Principles and Methods for Assessing Direct Immunotoxicity
Associated with Exposure to Chemicals
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
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
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.
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 an mercury, for instance, manifest immunosuppressive activity,
hypersensitivity, and autoimmunity (Lawrence et al., 1987). 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.
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 + +
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 mus be understood. Since knowledge of the structure and function of the immune system is growing rapidly.
2.2 Direct immunotoxicity in laboratory animals
The following are some illustrative examples of immunotoxic
chemicals.
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.
2.2.1.1 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.
2.2.1.2 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
diseases.
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