have a deficiency in only a single class (IgA deficiency
    as a primary defect or after phenytoin therapy).

           The concentration of immunoglobulins cannot be used as the
    sole criterion for a diagnosis of immunodeficiency. Diminished
    immunoglobulin concentrations can result from loss into the
    gastrointestinal tract as well as from decreased synthesis. An
    indication of loss can be obtained by measuring serum albumin, which
    is usually lost concomitantly.  Specific antibodies

           Antibody-mediated immunity can be assessed from antibody
    responses to common specific antigens (basal levels). Humoral immunity
    after immunization can be assessed in the same way. The response to
    antigenic stimulation with both protein and polysaccharide antigens
    must be defined if immunodeficiency is strongly suspected. Failure to
    respond to one or more classes of antigen has been observed in
    patients with normal or high levels of immunoglobulins, regardless of
    whether they have an isolated immunoglobulin class or subclass
    deficiency. Specifically, patients with the Wiskott-Aldrich syndrome
    may have normal or even elevated immunoglobulin concentrations, yet
    have multiple infections because of their failure to mount a specific
    immune response, especially when they are exposed predominantly to
    polysaccharide antigens.

            Natural antibodies: Isohaemagglutinins are naturally occurring
    antibodies to blood group A and B antigens found in all normal
    individuals except those with type AB red cells. By three years of
    age, 98% of normal persons with type A, B, or O blood have
    isohaemagglutinin titres of at least 1:16. Patients with the Wiskott-
    Aldrich syndrome may have normal immunoglobulin levels yet lack

    isohaemagglutinins as an indicator of their antibody-deficient state.
    Other natural antibodies that can be assayed include heterolysins
    (e.g. against sheep or rabbit red blood cells) and antistreptolysin.

            Antibody response to immunization: In order to test for
    T cell-dependent antibody responses, commercially available
    diphtheria-tetanus vaccine can be given in recommended doses. Blood is
    taken two weeks after each injection and tetanus and diphtheria
    antibodies are determined. In patients who have been immunized with
    diphtheria-tetanus or diphtheria-pertussis-tetanus vaccine, one
    booster injection is given before determination of antibodies. In
    testing for T cell-independent antibody responses, commercially
    available pneumococcal vaccine can be given in recommended doses.
    Three doses of killed poliomyelitis vaccine (1.0 ml intramuscularly,
    at intervals of two weeks) can also be used as the immunogen. Blood is
    taken two weeks after the last injection, and antibody is usually
    determined by virus neutralization. There is strong consensus that
    quantification of a primary immune response (antibody and/or cellular)
    after immunization is not only a very relevant test but also very
    sensitive. Although such tests are not routine in clinical immunology,
    they have been used successfully for determining immune integrity.
    While keyhole limpet haemocyanin has been used as the antigen,
    beneficial immunizations such as influenza vaccine linked to a marker
    epitope can also be used.

    5.6.3  Tests for inflammation and autoantibodies  C-Reactive protein

           Inclusion of an acute-phase reactant marker is helpful for
    clinical interpretation of other laboratory biomarkers such as
    autoantibodies. The concentration of C-reactive protein rises and
    falls to baseline values in direct proportion to tissue damage and is
    thus a sensitive indicator of the presence of a generalized
    inflammatory state. It offers the best example of an accepted,
    standardized procedure that can be used in large population studies,
    because it is less subject to transport variables than other
    procedures.  Antinuclear antibody

           Antinuclear antibody is a common autoantibody that may be
    associated with various rheumatic disorders and, classically, systemic
    lupus erythematosus. Several commercial kits are available to detect
    the presence of autoantibodies. Progressively greater titres increase
    the specificity for a disease. Positive sera should be titred at
    dilutions of > 1:40 to 1:640.  Rheumatoid factor

           Rheumatoid factor is an autoantibody to immunoglobulin (usually
    IgM) that occurs in a high percentage (50-70%) of individuals with
    classical rheumatoid arthritis but may also develop in a variety of
    other disorders, including infections and immunological diseases.
    Positive sera should be titred at dilutions of > 1:40 to 1:640.  Thyroglobulin antibody

           This antibody occurs in association with a variety of thyroid
    disorders but may be found without detectable thyroid dysfunction.
    Positive sera should be titred at dilutions of > 1:40 to 1:640.

    5.6.4  Tests for cellular immunity

           A variety of tests are commonly used to assess cell-mediated
    immunity, including enumeration of T cells and T-cell subsets,
    identification of delayed skin reactions, and measurement  in vitro
    of stimulation of lymphocytes to proliferate and form blast cells.
    Other tests are available to measure T-cell effector or regulatory
    function  in vitro. As for humoral immunity, a series of simple tests
    is available to screen for defects in cell-mediated immunity. The
    proportion of circulating T cells in a mononuclear cell preparation
    can be determined by immunofluorescence with CD2 or CD3 monoclonal
    antibodies. Normally, T cells constitute 55-80% of peripheral blood
    lymphocytes. The normal values reported for absolute numbers of
    circulating T cells are 1620-4320/mm3 during the first week up to
    18 months of life and 590-3090/mm3 after 18 months of age (Fleisher
    et al., 1975).  Flow cytometry

           Antibodies that may be used in immunological phenotyping are
    listed in Table 10 in section 4.1.2. Subsets of T cells have been
    defined with monoclonal antibodies specific to cell-surface antigens.
    The association of a particular T-cell subset with a given function
    has caused some confusion in the analysis of immunological data for
    patients with immunodeficiency states, as discussed in more detail in
    section For example, CD-positive cells have commonly been
    associated with helper functions, and CD8 cells have been associated
    with cytotoxic functions. This approach is an oversimplification,
    which became evident with the finding that CD and CD8 cells recognize
    foreign antigens in the context of MHC class II and class I antigens,
    respectively. Thus, the CD population contains helper cells, memory
    cells, and cytotoxic cells for targets bearing MHC class II molecules.
    The CD8 population contains cytotoxic cells and also cells that can
    recognize antigens presented by macrophages and cells that can augment

    the interaction of CD-positive cells with B cells. Abnormalities in
    the number of CD or CD8 cells, or their ratio, can be associated with
    abnormalities in the ability to recognize antigens and regulate T-cell
    function that can lead to immune incompetence or autoimmunity.  Delayed-type hypersensitivity

           The ability of patients to manifest pre-existing T-cell immunity
    has been evaluated  in vivo with a series of skin antigens that
    normally produce a delayed-type cutaneous hypersensitivity response.
    Because delayed cutaneous hypersensitivity, a localized immunological
    skin response, depends on functional thymus-derived lymphocytes, it is
    used in detecting T cell-mediated immunodeficiency. A device
    (Multitest(R), Institut Merieux, Lyon, France) is available that
    enables the simultaneous intradermal injection of seven standardized
    antigens and so overcomes the inconvenience of giving seven separate
    injections of antigens and the technical difficulty of ensuring their
    intradermal rather than subcutaneous injection. The Multitest(R),
    consisting of eight tined, preloaded heads, delivers a glycerol
    control and the seven test antigens dissolved in glycerol. The size of
    the induration produced by each antigen should be measured at 48h in
    two diameters: reactions of less than 2 mm are scored as negative. No
    reaction should be seen with the glycerol control. The test includes
    as antigens: tetanus, diphtheria, streptococcus, tuberculin,  Candida,
     Trichophyton, and Proteus.

           Recall of delayed-type hypersensitivity as a test for cell-
    mediated immune competence was assessed with the Multitest(R) device
    in 254 subjects. When 77 subjects were tested concurrently with the
    Multitest(R) and a conventional panel of six antigens (Frazer et
    al., 1985), similar results ( r = 0.65) were obtained with the two
    systems. The reproducibility of the Multitest(R) among three
    observers, who assessed the aggregate size of reactions in 45 subjects
    independently, was high ( r = 0.89); the correlation for the reaction
    score in 24 subjects tested twice, three months apart, was also high
    ( r = 0.88), demonstrating the suitability of the test for serial
    studies of immune function.  Proliferation of mononuclear cells in vitro

           A common test of lymphocyte function is measurement of the
    capacity of lymphocyte subsets to enlarge and convert into blast-like
    cells that synthesize DNA and incorporate thymidine after stimulation
     in vitro. In this test, lymphocytes can be activated by antigens
    (e.g. purified protein derivative,  Candida, streptokinase, tetanus,
    or diphtheria); allogeneic cells are also used in the one-way mixed
    lymphocyte culture to stimulate T-cell proliferation. T-Cell blast
    transformation can be assessed directly by measuring blastogenesis and
    proliferation of cells, expression of activation antigens (e.g. CD69
    or CD25 and HLA DR), and release of mediators. The blastogenic
    response is assessed as incorporation of 3H-thymidine, usually for

    16-24h, followed by cell precipitation on filter paper and liquid
    scintillation counting. Non-isotope assay alternatives are also
    available. The responses to various antigenic stimuli by different
    types of responding cells must be interpreted with caution. The mixed
    leukocyte reaction is the result of T-cell reactivity to MHC-encoded
    peptides displayed on the surface of B cells and monocytes. The
    T cells in the population of normal irradiated or mitomycin C-treated
    lymphocytes used as the stimulators can secrete factors that induce
    blastogenesis in the patient's lymphocytes. As this can be misleading,
    it is preferable to use B-cell lines or T cell-depleted normal cells
    as the stimulators.

    5.6.5  Tests for nonspecific immunity  Natural killer cells

           The differences between NK cell function, phenotyping for NK
    cells, and the cytology of large granular lymphocytes are described in
    section The identification of NK cells remains problematic
    owing to this apparent heterogeneity. The cells can be evaluated in
    ex-vivo/in-vivo tests with enriched peripheral blood mononuclear
    cells. The proportion of NK cells can be identified with appropriate
    monoclonal antibodies (see Table 10). Functional assays of NK activity
    involve the ability of the appropriate mononuclear cells to kill
    specific NK targets, such as the K562 cell, in which cell-mediated
    cytolysis  in vitro is quantified by release of 51Cr from the
    target cells.  Polymorphonuclear granulocytes

           Some defects of phagocytic function affect polymorphonuclear
    phagocytes. Neutrophil function depends on movement in response to
    chemotactic stimulus, adherence, endocytosis, and destruction of the
    ingested particles. Phagocyte mobility depends on the integrity of the
    cytoskeleton and contractile system. Defects in intracellular killing
    of ingested microorganisms usually result from failure of the
    'respiratory burst' that is critical to production of superoxide
    radicals and hydrogen peroxide. The organisms cultured from lesions of
    patients with this type of defect generally contain catalase and
    include staphylococci,  E. coli, Serratia marcescens, fungi, and
     Nocardia. Patients with defects in mobility, adherence, and
    endocytosis usually have infections of the skin, periodontitis, and
    intestinal or perianal fistulae; patients with normal endocytosis and
    defective killing tend to have chronic granulomas. Measurement of
    nitroblue tetrazolium dye reduction or chemiluminescence by actively
    phagocytosing leukocytes has been accepted as a standard measure for
    the adequacy of the respiratory burst. Recently, methods have been
    developed to measure the production of reactive oxygen intermediates
    by flow cytometry (Emmendorfer et al., 1994). Kits are commercially
    available for assessing phagocytic capacities and the production of
    reactive oxygen intermediates by phagocytes. Assays for bacterial

    killing yield highly variable results, depending on the bacterial
    species used in the assay, and are not recommended for routine use.
    The activation state of neutrophilic granulocytes can be assessed by
    flow cytometry with the antibodies CD11b, CD14, CD16, CD54, and CD64
    (Spiekermann et al., 1994). Activation of platelets can also be
    assessed by flow cytometry (Tschöpe et al., 1990)  Complement

           The classic complement system consists of nine components (C1-9)
    and a series of regulatory proteins (C1 inhibitor, C4 binding protein,
    and properdin factors). Many biological activities important in the
    inflammatory response and in host resistance to infection occur at
    various points in the classical and alternative pathways of complement
    activation. Three clinical states should raise a suspicion of
    deficiency in a complement component: systemic lupus erythematosus,
    recurrent infections of the type seen in hypogammaglobulinaemia in
    patients with normal immunoglobulin levels, and severe  Neisseria
    infection. Laboratory measurement of serum haemolytic complement
    (CH50) is a useful test. Serum haemolytic complement is usually absent
    and rarely above 10% of the normal value in inherited complement
    deficiencies, with the exception of hypercatabolism of C3. More
    detailed analysis of the complement system requires functional and
    antigenic measurements of the individual components, usually best
    performed in laboratories that specialize in the complement system.

    5.6.6  Clinical chemistry

           Assessment of clinical abnormalities in standard serum
    chemistry, such as liver function, renal function, glucose, and
    albumin, is indicated to facilitate reasonable interpretation of
    specific changes in the immune system as secondary to another

    5.6.7  Additional confirmatory tests

           After activation, mononuclear cells from peripheral blood
    express the genes that encode a series of interleukins and colony-
    stimulating factors. Activated T cells and monocytes synthesize and
    secrete IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, interferon, other
    cytokines such as tumour necrosis factor, and cytokine receptors.
    These cytokines are involved in the growth and differentiation of
    T and B cells, eosinophils, and basophils. The supernatants of
    mononuclear cells from peripheral blood stimulated by soluble
    phytohaemagglutinin can be assessed for IL-2 by determining their
    capacity to stimulate 3H-thymidine uptake by mouse IL-2-dependent,
    cultured T-cell lines. Other biological assays, radioimmunoassays, and
    ELISAs have been developed to quantify the production of lymphokines
    and colony-stimulating factors. With molecular cloning techniques,
    messenger RNA transcription by each of the lymphokines can be
    quantified after appropriate lymphocyte activation. In the assessment

    of lymphokines, T cells or peripheral blood mononuclear cells are
    usually activated with concanavalin A, pokeweed mitogen, or
    insolubilized CD3 antibodies; then, the appropriate assays are
    performed to quantify the specific lymphokine(s) produced in the
    culture media. Different patterns of lymphokine secretion have been
    observed with different subsets of T cells: INF gamma and IL-2 are
    produced by the Th1 subset, while IL-4, IL-5, and IL-10 are produced
    by the Th2 subset, as originally documented for cloned CD T-cell lines
    from mice (Mosmann & Coffman, 1989). Thus, an assay of the pattern of
    lymphokine production could be used in pinpointing the action of an
    immunotoxicant on a particular subset of immune cells.


    6.1  Introduction

           Publications on immunotoxicology published by IPCS and the
    European Union (Berlin et al., 1987; Dayan et al., 1990), the United
    States Office of Technology Assessment (1991), and the United States
    National Research Council (1992) demonstrate the growing interest and
    concern within scientific and public communities on the capacity of
    environmental agents to perturb normal immune processes. The
    incorporation of experimental data on toxicant-induced alterations in
    the immune system into evaluations of drugs, chemicals, and biological
    agents for human risk assessment has thus become increasingly common.
    For example, in the United States, the Environmental Protection Agency
    (Sjoblad, 1988) and Food and Drug Administration (Hoyle & Cooper,
    1990; Hinton, 1992) indicate the benefits of testing the
    immunosuppressive potential of biochemical pest control agents,
    antiviral drugs, and food additives. Furthermore, the Environmental
    Protection Agency has established reference doses (an estimate of the
    daily exposure of the human population that is likely to have no
    appreciable risk of deleterious effects during a lifetime), on the
    basis of data on the immunotoxicity of several compounds, including
    1,1,2-trichloroethane, 2,4-dichlorophenol, and dibutyltin oxide. The
    United States Agency for Toxic Substances and Disease Registry has
    derived 'minimal risk levels' for arsenic, dieldrin, nickel,
    1,2-dichloroethane, and 2,4-dichlorophenol on the basis of immune

           Risk assessment is a process whereby relevant biological, dose-
    response, and exposure data for a particular agent are analysed in an
    attempt to establish qualitative and quantitative estimates of adverse
    outcomes (Scala, 1991). Such data are sometimes used in the
    development of standards for regulating the manufacture, use, and
    release of chemicals into the environment (Kimmel, 1990). As defined
    by the United States National Academy of Science (US National Research
    Council, 1983), risk assessment comprises four steps: hazard
    identification, dose-response assessment, exposure assessment, and
    risk characterization. The process of assessing the risk of both
    cancer and non-cancer end-points, including immunotoxicity, may be
    adapted to this form.

           The first step, hazard identification, involves a qualitative
    evaluation of available human and animal data to determine whether a
    chemical agent poses a potential hazard. Consideration is given to
    dose, route, and duration of exposure. The precise quantitative
    relationships between changes in immune function or in the
    histological appearance of lymphoid organs and host resistance to
    infectious agents or neoplastic diseases are unclear. It can be
    assumed that any significant difference from appropriate controls in

    the ability of the immune system to respond to a challenge may evolve
    into an adverse effect and thus present a potential hazard. This
    applies to adaptive as well as nonadaptive responses.

           After hazard identification, 'dose-response' is assessed. For
    non-cancer toxicity, a no-observed-adverse-effect-level (NOAEL) is
    established for an adverse response of interest. This process is no
    different in immunotoxicology and is the same as for the other target
    organ systems. The NOAEL value is either obtained from the dose-
    response curve or is estimated from the lowest-observed-adverse-
    effect-level (LOAEL). Once the NOAEL has been determined, safety and
    uncertainty factors can be applied to allow for various uncertainties,
    such as species or interspecies variability, irreversible effects, and
    chronic exposure. The use of safety factors, however, should be
    flexible and should allow incorporation of any relevant information on
    the mechanism of action of the chemical under review. Ideally,
    however, dose-response relationships should be established from human
    epidemiological data that include the exposure levels expected on the
    basis of human contact with the agent in the environment. As
    illustrated in an assessment of developmental toxicity (Kimmel &
    Gaylor, 1988), use of the risk assessment and management paradigm of
    the United States National Academy of Sciences (US National Research
    Council, 1983) to non-cancer end-points such as immunotoxicity, offers
    some serious challenges. For example, since the presence or absence of
    an effect is based upon whether a statistically and/or biologically
    significant response is observed at a certain dose or doses, and since
    multiple assays are routinely conducted, the NOAEL will depend heavily
    on the sensitivity of the assay. The differences may be particularly
    exaggerated when continuous responses (i.e. the results of most immune
    function tests) are compared with categorical data, the latter being
    routinely expressed as proportions. For example, in experimental
    animals, changes in many immune functions may be statistically
    significant when they vary by as little as 15-25% from the control
    values. In contrast, host resistance, when expressed categorically
    (e.g. tumour frequency), must change by 80% to reach a comparable
    degree of significance, assuming group sizes of about 15 animals and
    effective doses of 20% in the control group. Furthermore, immune tests
    are often, but not always, interdependent (Luster et al., 1992), and
    individual or combinations of tests might have to be ranked in order
    of sensitivity and degree of interdependence before dose-response is
    assessed. This has not been done in the past.

           Exposure assessment (step 3) is done in parallel with hazard
    identification and dose-response assessment (Scala, 1991). It often
    involves field measurements and other estimates of human exposure,
    such as the composition and size of the population, biological or
    clinical effects and types, and the magnitude, frequency, and duration
    of exposure to the agent. Many of these parameters are difficult to
    determine accurately in a longitudinal study; even measurements of
    body burden can be misleading since the concentrations at the target

    organ (e.g. lymphoid tissue) are not determined. The problems in
    immune testing in humans are similar to those in testing other organs
    and systems and include differences in individual responses due to
    unique sensitivities (e.g. age, pregnancy, genetics) and confounding
    factors (e.g. smoking, stress, drugs).

           Risk characterization (step 4) is the aggregation of the three
    previous processes. It provides an estimate of the incidence of
    adverse effects in a population and the potential health problems. As
    part of risk characterization, the strengths and weaknesses of each
    component of the assessment are considered, with assumptions,
    scientific judgements, and, to the extent possible, estimated
    uncertainties. Most assessments of the risks presented by chemical
    agents have focused on the estimated incidence of cancer after
    lifetime exposure to a chemical at some unit dose, assuming that there
    is essentially no threshold for carcinogenicity. The assessment of
    non-cancer end-points, such as disorders of the neurological,
    developmental, and reproductive systems, is somewhat similar to that
    of cancer, in that it involves calculations that include both
    assumptions and uncertainties. For example, considerations in risk
    assessment include ranking the value of epidemiological against
    experimental data, extrapolations from high to low doses, from
    subchronic to chronic exposure, and from animals to humans, and
    appropriate use of mechanistic and pharmacokinetic data. Data from
    immunotoxicology, like those from developmental toxicology (Schwetz &
    Tyl, 1987), do not easily lend themselves to the mathematical models
    used in cancer risk assessment, which usually involve non-threshold
    models for genotoxic carcinogens. For more accurate assessment,
    mechanistic models will be required which include the concept of
    'threshold'. It is assumed that threshold levels exist below which no
    adverse immunological effect can be demonstrated. Complex mixtures of
    chemicals, in which each chemical occurs at a subthreshold
    concentration, may reach or exceed a threshold for immunotoxicity
    (Germolec et al., 1989), although problems associated with mixtures of
    compounds are not unique to the field of immunotoxicology.

           The approaches currently used by the US Environmental Protection
    Agency (1986) in extrapolating the risk for developmental toxicity
    have been outlined, and similar guidelines have been developed by IPCS
    (1994b). One method is to apply uncertainty factors to the NOEL or
    NOAEL for the most sensitive animal species tested. The uncertainty
    factor is usually composed of a 10-fold factor to account for
    interspecies differences and a 10-fold factor for intraspecies
    variability. If no NOEL is available, an additional 10-fold factor may
    be applied to the lowest-observed-effect level (LOEL). Another
    approach is to calculate a margin of safety, which is the NOEL divided
    by the estimated level of human exposure from all potential sources.
    The margin of safety can then be evaluated for adequacy to protect
    human health. There are several drawbacks to both of these approaches,
    the primary one being that they use only one point on the dose-
    response curve (NOEL or LOEL) and ignore the rest of the data. Also,

    since the variability around the NOEL and LOEL is usually not taken
    into account, these approaches may rely on poor studies, i.e. studies
    that result in a higher NOEL because of their limited ability to
    detect small changes over the background.

           Since the purpose of risk assessment is to make inferences about
    potential risk to human health, the most appropriate data are those
    derived from studies of humans; however, adequate data are seldom
    available, and most risk assessments are based on results obtained in
    experimental animals. In order to use these results, a number of
    assumptions must be made about their relevance to potential human
    health risk. Firstly, it is assumed that experimental animals respond
    to the agent of interest in a pharmacological and toxicological manner
    similar to that anticipated in humans (i.e. the test animals and
    humans metabolize the chemical similarly and have identical responses
    and toxicity at the target organ). Secondly, the immune system of the
    experimental species must be very similar to that of humans: the
    vertebrate immune system is highly conserved among higher vertebrate
    species, and the immune components and their interactions in mice,
    rats, and humans are closely similar. Thus, if toxicokinetic
    properties are similar, it is reasonable to test for potential adverse
    effects in humans using laboratory rodents.

           As immunosuppressive agents cannot ethically be administered to
    humans, quantitative comparisons of dose-responses in humans and
    experimental animals are limited (although it is possible to do so in
    hypersensitivity tests). Nonetheless, controlled human exposures have
    been studied and the results compared with the immune effects observed
    in animals. As an example, the immune effects of cyclosporin A in
    various species are compiled in Table 12, which shows that the mouse
    is much less sensitive to cyclosporin A than other species, in
    particular the rat, and that humans are slightly more sensitive than
    other species (Dean & Thurmond, 1987); however, for the most part,
    there was good qualitative and quantitative agreement between the
    species examined. Selgrade et al. (1995) compared phagocytosis by
    human and murine alveolar macrophages after exposure to ozone
     in vitro and  in vivo (Table 13): The effects of ozone on alveolar
    macrophage function in murine species are predictive of effects that
    occur in humans, and the effects on macrophage phagocytosis seen
     in vitro are predictive of those that occur  in vivo. Quantitative
    comparisons have also been made in mice and humans for the ability of
    UVR to inhibit the contact hypersensitivity response (Table 14). As
    described in section 2.2.11, exposure to UVB inhibits delayed-type
    hypersensitivity (Kripke et al., 1979). Noonan & Hoffman (1994)
    described three strains of mice with low, intermediate, and high
    susceptibility to UVR-induced immunosuppression, and Oberhelman et al.
    (1992) reported that suppression of the hypersensitivity response also
    occurs in humans and that the dose of radiation required to induce 50%
    suppression in fair-and dark-skinned individuals is similar to that
    required to inhibit the response in mice with high and intermediate
    susceptibility, respectively.

    Table 12.  Comparison of doses of cyclosporin A that suppress
               the immune response in various species

    Species          Response                          Cyclosporin A
                                                        (mg/kg body

    Mouse            Antibody production                   50-300
                     Cell-mediated immunity                100-300
                       (delayed-type hypersensitivity)
                     Graft-versus-host reaction            50-250

    Rat              Antibody production                   20-50
                     Graft-versus-host reaction            10-60

    Guinea-pig       Cell-mediated immunity                10-100
                       (delayed-type hypersensitivity)

    Dog              Cell-mediated immunity                15-30
                       (delayed-type hypersensitivity)

    Rhesus monkey    Antibody production                   50-250

    Human            Cell-mediated immunity                10-20

    Adapted from Dean & Thurmond (1987); White et al. (1994)

           Data from immunotoxicology also differ from those for
    carcinogenicity and possibly other non-cancer end-points because the
    immune system contains components with overlapping functions. For
    example, when an individual is exposed to an infectious agent,
    multiple immune components may work either independently or in concert
    to help defend the host; i.e. there is redundancy between immune
    functions. Furthermore, while a significant change in any immune
    function can be considered potentially deleterious, in that it may
    increase the risk of developing clinical disease, a change in immune
    function does not necessarily precipitate a disease or clinical health
    affect. That is, immunocompromised individuals function normally in
    the absence of infectious agents. Thus, immune function reserve and
    redundancy are relative terms, depending on the dose of infectious
    agent. This complicates dose-response assessment, and models should be
    developed that incorporate available information on the quantitative
    relationship between immune function and clinical disease and
    potential redundancy.

    Table 13.  Effect of exposure to ozone on phagocytosis by alveolar

    Treatment                     Phagocytic index (no. fluorescent
                                  particles ingested/100 macrophages)
                                  Mice                     Humans
                                  ----------------         -------------
                                  Mean      SE             Mean      SE

    Air in vitro                  369.2     26.4           386.7     50.5
                                  (n = 6)                  (n = 6)

    Ozone in vitro                291.7     17.4*          275.0     45.1*
                                  (n = 6)                  (n = 6)

    Suppression                   21%                      29%

    Air in vivo                   330.6     10.4           714.9     46.1
                                  (n = 4)                  (n = 10)

    Ozone in vivoa                194.0     19.7*          539.2     22.3*

                                  (n = 4)                  (n = 10)

    Suppression                   42%                      25%

    Suppression  corrected        28%                      25%
    for dosimetric differenceb

    Adapted from Selgrade et al. (1995)
    *  Significantly different from air control (P < 0.05; Student's
        t test)
    a  Mice were exposed to 0.8 ppm for 3 h; humans were exposed to
       0.08 ppm for 6.6 h while undergoing intermittent exercise.
    b  On the basis of studies using 18O, alveolar macrophages of
       mice exposed to 0.8 ppm ozone for 3 h receive roughly 1.5 times
       more ozone than those of humans exposed to 0.08 ppm ozone for 6.6 h
       while exercising moderately.

    Table 14.  Comparison of doses of ultraviolet radiation that cause
               50% suppression of contact sensitivity in mice and humans

    Mousea                                            Humanb
    ---------------------------------------------     --------------

    Sensitivity           kJ/m2        mJ/cm2         Skin type

    High (C57Bl)          0.7-2.3      70-230         Fair      100

    Intermediate (C3H)    4.7-6.9      470-690        Dark      225

    Low (BALB/c)          9.6-12.3     960-1230

    Adapted from Selgrade et al. (1995)
    a  Data from Noonan & Hoffman (1994)
    b  Data from Oberhelman et al. (1992)

           The increasing evidence that environmental contaminants affect
    wildlife populations has led to risk assessment at the level of the
    ecosystem; however, the limited evidence for immunotoxic effects in
    wildlife precludes an adequate understanding of the risks posed by
    current levels of environmental pollution. The demonstration of
    immunosuppression in harbour seals fed herring from the contaminated
    Baltic Sea in a semi-field experiment (De Swart et al., 1994; Ross et
    al., 1995) provided a first indication that ambient levels of
    contaminants in certain areas present an immunotoxic risk to mammalian
    wildlife occupying a high trophic level. These results may partially
    explain the severity of a series of unrelated epizootic viral episodes
    in various marine mammalian populations in coastal areas of Europe and
    North America (Dietz et al., 1989; Van Bressem et al., 1991). In
    similar semi-field experiments in a bottom-dwelling fish species,
    flounder exposed to contaminated sediments had more viral lymphocytic
    infection and liver tumours than controls (Vethaak & Wester, 1993).
    While the difficulties in conducting adequate immunological studies
    with wildlife may preclude an approach as comprehensive as that in
    humans, such semi-field strategies may provide the best available

           Should the application of field studies be possible, correlative
    approaches to immunotoxicology may substantiate an effect on the
    ecosystem. Such an approach was used to establish a correlation
    between the induction of mixed-function oxidases and pollutant burden

    (as measured by the toxic equivalence of organochlorine chemicals) in
    cormorant  (Phalocrocorax carbo) chicks collected from various sites
    in the Netherlands (van den Berg et al., 1994). A combination of
    laboratory experiments under controlled conditions, semi-field
    experiments under controlled conditions with exposure to environmental
    mixtures of pollutants, and correlative field studies is necessary to
    understand immunotoxicity in wildlife populations.

    6.2  Complements to extrapolating experimental data

    6.2.1  In-vitro approaches

           The complexities of the immune system and the requirement of
    many agents for metabolism and distribution in order to produce an
    immunotoxic response have resulted in the almost exclusive use of
    animal models  in vivo for assessing immunotoxicity. Culture systems
    have been used extensively, however, to study the mechanisms by which
    agents induce immunosuppression. In-vitro test systems with immune
    cells of human origin are particularly attractive, given the
    uncertainties in extrapolating the results of studies in experimental
    animals to humans and the accessibility of human peripheral blood
    cells. Although many of the immune cells obtained from human blood are
    immature forms, the large numbers and diverse populations (i.e.
    polymorphonuclear granulocytes, monocytes, NK cells, T cells, and
    B cells) that can be obtained and the ease of conducting challenge
    assays  in vitro provide an attractive alternative (or, preferably,
    adjunct) to more conventional studies in animals. Surprisingly few
    laboratories have conducted studies with immunosuppressive agents in
    which immune function responses in human immune cells are compared
    with thosein rodents  in vivo (Cornacoff et al., 1988; Luo et al.,
    1992; Wood et al., 1992; Lang et al., 1993). Althoughstudies in human
    cells  in vitro have been hampered by a lack of assays to assess
    primary antigen-specific immune responses, a relatively good
    interspecies correlation has been observed in the limited responses
    examined. Furthermore, some of these assays have been successfully
    modified to include metabolic fractions of liver homogenates (Shand,
    1975) or co-culture with primary hepatocytes (Kim et al., 1987) to
    allow for chemical metabolism.

    6.2.2  Parallelograms

           Interpretation of studies in experimental animals or  in vitro
    can be improved when even limited data on human exposure  in vivo are
    available, using a 'parallelogram' approach. In general, if a
    parallelogram can be constructed in which data are available for five
    of the six angles (Figure 37), it may be easier to predict the outcome
    at the remaining angle, at least qualitatively. For example, cytokine
    and phagocytic responses of alveolar macrophages or pulmonary
    epithelial cells after exposure to ozone  in vitro can be compared
    with the responses to ozone after exposure of humans and animals

    FIGURE 37

     in vivo. If the in-vitro data prove to be predictive indicators of
    the in-vivo effects in humans, more weight can be given to in-vitro
    studies with similar agents or with compounds that are too toxic to be
    assessed in clinical studies but for which data are available on both
    animals  in vivo and animals and humans  in vitro. A similar
    approach can be used to establish the relationship between acute and
    subchronic effects as a means of extrapolating from acute effects in
    humans to chronic effects, for which few data are usually available.
    Another situation in which this approach may be applicable is in
    extrapolating deficits in immune function to increased susceptibility
    to disease in animal models, as a means of interpreting the risk of
    disease in humans, for whom data on immune function but not infectious
    disease may be available.

    6.2.3  Severe combined immunodeficient mice

           Another approach, which warrants further exploration, is the use
    of SCID mice grafted with human immune cells. This model is described
    in section 4.5.3. In short, SCID mice have been successfully
    grafted with human fetal lymphoid tissue in order to study human
    haematopoiesis (McCune et al., 1988) or with human peripheral blood
    lymphocytes, which allow production of human immunoglobulins, to study
    secondary antibody responses (Mosier, 1990). Reconstituted mice have
    been used to study autoimmunity and the efficacy of antiviral
    therapeutic agents. There are still limitations to the use of these
    animals for immunotoxicology (Pollock et al., 1994).

    6.3  Host resistance and clinical disease

           A major limitation in assessing the risk of immunotoxicity is
    the difficulty in establishing quantitative relationships between
    immunosuppression and clinical diseases. The diseases are usually
    manifested as increases in the frequency, duration, or severity of
    infections, increased incidences of certain cancers, such as Kaposi's
    sarcoma and non-Hodgkin's lymphoma (malignancies often observed in
    immunosuppressed individuals), or an increased incidence of autoimmune

           Despite overwhelming experimental and clinical evidence that
    increases in the incidences of neoplastic and/or infectious diseases
    occur in animals and individuals with secondary immunodeficiency
    (Austin et al., 1989; Ehrke et al., 1989), neither the most
    appropriate immune end-points for predicting clinical disease nor the
    quantitative relationship between changes in immune function and
    impairment of host defence are clearly defined. For example, it would
    be useful to determine whether certain immune end-points (or quantity
    of changes) predict certain outcomes (e.g. increased susceptibility to
    influenza and decreased antibody responses). A better understanding of
    these relationships would be particularly beneficial for risk
    assessment, since changes in immune function are more readily
    quantifiable in populations at risk than are changes in the frequency

    or severity of infections. A particularly relevant question for risk
    estimation is whether increases in host susceptibility to challenge
    agents follow linear or 'threshold-like' models as a function of
    increased immunosuppression. While terms such as 'immune reserve' and
    'immunological redundancy' are applicable for individual responses, it
    is unclear how they would be applied to large populations. Since the
    potential outcomes of immunosuppression are increases in infections or
    neoplastic diseases and there is already a background incidence of
    these diseases in the population (Centers for Disease Control, 1991),
    it would be helpful to determine the additional frequency of disease
    that is associated with increased loss of immune function. Qualitative
    relationships are well established, but the quantitative relationship
    between immune function and clinical disease in humans has proved
    difficult to explore, owing, in part, to the complexity of the immune
    system, overlapping (i.e. redundant) immune responses, and variability
    in the virulence of infectious agents. Nonetheless, several studies
    have shown quantitative relationships in humans. For example, in
    longitudinal studies of a relatively large population, asymptomatic
    individuals with low NK cell activity were found to be at risk for
    developing upper respiratory infections (Levy et al., 1991). Larger
    population studies have been conducted in AIDS patients, as the
    depletion of CD4+ cells following HIV-1 infection is a clinical
    hallmark of the disease. The normal human range of CD4+ cells is
    800-1200 cells/µl, but this level generally declines to less than 500
    cells/µl within three to four years after HIV-1 infection and to 200
    cells/µl before overt opportunistic infections are seen (Masur et al.,
    1989; Phair et al., 1990). It has also been shown in seropositive
    individuals that a drop in CD4+ cells by 7% or more in a year
    increases the relative risk of developing AIDS (Burcham et al., 1991).

           Because of the uncertainties about the quantitative relationship
    between immune function and disease, there has been continuing
    interest in developing sensitive, reproducible experimental models of
    host resistance to define altered immune function after exposure to
    environmental agents. Most of these models were developed in mice and,
    to a lesser extent, in rats; they include bacterial, viral, protozoan,
    fungal, and syngeneic or semisyngeneic transplantable tumour cell
    models. Although the target organs and general host defence activities
    have been defined for most of these models, multiple immune and
    nonimmune, mechanisms are involved in resistance, making it difficult
    to determine the exact defect without assessing immune function
    responses to the challenge agent. For example, defence against
    extracellular organisms involves the interactions of T lymphocytes,
    B lymphocytes, macrophages, and polymorphonuclear granulocytes, in
    addition to a variety of cell-secreted products, whereas resistance to
    generalized infection from intracellular pathogens and neoplastic
    diseases is likely to involve macrophages, NK cells, and T cell-
    mediated immune processes.

           Although many host resistance assays are relatively simple to
    perform, they normally require large numbers of animals and appear to
    be less sensitive than immune function tests (Luster et al., 1993).
    Other studies have shown that host resistance assays are more
    sensitive than immune function tests (Vos et al., 1991; Burleson et
    al., in press). The dose of challenge agent used in experimental
    studies is important, since too low or too high a dose will not allow
    detection of changes in immunocompromised groups in comparison with
    controls (Selgrade et al., 1982; Luster et al., 1993). The sensitivity
    of a host resistance assay also depends on the end-point measured. For
    example, tests involving survival or tumour models (i.e. dichotomous)
    are by nature less sensitive than those with end-points that provide
    continuous data, such as enumeration of tumours, bacteria, or soluble
    immune activation markers, and several end-points in one model of
    infection, such as  T. spiralis. This is attributable partly to
    differences in the types of statistical analyses used to establish
    group differences. With dichotomous data, two approaches can be used.
    Some laboratories use a challenge dose that produces a response in
    10-30% of animals in the control group. An alternative is to use a
    dose slightly below that which would induce the desired response in
    any of the animals in the control group. The latter design increases
    the statistical power of data analysis. In most cases, extreme
    accuracy is needed in the delivery of the agent, to ensure that the
    administered dose of agent is only slightly smaller than that which
    will give the desired response. In either approach, statistical
    significance is heavily dependent on the dose of challenge agent and
    the number of animals in each treatment group: Table 15 shows that
    doubling the number of animals in a study greatly increases the
    ability to detect a significant change. Even more obvious is the
    increased ability to detect significant differences when the dose of
    the agent in the control group is lowered to a subclinical
    concentration. These hypothetical values demonstrate how statistically
    significant changes can be obtained in susceptibility assays by
    modifying the experimental design. In the first design, an effective
    dose of 30% is used in the control group (i.e. a concentration of
    agent that produces a response in 30% of normal animals) with 15
    animals per treatment group. In the second design, increasing the
    group size to 30 allows for even greater statistical significance. In
    the third design, a challenge inoculation is given which produces no
    effect in the control group (effective dose, 0), allowing for greater
    statistical significance.

           Two variables that influence the quantitative relationship
    between immune function and disease are the virulence and amount of
    the infectious agent. These remain a constant in most experimental
    studies but may vary between experiments as well as in the human
    population. For example, in the general population one can assume that
    an infectious disease such as influenza can develop in any individual,
    independently of their immune capacity or prior immunization, provided
    that the virulence or quantity of the challenging agent is sufficient
    to overwhelm the individual's defensive capacities. In Figure 38,

    FIGURE 38

        Table 15.  Chi-squared values (hypothetical)

    Treatment      Design 1                           Design 2                           Design 3
                   -------------------------------    -------------------------------    --------------------------------
                   No. affected/       One-tailed     No. affected/       One-tailed     No. affected/       One-tailed
                   no. tested          P value        no. tested          P value        no. tested          P value

    Control        3/15                -              6/30                -              0/15                -
    Dose 1         4//15               0.532          8/30                0.428          1/15                0.516
         2         5/15                0.411          10/30               0.273          2/15                0.274
         3         6/15                0.312          12/30               0.165          3/15                0.150
         4         7/15                0.233          14/30               0.096          4/15                0.084
         5         8/15                0.173          16/30               0.055          5/15                0.048*
         6         9/15                0.128          18/30               0.031*         6/15                0.028*
         7         10/15               0.094          20/30               0.017*         7/15                0.017*
         8         11/15               0.069          22/30               0.009*         8/15                0.010*
         9         12/15               0.051          24/30               0.005*         9/15                0.006*
        10         13/15               0.038*         26/30               0.003*         10/15               0.004*
        11         14/15               0.028*         28/30               0.002*         11/15               0.002*
        12         15/15               0.021*         30/30               0.001*         12/15               0.002*
        groups of mice pretreated with either vehicle (saline), 50 mg/kg
    cyclophosphamide (causing minimal immunosuppression), or 200 mg/kg
    cyclophosphamide (causing severe immunosuppression) were administered
    various numbers of PYB6 tumour cells. Even vehicle-treated mice
    developed a high frequency of tumours, provided that the challenge was
    sufficiently high (i.e. 8 × 104 tumour cells). In contrast, severely
    immunosuppressed mice (high dose of cyclophosphamide) developed an
    increased tumour frequency at all challenge levels of PYB6 tumour
    cells. The groups treated with the low dose of cyclophosphamide were
    of particular interest, since evidence of increased susceptibility
    appeared but only as a function of the tumour cell concentration.
    Assuming that these observations are applicable to human populations,
    even small changes in immune function could increase the likelihood of

          As indicated earlier, while experimental data have been used
    occasionally in risk assessment, most immunotoxicological data have
    focused on hazard identification. Although comparative data on the
    effects of specific immunotoxic agents in humans and animals are
    limited, other factors contribute to the minimal use of these data in
    risk assessment, including the concern that immunotoxicity testing has
    often been conducted without full knowledge of its predictive value in
    humans or its quantitative relationship to immune-mediated diseases.

          Luster et al. (1988) reported on the design and content of a
    screening battery involving a tier approach for detecting potential
    immunosuppressive compounds in mice. This battery has been used to
    examine a variety of compounds, and the database, generated on over 50
    compounds, has been analysed in an attempt to improve the accuracy and
    efficiency of tests for screening chemicals for immunosuppression and
    to identify better those tests that predict experimentally induced
    immune-mediated diseases (Luster et al., 1992, 1993). Specifically,
    attempts have been made to develop a 'streamlined' test configuration
    for accurately predicting immunotoxic agents and to establish models
    that could be used to provide insight into the qualitative and
    quantitative relationships between the immune and host resistance
    assays commonly used to examine potential immunotoxic chemicals in
    experimental animals. While the analyses had a number of limitations,
    several conclusions can be drawn from the results:

          (1) With this particular testing configuration, examination of
    only two or three immune parameters was needed in order to identify
    potential immunotoxicants. Lymphocyte enumeration and quantification
    of the T cell-dependent antibody response appeared to be particularly
    useful. Furthermore, some commonly employed measures (e.g. leukocyte
    counts, lymphoid organ weights), while probably good predictors of
    immunotoxicity, are apparently not as sensitive as the other tests.
    Obviously, inclusion of additional tests that are not part of the
    original battery may improve the prediction of immunotoxicity.

          (2) A good correlation was found between changes in immune tests
    and altered host resistance, in that there was no instance in which
    host resistance was altered without a significant change in the immune
    test(s). In many instances, however, immune changes were observed in
    the absence of detectable changes in host resistance (Table 16),
    indicating that immune tests are generally more sensitive than host
    resistance assays.

          (3) No single immune test could be considered highly predictive
    for altered host resistance; however, many of the tests were good
    indicators, while others, such as leukocyte counts and proliferative
    response to lipopolysaccharide, were relatively poor indicators of a
    change in host resistance. Some of the tests that showed the highest
    association with host resistance were those described previously as
    the best indicators of immunotoxicity, such as the plaque-forming
    assay and surface markers, but also included tests such as delayed-
    type hypersensitivity and thymic weight.

          (4) Regression modelling, using a large data set on one chemical
    agent, indicated that most, but not all, of the immune function-host
    resistance relationships follow a linear model. It was not possible,
    however, to establish linear or threshold models for most of the
    chemicals studied when the data from all 50 chemicals were combined;
    thus, a more mechanistically based mathematical model will have to be
    developed. A similar conclusion was drawn on the basis of a limited
    data set collected by the Environmental Protection Agency (Selgrade et
    al., 1992), in which changes in NK cell activity were correlated with
    changes in susceptibility to cytomegalovirus in a murine model. It is
    impossible, at present, to determine how applicable these analyses
    will be for immunotoxic compounds with different immune profiles;
    however, as more analyses become available, the ability to estimate
    potential clinical effects accurately from the results of
    immunological tests should increase.

        Table 16.  Association between the results of host resistance models and immune tests

    Challenge                       No. of       Frequencya
    agent                           tests                                                     
                                                 Specificity    Sensitivity    Concordance
                                                 (-/-)          (+/+)          (Total)

    Listeria monocytogenes          34           100            52             65**
    PYB6 tumour                     24           100            39             54
    Streptococcus pneumoniae        19           100            38             58
    B16F10 melanoma                 19           100            40             68
    Plasmodium yoelii               11           100            38             55
    Influenza                       9            100            17             44

    Any of the aboveb               46           100            68             78*

    From Luster et al. (1993)
    *  Agreement statistically significant at P < 0.05
    a  Frequency is defined as: specificity, the percentage of non-immunotoxic
       chemicals with no effect on the host resistance models; sensitivity,
       the percentage of potentially immunotoxic chemicals causing a change
       in a host resistance model; concordance, percentage of qualitative agreement
    b  Frequency calculated on the basis of all of the host resistance models used to
       study an agent

     Accessory cell. Passenger cell (leukocyte, mainly monocytes) or
    stationary cell (reticulum cell, epithelial cell, endothelial cell)
    that aids T or B lymphocytes in inducing immunological reactions,
    either by direct contact or by releasing factors; normally expresses
    MHC class II molecules

     Acquired immunity. A state of protection against pathogen-induced
    injury, with rapid immune elimination of pathogenic invaders; due to
    previous immunization or vaccination

     Activation. The process of going from a resting or inactive state to
    a functionally active state, of leukocytes (lymphocytes, monocytes) or
    proteins (complement, coagulation)

     Acute-phase protein. Non-antibody humoral factor that emerges in
    increasing amounts in the circulation shortly after induction of an
    inflammatory response; e.g. alpha 2-macroglobulin, C-reactive protein,
    fibrinogen, alpha 1-antitrypsin, and complement components

     Adaptive immunity. A state of specific acquired protection against
    pathogenic invaders, induced by immunization or vaccination

     Addressin. Receptor for lymphocytes on endothelial cells of venules,
    involved in homing of cells in lymphoid tissue; belongs to the
    immunoglobulin gene superfamily, integrins, and selectins

     Adenoid. See  Tonsil

     Adhesion receptors Molecule involved in cellular adhesion between
    passenger cells and the extracellular stationary matrix (endothelium,
    connective tissue); comprises three main families; member of the
    immunoglobulin gene superfamily, integrins, and selectins

     Adjuvant. Material that enhances an immune response

     Adoptive immunity or tolerance. Transfer of a state of immunity or
    tolerance via cells or serum from an immune or tolerant individual to
    a naive individual

     Affinity. Binding strength of an antibody-combining site to an
    antigenic determinant (epitope); expressed as an association constant

     Agglutination. Process of aggregation of visible antigenic particles
    (e.g. erythrocytes) mediated by antibodies directed towards the

     Allele. One or more genes at the same chromosomal locus which
    control alternative forms (phenotypes) of a particular inherited

     Allergen. Antigen that induces an allergic or hypersensitivity
    reaction, resulting in immune-mediated or nonimmune-mediated tissue
    damage; restricted mainly to immediate hypersensitivity or
    anaphylactic reactions

     Allergy. State of altered immunity, resulting in hypersensitivity
    reaction on contact with antigen or allergen; often restricted to
    immediate hypersensitivity or anaphylaxis

     Alloantigen. Antigen that differs between different (not inbred)
    individuals within one species

     Allogeneic. Genetically different phenotypes in different (not
    inbred) individuals within one species; opposite of isogeneic, or

     Allotype. Genetically different antigenic determinants on protein of
    (not inbred) individuals within one species

     alpha Chain. First chain of a multimeric receptor molecule: in
    immunoglobulin molecules, the a heavy chain forming the IgA class; in
    T-cell receptor molecules, one of the chains forming the dimeric
    alpha-ß receptor molecule; in MHC class I molecules, the main
    polypeptide chain associated with the ß2-microglobulin molecule; in
    MHC class II molecules, one of the chains in the dimeric molecule

     Alternative pathway of complement activation. Activation of
    complement pathway by substances other than antigen-antibody
    complexes; involves factor B, properdin, and complement component C3

     Anaphylatoxin. Activated components of complement components C3 and
    C5 (C3a and C5a, respectively), which induce anaphylactic reactions by
    activating mast cells and basophilic granulocytes

     Anaphylaxis or anaphylactic reaction. Local or systemic immediate
    hypersensitivity reaction initiated by mediators released after
    immunological stimulation; symptoms can be a drop in blood pressure
    related to vascular permeability and vascular dilatation, and
    obstruction of airways related to smooth muscle contraction or

     Anergy. State of unresponsiveness to antigenic stimulation, due to
    the absence of responding elements or the loss of capacity of existing
    elements to mount a reaction; synonym for tolerance

     Antibody. Immunoglobulin molecule produced in response to immunization
    or sensitization, which specifically reacts with antigen  Antibody-dependent
    cell-mediated cytotoxicity. Cytotoxic reaction in which an antibody forms
    the bridge between the cytotoxic cell (lymphocyte, macrophage) and the
    target cell

     Antigen. Any substance that induces a specific immunological

     Antigen-binding site (paratope). Part on an antibody molecule that
    binds antigen (antigenic determinant, or epitope); part of the T-cell
    receptor that binds the complex of antigen and MHC molecule

     Antigenic determinant (epitope). Part of an antigen that binds to
    antibody or T-cell receptor (the latter in combination with MHC

     Antigenicity. Capacity to react with components of the specific
    immune system (antibody, receptors on lymphocytes)

     Antigen presentation. Process of enabling lymphocytes to recognize
    antigen on a specific receptor on the cell surface. For presentation
    to T lymphocytes, includes intracellular processing and complexing of
    processed peptides with MHC molecule on the cell membrane of the
    antigen-presenting cell. For presentation to B lymphocytes, can
    include formation of immune complexes (in germinal centres)

     Antigen-presenting cell. Cell that presents antigen to lymphocytes,
    making possible specific recognition by receptors on the cell surface.
    In a more restricted way, used to describe MHC class II-positive
    (accessory) cells which can present (processed) antigenic peptides
    complexed with MHC class II molecules to T helper-inducer lymphocytes.
    Includes macrophage populations (in particular, Langerhans cells and
    dendritic or interdigitating cells), B lymphocytes, activated T
    lymphocytes, certain epithelia (after MHC class II antigen induction
    by e.g. interferon gamma); others are follicular dendritic cells, not
    of bone-marrow origin, which present antigen in the form of immune
    complexes to B cells in germinal centres of peripheral lymphoid
    tissue; marginal zone macrophages in splenic marginal zone, which
    present antigen, without contact with T helper cells, to B cells at
    this location (T cell-independent response, e.g. to bacterial
    polysaccharide). In rodent skin epidermis, a dendritic epidermal cell,
    of T-cell origin, has an antigen-presenting function.

     Antigen receptor. Multichain molecule on lymphocytes, to which
    antigens bind. For B lymphocytes, an immunoglobulin molecule that
    recognizes nominal antigen; for T lymphocytes, a T-cell receptor
    molecule that recognizes antigenic peptide in combination with the
    polymorphic determinant of an MHC molecule

     Antinuclear antibody. Antibody directed to nuclear antigen; can have
    various specificities (e.g. to single- or double-stranded DNA or
    histone proteins); frequently observed in patients with rheumatoid
    arthritis, scleroderma, Sjögren's syndrome, systemic lupus
    erythematosus, and mixed connective tissue disease. Also called
    antinuclear factor

     Antiserum. Serum from an individual that contains antibodies to a
    given antigen

     Aplasia. Absence of tissue structure or cellular component, either
    congenital or acquired

     Apoptosis (programmed cell death). Process whereby the cell kills
    itself after activation, by Ca2+-dependent endonuclease-induced
    chromosomal fragmentation into fragments of about 200 base-pairs

     Appendix. Lymphoid organ in the gastrointestinal tract, at the
    junction of ileum and caecum; forms part of gut-associated lymphoid

     Arthus reaction. Inflammatory response, generally in skin, induced
    by immune complexes formed after injection of antigen into an
    individual that contains antibodies

     Asthma. Chronic inflammatory disease characterized by bronchial
    hyperresponsiveness to various stimuli

     Atopy. In general terms, 'unwanted reactivity'; used mostly to
    describe the state of general systemic or local hypersensitivity
    reactions related to genetic predisposition

     Auto-antibody. Antibody to component in the individual itself

     Auto-antigen. Antigen to which an autoimmune reaction is directed

     Autoimmunity. A state of immune reactivity towards self

     Autologous. Derived from self; components of an immunological
    reaction (e.g. antibody, lymphocytes, grafted tissue) from the same
    individual; opposite of heterologous

     Avidity. Binding strength between antibody and antigen, or receptor
    and ligand; for antibody, represents the product of more than one
    interaction between antigen-binding site and antigenic determinant

     Bacteraemia. Presence of bacteria in blood

     Basophilic granulocyte. Polymorphonuclear leukocyte that contains
    granules with acid glycoproteins stained by basic (blue) dyes; after
    release, glycoproteins induce anaphylactic reactions

     B-Cell growth and differentiation factor, B-cell growth factor, and
     B-cell stimulating factor. See  Interleukin-4 and -5

     ß Chain. In T-cell receptor molecules, one of the chains forming the
    dimeric alpha-ß receptor molecule; in MHC class II molecules, one of
    the chains in the dimeric molecule

     ß2-Microglobulin. A peptide of 12 kDa, which forms part of MHC class
    I molecule

     B Lymphocyte or cell. Lymphocytes that recognize nominal antigen by
    immunoglobulin (antibody) surface receptor (on virgin B cell, IgM and
    IgD) and, after activation, proliferate and differentiate into
    antibody-producing plasma cells. During a T-dependent process, there
    is immunoglobulin class switch (IgM into IgG, IgA, IgD, or IgE), with
    maintenance of the antigen-combining structure. For T-independent
    antigens, cells differentiate only in IgM-producing plasma cells. B
    Lymphocytes originate from precursor cells in bone marrow; in avian
    species, they undergo maturation in the bursa of Fabricius (B, bursa-
    dependent); in mammals, in the bone marrow

     B Lymphocyte area. That part of an lymphoid organ or tissue that is
    occupied by B lymphocytes, e.g. follicles in peripheral lymphoid
    tissue, marginal zone in spleen

     Birbeck granule. Rod-shaped structure with rounded end,
    approximately 6 nm thick, found in the cytoplasm of Langerhans cells
    in the epidermis, and interdigitating dendritic cells in T-lymphocyte
    area of lymphoid tissues

     Blast cell. Large cell (about 15 µm or more) with dispersed nuclear
    chromatin and cytoplasm rich in ribosomes; in an active stage of the
    cell cycle before mitosis

     Blast transformation. Process of activation of lymphocytes into cell
    cycle and to form blastoid cells before mitosis

     Blocking antibody. Antibody that can interfere with another antibody
    or with reactive cells in binding antigen, thereby preventing effector
    reactions (often used in association with an allergic reaction or
    tissue damage)

     Bone marrow. Soft tissue in hollow bones, containing haematopoietic
    stem cells and precursor cells of all blood cell subpopulations
    (primary lymphoid organ); major site of plasma cell and antibody
    production (secondary lymphoid organ)

     Booster. Dose of antigen given after immunization or sensitization
    to evoke a secondary response

     Bradykinin. Peptide of nine amino acids split from alpha 2-
    macroglobulin by the enzyme kallikrein; causes contraction of smooth

     Bronchus-associated lymphoid tissue. Lymphoid tissue located along
    the bronchi, considered to represent the location of presentation of
    antigens entering the airways; contributes to mucosa-associated
    lymphoid tissue

     Bursa equivalent. Site where B-cell precursors undergo maturation
    into immunocompetent cells in non-avian species; bone marrow in adult

     Bursa of Fabricius. Primary lymphoid organ in avian species, located
    in cloaca, with an epithelial reticulum, where precursors of B
    lymphocytes from the bone marrow undergo maturation into
    immunocompetent cells and then move to peripheral lymphoid organs

     C (constant) gene. Gene that encodes  the constant part of 
    immunoglobulin chains or T-cell receptor chains (e.g. Cµ, Cdelta
    for immunoglobulin heavy chains, Ckappa for immunoglobulin kappa
    light chain, Calpha for T-cell receptor alpha chain)

     C (constant) region. Region at carboxy terminal of immunoglobulin
    chains or T-cell receptor chains, identical for a given immunoglobulin
    class or subclass or for a given T-cell receptor chain; encoded by C
    genes in DNA

     Cachectin. See  Tumour necrosis factor

     CD (cluster of differentiation). Group of (monoclonal mouse)
    antibodies that react to identical leukocyte surface molecules in
    humans (but not necessarily to identical epitopes), on the basis of
    comparative evaluations during international workshops and transferred
    to other species by analogy. Does not include MHC or immunoglobulin

     CD3 molecule. Molecule consisting of at least four invariant
    polypeptide chains, present on the surface of T lymphocytes associated
    with the T-cell receptor; mediates transmembrane signalling (tyrosine
    phosphorylation) after antigen binding to T-cell receptor

     CD4 molecule. Glycoprotein of 55 kDa on the surface of T lymphocytes
    and part of monocytes and macrophages. On mature T cells, restricted
    to T helper (inducer) cells; has an accessory function to antigen
    binding by T-cell receptors, by binding to a non-polymorphic
    determinant of MHC class II molecule

     CD8 molecule. Complex of dimers or higher multimers of 32-34 kDa
    glycosated polypeptides linked by disulfide bridges, on the surface of
    T lymphocytes. On mature T cells, the presence is restricted to
    T cytotoxic-suppressor cells; has an accessory function to antigen
    binding by the T-cell receptor, by binding to a non-polymorphic
    determinant of MHC class I molecule

     Cell-mediated immunity. Immunological reactivity mediated by
    T lymphocytes

     Central (primary) lymphoid organ. Lymphoid organ in which precursor
    lymphocytes differentiate and proliferate in close contact with the
    microenvironment, to form immunocompetent cells; not antigen-driven
    but can be influenced by mediators produced as a result of antigen

     Centroblast. Intermediately differentiated B lymphocyte present in
    germinal centres of follicles in lymphoid tissue; a medium to large,
    12-18-µm cell, with a round to ovoid nucleus that has moderately
    condensed heterochromatin and medium-sized nucleoli close to the
    nuclear membrane, medium-sized to broad cytoplasm containing many
    polyribosomes and a variable amount of rough endoplasmic reticulum

     Centrocyte. Intermediately differentiated B lymphocyte present in
    germinal centres of follicles in lymphoid tissue; medium-sized,
    8-12-µm lymphoid cell with irregular nucleus with condensed
    heterochromatin; small cytoplasm containing a few organelles

     CH50. The amount of serum (or dilution of serum) that is required to
    lyse 50% of erythrocytes in a standard haemolytic complement assay

     Chemiluminescence. Luminescence produced by direct transformation of
    chemical energy into light energy

     Chemotactic factor. Substance that attracts cells to inflammatory

     Chemotaxis. Process of attracting cells to a given location, where
    they contribute to an inflammatory lesion

     Class I MHC molecule. Molecule coded by the A, B, or C locus in the
    HLA complex, the K and D locus in the mouse H2 complex, and less well
    defined MHC I gene loci in other species, in association with the
    ß2-microglobulin molecule. Two-chain molecule occurring on all
    nucleated cells, without allelic exclusion

     Class II MHC molecule. Molecule coded by the D (DR, DP, DQ) locus in
    the HLA complex, the I-A and I-E locus in the mouse H2 complex, and
    less well defined MHC II gene loci in other species, comprising an
    alpha and a ß chain (intracellular, associated with an 'invariant'
    chain). Two-chain molecule occurring, without allelic exclusion, on B
    lymphocytes, activated T lymphocytes, monocytes-macrophages,
    interdigitating dendritic cells, some epithelial and endothelial cells
    (variable, dependent on species and state of activation); antigen-
    presenting cells

     Class switch. See  Immunoglobulin class switch

     Classical pathway of complement activation. Activation of complement
    pathway by antigen-antibody complexes, starting with complement
    component C1 and ending with complement component C3

     Clone. Population of cells that emerge from a single precursor cell;
    within T or B lymphocytes, cells with a fixed rearrangement of genes
    coding for T-cell receptor or immunoglobulin

     Clonal expansion. Proliferation of cells that have a genetically
    identical constitution; when uncontrolled, may result in tumour

     Colony-stimulating factor. Substance that supports clonal cell
    growth of haematopoietic cells

     Complement. Group of about 20 proteinase precursors that activate
    and split each other, in sequential order. The various components are
    present in inactive (precursor) form, except for C3, which in a normal
    state shows a low turnover (major split products C3a and C3b). The
    split products are either bound to the activating substance (immune
    complex or antibody-coated cell) or are released as active mediators.
    The classical cascade starts by activation of component C1,
    subsequently C4, C2, and C3, and is initiated by (IgG/IgM) immune
    complexes. The alternative cascade starts by activation of C3b and
    factor B, subsequently factors D and C3, and is initiated by nonimmune
    specific activators like microbial polysaccharides and some
    'allergens'. C3 split products (C3b) activate the amplification loop,
    in which factors D and B are also used, activate C5, and thereafter
    the terminal cascade C6, C7, C8, and C9, which attack the cell
    membrane and kill the cell (microorganism). The cascade is under the
    control of various inhibitors. Major effects of complement split
    products are adherence to receptors on phagocytes (C3b, C3d); mediator
    activity, like chemoattraction of inflammatory cells, vasodilatation,
    increased vascular permeability, and smooth muscle contraction
    (C3a, C5a); cell lysis by membrane lesions (C6-C9)

     Complement fixation. Binding and consumption of complement by
    antigen-antibody complexes; often used in association with assays for
    complement activity

     Complement receptor (CR). Cell surface molecule that can bind
    activated complement components in e.g. antigen-antibody complexes;
    CR1 (CD35) is a receptor for C3b, present on B lymphocytes, monocytes
    and macrophages, granulocytes, and erythrocytes; CR2 (CD21) is a
    receptor for C3d, present on mature B lymphocytes; CR3 (CD11b/CD18) is
    a receptor for C3b present on macrophages, granulocytes, natural
    killer cells, and a subset of CD5+ B lymphocytes; CR4 (CD11c/CD18)
    is a receptor for C3b present on monocytes, macrophages, granulocytes,
    and natural killer cells

     Contact sensitivity. Hypersensitivity reaction evoked in skin by
    placing sensitizing agents or substances on skin

     Cords of Billroth. Medullary cords in spleen

     Corona. See  Mantle

     Cortex. Outer parenchymal layer of organs

     Cross-reactivity. Reactivity of antigen-specific elements (T lymphocytes
    sensitized by T-cell receptor, B lymphocytes by antibody; antibody
    molecules) towards antigens other than those used in original
    sensitization, owing to shared antigenic epitopes on different
    antigenic molecules; also used to describe reactions towards antigenic
    determinants other than those originally used in sensitization, due to
    similarities in structure

     Cytokine. Biologically active peptide, synthesized mainly by
    lymphocytes (lymphokines) or monocytes and macrophages (monokines);
    modulates the function of cells in immunological reactions; include
    interleukins. Some (pleotrophic cytokines) have a broad spectrum of
    biological action, including neuromodulation, growth factor activity,
    and proinflammatory activity

     Cytokine receptor. Ligand for cytokine on target cell, acts in
    signal transduction through the cell membrane; many are multichain
    molecules belonging to different receptor families

     Cytolytic antibody. Antibody that can mediate lysis of the target to
    which it is directed, either in combination with complement or as
    bridge between cytotoxic cell and target

     Cytotoxic cell (killer cell). Effector T cell, natural killer cell,
    or activated macrophage; kills target cells and tissue extracellularly
    after binding; mediated by release of substances from cytolytic
    granules (including serine esterase, cytolysin, and perforins)

     Cytotoxic reaction. Effector reaction of antibody or cells, resulting
    in lysis of target cell or tissue

     Cytotoxic T lymphocyte. Subpopulation of T lymphocytes with CD8
    phenotype; after recognition of antigen in an MHC class I-restricted
    manner, differentiates from precursor to effector cytotoxic cell and
    subsequently kills target cells

     Degranulation. Process of fusion of cytoplasmic granules with cell
    membrane, whereby the content of the granules is released into the
    extracellular space; mainly used in association with immediate
    hypersensitivity reactions

     Delayed-type hypersensitivity. Inflammatory lesion mediated by
    effector T lymphocytes or their products, with attraction mainly of
    macrophages towards the inflammatory lesion. Term originates from the
    classical skin reaction after challenge of a sensitized individual;
    maximal (wheal and flare) response reached within 24-72 h

     Delayed-type hypersensitivity T cell. Subpopulation of T lymphocytes
    with CD4 phenotype; after recognition of antigen in an MHC class 
    II-restricted manner, secretes mediators involved in inflammatory
    responses, e.g. INF gamma and tumour necrosis factor

     delta Chain. In immunoglobulin molecules, delta heavy chain forming
    the IgD immunoglobulin class; in T-cell receptor molecules, one of the
    chains forming the dimeric gamma-delta receptor molecule; one of the
    chains in the CD3 molecule associated with the T-cell receptor

     Dendritic cell. Cell in tissue that shows elongations or protrusions
    of cytoplasm into the parenchyma. Often used in a restricted manner to
    designate a type of antigen-presenting cell, of which two categories
    exist: one of bone-marrow origin belonging to the macrophage lineage,
    including Langerhans cells in skin and interdigitating dendritic cells
    in T-cell areas of lymphoid tissue and a very small leukocyte
    population in blood; the second of tissue parenchymal origin
    (presumably pericytes around blood vessels), the follicular dendritic
    cells in B-cell areas (follicles) of lymphoid tissue

     Dendritic epidermal cell. T Cell in the epidermis that has dendritic
    morphology; has antigen-presenting function but is not a (macrophage-
    related) Langerhans cell. Occurs in rodents but not in humans;
    contributes to skin immune system

     Dermal immune system. See  Skin immune system

     Desensitization. Induction of anergy or tolerance to allergic
    substances by active intervention in immune reactivity (exhaustion of
    reactive elements or induction of blocking phenomena)

     Diapedesis. Passage of cells through blood vessel walls into tissue
    parenchyma, mediated by constriction of endothelial cells

     D (diversity) gene. Gene that encodes the variable part of
    immunoglobulin heavy chains, or T-cell receptor alpha, ß, or gamma
    chain (e.g. DH1-DHn for immunoglobulin heavy chains, Ddelta
    1-Vdelta n for T-cell receptor alpha or delta chain)

     Domain. Part of polypeptide chain folded to a relatively rigid
    globular tertiary structure fixed by disulfide bonds. Molecules of the
    immunoglobulin gene superfamily have a tertiary domain-like structure:
    each domain is about 110 amino acids long and arranged in a sandwich
    of two sheets of anti-parallel ß strands. See also  Homologous,

     Ectoderm. Outermost of the three cellular layers of the embryo;
    produces the epidermis and neuronal tissue

     Eczema. Superficial inflammation in skin, involving primarily the
    epidermis; characterized by redness, itching, minute papules and
    vesicles, weeping, oozing, and crusting. Histological changes include
    microvesiculation and oedema of the epidermis and an infiltrate of
    lymphocytes and macrophages in the dermis

     Effector cell. General term to describe a cell that mediates a
    function after a stage of activation, differentiation, and

     Endocytosis. Process of uptake of material by a cell; special forms
    are phagocytosis and pinocytosis

     Endoderm. Innermost of the three cellular layers of the embryo;
    produces the gastrointestinal lining and some internal organs, such as
    liver and pancreas

     Endoplasmic reticulum. Membrane-like structure in cell cytoplasm;
    site of protein synthesis

     Endothelium. Cells that line blood vessels; exert a major function
    in traffic of leukocytes from blood into tissue, by altered expression
    of adhesion molecules (modulation of numbers of receptors; maturation
    and activation resulting in altered glycosylation, expression of new
    ligands or altered ligand binding affinity; change in cytoskeleton
    organization). A special endothelial cell type occurs in T-lymphocyte
    areas of lymphoid tissue in the high endothelial (postcapillary)

     Endotoxin. Lipopolysaccharide from the cell wall of Gram-negative
    bacteria; has toxic, pyrogenic, and immunoactivating effects

     Enzyme-linked immunosorbent assay. Immunoenzymetric assay based on
    the use of antigens or antibodies labelled with a specific enzyme;
    combines the virtues of solid-phase technology and enzyme-labelled
    immunoreagents. The antigen-antibody complex is determined by an
    enzyme assay involving the incubation of the complex with an
    appropriate substrate of the enzyme.

     Eosinophil chemotactic factor. Acidic tetrapeptide of 0.5 kDa
    produced by mast cells (preformed mediator); attracts eosinophils to
    the site of inflammation

     Eosinophilia. State of increased proportions of eosinophilic
    granulocytes in blood

     Eosinophilic granulocyte. Polymorphonuclear leukocyte that contains
    granules with basic proteins stained by acidophilic (red) dyes; after
    release, the proteins modulate inflammatory reactions

     Epithelium. Cells covering the surface of the body and forming the
    first line of defence against pathogenic invaders. Reticular
    epithelium forming the stroma of tissue occurs in thymus (in avian
    species in the bursa of Fabricius); these cells have a major function
    in processing precursor cells to immunocompetent lymphocytes

     Epithelioid cell. Cell of macrophage origin in chronic inflammatory
    lesions, which resembles an epithelial cell morphologically

     Epitope (antigenic determinant). Part of antigen that binds to
    antibody or T-cell receptors (the latter in combination with MHC

     epsilon Chain (see also  alpha Chain). In immunoglobulin molecules,
    the epsilon heavy chain forming the IgE immunoglobulin class; one of
    the chains in the CD3 molecule associated with the T-cell receptor

     Erythema. Redness of skin produced by congestion of blood
    capillaries due to dermal arterial vasodilatation

     Erythrocyte. Red blood cell; a bone marrow-derived blood cell
    component involved in oxygen transport to tissue; contains a nucleus
    in distinct avian species like chickens but does not have a nucleus in

     Exudation. Inflammation in tissue; contains blood cells and fluid
    comprising serum proteins of high relative molecular mass

     Ex vivo/in vitro. An assay method in which the effects of a
    xenobiotic are evaluated  in vitro in cells isolated from an animal
    or human exposed to the compound of interest

     Fab fragment. Part of an antibody molecule in which monovalent
    binding of an antigenic determinant occurs; formed by the three-
    dimensional structure of variable parts (domain) of one heavy and one
    light chain and the adjacent part of the constant part (constant
    domain); ab, antigen binding

     F(ab')2 fragment. Part of an antibody molecule in which divalent
    binding of antigenic determinants occurs; formed by the
    three-dimensional structure of both Fab fragments

     Fc fragment. Part of an antibody molecule formed by the three-
    dimensional structure of the constant part (constant domains) of the
    heavy chains (except that adjacent to the variable domain), involved
    in antibody effector functions; c, crystallizable

     Fc receptor. Structure on leukocytes (lymphocytes, monocytes,
    macrophages, granulocytes) that mediates binding of immunoglobulin or
    antibody, alone or after forming aggregates in antigen-antibody
    complexes. Receptors for IgE (Fc epsilon) occur on mast cells and
    basophilic granulocytes and are involved in immediate hypersensitivity
    reactions; receptors for IgG are of three classes: low-affinity FcR
    III, CD16, on natural killer cells, monocytes, macrophages, and
    granulocytes; low-affinity FcR II, CD32, on B cells, myeloid cells,
    Langerhans cells, and interdigitating dendritic cells; high-affinity
    FcR III, CD64, on monocytes and macrophages

     Follicle. Round to oval structure in lymphoid tissue, where B cells
    are lodged. Primary follicles contain only small resting B cells;
    secondary follicles comprise a pale-stained germinal centre, with
    centrocytes and centroblast, and contain B lymphocytes in a state of
    activation or proliferation and macrophages, the stroma consisting of
    follicular dendritic cells. The germinal centre is surrounded by a
    mantle with small B lymphocytes

     Follicular dendritic cell. Cell forming the stationary micro-
    environment of germinal centres of follicles in lymphoid tissue;
    elongated, often binucleated cell with long branches extending between
    germinal centre cells and forming a labyrinth-like structure; linked
    by desmosomes. Of local parenchymal origin, presumably from pericytes
    surrounding blood vessels. Its main function is presentation of
    antigen, trapped as immune complex in the labyrinth, to B lymphocytes.

     gamma Chain (see also  alpha Chain). In immunoglobulin molecules,
    the gamma heavy chain forming the IgG immunoglobulin class; in T-cell
    receptor molecules, one of the chains forming the dimeric gamma-delta
    receptor molecule; one of the chains in the CD3 molecule associated
    with the T-cell receptor

     gamma-delta T cell. T Lymphocyte with an antigen receptor composed
    of a gamma and a delta chain associated with CD3 transmembrane
    molecule; develops in part inside the thymus (including intrathymic
    selection), in part outside the thymus. Has a major role as a
    cytotoxic cell in the first phase of the (innate) immune response,
    e.g. in rodents in the mucosal epithelium

     Gammaglobulin. Part of serum proteins that move towards the negative
    electrode (gamma fraction) upon electrophoresis; contains

     Gene rearrangement. For immunoglobulin and T-cell receptor, the
    process whereby the germline chromosomal genomic structure of variable
    (diversity), joining, and constant segments recombine to form a
    specific V-(D-)J-C combination, enabling transcription into mRNA and
    translation into protein. The V-(D-)J combination of different chains
    determines the specificity of the receptor (immunoglobulin or T-cell

     Germinal centre. The pale-staining centre in follicles of lymphoid
    tissue, where B lymphocytes are activated by antigen in a
    T lymphocyte-dependent manner and subsequently go into proliferation
    and differentiation, acquiring the morphology of centroblasts,
    centrocytes, and plasma cells. Has a special microenvironment made up
    of follicular dendritic cells and large macrophages (tingible body or
    starry-sky macrophages)

     Glomerulonephritis. Inflammation of glomeruli in kidney, often
    associated with deposition of immune complexes along the glomerular
    basement membrane or in the mesangium, and influx of polymorpho-
    nuclear granulocytes

     Golgi apparatus. Tubular structures in cytoplasm, involved in
    secretion of synthesized proteins

     Granulocyte colony-stimulating factor. Synthesized by T lymphocytes
    and macrophages, epithelial cells, fibroblasts, and endothelial cells;
    supports growth of granulocyte progenitors, in synergism with IL-3 and
    granulocyte-macrophage colony-stimulating factor of monocyte-
    macrophage progenitors

     Granulocyte-macrophage colony-stimulating factor. Produced by T
    lymphocytes, endothelial cells, macrophages, and lung cells; supports
    growth and differentiation of macrophages and granulocyte progenitors;
    activates macrophages and polymorphonuclear macrophages to become
    tumoricidal and produce superoxide anion

     Granuloma. Chronic inflammatory reaction in tissue comprising macro-
    phages (epitheloid cells), lymphocytes, and fibroblasts; formed in a
    cell-mediated response towards poorly degradable material, in
    immunological reactions as part of a delayed-type hypersensitivity

     Gut-associated lymphoid tissue. Lymphoid organs and tissue located
    along the gastrointestinal tract, presumed to be a first location of
    presentation of antigens entering through the digestive tract;
    comprises Peyer's patches, appendix, in part mesenteric lymph nodes,
    adenoids, and tonsils; contributes to the mucosa-associated lymphoid

     H-2. Major histocompatibility complex of mouse

     Haemagglutinin. Antibody or substance that induces agglutination of

     Haematopoiesis. Production of cells of blood; subdivided into
    erythropoiesis, lymphopoiesis, and myelopoiesis

     Haematopoietic malignancy. Malignancy of blood-forming cells

     Haemolysis. Process of lysis of erythrocytes, with release of

     Haemolytic agent (haemolysin). Antibody or substance that induces
    lysis of erythrocytes

     Haemopoietin. Growth factor that induces production of distinct
    types of blood cells; also enhances the function of the mature cells

     Haplotype. Phenotype of inherited characteristic on closely linked
    genes on one chromosome

     Hapten. Structure around one antigenic determinant, which itself
    does not evoke an immune response unless coupled to a carrier
    substance but can react with the products (antibodies, cells) of an
    immune response

     Hassall's corpuscle. Epithelial aggregate in onion-like structure,
    often with debris of other cells; in the medulla of the thymus,
    surrounded by large epithelial cells secreting thymic hormones; does
    not occur in rodent thymus

     Heat-shock protein. Family of proteins (60-90 kDa) with conserved
    sequence in evolution; play a prime role in regulation and transport
    of intracellular proteins. Expression is upregulated when cells are
    under 'stress' (originally induced by heating), such as inflammatory
    conditions, and may act as autoantigen in triggering and perpetuating
    an auto-immune response

     H (heavy) chain. One of the 45-kDa polypeptide chains in
    immunoglobulin molecules, consisting of a variable domain and three
    constant domains (four constant domains in the 55-kDa µ chain). The
    combination of the constant part of two heavy chains (alpha, ß, delta,
    gamma, or µ) forms the immunoglobulin class-associated part of the
    molecule (IgA, IgD, IgE, IgG, or IgM class)

     Helper (inducer) T cell. Cell in a subpopulation of T lymphocytes,
    with CD4 phenotype; after recognizing antigen in an MHC class 
    II-restricted manner, induces immunological reactions, secretes
    interleukins, and cooperates (supports) B lymphocytes, cytotoxic
    T-cell precursors, and macrophages

     Helper T cell subpopulations. Th1 and Th2: Th1 cells produce
    interleukin-2 and interleukin-3, interferon gamma, tumour necrosis
    factor alpha and ß, and granulocyte-macrophage colony stimulating
    factor, and function in induction of delayed-type hypersensitivity,
    macrophage activation, and IgG2a synthesis. Th2 cells produce
    interleukin-3, interleukin-4, and interleukin-5, tumour necrosis
    factor alpha and granulocyte-macrophage colony stimulating factor, and
    function in induction of IgG1, IgA, and IgE synthesis and induction of
    eosinophilic granulocytes

     Heterologous. Derived from foreign source or species; components of
    an immunological reaction (e.g. antibody, lymphocytes, grafted tissue)
    derived from another individual of the same species or another
    species; opposite of autologous

     Heterophilic antigen. Antigen in unrelated species; can be directed
    towards xenogeneic immune reactivity; often has carbohydrate
    structure; opposite of homocytotropic antibody

     High endothelial (postcapillary) venule. Specialized blood vessels
    in T-lymphocyte area of lymphoid tissue, through which circulating
    lymphocytes pass into the parenchyma

     Hinge region. Stretch in immunoglobulin molecule between Fab and Fc
    fragments (first constant domain and other constant domains of the
    heavy chain), where the quaternary structure of the molecule is not
    rigid but flexible; bending of the hinge region after antigen binding
    serves as a signal transduction, resulting in effector reactions

     Histamine. ß-Imidazolylethylamine; component of granules in mast
    cells and basophilic granulocytes that is released upon activation and
    induces immediate hypersensitivity reaction, e.g. vasodilatation,
    vascular permeability, smooth muscle contraction, and broncho-

     Histiocyte (histiocytic reticulum cell). Monocyte in tissue. See

     Histiocytosis. Increase in proportion of macrophages in tissue

     HLA, human leukocyte antigen. Major histocompatibility complex of

     Homocytotropic antibody. Antibody that binds preferentially to cells
    from the same species rather than to cells from other species;
    opposite of heterologous antibody

     Homologous, Homology. Similarity in primary structure between
    substances; homology region is a synonym for domain

     Host defence. Ability of an individual or species to protect itself
    against opportunistic agents and to eliminate certain tumours and
    exogenous agents such as (micro)organisms, viruses, and particles that
    can cause disease

     Hot spot. See  Hypervariable region

     Humoral immunity. Immunological reactivity mediated by antibody

     Hybridoma. Transformed cell line or cell clone formed by fusion of
    two different parental cell lines or clones

     Hyperplasia. Reversible increase in cell number, usually as the
    result of a physiological stimulus or persistent cell injury due to
    irritating compounds

     Hypersensitivity. Increased reactivity or sensitivity; in
    immunological reactions, often associated with tissue destruction

     Hypervariable region. Amino acid sequences in the variable regions
    of antibody molecules or T-cell receptor chains where variability is
    highest and which together form the antigen-binding site. Synonym for
    hot spot

     Hypoplasia. Reversible decrease in cell number, usually as a result
    of a physiological stimulus

     Ia antigen. MHC class II cell surface molecule

     Idiotype. Antigenic determinant of variable domain of immunoglobulin
    molecules or T-cell receptor

     Immediate hypersensitivity. Inflammatory response that occurs within
    minutes after exposure to allergen; caused by physical or
    immunological stimulus, with vascular dilatation, increased vascular
    permeability, and oedema as the main effects. Term originates from the
    classical skin reaction after challenge of a sensitized individual in
    skin, which takes 20-30 min to reach maximal (wheal and flare)
    response and is mimicked by injection of mediator only (histamine)

     Immune adherence. Binding of antigen-antibody complexes (antibody-
    coated particles) to erythrocytes, platelets, or leukocytes; mediated
    by activation of complement C3

     Immune complex. Complex between antigen and antibody

     Immune elimination. Rapid clearance of pathogen from the circulation
    by components of the immune system; often used in association with
    antibody molecules (removal by immune complex formation and

     Immune exclusion. Process whereby entry of pathogens at mucosal
    surfaces is prevented by the action of specific (secretory IgA)

     Immune interferon. Former name for interferon gamma

     Immune surveillance. Function (still hypothetical) of the immune
    system in preventing or eliminating cells after malignant
    transformation to a neoplastic process

     Immunity. State of protection against pathogen-induced injury, with
    fast immune elimination of pathogenic invaders due to previous antigen
    contact or a special acquired state of responsiveness

     Immunization (vaccination). Active intervention resulting in
    immunity; used mainly in the context of presentation of (inactivated
    or attenuated, nonpathogenic) substance to induce immunological
    memory. Passive immunization is the adoptive transfer of immune system
    components after previous contact with the pathogen and is performed
    mainly with antibodies

     Immunoblast. Intermediately differentiated B lymphocyte in lymphoid
    tissue; a large, 15-20 µm, round-to-spherical cell with a rounded
    euchromatic nucleus. The abundant cytoplasm contains many ribosomes,
    well-developed rough endoplasmic reticulum and Golgi complex

     Immunocompetence. Capacity of B or T lymphocytes to specifically
    recognize antigen, resulting in a specific immunological reaction

     Immunodeficiency. Defects in the immune system resulting in
    decreased or absent reactivity to pathogens. Primary immunodeficiency
    is mainly intrinsic defects in the differentiation of T or B
    lymphocytes and can be congenital or acquired. Secondary
    immunodeficiency is defects of which the cause is outside the immune
    system (malnutrition; stress; protein loss after burns, nephrotic
    syndrome, or intestinal bleeding; viral infection; therapy with
    immunosuppressive or cytostatic drugs; irradiation).

     Immunogen. A substance that can induce an immunological reaction

     Immunogenicity. Capacity to evoke an immune response

     Immunoglobulin. Formerly the electrophoretically-defined
    gammaglobulin (in blood) but is also present in the ß fraction;
    synthesized by plasma cells. The basic subunit consists of two
    identical heavy chains (about 500 amino acid residues, organized into
    four homologous domains; for µ chain in IgM, about 600 amino acid
    residues, organized into five homologous domains) and two identical
    light chains (about 250 amino acid residues organized into two
    homologous domains), each consisting of a variable domain and one to
    three constant domains (in the µ chain, four constant domains). The
    antigen-binding fragment (Fab) consists of variable domains of heavy
    and light chains (two per basic subunit). Five classes of
    immunoglobulins exist, which differ according to heavy chain type
    (constant domains): IgG (major immunoglobulin in blood), IgM
    (pentamer, consisting of five basic units), IgA (major immunoglobulin
    in secretions; present mainly as a dimeric molecule), IgD (major
    function, receptor on B lymphocytes), and IgE. Effector functions
    after antigen binding are mediated by constant domains of the heavy
    chain (Fc part of the molecule) and include complement activation
    (IgG, IgM), binding to phagocytic cells (IgG), sensitization and
    antibody-dependent cell-mediated cytotoxicity (IgG), adherence to
    platelets (IgG), sensitization and degranulation of mast cells and
    basophils (IgE). IgA lacks these effector functions and acts mainly in
    immune exclusion (prevention of entry in the body) at secretory
    surfaces ('antiseptic paint').

    I mmunoglobulin class. Subfamily of immunoglobulins, based on
    difference in heavy chain. Five classes exist: IgA, secretory
    immunoglobulin, dimeric; IgD, immunoglobulin on B cells that acts as
    antigen receptor; IgE, immunoglobulin fixed to mast cells and
    basophilic granulocytes, involved in immediate hypersensitivity
    reactions; IgG, main immunoglobulin in circulation; IgM, pentameric
    immunoglobulin with optimal agglutinating capacity, produced on first
    antigen contact

     Immunoglobulin class switch. Process whereby synthesis of IgM
    antibody changes into synthesis of antibody of another immunoglobulin
    class, with maintenance of the same variable part of the
    immunoglobulin molecule. At the genomic level, this includes gene
    rearrangement, with an exchange of a constant gene segment to a fixed
    V-D-J gene segment combination. This switch is thought to occur in
    germinal centres of follicles in lymphoid tissue, during the change of
    a primary into a secondary response, and is under the control of
    cytokines (switch factors)

     Immunoglobulin gene superfamily. Group of molecules including
    immunoglobulins, T-cell receptors, MHC molecules, and others, like the
    lymphocyte function-related antigens LFA-2 (CD2) and LFA-3 (CD58), the
    intercellular adhesion molecules ICAM-1 (CD54) and ICAM-2, the
    vascular cell adhesion molecule VCAM-1, the neural cell adhesion
    molecule NCAM (CD56), and the CD4 and CD8 molecules, which have a
    similar tertiary basic domain-like structure, in which each domain is
    about 110 amino acids long and stabilized by a disulfide bridge. These
    molecules are known to be important for specific recognition and
    adhesion functions

     Immunoglobulin light chain type. Defines the light chain in the
    immunoglobulin unit, either kappa or lamda, each defined at the
    germline DNA level by individual constant (C), joining (J), and
    variable (V) gene segments

     Immunoglobulin subclass. Subfamily within a distinct immunoglobulin
    class, based on subtle differences in heavy chain. For instance, in
    humans there are two IgA subclasses, IgA1 (alpha1 heavy chain) and
    IgA2 (alpha 2 heavy chain), and four IgG subclasses, IgG1-IgG4
    (gamma 1-gamma 4 heavy chain). In rodents, these are designated IgG1
    (gamma 1), IgG2a (gamma 2a), IgG2b (gamma 2b), and IgG3 (gamma 3)

     Immunological memory. Acquired state of the immune system after
    first contact with antigen, whereby the reaction upon subsequent
    contact is faster, more intense, and of higher affinity. For antibody
    response, associated with an immunoglobulin class switch and 'affinity
    maturation' (by somatic mutation)

     Immunosuppression. Prevention or diminution of the immune response
    by administration of antineoplastic or antimetabolic drugs,
    antilymphocyte serum, or exposure to e.g. environmental chemicals or
    microorganisms (viruses)

     Immunotoxicant. Drug, chemical, or other agent that is toxic to
    cells or other components of the immune system

     Inducer (helper) T cell. See  Helper (inducer) T cell

     Inflammation. Process whereby blood proteins or leukocytes enter
    tissue in response to or in association with infection or tissue

     Inflammatory cell. General description of cells in an inflammatory
    infiltrate; in acute inflammation, mainly polymorphonuclear
    leukocytes; in chronic inflammation, mainly lymphocytes and

     Innate immunity. State of protection against pathogen-induced
    injury; does not require previous immunization or vaccination

     Innocent bystander. Cell or tissue component that is destroyed by an
    immunological reaction specifically directed against a unrelated

     Integrin. Family of heterodimeric molecules sharing a ß chain (ß1,
    ß2, ß3, about 750 amino acids long), each with a different alpha chain
    (about 1100 amino acids long), with a major function in cell adhesion
    and migration. Form a protein family rather than a superfamily on the
    basis of strong structural and functional similarities. Examples:
    leukocyte function-related antigen LFA-1 (alpha L/ß1, CD11a/CD18;
    receptor for intercellular adhesion molecules ICAM-1, ICAM-2, and
    ICAM-3); Mac-1 (alpha M/ß2, CD11b/CD18; complement C3 receptor, CR3);
    p150,95 (alpha X/ß2, CD11c/CD18); very late antigens VLA-1
    (alpha 1/ß1, CD49a/CD29; laminin, collagen receptor), VLA-2
    (alpha 2/ß1, CD49b/CD29; laminin, collagen receptor), VLA-3
    (alpha 3/ß1, CD49c/CD29; laminin, collagen, fibronectin receptor),
    VLA-4/LPAM-1 (alpha 4/ß1, CD49d/CD29; receptor for fibronectin and
    VCAM-1), VLA-5 (alpha 5/ß1, CD49e/CD29, fibronectin receptor), and
    VLA-6 (alpha 6/ß1, CD49f/CD29; laminin receptor, and alpha V/ß1,
    CD51/CD29; vitronectin receptor); LPAM-2 (alpha 4/ßp, CD49d/.., or
    alpha 4/ß7)

     Interdigitating dendritic cell. Leukocyte belonging to the monocyte-
    macrophage cell lineage, present in T-cell areas in lymphoid organs;
    has a major function in presentation of antigen (MHC class 
    II-restricted) to helper-inducer T lymphocytes. Cytoplasm contains
    characteristic rod-like structures called Birbeck granules. Its
    equivalent in epidermis is the Langerhans cell, and that in lymph, the
    veiled macrophage.

     Interferon. Low-relative-molecular-mass substance produced mainly
    during viral infection by leukocytes (IFN alpha), fibroblasts (IFNß),
    and lymphocytes (IFN gamma); has a major function in interfering with
    viral replication

     Interferon-alpha. Produced by leukocytes; stimulates B cells to
    proliferate and differentiate; stimulates natural killer cells and
    increases cytotoxic T cell generation, but blocks T-cell proliferation
    and lymphokine-activated killer activity; stimulates macrophage
    accessory activity and enhances Fc receptor expression and MHC class I
    and II expression on various cell types; induces antiviral state in
    cells and is cytostatic for tumour cells, inhibits fibroblast and
    adipocyte differentiation, and enhances promyelocytic and monoblastic
    cell differentiation

     Interferon-ß. Produced by fibroblasts and epithelia; activity
    similar to that of IFN alpha

     Interferon-gamma. Produced by T cells; induces antiviral state and
    is cytostatic for tumour cells; enhances MHC class I and II expression
    on various cell types, is antagonistic to interleukin-4 in IgE/IgG1
    synthesis, and stimulates IgG2a synthesis; activates macrophages to
    become cytolytic and enhances natural killer and lymphokine-activated
    killer activity.

     Interfollicular area. Area between follicles in lymphoid tissue,
    where mainly small T lymphocytes are lodged; recognized by presence of
    high endothelial venules. In lymph nodes, located in the outer cortex
    and continuous with the paracortex

     Interleukin. Immunoregulatory protein, also known as lymphokine,
    monokine, interferon, or cytokine. Generally, low relative molecular
    mass (< 80 kDa) and frequent glycosylation; regulates immune cell
    function and inflammation by binding to specific cell surface
    receptors; transient and local production; acts in paracrine or
    autocrine manner, with stimulatory or blocking effect on growth and
    differentiation; very potent, functions at picomolar concentrations.
    Represents an extensive series of mediators (interleukins 1-12), with
    a wide range of overlapping functions. Other mediators in this series
    are c-kit ligand, interferon, tumour necrosis factor, and transforming
    growth factor

     Interleukin 1. Comprises two forms, IL-1 alpha and IL-1ß; produced
    mainly by cells of the mononuclear phagocytic system (macrophages),
    astrocytes, endothelium, and some epithelia, following stimulation by
    e.g. microorganisms, immune complexes, or particulate compounds.
    IL-1 alpha is mainly cell-associated; IL-1ß is released. IL-1 has
    (together with IL-6 and tumour necrosis factor) multiple effects in
    the systemic acute-phase response and in local acute and chronic
    inflammation: it stimulates T (helper) cells to synthesize IL-2 and
    IL-2 receptors, interferon gamma, and other lymphokines, B cells
    (proliferation and differentiation), neutrophils, and natural killer
    cells; stimulates monocytes and macrophages to produce IL-1, IL-6, and
    tumour necrosis factor; acts in the acute-phase response by inducing
    synthesis of acute-phase proteins in liver and reducing cytochrome
    P450 synthesis; induces natriuresis in kidney, insulin production in
    pancreas ß cells, muscular proteolysis ('easy' energy generation) in
    muscle cells, slow-wave sleep in cerebral cortex; raises the
    temperature set-point (fever) in hypothalamus; stimulates
    haematopoiesis and prostaglandin synthesis by various cell types
    (fibroblasts, macrophages, endothelium); inhibits gastric motility
     in vitro ; induces collagenase production by synovial cells and
    osteoclasts, and antiviral state; inhibits gastric smooth muscle
     in vitro ; is cytostatic for tumour cells and activates endothelium

     Interleukin 2. Synthesized by T helper cells after activation;
    stimulates (autocrine) T cells to divide and release mediators,
    B cells to proliferate and differentiate; activates monocytes and
    natural killer cells; stimulates lymphokine-activated killer cells;
    promotes generation of T helper 1 cells.

     Interleukin 3. Formerly called multi-colony-stimulating factor;
    synthesized by T helper cells; promotes growth of pluripotent
    haematopoietic progenitor cells to granulocytes (eosinophilic,
    basophilic, neutrophilic), mast cells, macrophages, megakaryocytes,
    and, together with erythropoietin, to normoblasts and erythrocytes;
    activates eosinophils and mast cells; stimulates haematopoiesis and
    B-cell differentiation; blocks lymphokine-activated killer cells

     Interleukin 4. Formerly called B-cell growth factor or B-cell
    stimulating factor; synthesized by T helper and B cells; stimulates
    IgE and IgG1 production by B cells and enhances MHC class II and IgE
    receptor expression on B cells; acts in synergism with IL-2 in killer
    cell generation, is mitogenic for T cells, and activates macrophages.
    It is the dominant interleukin in generating T helper 2 cells, with a
    negative feedback on the generation of T helper 1 cells.

     Interleukin 5. Formerly called T-cell replacing factor or B-cell
    growth and differentiation factor II; synthesized by T helper cells;
    activates B cells and eosinophils, and stimulates IgA production by
    B cells

     Interleukin 6. Formerly called interferon ß2; synthesized by T
    cells, monocytes, endothelial cells, fibroblasts, and smooth muscle
    cells, among others, during inflammatory reactions; stimulates T and B
    cells to proliferate and differentiate; has properties similar to IL-1
    and acts synergistically with it in the acute-phase response (fever,
    synthesis of acute-phase proteins); synergizes with IL-3 in promoting
    haematopoietic progenitor cell proliferation; inhibits production of
    IL-1 and tumour necrosis factor by monocytes

     Interleukin 7. Formerly called lymphopoietin; synthesized by bone-
    marrow stroma; induces growth of immature T and B lymphocytes

     Interleukin 8. Formerly called neutrophil-activating protein;
    synthesized by monocytes and various tissue cells in response to
    inflammatory stimuli; performs chemotaxis of neutrophilic granulocytes
    and subsequent granule exocytosis and respiratory burst; induces
    increased expression of adhesion molecules CD11b/CD18 (complement C3
    receptor CR3) and promotes vascular leakage. Endothelium-derived IL-8
    inhibits adhesion of neutrophilic granulocytes induced by IL-1

     Interleukin 9. Synthesized by T lymphocytes; stimulates growth of
    erythroid and megakaryocyte precursors and promotes (mucosal) mast-
    cell growth; acts synergistically with IL-4 in modulating IgE and IgG

     Interleukin 10. Synthesized by T and B lymphocytes; inhibits
    mediator synthesis (IL-2, IL-3, tumour necrosis factor, interferon
    gamma, granulocyte-macrophage colony-stimulating factor) by T helper
    1 cells, inhibits mediator synthesis (IL-1 alpha, IL-1ß, IL-6, IL-8,
    and tumour necrosis factor alpha) by monocytes; stimulates IL-2-
    dependent growth and cytotoxicity of cytotoxic T cells and stimulates
    mast cell growth together with IL-2 or IL-3 and IL-4; induces MHC
    class II antigen expression on B cells, but down-regulates MHC class
    II on monocytes; promotes generation of T helper 2 cells

     Interleukin 11. Synthesized by fibroblasts and bone-marrow stromal
    cells; resembles IL-6 in function: stimulates haematopoietic cell
    growth and differentiation (myeloid, erythroid, megakaryocyte
    lineage); enhances T-cell-dependent antibody response; and suppresses
    adipocyte differentiation and lipoprotein lipase production

     Interleukin 12. Also called natural killer cell stimulatory factor;
    synthesized by monocytes-macrophages, B cells, and accessory cells, in
    response to bacteria or parasites; stimulates T-lymphocyte
    proliferation, activates natural killer cells, and stimulates
    lymphokine-activated killer activity; synergizes with IL-2 in
    activation of cytotoxic lymphocytes; induces production of interferon
    gamma and other cytokines by lymphocytes. It is the dominant
    interleukin in generating T helper 1 cells and has a negative feedback
    on the generation of T helper 2 cells.

     in vitro. In the context of this book, exposure of cells or cell
    systems to the immunotoxic agent  in vitro. If the donors of cells or
    cell systems are exposed but these are analysed  in vitro, the term
     ex vivo/in vitro is used

     Isohaemagglutinin. Antibodies mainly of the IgM class that react
    with (carbohydrate) antigens on erythrocytes from individuals of the
    same species, resulting in agglutination  in vitro

     Isologous. Synonym for isogeneic

     Isotype. Antigenic determinant that defines class or subclass of
    immunoglobulin molecules

     J (joining) chain. A 15-kDa polypeptide chain that acts
    intracellularly to combine (identical) IgA or IgM immunoglobulin
    units, consisting of two heavy and two light chains, to form a dimeric
    IgA or a pentameric IgM molecule

     J (joining) genes. Genes that encode  the variable part of
    immunoglobulin  or T-cell receptor chains (e.g. JH1-JHn for
    immunoglobulin heavy chains, Jkappa 1-Jkappa n for immunoglobulin
    kappa light chain, Jalpha 1-Jalpha n for T-cell receptor alpha

     Kallikrein (kininogenase). Arises in tissue fluids after cleavage of
    prekallikrein; acts on kininogen to produce kinins, resulting in
    immediate hypersensitivity reaction, e.g. vasodilatation and oedema.
    It is a preformed mediator present in mast cell granules

     kappa Chain. In immunoglobulin molecules, the kappa light chain  
    forms the light chain type of the molecule

     Keratinocyte. Epithelial cell in the epidermis; in some
    circumstances, can manifest antigen presentation and produce
    immunoregulatory cytokines; hence belongs to the skin immune system

     Killer cell (K cell). See  Cytotoxic cell

     Kinin system. Humoral amplification system involved in inflammation,
    whereby substrate proteins become active after enzymatic cleavage;
    cause vasodilatation, increased vascular permeability, hypotension,
    and contraction of smooth muscle

     c-Kit ligand. Also called stem cell growth factor or mast cell
    growth factor; synthesized by various stromal cells, fibroblasts, and
    liver cells; stimulates growth of early pluripotent progenitor cells
    and that of myeloid, erythroid, and lymphoid progenitors in synergy
    with interleukin-1, -3, -6, -7, and granulocyte-macrophage colony-
    stimulating factor; promotes growth of mast cells

     Kupffer cells. Macrophages on or between endothelial cells lining
    the sinusoids of the liver

     lamba Chain. In immunoglobulin  molecules, the lamba light chain    
    forms the light chain type of the molecule

     Lamina propria. Thin layer of connective tissue under the villous
    epithelium of the gastrointestinal tract; site of plasma cells,
    producing mainly dimeric IgA, including J chain

     Langerhans cell. Leukocyte belonging to the monocyte-macrophage cell
    lineage, present in skin epidermis; has a major function in uptake and
    processing of antigen, followed by presentation (MHC class II
    restricted) to T helper lymphocytes. Cytoplasm contains characteristic
    rod-like structures, Birbeck granules. Its equivalent in lymphoid
    tissue is the interdigitating dendritic cell, and that in lymph,
    veiled macrophage; forms part of the skin immune system

     Large granular lymphocyte. Intermediate-sized, 10-12-µm lymphocyte
    with a kidney-shaped nucleus and prominent, large, azurophilic
    granules in the cytoplasm; occurs in the circulation and in tissue and
    has a major function as a natural killer cell; forms a heterogeneous
    population with either T markers or monocyte-macrophage markers.

     Lectin. Plant-derived substance that binds to lymphocytes and can
    induce cell proliferation; some also bind to other haematopoietic

     Leukaemia. Neoplasia of lymphoid cells in blood or bone marrow

     Leukocyte. Bone marrow-derived white blood cell, including cells in
    the lymphoid, myeloid, and monocyte lineages; sometimes used to
    describe only granulocytes

     Leukocytosis. Increased proportion of leukocytes in blood

     Leukopenia, leukocytopenia. Reduced proportion of leukocytes in

     Leukotriene. Formerly called slow-reacting substance of anaphylaxis;
    products of arachidonic acid metabolism following the lipoxygenase
    pathway, which act as mediators in the immediate hypersensitivity
    reaction, mainly as chemoattractants for granulocytes and monocytes,
    and in smooth muscle contraction; newly synthesized by mast cells upon

     L (light) chain. One of the 22-kDa polypeptide chains in
    immunoglobulin molecules, consisting of a variable domain and a
    constant domain. The light chain, either kappa or lamba, determines
    the light chain type of the immunoglobulin molecule

     Ly antigen. T Lymphocyte antigen in mice

     Lymph. Fluid in lymphatic vessels

     Lymph node. Secondary (peripheral) lymphoid organ, the main function
    of which is to filter lymphatic vessels. Present throughout the body,
    at connecting places of lymphatics and blood vessels; forms a major
    site of encounter between pathogenic substances in the lymph and
    lymphocytes entering from blood vessels, and subsequent initiation of
    antigen-specific immunological reactions

     Lymphatic. Vessel that collects fluid from interstitial spaces and
    goes via lymph nodes (filtering) to the thoracic duct and blood

     Lymphocyte. Cell belonging to the lymphoid lineage of bone marrow-
    derived haematopoietic cells. In a restricted way, the designation of
    a small, resting or recirculating mononuclear cell in blood or
    lymphoid tissue, which measures about 7-8 µm, has a round nucleus
    containing densely aggregated chromatin, and little cytoplasm. Plays a
    key role in immune reactions by specific recognition of antigens

     Lymphocytosis. Increased proportions of lymphocytes in blood

     Lymphoid organ. Tissue in the body where cells of the immune system,
    mainly lymphocytes, are lodged in an organized microenvironment,
    either in a resting state or in a state of activation,
    differentiation, or proliferation. Includes bone marrow, thymus, lymph
    nodes, spleen, and mucosa-associated lymphoid tissue

     Lymphokine. Hormonal substance synthesized by lymphocytes, which
    modulates the function of cells in immunological reactions

     Lymphoma. Neoplasia of lymphoid cells in tissue

     Lymphopenia, lymphocytopenia. Reduced proportions of lymphocytes in

     Lymphotoxin. Former name for tumour necrosis factor ß; lymphokine
    synthesized by T lymphocytes, which kills selected target cells

     Lysosome. Granule present in many cell types that contain hydrolytic
    enzymes; also performs intracellular degradation of pathogens after

     Lysozyme (muramidase). A low-relative-molecular-mass, cationic
    enzyme present in tissue fluids and secretions, which degrades
    mucopeptides of bacterial cell walls

     Macroglobulin. Glycoprotein of relative molecular mass > 200 kDa

     Macrophage. Large, 12-20-µm bone marrow-derived mononuclear cell in
    the monocyte-macrophage lineage, present in tissue, and forming the
    mononuclear phagocytic system. Its reniform nucleus usually has
    pronounced peripheral condensation of nuclear chromatin; its cytoplasm
    contains a great variety of cell organelles, including rough
    endoplasmic reticulum, mitochondria, ribosomes, lysosomes, and Golgi
    complex. Has a major function in (chronic) inflammatory reactions, by
    virtue of its phagocytic capacity, with immunoglobulin Fc and
    complement C3 receptors which bind to immune complexes. Macrophages
    develop into killer cells after activation by e.g. T-cell factors and
    can mediate antibody-dependent cell-mediated cytotoxicity; also
    functions as an accessory cell in induction of immune responses
    (antigen presentation, mediator secretion). Macrophages in blood are
    called monocytes. Subtypes with special functions are interdigitating
    dendritic cells: T-cell area of lymphoid tissue, Langerhans cell
    (skin), Kupffer cells (liver), metallophilic macrophages (spleen),
    microglia (brain), osteoclasts (bone), tingible body macrophage
    (starry-sky macrophage), veiled macrophage (lymph).

     Macrophage colony-stimulating factor. Synthesized mainly by
    endothelial cells and fibroblasts, and possibly macrophages; supports
    growth of monocyte-macrophage progenitors.

     Major histocompatibility complex (MHC). Set of genes that codes for
    tissue compatibility markers, which are targets in allograft rejection
    and hence determine the fate of allografts; plays a central role in
    control of cellular interactions during immunological reactions.
    Tissue compatibility is coded by classes I and II loci (see  Class I
    and  Class II MHC molecule). Genes within or closely linked to MHC
    control certain complement components (MHC class III genes). The MHC
    complex of humans is HLA, that of mice H-2, and that of rats, RT-1.

     Mantle (corona). Zone in secondary follicles surrounding the central
    germinal centre, densely packed with small resting B lymphocytes

     Marginal zone. Outer layer of white pulp in spleen, surrounding
    follicles, and periarteriolar lymphocyte sheath; separated from these
    by the marginal sinus; populated by intermediate-sized, slightly
    pyroninophilic B cells which have a major function in the T cell-
    independent antibody response. The microenvironment manifests a
    special type of macrophage, the marginal metallophilic macrophage

     Margination. Adherence of blood leukocytes to endothelium during
    inflammatory reactions

     Mast cell. A bone marrow-derived polymorphonuclear leukocyte present
    in tissue; has a major function in immediate hypersensitivity
    reactions; has a round or oval nucleus and abundant cytoplasm with
    basophilic (blue) granules stained by metachromatic dyes; granules
    contain mediators of immediate hypersensitivity reactions, e.g.
    heparin, histamine, serotonin, tryptase, kallikrein, and
    chemoattractants for neutrophilic and eosinophilic granulocytes; has a
    high-affinity receptor for IgE. After activation (physical stimuli,
    cross-linking via allergen-IgE-IgE receptor), there is immediate
    granule release and synthesis of other mediators, including
    prostaglandins, thromboxanes, leukotrienes, and platelet-activating
    factor. The cell can exert modulatory activity by secreting cytokines
    such as IL-3, IL-6, and tumour necrosis factor. Two subtypes exist, in
    the mucosa and in connective tissue; the equivalent in the circulation
    is the basophilic granulocyte

     Mast cell growth factor. See  Interleukin 9

     Medulla. Inner parenchymal layer of organs

     Medullary cord. Parenchyma in medulla of lymph nodes separating
    lymphatic sinusoids

     Megakaryocyte. Large, multinucleated giant cell, precursor of blood
    platelets, formed by separation of portions of membrane-bound
    cytoplasm; occurs in haematopoietic tissue, including bone marrow

     Memory. See  Immunological memory

     Mesoderm. Middle of the three cellular layers of the embryo;
    produces connective tissue and blood cells

     Metallophilic macrophage. Subtype of macrophage identified by silver
    impregnation, present at the inner border of the marginal zone in

     MHC restriction. Immunological reactions can occur only in
    associated recognition with the polymorphic determinant of a given MHC
    molecule and not with that of another MHC molecule. Applies to T
    lymphocytes with an alpha-ß T-cell receptor, which recognizes
    antigenic peptides in combination with the polymorphic determinant of
    MHC molecules, and part of the T cell population with the gamma-delta
    T-cell receptor.

     Microglia. Macrophages in central nervous system

     Migration inhibitory factor. A lymphokine that inhibits the movement
    of macrophages

     Milky spot. Aggregate of lymphoid cells in omentum, macroscopically
    visible as a small white spot; not organized tissue but rather the
    product of immune stimulation in that area of the body

     Minor histocompatibility antigen. Ill-defined histocompatibility
    marker not encoded by the MHC, which is a target in allograft
    reactions (apart from products of the MHC)

     Mitogen. Substance that activates resting cells to transform and

     Monoclonal. Derived from a single clone. For T and B lymphocytes, a
    cell population in which all cells have a distinct V-D-J gene
    rearrangement product (as seen in lymphoma and leukaemia). Monoclonal
    antibodies are products of hybridomas prepared after fusion of
    antibody-producing cells and a transformed (non-producing)
    plasmacytoid cell line.

     Monocyte. Large, 10-15-µm bone marrow-derived mononuclear cell in
    the monocyte-macrophage lineage, present in the blood and in lymphatic

     Monokine. Hormonal substance synthesized by monocytes-macrophages;
    modulates the function of cells in immunological reactions

     Mononuclear cell. Leukocyte with a single rounded nucleus, e.g.
    lymphocytes and monocytes-macrophages

     Mononuclear phagocytic system. Formerly called reticuloendothelial
    system; composite of phagocytic cells in the body, including monocytes
    and tissue macrophages. Main populations are Kupffer cells in liver,
    microglia in the central nervous system, macrophages in red pulp of
    spleen, alveolar macrophages in lung, and, after induction, peritoneal
    macrophages in the peritoneal cavity; others are mesangial macrophages
    in kidney and osteoclasts in bone

     µ Chain. In immunoglobulin molecules, the µ heavy chain forming the
    IgM immunoglobulin class

     Mucosa-associated lymphoid tissue. Lymphoid tissue or organs in
    immediate contact with the mucous-secreting mucosal layer in nasal
    cavity and nasopharynx (nasal-associated lymphoid tissue), airways
    (bronchus-associated lymphoid tissue), and intestinal tract (gut-
    associated lymphoid tissue). Serves as the immunological defence at
    secretory surfaces, to some extent independent of the systemic
    (internal) response; includes IgA synthesis by plasma cells in the
    lamina propria and excretion into the lumen

     Multiple myeloma. Tumour of plasma cells in bone marrow

     Muramidase. See  Lysozyme

     Myeloblast. Immature precursor cell in the lineage of polymorpho-
    nuclear cells (granulocytes, mast cells)

     Nasal-associated lymphoid tissue. Lymphoid organs or tissue located
    in the nasal cavity and nasopharynx, presumed to be a first location
    for presentation of antigens entering through the nose; contributes to
    the mucosa-associated lymphoid tissue

     Natural antibody. Antibody in serum of individuals with no previous
    exposure to the corresponding antigen; often generated by contact with
    cross-reacting agents, e.g. bacterial products; often restricted to
    antibodies that react to xenogeneic antigens

     Natural killer cell. Leukocyte with a limited repertoire to
    recognize antigen; can kill target cells without prior sensitization;
    can be of lymphoid or monocyte-macrophage origin. Large granular
    lymphocytes are the main population

     Necrosis. Death of tissue and cells

     Neoantigen. New antigen appearing on cells or tissue during
    malignant transformation or (viral) infection

     Neoplasia. Uncontrolled malignant transformation of cells resulting
    in tumour formation

     Neutralization. Process whereby a pathogenic substance becomes
    inactivated by effector components (antibodies) of the immunological

     Neutropenia. Reduced proportions of neutrophilic granulocytes in

     Neutrophil chemotactic factor. Preformed mediator with a relative
    molecular mass > 750 kDa, present in granules of mast cells and
    basophilic granulocytes; released after activation and attracts
    neutrophilic granulocytes to the site of inflammation or

     Neutrophilic granulocyte. Polymorphonuclear leukocyte that contains
    granules stained by neither acidophilic nor basophilic dyes; can
    phagocytose immune complexes by receptor-mediated endocytosis,
    followed by intracellular degradation in lysosomes. Degranulation
    releases catepsins and lysosomal enzymes, resulting in tissue damage.

     Nonspecific immunity. Immunity induced by non-immunological
    mechanisms, for instance by action of complement, lysozyme,
    phagocytosis, or interferon

     Oedema. Swelling of tissue due to extravasation of fluid from the
    intravascular space following increase in vascular permeability

     Ontogeny. Life cycle of an organism; in immunological terms, often
    used to describe the process whereby the immune system develops

     Opsonization. Adherence of pathogen to phagocytic cell due to action
    of antibody or activated complement

     Osteoclast. Macrophage in bony tissue involved in bone resorption

     Paracortex. Area in the inner cortex of lymph node where T
    lymphocytes are lodged; recognized by the presence of high endothelial
    venules; continuous with interfollicular areas in the outer cortex on
    one side and with the medullary cords on the other

     Paratope (antigen-binding site). Part of antibody that binds antigen
    (antigenic determinant, or epitope); part of T-cell receptor that
    binds the complex of antigen and MHC molecule

     Periarteriolar lymphocyte sheath. Area in white pulp of spleen
    surrounding the central artery, populated mainly by small
    T lymphocytes

     Peripheral (secondary) lymphoid organ. Lymphoid organ in which
    immunocompetent lymphocytes recognize antigen, subsequently initiate
    immunological reactions, and produce effector elements of these

     Peyer's patch. Lymphoid tissue in wall of small intestine,
    particularly ileum, separated from the gut lumen by a domed area and
    an epithelial layer ('dome' epithelium); forms part of gut-associated
    lymphoid tissue; main function is initiation of immunological
    reactions towards pathogens entering through dome epithelium

     Phagocytosis. Uptake of material >1 µm by cells, by receptor-
    mediated endocytosis, by cells of the mononuclear phagocytic system;
    requires Fc receptors, with accessory help of complement receptors; is
    blocked by cytochalasins. Occurs via a 'zipper' mechanism, in which
    the opsonized particle (coated with antibody or complement) becomes
    enclosed by the cell membrane of the phagocyte; a second mechanism
    involves oxidative burst, with formation of superoxide anion, peroxide
    anion, and hydroxyl radicals, which kill or degrade the phagocytosed

     Phagolysosome. Membrane-bound cytoplasmic vesicle formed by fusion
    of a phagosome and a lysosome

     Phagosome. Membrane-bound vesicle in phagocytic cells containing
    phagocytosed material

     Pharmacokinetics. Fate of drugs or chemicals in the body over time,
    including the processes of absorption and distribution in tissues,
    biotransformation, and excretion

     Phenotype. Characteristic of a distinct cell or individual,
    reflecting the expression of a genetically determined property

     Phenotypic marker. Expressed characteristic(s), for instance an
    antigenic determinant, of a given cell or molecule, associated with
    function or specificity

     Phylogeny. Evolutionary history of a particular species

     Pinocytosis. Uptake of material < 1 µm by cells; often restricted
    to a receptor-mediated process in leukocytes of the monocyte-
    macrophage series, e.g. uptake of lipoproteins and viruses into
    clathrin-coated vesicles

     Plasma. Fluid of uncoagulated blood after removal of cells

     Plasma cell. Terminally differentiated B lymphocyte that synthesizes
    and secretes immunoglobulin; these medium-sized, 10-15-µm cells have a
    small excentric nucleus, with heterochromatin organized in a
    'cartwheel'-like structure, and abundant cytoplasm filled with rough
    endoplasmic reticulum.

     Platelet. Small bone marrow-derived cytoplasmic fragment in blood
    responsible for coagulation; main role is to block damaged vessel
    walls and prevent haemorrhage, by clumping and aggregation; contains
    heparin and serotonin, which contribute after release to the acute
    vascular response in hypersensitivity reactions and produce oxygen

     Platelet-activating factor. Low-relative-molecular-mass phospholipid
    generated from alkyl phospholipids in mast cells, basophilic and
    neutrophilic granulocytes, and monocytes-macrophages, which mediates
    microthrombus formation of platelets in hypersensitivity reactions

     Polyclonal. Derived from many different clones; for T and B
    lymphocytes, cell populations in which the cells have different V-D-J
    gene rearrangement products. Polyclonal activation is stimulation of
    multiple lymphocyte clones, resulting in a heterogeneous response

     Polymorphonuclear granulocyte. Leukocyte of bone-marrow origin, with
    a lobulated nucleus, involved in acute inflammatory reactions. Main
    subsets are basophilic, eosinophilic, and neutrophilic granulocytes
    (different cytoplasmic granule colours after haematological staining).
    Contributes to (acute) inflammatory reactions after attraction by
    specific (immune complex-mediated) or nonspecific stimuli (including
    complement components); after activation, releases granules containing
    various hydrolytic enzymes

     Postcapillary venule. Small blood vessel though which blood flows
    after leaving the capillaries before reaching veins; often the site
    where inflammatory cells leave the circulation and enter the tissue

     Primary (central) lymphoid organ. Lymphoid organ where precursor
    lymphocytes differentiate and proliferate in close contact with the
    microenvironment to form immunocompetent cells; not antigen-driven but
    can be influenced by mediators produced as a result of antigen

     Primary follicle. See  Follicle

     Primary response. Immunological reaction after first contact with
    antigen, resulting in generation of immunological memory

     Programmed cell death. See  Apoptosis

     Prostaglandin. Aliphatic acid produced by arachidonic acid
    metabolism following the cyclo-oxygenase pathway; synthesized by mast
    cells after activation; mediates immediate hypersensitivity reactions,
    mainly smooth muscle contraction or bronchoconstriction; also
    decreases the threshold for pain

     Prothymocyte. Precursor of T lymphocytes in bone marrow before
    moving to the thymus, or present in the thymus just before intrathymic

     Pyrogen. Substance that increases the temperature in the central
    nervous system, resulting in fever; examples are bacterial endotoxin
    and IL-1

     Reactive oxygen intermediate. Reactive species of oxygen produced
    e.g. by phagocytes (granulocytes and monocyte-macrophages) in response
    to phagocytic stimuli like bacteria

     Reagin. Former designation of IgE class antibody

     Recall antigen. Antigen used to elicit a response from an individual
    already sensitized to that antigen; may be one that the host has
    knowingly been sensitized to or, in humans, one that it is assumed
    that most individuals have been sensitized to

     Red pulp. Area in spleen comprising venous sinuses filled with blood
    and splenic cords; venous sinuses mainly contain erythrocytes
    surrounded by endothelial cells; cords comprise macrophages,
    lymphocytes, and occasionally megakaryocytes, but other types of blood
    cells can also be present. Main function is phagocytosis of
    particulate material and removal of old erythrocytes from blood. In
    rodents, the red pulp can also be a site of haematopoiesis.

     Reticuloendothelial system. See  Mononuclear phagocytic system

     Repertoire. All specific antigen-recognizing capacities (diversity)
    within a population of T or B lymphocytes

     RT-1. The major histocompatibility complex of rats

     Secretory immunoglobulin. Immunoglobulin encountered in secretions
    like tears, saliva, and jejunal juice; concerns mainly secretory IgA,
    a dimer of the basic four-chain immunoglobulin structure, linked by a
    J chain and surrounded by a secretory piece molecule.

     Secretory piece. A 70-kDa molecule produced by epithelial cells
    covering mucosa-associated lymphoid tissue; functions as a receptor
    for IgA or IgM, thereby facilitating intercellular transport of these
    molecules into the lumen. During this process, the secretory piece
    becomes associated with the immunoglobulin, thereby enhancing its
    stability in nonphysiological conditions of secretory fluid

     Secondary follicle. See  Follicle

     Secondary (peripheral) lymphoid organ. Lymphoid organ in which
    immunocompetent lymphocytes recognize antigen, subsequently initiate
    immunological reactions, and produce effector elements of those

     Secondary response. Response after first contact (immunization,
    primary response) with an antigen, based on the presence of
    immunological memory; characterized as faster, more intense, and of
    higher affinity; for the antibody response, associated with an
    immunoglobulin class switch

     Selectin. Cell surface glycoprotein that has a prominent function in
    the interaction between lymphocytes, monocytes, neutrophilic
    granulocytes, and endothelium. They share an N-terminal domain of
    approximately 120 amino acids that is homologous to many Ca2+-
    dependent animal lectins and binds to carbohydrates. Examples are
    L-selectin (MEL-14, LAM-1, present on leukocytes; adherence of
    endothelial cells, role in lymphocyte recirculation and neutrophil and
    leukocyte inflammation); E-selectin (ELAM-1, present on endothelium;
    adherence of monocytes, neutrophils, and T cells; role in
    inflammation); P-selectin (PADGEM, GMP-140, CD62, present on platelets
    and endothelium; adherence of monocytes, neutrophils, and T cells;
    role in inflammation).

     Self-MHC restriction. See  MHC restriction; applies to MHC
    molecules of the individual

     Sensitization. Induction of specialized immunological memory in an
    individual by exposure to antigen

     Serotonin. 5-Hydroxytryptamine; catecholamine with relative molecular
    mass of 176 Da; preformed mediator of immediate hypersensitivity
    reactions, present in granules of mast cells and in platelets. After
    activation, is released and mediates vasodilatation and increased
    vascular permeability

     Serum. Fluid of blood after coagulation (removal of fibrinogen) and
    removal of cells

     Serum sickness. Systemic vasculitis, glomerulonephritis, or arthritis
    due to immune complex formation after the reaction between antibody and
    injected foreign antigen (serum)

     Skin-associated lymphoid tissue. See  Skin immune system

     Skin immune system. Combination of immune system components and
    their function, present in skin; antigen presentation by Langerhans
    cells, by dendritic epidermal cells, and in some conditions by
    keratinocytes; immunoregulation by e.g. keratinocyte-derived
    cytokines, and distinct dermatotropic T-cell populations

     Slow-reacting substance of anaphylaxis. See  Leukotriene

     Somatic mutation. Small changes in genes resulting in alterations in
    amino acids built into protein chains. For immunoglobulin molecules,
    changes in diversity of antigen-binding site (variable region)

     Spleen. Lymphoid organ in the left abdominal cavity, for filtering
    blood; main function is phagocytosis of particles from blood, removal
    of old erythrocytes in red pulp, and initiation of immunological
    reactions in white pulp. The marginal zone of the white pulp serves as
    the main site of T cell-independent antibody formation.

     Starry-sky macrophage. See  Tingible body macrophage

     Stem cell. Multipotential, self-renewing precursor cell of all
    haematopoietic cell lineages, present in bone marrow

     Stem-cell growth factor (synonym for  c-Kit ligand). An interleukin
    that supports continuous growth of mast cells and augments the
    response of progenitor cells to stem growth factors; interacts via
     c-kit proto-oncogene

     Subcapsular sinus. Area in lymph node just under the capsule and
    surrounding the cortex, which is connected with afferent lymphatics,
    and through cortical (peritrabecular) sinuses with medullary sinuses;
    contains dendritic macrophages

     Superantigen. Antigenic moiety that, in MHC-restricted presentation
    to T lymphocytes, is not present in the 'groove' made by the
    quaternary structure of the MHC molecule but is complexed with the MHC
    molecule. Examples are the endogenous viral-encoded Mlsa (minor
    lymphocyte stimulatory) antigen, which is present in certain mouse
    strains, and  Staphylococcus enterotoxin A

     Suppressor T cell. Subpopulation of T lymphocytes with CD8 phenotype;
    after recognition of antigen in an MHC class I-restricted manner,
    suppresses immunological reactions, in part by cytotoxic activity

     Systemic lupus erythematosus. Chronic, remitting, relapsing,
    inflammatory, and often febrile multisystemic disorder of connective
    tissues, with possible involvement of the central nervous system,
    skin, joints, kidneys, and serosal membranes; can be acute or
    insidious in onset. The etiology is unknown but is thought to follow a
    failure of the regulatory mechanisms of the immune system that sustain
    self-tolerance. Many drugs and chemicals can induce lupus-like
    symptoms (drug-induced lupus erythematosus)

     T-Cell receptor. Heterodimeric molecule on the surface of the T
    lymphocyte that recognizes antigen. The polypeptide chains have a
    variable and a constant part, and can be alpha, ß, gamma, or delta.
    The alpha-ß T-cell receptor occurs on most T cells and recognizes
    antigenic peptides in combination with the polymorphic determinant of
    MHC molecules (self-MHC restricted). The gamma-delta T-cell receptor
    occurs on a small subpopulation, e.g. in mucosal epithelium, and can
    recognize antigen in a non-MHC restricted manner. The T-cell receptor
    occurs exclusively with the CD3 molecule, which is thought to mediate
    transmembrane signalling.

     T-Dependent antigen. Antigen for which antibody formation requires
    T cells.

     Terminal pathway of complement activation. Activation of complement
    components C6-C9, with formation of the membrane attack complex and
    subsequent lysis of the cell

     Tingible body macrophage (starry-sky macrophage). Large macrophage
    in cortex of thymus and germinal centres of follicles in lymphoid
    tissue, filled with condensed nuclear material with high affinity for
    dyes; has a major function in phagocytosis, presumably of apoptotic

     T Lymphocyte or cell. Lymphocyte that induces, regulates, and
    effects specific immunological reactions after stimulation by antigen,
    mostly in the form of processed antigen complexed with MHC product on
    an antigen-presenting cell. They originate from precursors in the bone
    marrow and undergo maturation in the thymus (T, thymus-dependent).
    Most T lymphocytes recognize antigen by a heterodimeric alpha-ß
    surface receptor molecule associated with the CD3 molecule, which
    mediates transmembrane signalling. Subsets include helper-inducer and
    suppressor-cytotoxic cells.

     T-Lymphocyte area. That part of a lymphoid organ or tissue that is
    occupied by T lymphocytes, e.g. paracortex or interfollicular area in
    lymph node, periarteriolar lymphocyte sheath in spleen

     Thrombocyte. See  Platelet

     Thrombocytopenia. Reduced proportion of platelets in blood

     Thromboxane. Product of arachidonic acid following the cyclo-
    oxygenase pathway; synthesized by mast cells after activation and
    mediates immediate hypersensitivity reactions, mainly smooth muscle
    contraction, bronchoconstriction, and platelet aggregation

     Thymocyte. Lymphocyte in the thymus

     Thymoma. Tumour of the thymus; neoplastic cell is an epithelial cell

     Thymus. Central lymphoid organ located dorsal to the cranial part of
    the sternum in the thorax, comprising two lobes, each consisting of
    many lobules. Its main function is generation of immunocompetent
    T lymphocytes from prothymocytes from the bone marrow

     Tolerance. State of unresponsiveness to antigenic stimulation, due
    to the absence of responding elements or loss of capacity of existing
    elements to mount a reaction. Synonym for anergy

     Tolerogen. Antigen that evokes tolerance

     Tonsil. Organized mucosa-associated lymphoid tissue in
    oronasopharynx. Adenoids  strictu sensu are also tonsils. The main
    function is initiation of immunological reactions towards pathogens
    entering through the mouth. Contributes in part to the gut-associated
    lymphoid tissue. Together with lymphoid aggregates in oronasopharynx,
    these tissues form the ring of Waldeyer

     Transforming growth factor ß. Mediator synthesized by lymphocytes or
    macrophages, with a function in down-regulation of immune reactions;
    suppresses T- and B-lymphocyte growth, IgM and IgG production, and
    down-regulates MHC class II expression; interferes with production of
    tumour necrosis factor and adhesion of granulocytes to endothelial
    cells; is chemotactic for monocytes and induces interleukin-1 and
    interleukin-6 expression

     Transudation. Transfer of fluid and low-relative-molecular-mass
    proteins from intravascular to extravascular tissue during
    inflammatory processes

     Tryptase. Proteolytic enzyme present in granules of mast cells;
    released after activation and activates complement component C3, with
    formation of the anaphylatoxin C3a

     Tumour necrosis factor. General mediator of inflammation and septic
    shock; formerly named cachectin and lymphotoxin. Two forms: alpha and
    ß, both produced by monocytes-macrophages, TNF-ß also by T lymphocytes
    and natural killer cells. Has activity similar to interleukin-1 and
    acts synergistically with it; promotes antiviral state and is
    cytotoxic for tumour cells; stimulates granulocytes and eosinophils,
    activates macrophages to interleukin-1 synthesis, stimulates B cells
    to proliferate and differentiate, T cells to proliferate,interleukin-2
    receptor synthesis, and interferon gamma synthesis; induces
    fibroblasts to synthesize prostaglandin and proliferate; induces fever
    and synthesis of acute-phase proteins; reduces cytochrome P450
    synthesis; activates endothelium and promotes adherence of
    neutrophilic granulocytes to endothelium; induces cell adhesion
    molecules like lymphocyte function-associated antigens LFA-1 and
    LFA-3, ICAM-1, and ELAM-1; inhibits gastric motility  in vitro;
    reduces lipoprotein lipase synthesis by adipocytes; and activates
    osteoclasts to bone resorption

     Urticaria. Transient eruption of skin characterized by erythematous
    or oedematous swelling (wheal) of the dermis or subcutaneous tissue

     Vaccination. See  Immunization

     Valency. Number of antigenic determinants or ligands that can bind
    to one antibody molecule or receptor

     V (variable) gene. Gene that encodes the variable part of
    immunoglobulin or T-cell receptor chains (e.g. VH1-VHn for
    immunoglobulin heavy chains, Vkappa 1-Vkappa n for immunoglobulin
    kappa light chain, Valpha 1-Valpha n for T-cell receptor alpha

     Variable gene family. Groups of germline V genes (which encode
    immunoglobulin chains or T-cell receptor genes) that have more than
    about 80% nucleotide sequence identity

     V (variable) region. Region at the amino terminal of immunoglobulin
    or T-cell receptor chains, which contributes to the antigen-binding
    site of the molecule. Encoded by V (variable), D (diversity), and J
    (joining) genes in DNA

     Vasoconstriction. Contraction of capillary venules, resulting in
    decreased blood flow

     Vasodilatation. Dilatation of capillary venules, resulting in
    increased blood flow through capillaries and lowering of local blood

     Veiled macrophage. Leukocyte belonging to the monocyte-macrophage
    lineage, present in lymph; has a major function in uptake and
    processing of antigen, followed by presentation (MHC class II-
    restricted) to helper-inducer T lymphocytes. Cytoplasm contains
    characteristic rod-like structures, Birbeck granules. Its equivalent
    in lymphoid tissue is the interdigitating dendritic cell, and that in
    skin is the Langerhans cell

     Waldeyer's ring. Lymphoid tissue of tonsils and adenoids located
    around the junction of the pharynx and oral cavity in humans and
    domestic animals. Main function is initiation of immunological
    reactions towards pathogens entering through the mouth. Contributes to
    the gut-associated lymphoid tissue

     White blood cell. Polymorphonuclear leukocyte, lymphocyte, or
    monocyte in peripheral blood

     White pulp. Area in spleen around central arterioles where lymphoid
    cells reside. Comprises three major compartments: the periarteriolar
    lymphocyte sheath, follicles, and marginal zone

     Xenobiotic. Chemical or substance that is foreign to the biological

     Xenogeneic. Genetically different phenotypes in individuals of
    different species; opposite of allogeneic, or isogeneic

     zeta Chain (see  epsilon Chain). One of the chains in the CD3
    molecule associated with the T-cell receptor


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    1.  RESUME

    1.    Le système immunitaire a évolué de manière à contrer les
    atteintes que peuvent porter à l'intégrité du soi des microorganismes
    ou des cellules ayant échappé au contrôle de l'organisme. Une
    intrusion xénobiotique peut perturber le fonctionnement du système
    immunitaire et c'est la reconnaissance de cet état de choses qui a
    permis les progrès de ces vingt dernières années. Toute une
    méthodologie expérimentale a été développée et validée
    (essentiellement sur des rongeurs) par des études impliquant une
    multitude de laboratoires. La présente monographie examine les
    fonctions et l'histophysiologie du système immunitaire et présente les
    données nécessaires à la compréhension et à l'interprétation des
    modifications pathologiques provoquées par les agressions
    immmunotoxiques. L'accent est mis sur le système immunitaire de
    l'homme et des rongeurs, mais sans négliger d'autres espèces, les
    poissons en particulier, auxquelles des études immunotoxicologiques
    ont été consacrées. Il importe, pour comprendre l'impact des effets
    immunotoxiques, de connaître la pathophysiologie du système
    immunitaire, et notamment la sensibilité variable de ses constituants,
    les modifications subies par les organes lymphoïdes et la réversibilité
    de ces modifications.

    2.    L'immunosuppression et l'immunostimulation ont toutes deux des
    conséquences sur le plan clinique. On a établi une corrélation entre
    les états d'immunodéficience ou d'immunodépression grave que l'on
    observe par exemple chez des patients greffés ou sous traitement
    cytostatique, et l'accroissement de l'incidence des maladies
    infectieuses (opportunistes en particulier) et du cancer. L'exposition
    à des substances chimiques immunotoxiques présentes dans l'environnement
    peut toutefois donner lieu à des formes plus subtiles d'immunodépression,
    difficiles à déceler, qui entraînent une augmentation de l'incidence de
    maladies infectieuses comme la grippe ou le rhume. Des travaux effectués
    sur des animaux de laboratoire et sur l'homme montrent que de nombreux
    produits chimiques présents dans l'environnement dépriment la réponse
    immunitaire. Ces substances xénobiotiques immunotoxiques ne se limitent
    pas à untype particulier de composé chimique ou d'agent physique. Il y
    a, parmi les médicaments, les pesticides, les solvants, les hydro-
    carbures halogénés, les hydrocarbures aromatiques et les métaux, des
    substances susceptibles d'altérer le système immunitaire et le
    rayonnement ultra-violet a également cette propriété. L'administration,
    à des fins thérapeutiques, d'agents immunostimulants, peut avoir des
    effets nocifs et certaines substances (béryllium, silice, hexa-
    chlorobenzène) présentes dans l'environnement, ont des propriétés
    immunostimulantes qui peuvent se traduire par des effets cliniques.

    3.    Du fait de sa complexité, le système immunitaire offre une
    multiplicité de cibles potentielles à l'action de ces agents avec
    toutes sortes de séquelles pathologiques. Les premières stratégies
    élaborées par les immunotoxicologues ont consisté à choisir et à
    mettre en oeuvre une batterie de tests multiphasiques sur animaux de
    laboratoire afin d'identifier les agents immunosuppresseurs ou
    immunostimulants. Ces batteries de tests peuvent varier selon
    l'organisme ou le laboratoire qui les mettent en oeuvre ou encore
    selon l'espèce animale utilisée, mais elles comportent toutes un ou
    plusieurs des éléments suivants: modification du poids ou de
    l'histologie des organes lymphoïdes; examen des leucocytes du sang
    périphérique, des cellules du tissu lymphoïde ou de la moelle osseuse
    à la recherche de modifications; intégrité de la fonction des cellules
    effectrices et régulatrices et modification éventuelle de la
    sensibilité à une exposition à des agents infectieux ou à des cellules

          La directive expérimentale originale No 407 de l'Organisation de
    coopération et de développement économiques, publiée en 1981, n'avait
    pas pour but la mise en évidence d'une immunotoxicité potentielle,
    aussi a-t-elle été modifiée pour la rendre plus adaptée à
    l'identification des substances immunotoxiques. Des systèmes d'épreuve
    multiphasiques permettant une investigation plus large de
    l'immunotoxicité ont été conçus par l'US National Toxicology Program,
    l'Institut Néerlandais de la Santé Publique et de la Protection de
    l'Environnement, l'US Environmental Protection Agency (Office of
    Pesticides), ainsi que par le Center for Food Safety andApplied
    Nutrition de l'US Food and Drug Administration. Des études ont été
    effectuées sur des souris et, à un moindre degré, sur des rats afin
    d'évaluer la spécificité, la précision (reproductibilité), la
    sensibilité, l'exactitude et la pertinence, pour l'appréciation du
    risque sanitaire, de diverses mesures de l'état immunitaire. On a
    entrepris la validation interlaboratoires, au niveau international, de
    toutes ces méthodes, dans le cadre de l'Etude collective
    internationale sur l'immunotoxicité organisée par le PISC, l'Union
    européenne et le Bundesinstitut für Gesundheitllichen
    Verbraucherschutz und Veterinärmedizin. Des études analogues ont été
    menées sur des rats Fischer 344 à propos de la cyclosporine A.

    4.    Les épreuves utilisées dans le système multiphasique sont
    décrites à la section 3 avec les raisons qui ont guidé ce choix et un
    exposé des difficultés que l'on peut rencontrer dans leur mise en
    oeuvre. Bien que conçus à l'origine pour des études sur des rats et
    des souris, certains de ces protocoles ont pu être utilisés avec fruit
    pour des travaux d'immunotoxicologie portant sur d'autres espèces,
    notamment des primates non humains, des mammifères marins, des chiens,
    des oiseaux et des poissons.

          Lorsqu'on se propose de déterminer dans quelle mesure un agent
    environnemental ou un médicament est susceptible d'avoir une influence
    nocive sur le système immunitaire, il faut prendre en considération un
    certain nombre de facteurs. Il s'agit notamment du choix d'un modèle
    animal et de variables d'exposition appropriés, de la prise en compte
    des paramètres toxicologiques généraux, de la pertinence biologique
    des points d'aboutissement retenus, de la mesure de grandeurs dûment
    validées et de la mise en oeuvre d'un système d'assurance de la
    qualité. Les conditions expérimentales doivent tenir compte des
    modalités de l'exposition humaine (voie de pénétration et
    concentration ou intensité) et de toute information disponible d'ordre
    toxicodynamique ou toxicocinétique. Les doses et la taille des
    échantillons doivent être choisies de manière à permettre l'obtention
    de bonnes courbes dose-réponse et la détermination de la dose sans
    effet (nocif ou non) observable. Cesstratégies sont améliorées en
    permanence afin de permettre une meilleure prévision des situations
    susceptibles d'entraîner une pathologie. En outre, on devrait pouvoir
    disposer de techniques qui faciliteraient l'étude du mode d'action des
    agents en cause. Il pourrait s'agir de méthodes  in vitro, de l'étude
    des réponses immunitaires locales (par exemple au niveau de la peau,
    des poumons et de l'intestin), de techniques de biologie moléculaire
    et de l'utilisation d'animaux génétiquement modifiés.

    5.    Mettre en évidence des modifications d'ordre immunologique après
    une exposition à des composés potentiellement immunotoxiques est plus
    complexe chez l'homme que chez l'animal de laboratoire. Les
    possibilités d'expérimentation sont limitées, il est difficile
    d'établir le niveau d'exposition à l'agent en cause (c'est-à-dire la
    dose) et de plus, l'état immunitaire des populations est extrêmement
    hétérogène. Cette hétérogénéité trouve son origine dans un certain
    nombre de facteurs: âge, sexe, race, gravidité, stress et aptitude à y
    faire face, pathologies et états infectieux cocomitants, état
    nutritionnel, tabagisme et prise de certains médicaments. L'intérêt de
    telle ou telle étude pour l'évaluation du risque est conditionné par
    un élément important, à savoir le concept épidémiologique qui la sous-
    tend. La plupart du temps on procède à une étude transversale, qui
    consiste à déterminer l'exposition et la morbidité à un moment donné
    ou sur une courte période. On compare ensuite la fonction immunitaire
    des sujets exposés à celle d'un groupe analogue de sujets non exposés.
    Ce genre d'étude peut receler un certain nombre de pièges.

          Nombre des altérations que produit l'exposition à une substance
    chimique ne se manifestent chez l'homme que de manière subtile et
    sporadique, aussi faut-il étudier des populations récemment exposées
    et utiliser des épreuves sensibles.

          La plupart des épreuves concernant l'immunité spécifique (à
    médiation cellulaire ou humorale), l'immunité non spécifique et les
    processus inflammatoires ont été conçues pour rechercher des anomalies
    chez des patients atteints d'une déficience immunitaire et ne sont pas

    forcément capables de déceler les modifications subtiles provoquées
    par les substances chimiques. Le PISC, les Centers for Disease Control
    et l'Académie des Sciences des Etats-Unis ont, chacun de leur côté,
    défini une méthodologie pour évaluer les modifications du système
    immunitaire quipourraient résulter d'une exposition à des produits
    immunotoxiques mais les épreuves proposées demandent encore à être

    6.    L'évaluation du risque consiste à analyser les données
    pertinentes sur un agent donné (effets biologiques, relations dose-
    réponse et exposition) pour tenter d'obtenir une estimation
    qualitative et quantitative des diverses conséquences nocives de la
    présence de cet agent. Elle se caractérise par quatre phases
    principales: reconnaître le danger, établir la relation dose-réponse,
    évaluer l'exposition et caractériser le risque. Jusqu'ici,
    l'immunotoxicologie s'est essentiellement attachée à reconnaître le
    danger et, dans une certaine mesure, à établir des relations dose-
    réponse, mais très peu d'études ont été consacrées à l'évaluation du
    risque ou à sa caractérisation.

          Comme dans d'autres domaines de la toxicologie, des incertitudes
    demeurent qui sont susceptibles de gêner l'interprétation des données
    immunotoxicologiques dans l'optique du risque pour la santé humaine.
    Les deux questions qui sont actuellement les plus problématiques -
    l'extrapolation à l'organe dans son ensemble des effets constatés sur
    la cellule et l'extrapolation à l'homme des données obtenues sur
    l'animal - valent pour la plupart des points d'aboutissement des
    effets, cancer excepté. Le premier problème tient aux incertitudes que
    comporte l'établissement d'une relation quatitative entre les
    modifications subies par la fonction immunitaire d'un individu et
    l'altération de sa résistance aux maladies infectieuses et
    néoplasiques. Le deuxième est lié à l'incertitude qui entache
    l'évaluation du risque pour la santé humaine à partir des résultats
    obtenus sur des animaux de laboratoire.

          L'évaluation du risque a pour finalité de protéger la santé
    humaine et l'environnement. Il faut donc que le choix des modéles
    expérimentaux soit judicieux. La toxicocinétique de la substance
    étudiée et la réponse immunitaire suscitée dans le modèle doivent
    pouvoir être comparées à celles qu'on observerait chez l'homme.

          Pour tirer une limite d'exposition des résultats expérimentaux,
    il est convenu d'appliquer un facteur d'incertitude aux données
    d'évaluation du risque. Cette convention ne tient pas compte de la
    réserve fonctionnelle ou de la redondance du système immunitaire. Une
    méthode plus récente d'évaluation du risque consiste à ultiliser des
    modèles  in vitro en complément aux études sur animaux de laboratoire.
    Cette méthode a l'avantage de permettre une extrapolation plus fidèle
    de l'animal à l'homme et de ne nécessiter qu'un minimum d'animaux.
    Elle permet également de pallier l'absence de données dans les cas où

    des considérations d'éthique limitent l'expérimentation sur l'homme.
    Le Chapitre 6 donne deux exemples de situation où les données obtenues
     in vitro permettent de lever en partie les incertitudes dans
    l'évaluation du risque dû à l'exposition à l'ozone et au rayonnement
    ultra-violet. L'utilisation des données immunotoxicologiques dans
    l'évaluation du risque reste limitée par la difficulté d'établir des
    relations quantitatives entre l'immunodépression et les manifestations
    cliniques d'une pathologie donnée.


    1.    El sistema inmunitario ha evolucionado para hacer frente a las
    amenazas a la integridad del organismo vivo provenientes de
    microorganismos o de células que han escapado a los mecanismos de
    control del organismo. El reconocimiento de que las sustancias
    xenobióticas pueden trastornar el funcionamiento del sistema
    inmunitario ha llevado a avances en el campo de la inmunotoxicología
    durante los dos últimos decenios. Se han formulado métodos
    experimentales (empleando principalmente especies de roedores), que
    han sido validados en estudios multilaboratorio. En esta monografía se
    examinan la función y la histofisiología del sistema inmunitario,
    presentándose la información necesaria para la comprensión e
    interpretación de los cambios patológicos causados por las agresiones
    inmunotóxicas. Si bien se hace hincapié en los sistemas inmunitarios
    del ser humano y de las especies de roedores, se hace referencia a
    otras especies, incluidos los peces, que han sido objeto de estudios
    inmunotoxicológicos. La fisiopatología del sistema inmunitario,
    incluidas la sensibilidad variable de sus componentes, las
    alteraciones de los órganos linfoides y la reversibilidad de los
    cambios, es importante para comprender las repercusiones de la

    2.    Tanto la inmunosupresión como la inmunoestimulación tienen
    consecuencias clínicas. Se ha observado que los estados de
    inmunodeficiencia y de inmunosupresión grave, como los que se pueden
    presentar en casos de trasplante y de terapia citostática, van
    acompañados de mayor incidencia de enfermedades infecciosas
    (especialmente las oportunistas) y de cáncer. Con todo, cabe prever
    que la exposición a los productos químicos inmunotóxicos enel medio
    ambiente dará origen a formas más sutiles de inmunosupresión cuya
    detección podría resultar difícil, lo que se traduciría en una mayor
    incidencia de infecciones tales como la gripe y el resfriado común.
    Estudios realizados con animales de laboratorio y seres humanos han
    mostrado que muchos de los productos químicos presentes en el medio
    ambiente provocan la supresión de la respuesta inmunitaria. Las
    sustancias xenobióticas inmunotóxicas no se limitan a una clase
    determinada de productos químicos. Entre los compuestos que tienen
    efectos nocivos para el sistema inmunitario se cuentan fármacos,
    plaguicidas, disolventes, hidrocarburos halogenados y aromáticos y
    metales; la radiación ultravioleta puede resultar también
    inmunotóxica. La administración terapéutica de agentes
    inmunoestimulantes puede provocar reacciones adversas; asimismo,
    algunos de los productos químicos presentes en el medio ambiente que
    poseen propiedades inmunoestimulantes (berilio, sílice,
    hexaclorobenceno) pueden tener consecuencias clínicas.

    3.    La complejidad del sistema inmunitario lleva aparejadas
    multiplicidad de posibles puntos vulnerables y secuelas patológicas.
    Los métodos iniciales ideados por los inmunotoxicólogos que realizan
    investigaciones sobre toxicología y evaluación de la inocuidad
    consistían en seleccionar y aplicar una serie de valoraciones
    escalonadas para identificar los agentes inmunosupresores e
    inmunoestimulantes en animales de laboratorio. Si bien la
    configuración de esas series de análisis puede variar según la
    institución o el laboratorio en que se llevan a cabo, así como según
    las especies de animales empleadas, en todas se mide unoo más de los
    siguientes parámetros: alteraciones del peso y de la histología de los
    órganos linfoides; cambios en la celularidad del tejido linfoide, de
    los leucocitos en la sangre periférica y/o de la médula ósea;
    trastornos de la función celular a nivel de los efectores o de la
    regulación, y alteración de la sensibilidad a la amenaza que presentan
    los agentes infecciosos o las células tumorales.

          La Directriz de pruebas inicial No 407 de la Organización de
    Cooperación y Desarrollo Económicos, publicada en 1981, no preveía la
    detección de los riesgos de inmunotoxicidad, y se han propuesto
    modificaciones destinadas a aumentar la utilidad de esa Directriz para
    la identificación de las sustancias inmunotóxicas. El Programa
    Nacional de Toxicología de los Estados Unidos, el Instituto Nacional
    de Salud Pública y Protección del Medio Ambiente de los Países Bajos,
    la Oficina de Plaguicidas de la Agencia para la Protección del Medio
    Ambiente de los Estados Unidos y el Centro de Seguridad de los
    Alimentos y Nutrición Aplicada de la Administración de Alimentos y
    Medicamentos de los Estados Unidos han elaborado sistemas de pruebas
    escalonadas para la investigación en mayor escala de los riesgos de
    inmunotoxicidad. Se han realizado estudios con ratones, y en menor
    medida con ratas, de diferentes indicadores del estado inmunológico
    con el fin de determinar su especificidad, precisión (reproducibilidad),
    sensibilidad, exactitud y pertinencia para la evaluación de los riesgos
    para la salud humana. Los métodos han sido objeto de validaciones
    internacionales interlaboratorios en el marco del Estudio Internacional
    en Colaboración sobre Inmunotoxicidad del IPCS, la Unión Europea y el
     Bundesinstitut für Gesundheitlichen Verbraucherschutz und
     Veterinärmedizin, yen estudios sobre la ciclosporina A en ratas
    Fisher 344.

    4.    Las pruebas empleadas en los programas de verificaciones
    escalonadas se describen en la Sección 3, en la que se indican la
    razón de ser de su selección y las complejidades que entraña su
    realización. Si bien esos protocolos fueron diseñados para estudios
    realizados con ratas y ratones, algunos de ellos se han aplicado con
    buenos resultados al estudio de la inmunotoxicidad en otras especies
    animales, incluidos primates no humanos, mamíferos marinos, perros,
    aves y peces.

          A la hora de evaluar las posibles repercusiones negativas de un
    agente ambiental o fármaco sobre el sistema inmunitario de los
    animales experimentales deberán considerarse diversos factores. Entre
    ellos cabe señalar: la selección de los modelos y las variables de
    exposición apropiados para los animales, la inclusión de parámetros
    toxicológicos generales, la comprensión de la importancia de los
    parámetros objeto de medición, el empleo de medidas validadas y el
    control de la calidad. En las condiciones experimentales deberán
    tenerse en cuenta las vías y el nivel posibles de exposición del ser
    humano, así como toda la información disponible sobre toxicodinámica y
    toxicocinética. Las dosis y el tamaño de las muestras deberán
    seleccionarse de manera que se puedan obtener curvas de dosis-
    respuesta bien definidas, además del nivel sin efectos adversos
    observados y del nivel sin efectos observados. Los métodos se
    perfeccionan continuamente para poder predecir mejor las condiciones
    que podrían ser causantes de enfermedades. Además, deberán elaborarse
    técnicas que contribuyana la identificación de mecanismos de acción;
    éstos podrían incluir los métodos  in vitro, el examen de las
    respuestas inmunitarias locales (por ejemplo, en la piel, los pulmones
    y los intestinos), y el empleo de las técnicas de la biología
    molecular y de animales modificados genéticamente.

    5.    La detección de los cambios inmunológicos ocurridos tras la
    exposición a compuestos que podrían ser inmunotóxicos resulta más
    complicada en el ser humano que en los animales de laboratorio. Las
    posibilidades de realizar pruebas son limitadas; los niveles de
    exposición al agente (es decir, la dosis) son difíciles de establecer
    y el estado inmunitario de las poblaciones es sumamente heterogéneo.
    La edad, la raza, el sexo, la gestación, el estrés agudo y la
    capacidad para hacerle frente, las enfermedades e infecciones
    coexistentes, el estado nutricional, el humo del tabaco y algunos
    medicamentos se cuentan entre los factores que contribuyen a esa

          El diseño de los estudios epidemiológicos es un factor importante
    para determinar la utilidad de un estudio determinado para la
    evaluación de riesgos. El tipo de diseño empleado más frecuentemente
    en materia de inmunotoxicidad son los estudios transversales, en los
    que se miden las condiciones de exposición y el estado de la
    enfermedad en un momento dado, o durante un periodo breve. A
    continuación se compara la función inmunitaria de los sujetos
    «expuestos» con la de un grupo comparable de individuos
    «no expuestos». Ese tipo de diseño lleva aparejados posibles escollos.

          Como muchos de los cambios observados en la respuesta inmunitaria
    de los seres humanos tras su exposición a un productoquímico podrían
    ser esporádicos y sutiles, habrá que estudiar las poblaciones que han
    estado expuestas recientemente empleando pruebas de gran sensibilidad

    para la evaluación del sistema inmunitario. Las conclusiones sobre los
    efectos inmunotóxicos deberán estar basadas en las variaciones
    detectadas no en un parámetro aislado sino en el perfil inmunológico
    del individuo o de la población.

          La mayoría de las pruebas existentes para determinar la inmunidad
    específica (de base celular y humoral), la inmunidad no específica y
    la inflamación han sido concebidas para detectar las alteraciones
    inmunitarias en pacientes que padecen de inmunodeficiencia y no
    siempre resultan adecuadas para detectar las alteraciones sutiles
    provocadas por los productos químicos presentes en el medio ambiente.
    El IPCS, los Centros de Control de Enfermedades y la Academia de
    Ciencias de los Estados Unidos han descrito procedimientos para
    evaluar los cambios que ocurren en el sistema inmunitario del ser
    humano como consecuencia de la exposición a sustancias inmunotóxicas;
    con todo, las pruebas descritas deberán ser evaluadas con este fin.

    6.    La evaluación de riesgos es un proceso en el que se analizan la
    información pertinente sobre los efectos biológicos, las relaciones
    dosis-respuesta y la exposición a un agente determinado con miras a
    establecer estimaciones cualitativas y cuantitativas de los resultados
    adversos. Por regla general, la evaluación de los riesgos supone
    cuatro pasos fundamentales: identificación de los riesgos; evaluación
    de la relación dosis-respuesta; evaluación de la exposición y
    caracterización de los riesgos. Hasta ahora, lainmunotoxicología se ha
    centrado principalmente en la identificación de los riesgos y, en
    cierta medida, en la evaluación de las relaciones dosis-respuesta; muy
    contados han sido los estudios que han incluido la evaluación de la
    exposición o la caracterización de los riesgos.

          Al igual que ocurre en otros campos de la toxicología, existen
    incertidumbres que podrían afectar a la interpretación de los datos
    sobre inmunotoxicidad en cuanto a los riesgos para la salud humana.
    Las dos cuestiones más problemáticas - la extrapolación de los efectos
    observados en células individuales a todo un órgano, o a niveles
    superiores, y la extrapolación al ser humano de los resultados
    obtenidos en experimentos con animales - son comunes a la mayoría de
    los parámetros de valoración no relacionados con el cáncer. La primera
    obedece a las incertidumbres vinculadas al establecimiento de una
    relación cuantitativa entre los cambios observados en la función
    inmunitaria del individuo y la perturbación de la resistencia a las
    infecciones y enfermedades neoplásicas. La segunda cuestión es
    consecuencia de las incertidumbres que lleva aparejadas la evaluación
    de los riesgos para la salud humana basándose en los estudios
    realizados con animales de laboratorio.

          El objetivo fundamental de la evaluación de los riesgos es la
    protección de la salud de los seres humanos y del medio ambiente. Por
    lo tanto, deberán seleccionarse sistemas modelo idóneos. La
    toxicocinética del material de prueba y la índole y magnitud de la
    respuesta inmunitaria generada en el modelo deberán ser comparables a
    la de los seres humanos.

          Habitualmente, en la evaluación de los riesgos se emplean
    factores empíricos de incertidumbre para determinar el límite de
    exposición aceptable a partir de los resultados experimentales. Ese
    procedimiento no toma en cuenta la reserva funcional ni la redundancia
    del sistema inmunitario. Un adelanto más reciente en materia de
    evaluación de riesgos es el empleo de modelos  in vitro como
    complemento de los estudios realizados con animales de laboratorio.
    Ese procedimiento tiene la ventaja de que permite aumentar la
    exactitud de la extrapolación al ser humano de los resultados
    obtenidos en los experimentos realizados con animales, reduciendo al
    mínimo el número de animales necesarios; asimismo, permite colmar la
    brecha entre ambos tipos de información, sobre todo en los casos en
    que los experimentos con seres humanos se ven limitados por
    consideraciones de índole ética. En el capítulo 6 se presentan dos
    ejemplos de cómo la información  in vitro permite reducir las
    incertidumbres en materia de evaluación de riesgos relacionadas con la
    exposición al ozono y a la radiación ultravioleta. La dificultad para
    establecer relaciones cuantitativas entre la inmunosupresión y las
    enfermedades clínicas ha limitado el empleo de los datos
    inmunotoxicológicos en la evaluación de los riesgos.

    See Also:
       Toxicological Abbreviations