INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY
ENVIRONMENTAL HEALTH CRITERIA 180
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
venture of the United Nations Environment Programme, the International
Labour Organisation, and the World Health Organization. The main
objective of the IPCS is to carry out and disseminate evaluations of
the effects of chemicals on human health and the quality of the
environment. Supporting activities include the development of
epidemiological, experimental laboratory, and risk-assessment methods
that could produce internationally comparable results, and the
development of manpower in the field of toxicology. Other activities
carried out by the IPCS include the development of know-how for coping
with chemical accidents, coordination of laboratory testing and
epidemiological studies, and promotion of research on the mechanisms
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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CONTENTS
NOTE TO READERS OF THE CRITERIA MONOGRAPHS
PREAMBLE
WHO TASK GROUP MEETING ON PRINCIPLES AND METHODS FOR ASSESSING DIRECT
IMMUNOTOXICITY ASSOCIATED WITH EXPOSURE TO CHEMICALS
PRINCIPLES AND METHODS
ABBREVIATIONS
SUMMARY AND RECOMMENDATIONS
1. INTRODUCTION TO IMMUNOTOXICOLOGY
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
1.2.1.1 Encounter and recognition
1.2.1.2 Specificity
1.2.1.3 Choice of effector reaction; diversity
of the answer
1.2.1.4 Immunoregulation
1.2.1.5 Modifying factors outside the immune
system
1.2.1.6 Immunological memory
1.2.2. Histophysiology of lymphoid organs
1.2.2.1 Overview: structure of the immune system
1.2.2.2 Bone marrow
1.2.2.3 Thymus
1.2.2.4 Lymph nodes
1.2.2.5 Spleen
1.2.2.6 Mucosa-associated lymphoid tissue
1.2.2.7 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. HEALTH IMPACT OF SELECTED IMMUNOTOXIC AGENTS
2.1. Description of consequences on human health
2.1.1. Consequences of immunosuppression
2.1.1.1 Cancer
2.1.1.2 Infectious diseases
2.1.2. Consequences of immunostimulation
2.2. Direct immunotoxicity in laboratory animals
2.2.1. Azathioprine and cyclosporin A
2.2.1.1 Azathioprine
2.2.1.2 Cyclosporin A
2.2.2. Halogenated hydrocarbons
2.2.2.1
2,3,7,8-Tetrachlorodibenzo- para-dioxin
2.2.2.2 Polychlorinated biphenyls
2.2.2.3 Hexachlorobenzene
2.2.3. Pesticides
2.2.3.1 Organochlorine pesticides
2.2.3.2 Organophosphate compounds
2.2.3.3 Pyrethroids
2.2.3.4 Carbamates
2.2.3.5 Dinocap
2.2.4. Polycyclic aromatic hydrocarbons
2.2.5. Solvents
2.2.5.1 Benzene
2.2.5.2 Other solvents
2.2.6. Metals
2.2.6.1 Cadmium
2.2.6.2 Lead
2.2.6.3 Mercury
2.2.6.4 Organotins
2.2.6.5 Gallium arsenide
2.2.6.6 Beryllium
2.2.7. Air pollutants
2.2.8. Mycotoxins
2.2.9. Particles
2.2.9.1 Asbestos
2.2.9.2 Silica
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
2.3.1.1 Fish
2.3.1.2 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. STRATEGIES FOR TESTING THE IMMUNOTOXICITY OF CHEMICALS IN ANIMALS
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
immunotoxicity
3.3.1. Species selection
3.3.2. Systemic immunosuppression
3.3.3. Local suppression
4. METHODS OF IMMUNOTOXICOLOGY IN EXPERIMENTAL ANIMALS
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
4.2.4.1 Spleen immunoglobulin M and
immunoglobulin G plaque-forming cell
assay to the T-dependent antigen, sheep
red blood cells
4.2.4.2 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
4.2.10.1 Listeria monocytogenes
4.2.10.2 Streptococcus infectivity models
4.2.10.3 Viral infection model with mouse and rat
cytomegalovirus
4.2.10.4 Influenza virus model
4.2.10.5 Parasitic infection model with
Trichinella spiralis
4.2.10.6 Plasmodium model
4.2.10.7 B16F10 Melanoma model
4.2.10.8 PYB6 Carcinoma model
4.2.10.9 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
4.3.3.1 Fish
4.3.3.2 Chickens
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. ESSENTIALS OF IMMUNOTOXICITY ASSESSMENT IN HUMANS
5.1. Introduction: Immunocompetence and immunosuppression
5.2. Considerations in assessing human immune status related to
immunotoxicity
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
5.6.2.1 Immunoglobulin concentration
5.6.2.2 Specific antibodies
5.6.3. Tests for inflammation and autoantibodies
5.6.3.1 C-Reactive protein
5.6.3.2 Antinuclear antibody
5.6.3.3 Rheumatoid factor
5.6.3.4 Thyroglobulin antibody
5.6.4. Tests for cellular immunity
5.6.4.1 Flow cytometry
5.6.4.2 Delayed-type hypersensitivity
5.6.4.3 Proliferation of mononuclear cells in
vitro
5.6.5. Tests for nonspecific immunity
5.6.5.1 Natural killer cells
5.6.5.2 Polymorphonuclear granulocytes
5.6.5.3 Complement
5.6.6. Clinical chemistry
5.6.7. Additional confirmatory tests
6. RISK ASSESSMENT
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
7. SOME TERMS USED IN IMMUNOTOXICOLOGY
REFERENCES
RESUME
RESUMEN
NOTE TO READERS OF THE CRITERIA MONOGRAPHS
Every effort has been made to present information in the Criteria
monographs as accurately as possible without unduly delaying their
publication. In the interest of all users of the Environmental Health
Criteria monographs, readers are requested to communicate any errors
that may have occurred to the Director of the International Programme
on Chemical Safety, World Health Organization, Geneva, Switzerland, in
order that they may be included in corrigenda.
* * *
A detailed data profile and a legal file can be obtained from the
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
Protection.
Environmental Health Criteria
PREAMBLE
Objectives
The WHO Environmental Health Criteria Programme was initiated in
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The first Environmental Health Criteria (EHC) monograph, on
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WHO TASK GROUP MEETING ON PRINCIPLES AND METHODS FOR ASSESSING DIRECT
IMMUNOTOXICITY ASSOCIATED WITH EXPOSURE TO CHEMICALS
Members
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,
Netherlands)
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
Observers
IUTOX
Dr P. Montuschi, Department of Pharmacology, Catholic University of
the Sacred Heart, Rome, Italy
ECETOC
Dr R.W.R. Crevel, Environmental Safety Laboratory, Unilever Research
and Engineering, Sharnbrook, Bedfordshire, United Kingdom
Secretariat
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
ENVIRONMENTAL HEALTH CRITERIA PRINCIPLES AND METHODS FOR ASSESSING
DIRECT IMMUNOTOXICITY ASSOCIATED WITH EXPOSURE TO CHEMICALS
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
ABBREVIATIONS
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
SUMMARY
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
characterization.
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
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
established.
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. INTRODUCTION TO IMMUNOTOXICOLOGY
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.,
1994).
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
rodents
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.
1.2.1.1 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) + ? ? ? ?
Agnatha
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) ? - - - - -
Agnatha
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
cells
Small T B
Tunicata (sea squirts) + - - - +
Agnatha
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) - - - -
Agnatha
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
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.
1.2.1.2 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.
1.2.1.3 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).
1.2.1.4 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.
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).
1.2.1.5 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).
1.2.1.6 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,
1989).
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
1.2.2.1 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 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
Fabricius.
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
1.2.2.7).
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).
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.
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
Thymus
Cortex Reticular epithelium, immature Generation of T-cell competence,
T cells T-cell receptor rearrangement, positive
selection (MHC restriction), negative
selection (autoreactive cells), phenotypic
changes
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
lumen
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 1.2.2.3
and 1.2.2.4.
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