The functions of the immune system are to protect the body from invading infectious agents and to provide immune surveillance against arising tumour cells. It has a first line of defence that is non-specific and that can initiate effector reactions itself, and an acquired specific branch, in which lymphocytes and antibodies carry the specificity of recognition and subsequent reactivity towards the antigen.

Immunotoxicology has been 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).

When the immune system acts as a passive target of chemical insults, the result can be decreased resistance to infection and certain forms of neoplasia, or immune disregulation/stimulation that can exacerbate allergy or auto-immunity. In the case that the immune system responds to the antigenic specificity of the xenobiotic or host antigen modified by the compound, toxicity can become manifest as allergies or autoimmune diseases.

Animal models to investigate chemical-induced immune suppression have been developed, and a number of these methods are validated (Burleson, Munson, and Dean 1995; IPCS 1996). For testing purposes, a tiered approach is followed to make an adequate selection from the overwhelming number of assays available. Generally, the objective of the first tier is to identify potential immunotoxicants. If potential immunotoxicity is identified, a second tier of testing is performed to confirm and characterize further the changes observed. Third-tier investigations include special studies on the mechanism of action of the compound. Several xenobiotics have been identified as immunotoxicants causing immunosuppression in such studies with laboratory animals.

The database on immune function disturbances in humans by environmental chemicals is limited (Descotes 1986; NRC Subcommittee on Immunotoxicology 1992). The use of markers of immunotoxicity has received little attention in clinical and epidemiological studies to investigate the effect of these chemicals on human health. Such studies have not been performed frequently, and their interpretation often does not permit unequivocal conclusions to be drawn, due for instance to the uncontrolled nature of exposure. Therefore, at present, immunotoxicity assessment in rodents, with subsequent extrapolation to man, forms the basis of decisions regarding hazard and risk.

Hypersensitivity reactions, notably allergic asthma and contact dermatitis, are important occupational health problems in industrialized countries (Vos, Younes and Smith 1995). The phenomenon of contact sensitization was investigated first in the guinea pig (Andersen and Maibach 1985). Until recently this has been the species of choice for predictive testing. Many guinea pig test methods are available, the most frequently employed being the guinea pig maximization test and the occluded patch test of Buehler. Guinea pig tests and newer approaches developed in mice, such as ear swelling tests and the local lymph node assay, provide the toxicologist with the tools to assess skin sensitization hazard. The situation with respect to sensitization of the respiratory tract is very different. There are, as yet, no well-validated or widely accepted methods available for the identification of chemical respiratory allergens although progress in the development of animal models for the investigation of chemical respiratory allergy has been achieved in the guinea pig and mouse.

Human data show that chemical agents, in particular drugs, can cause autoimmune diseases (Kammüller, Bloksma and Seinen 1989). There are a number of experimental animal models of human autoimmune diseases. Such comprise both spontaneous pathology (for example systemic lupus erythematosus in New Zealand Black mice) and autoimmune phenomena induced by experimental immunization with a cross-reactive autoantigen (for example the H37Ra adjuvant induced arthritis in Lewis strain rats). These models are applied in the preclinical evaluation of immunosuppressive drugs. Very few studies have addressed the potential of these models for assessment of whether a xenobiotic exacerbates induced or congenital autoimmunity. Animal models that are suitable to investigate the ability of chemicals to induce autoimmune diseases are virtually lacking. One model that is used to a limited extent is the popliteal lymph node assay in mice. Like the situation in humans, genetic factors play a crucial role in the development of autoimmune disease (AD) in laboratory animals, which will limit the predictive value of such tests.

The Immune System

The major function of the immune system is defence against bacteria, viruses, parasites, fungi and neoplastic cells. This is achieved by the actions of various cell types and their soluble mediators in a finely tuned concert. The host defence can be roughly divided into non-specific or innate resistance and specific or acquired immunity mediated by lymphocytes (Roitt, Brostoff and Male 1989).

Components of the immune system are present throughout the body (Jones et al. 1990). The lymphocyte compartment is found within lymphoid organs (figure 1). The bone marrow and thymus are classified as primary or central lymphoid organs; the secondary or peripheral lymphoid organs include lymph nodes, spleen and lymphoid tissue along secretory surfaces such as the gastrointestinal and respiratory tracts, the so-called mucosa-associated lymphoid tissue (MALT). About half of the body’s lymphocytes are located at any one time in MALT. In addition the skin is an important organ for the induction of immune responses to antigens present on the skin. Important in this process are epidermal Langerhans cells that have an antigen-presenting function.

Figure 1. Primary and secondary lymphoid organs and tissues

Phagocytic cells of the monocyte/macrophage lineage, called the mononuclear phagocyte system (MPS), occur in lymphoid organs and also at extranodal sites; the extranodal phagocytes include Kupffer cells in the liver, alveolar macrophages in the lung, mesangial macrophages in the kidney and glial cells in the brain. Polymorphonuclear leukocytes (PMNs) are present mainly in blood and bone marrow, but accumulate at sites of inflammation.

Non-specific defence

A first line of defence to micro-organisms is executed by a physical and chemical barrier, such as at the skin, the respiratory tract and the alimentary tract. This barrier is helped by non-specific protective mechanisms including phagocytic cells, such as macrophages and polymorphonuclear leukocytes, which are able to kill pathogens, and natural killer cells, which can lyse tumour cells and virus-infected cells. The complement system and certain microbial inhibitors (e.g., lysozyme) also take part in the non-specific response.

Specific immunity

After initial contact of the host with the pathogen, specific immune responses are induced. The hallmark of this second line of defence is specific recognition of determinants, so-called antigens or epitopes, of the pathogens by receptors on the cell surface of B- and T-lymphocytes. Following interaction with the specific antigen, the receptor-bearing cell is stimulated to undergo proliferation and differentiation, producing a clone of progeny cells that are specific for the eliciting antigen. The specific immune responses help the non-specific defence presented to the pathogens by stimulating the efficacy of the non-specific responses. A fundamental characteristic of specific immunity is that memory develops. Secondary contact with the same antigen provokes a faster and more vigorous but well-regulated response.

The genome does not have the capacity to carry the codes of an array of antigen receptors sufficient to recognize the number of antigens that can be encountered. The repertoire of specificity develops by a process of gene rearrangements. This is a random process, during which various specificities are brought about. This includes specificities for self components, which are undesirable. A selection process that takes place in the thymus (T cells), or bone marrow (B cells) operates to delete these undesirable specificities.

Normal immune effector function and homeostatic regulation of the immune response is dependent upon a variety of soluble products, known collectively as cytokines, which are synthesized and secreted by lymphocytes and by other cell types. Cytokines have pleiotropic effects on immune and inflammatory responses. Cooperation between different cell populations is required for the immune response—the regulation of antibody responses, the accumulation of immune cells and molecules at inflammatory sites, the initiation of acute phase responses, the control of macrophage cytotoxic function and many other processes central to host resistance. These are influenced by, and in many cases are dependent upon, cytokines acting individually or in concert.

Two arms of specific immunity are recognized—humoral immunity and cell-mediated or cellular immunity:

Humoral immunity. In the humoral arm B-lymphocytes are stimulated following recognition of antigen by cell-surface receptors. Antigen receptors on B-lymphocytes are immunoglobulins (Ig). Mature B cells (plasma cells) start the production of antigen-specific immunoglobulins that act as antibodies in serum or along mucosal surfaces. There are five major classes of immunoglobulins: (1) IgM, pentameric Ig with optimal agglutinating capacity, which is first produced after antigenic stimulation; (2) IgG, the main Ig in circulation, which can pass the placenta; (3) IgA, secretory Ig for the protection of mucosal surfaces; (4) IgE, Ig fixing to mast cells or basophilic granulocytes involved in immediate hypersensitivity reactions and (5) IgD, whose major function is as a receptor on B-lymphocytes.

Cell-mediated immunity. The cellular arm of the specific immune system is mediated by T-lymphocytes. These cells also have antigen receptors on their membranes. They recognize antigen if presented by antigen presenting cells in the context of histocompatibility antigens. Hence, these cells have a restriction in addition to the antigen specificity. T cells function as helper cells for various (including humoral) immune responses, mediate recruitment of inflammatory cells, and can, as cytotoxic T cells, kill target cells after antigen-specific recognition.

Mechanisms of Immunotoxicity

Immunosuppression

Effective host resistance is dependent upon the functional integrity of the immune system, which in turn requires that the component cells and molecules which orchestrate immune responses are available in sufficient numbers and in an operational form. Congenital immunodeficiencies in humans are often characterized by defects in certain stem cell lines, resulting in impaired or absent production of immune cells. By analogy with congenital and acquired human immunodeficiency diseases, chemical-induced immunosuppression may result simply from a reduced number of functional cells (IPCS 1996). The absence, or reduced numbers, of lymphocytes may have more or less profound effects on immune status. Some immunodeficiency states and severe immunosuppression, as can occur in transplantation or cytostatic therapy, have been associated in particular with increased incidences of opportunistic infections and of certain neoplastic diseases. The infections can be bacterial, viral, fungal or protozoan, and the predominant type of infection depends on the associated immunodeficiency. Exposure to immunosuppressive environmental chemicals may be expected to result in more subtle forms of immunosuppression, which may be difficult to detect. These may lead, for example, to an increased incidence of infections such as influenza or the common cold.

In view of the complexity of the immune system, with the wide variety of cells, mediators and functions that form a complicated and interactive network, immunotoxic compounds have numerous opportunities to exert an effect. Although the nature of the initial lesions induced by many immunotoxic chemicals have not yet been elucidated, there is increasing information available, mostly derived from studies in laboratory animals, regarding the immunobiological changes which result in depression of immune function (Dean et al. 1994). Toxic effects might occur at the following critical functions (and some examples are given of immunotoxic compounds affecting these functions):

•  development and expansion of different stem cell populations (benzene exerts immunotoxic effects at the stem cell level, causing lymphocytopenia)
•  proliferation of various lymphoid and myeloid cells as well as supportive tissues in which these cells mature and function (immunotoxic organotin compounds suppress the proliferative activity of lymphocytes in the thymic cortex through direct cytotoxicity; the thymotoxic action of 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) and related compounds is likely due to an impaired function of thymic epithelial cells, rather than to direct toxicity for thymocytes)
•  antigen uptake, processing and presentation by macrophages and other antigen-presenting cells (one of the targets of 7,12-dimethylbenz(a)anthracene (DMBA) and of lead is antigen presentation by macrophages; a target of ultraviolet radiation is the antigen-presenting Langerhans cell)
•  regulatory function of T-helper and T-suppressor cells (T-helper cell function is impaired by organotins, aldicarb, polychlorinated biphenyls (PCBs), TCDD and DMBA; T-suppressor cell function is reduced by low-dose cyclophosphamide treatment)
•  production of various cytokines or interleukins (benzo(a)pyrene (BP) suppresses interleukin-1 production; ultraviolet radiation alters production of cytokines by keratinocytes)
•  synthesis of various classes of immunoglobulins IgM and IgG is suppressed following PCB and tributyltin oxide (TBT) treatment, and increased after hexachlorobenzene (HCB) exposure).
•  complement regulation and activation (affected by TCDD)
•  cytotoxic T cell function (3-methylcholanthrene (3-MC), DMBA, and TCDD suppress cytotoxic T cell activity)
•  natural killer (NK) cell function (pulmonary NK activity is suppressed by ozone; splenic NK activity is impaired by nickel)
•  macrophage and polymorphonuclear leukocyte chemotaxis and cytotoxic functions (ozone and nitrogen dioxide impair the phagocytic activity of alveolar macrophages).

Allergy

Allergy may be defined as the adverse health effects which result from the induction and elicitation of specific immune responses. When hypersensitivity reactions occur without involvement of the immune system the term pseudo-allergy is used. In the context of immunotoxicology, allergy results from a specific immune response to chemicals and drugs that are of interest. The ability of a chemical to sensitize individuals is generally related to its ability to bind covalently to body proteins. Allergic reactions may take a variety of forms and these differ with respect to both the underlying immunological mechanisms and the speed of the reaction. Four major types of allergic reactions have been recognized: Type I hypersensitivity reactions, which are effectuated by IgE antibody and where symptoms are manifest within minutes of exposure of the sensitized individual. Type II hypersensitivity reactions result from the damage or destruction of host cells by antibody. In this case symptoms become apparent within hours. Type III hypersensitivity, or Arthus, reactions are also antibody mediated, but against soluble antigen, and result from the local or systemic action of immune complexes. Type IV, or delayed-type hypersensitivity, reactions are effected by T-lymphocytes and normally symptoms develop 24to 48hours following exposure of the sensitized individual.

The two types of chemical allergy of greatest relevance to occupational health are contact sensitivity or skin allergy and allergy of the respiratory tract.

Contact hypersensitivity. A large number of chemicals are able to cause skin sensitization. Following topical exposure of a susceptible individual to a chemical allergen, a T-lymphocyte response is induced in the draining lymph nodes. In the skin the allergen interacts directly or indirectly with epidermal Langerhans cells, which transport the chemical to the lymph nodes and present it in an immunogenic form to responsive T-lymphocytes. Allergen- activated T-lymphocytes proliferate, resulting in clonal expansion. The individual is now sensitized and will respond to a second dermal exposure to the same chemical with a more aggressive immune response, resulting in allergic contact dermatitis. The cutaneous inflammatory reaction which characterizes allergic contact dermatitis is secondary to the recognition of the allergen in the skin by specific T-lymphocytes. These lymphocytes become activated, release cytokines and cause the local accumulation of other mononuclear leukocytes. Symptoms develop some 24 to 48 hours following exposure of the sensitized individual, and allergic contact dermatitis therefore represents a form of delayed-type hypersensitivity. Common causes of allergic contact dermatitis include organic chemicals (such as 2,4-dinitrochlorobenzene), metals (such as nickel and chromium) and plant products (such as urushiol from poison ivy).

Respiratory hypersensitivity. Respiratory hypersensitivity is usually considered to be a Type I hypersensitivity reaction. However, late phase reactions and the more chronic symptoms associated with asthma may involve cell-mediated (Type IV) immune processes. The acute symptoms associated with respiratory allergy are effected by IgE antibody, the production of which is provoked following exposure of the susceptible individual to the inducing chemical allergen. The IgE antibody distributes systemically and binds, via membrane receptors, to mast cells which are found in vascularized tissues, including the respiratory tract. Following inhalation of the same chemical a respiratory hypersensitivity reaction will be elicited. Allergen associates with protein and binds to, and cross-links, IgE antibody bound to mast cells. This in turn causes the degranulation of mast cells and the release of inflammatory mediators such as histamine and leukotrienes. Such mediators cause bronchoconstriction and vasodilation, resulting in the symptoms of respiratory allergy; asthma and/or rhinitis. Chemicals known to cause respiratory hypersensitivity in man include acid anhydrides (such as trimellitic anhydride), some diisocyanates (such as toluene diisocyanate), platinum salts and some reactive dyes. Also, chronic exposure to beryllium is known to cause hypersensitivity lung disease.

Autoimmunity

Autoimmunity can be defined as the stimulation of specific immune responses directed against endogenous “self” antigens. Induced autoimmunity can result either from alterations in the balance of regulatory T-lymphocytes or from the association of a xenobiotic with normal tissue components such as to render them immunogenic (“altered self”). Drugs and chemicals known to incidentally induce or exacerbate effects like those of autoimmune disease (AD) in susceptible individuals are low molecular weight compounds (molecular weight 100 to 500) that are generally considered to be not immunogenic themselves. The mechanism of AD by chemical exposure is mostly unknown. Disease can be produced directly by means of circulating antibody, indirectly through the formation of immune complexes, or as a consequence of cell-mediated immunity, but likely occurs through a combination of mechanisms. The pathogenesis is best known in immune haemolytic disorders induced by drugs:

•  The drug can attach to the red-cell membrane and interact with a drug-specific antibody.
•  The drug can alter the red-cell membrane so that the immune system regards the cell as foreign.
•  The drug and its specific antibody form immune complexes that adhere to the red-cell membrane to produce injury.
•  Red-cell sensitization occurs due to the production of red-cell autoantibody.

A variety of chemicals and drugs, in particular the latter, have been found to induce autoimmune-like responses (Kamüller, Bloksma and Seinen 1989). Occupational exposure to chemicals may incidentally lead to AD-like syndromes. Exposure to monomeric vinyl chloride, trichloroethylene, perchloroethylene, epoxy resins and silica dust may induce scleroderma-like syndromes. A syndrome similar to systemic lupus erythematosus (SLE) has been described after exposure to hydrazine. Exposure to toluene diisocyanate has been associated with the induction of thrombocytopenic purpura. Heavy metals such as mercury have been implicated in some cases of immune complex glomerulonephritis.

Human Risk Assessment

The assessment of human immune status is performed mainly using peripheral blood for analysis of humoral substances like immunoglobulins and complement, and of blood leukocytes for subset composition and functionality of subpopulations. These methods are usually the same as those used to investigate humoral and cell-mediated immunity as well as nonspecific resistance of patients with suspected congenital immunodeficiency disease. For epidemiological studies (e.g., of occupationally exposed populations) parameters should be selected on the basis of their predictive value in human populations, validated animal models, and the underlying biology of the markers (see table 1). The strategy in screening for immunotoxic effects after (accidental) exposure to environmental pollutants or other toxicants is much dependent on circumstances, such as type of immunodeficiency to be expected, time between exposure and immune status assessment, degree of exposure and number of exposed individuals. The process of assessing the immunotoxic risk of a particular xenobiotic in humans is extremely difficult and often impossible, due largely to the presence of various confounding factors of endogenous or exogenous origin that influence the response of individuals to toxic damage. This is particularly true for studies which investigate the role of chemical exposure in autoimmune diseases, where genetic factors play a crucial role.

Table 1. Classification of tests for immune markers

As adequate human data are seldom available, the assessment of risk for chemical-induced immunosuppression in humans is in the majority of cases based upon animal studies. The identification of potential immunotoxic xenobiotics is undertaken primarily in controlled studies in rodents. In vivo exposure studies present, in this regard, the optimal approach to estimate the immunotoxic potential of a compound. This is due to the multifactoral and complex nature of the immune system and of immune responses. In vitro studies are of increasing value in the elucidation of mechanisms of immunotoxicity. In addition, by investigating the effects of the compound using cells of animal and human origin, data can be generated for species comparison, which can be used in the “parallelogram” approach to improve the risk assessment process. If data are available for three cornerstones of the parallelogram (in vivo animal, and in vitro animal and human) it may be easier to predict the outcome at the remaining cornerstone, that is, the risk in humans.

When assessment of risk for chemical-induced immunosuppression has to rely solely upon data from animal studies, an approach can be followed in the extrapolation to man by the application of uncertainty factors to the no observed adverse effect level (NOAEL). This level can be based on parameters determined in relevant models, such as host resistance assays and in vivo assessment of hypersensitivity reactions and antibody production. Ideally, the relevance of this approach to risk assessment requires confirmation by studies in humans. Such studies should combine the identification and measurement of the toxicant, epidemiological data and immune status assessments.

To predict contact hypersensitivity, guinea pig models are available and have been used in risk assessment since the 1970s. Although sensitive and reproducible, these tests have limitations as they depend on subjective evaluation; this can be overcome by newer and more quantitative methods developed in the mouse. Regarding chemical-induced hypersensitivity induced by inhalation or ingestion of allergens, tests should be developed and evaluated in terms of their predictive value in man. When it comes to setting safe occupational exposure levels of potential allergens, consideration has to be given to the biphasic nature of allergy: the sensitization phase and the elicitation phase. The concentration required to elicit an allergic reaction in a previously sensitized individual is considerably lower than the concentration necessary to induce sensitization in the immunologically naïve but susceptible individual.

As animal models to predict chemical-induced autoimmunity are virtually lacking, emphasis should be given to the development of such models. For the development of such models, our knowledge of chemical-induced autoimmunity in humans should be advanced, including the study of genetic and immune system markers to identify susceptible individuals. Humans that are exposed to drugs that induce autoimmunity offer such an opportunity.

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