Biologic Markers in Immunotoxicology (1992)

This chapter deals with autoimmune diseases caused by exposure to environmental chemicals. The first portions discuss current understanding of the problem, that is, the definition, scope, and relative contributions of innate genetic susceptibility to environmental agents in producing autoimmune diseases. The next section deals with the possible mechanisms by which xenobiotics can induce autoimmunity. It includes a consideration of the lessons that can be learned from the study of animal models. The end of the chapter discusses the availability of potential biologic markers of autoimmunity and of autoimmune diseases.

Autoimmune diseases are those in which an individual's own immune system attacks one or more tissues or organs, resulting in functional impairment, inflammation, and sometimes permanent tissue damage. Autoimmune diseases result from the loss of immune tolerance to self-antigens, and an immune response to one or more relevant tissue antigens can be demonstrated. Sometimes, however, when no specific antigen can be detected, an autoimmune process can be inferred if there is inflammation of a tissue or group of tissues that show no evidence of infection and if the condition responds to immunosuppressive therapy. Exposure to xenobiotic substances is associated with certain autoimmune diseases; Table 4-1 provides a partial list.

The autoimmune response can produce disease directly by means of circulating antibody, indirectly through the formation of immune complexes, or as a consequence of cell-mediated immunity. In most cases, more than one pathogenetic mechanism manifests itself. In myasthenia gravis or α-methyldopa-induced autoimmune hemolytic anemia, antibodies against the acetylcholine receptor at the neuromuscular junction or the red blood cell membrane can be confirmed. One example of prominent cellular immunity is autoimmune thyroiditis, in which the lymphocytes infiltrate the thyroid gland. Immune complexes also can be involved, as in lupus nephritis.

Autoimmune diseases vary widely in different populations, displaying both geographic

and temporal variations. The prevalence of some putative autoimmune diseases is listed in Table 4-2. Crohn's disease was not described until 1932 (Crohn et al., 1932). Rheumatoid arthritis is also relatively new and is less common than other forms of arthritis manifested in Old World skeletal remains (Woods and Rothschild, 1988). The

incidence of Crohn's disease is increasing in several geographic areas. In central Israel, the incidence increased from 0.33/100,000 in 1970 to 3.1/100,000 in 1979 (Fireman et al., 1989). Over similar periods, increases have been documented in Alberta, Canada (Pinchbeck et al., 1988), and in northern Europe (Binder, 1988), suggesting a xenobiotic or infectious environmental contribution. Trends and geographic variations are useful, if difficult, in clarifying the epidemiology and etiology of autoimmune disease.

There is a familial clustering of many autoimmune diseases, suggesting a genetic predisposition. The human leukocyte antigen (HLA) system, which is a classification of cell-surface glycoproteins on lymphocytes and macrophages, is an important marker of susceptibility for 40 or more diseases. The influence of this system can play a dominant role in the development of autoimmune disease. The association between the HLA-B27 (class I antigen) haplotype and ankylosing spondylitis (suspected to be of autoimmune origin) is a striking example. Ninety percent of patients and 9% of normal subjects having this haplotype have a relative risk of 87 (87 times the normal rate of disease). The risk varies from country to country, and in the United States there is a significant

racial difference; HLA-B27-positive blacks have a lower risk than whites (McDevitt, 1985).

For rheumatoid arthritis, as for most other autoimmune diseases, the role of genetics is much less pronounced; 50% of patients and 20% of healthy persons have the HLA-DR4 haplotype for a relative risk of 4 (McDevitt, 1985). The mechanism by which this haplotype influences susceptibility to autoimmune disease is still unknown, but several possibilities have been proposed: The class II MHC (major histocompatibility complex) product binds the autoantigen firmly and presents it to the reactive T cell; the class II MHC product expressed by the thymic epithelium shapes the T-cell repertoire; or an unknown disease-susceptibility gene is in linkage disequilibrium with the class II MHC gene.

A number of other genetic traits are associated with an increased prevalence of autoimmune disease. Studies of human populations have shown that certain immunoglobulin allotypes (genetically determined markers on immunoglobulin molecules) are associated with a greater risk of developing particular autoimmune diseases, such as Graves' disease and insulin-dependent diabetes mellitus. Polymorphisms of the β chain of the T-cell receptor, as determined indirectly by restriction fragment length analysis of the genes, also seem to be associated with susceptibility to some autoimmune diseases (Demaine and Welsh, 1988).

Experimentally induced autoimmune diseases require that particular genes encode the variable regions of the T-cell receptor (Heber-Katz, 1990). If such findings pertain to humans, an important new biologic marker will have been discovered.

Several autoimmune diseases associated with exposure of susceptible individuals to organic chemicals are listed in Table 4-2 (Bigazzi, 1988; Kammüller et al., 1988). It is clear that illnesses that meet the diagnostic criteria for systemic lupus erythematosus (SLE) can occur after exposure to a number of pharmaceuticals, including hydralazine, procainamide, phenytoin, and isoniazid (Weinstein, 1980). With hydralazine-induced lupus, susceptibility has been linked to the patient's rate of acetylation of the drug (Godeau et al., 1973; Perry, 1973; Reidenberg and Martin, 1974; Strandberg et al., 1976; Batchelor et al., 1980). Because many cases of lupus are not related to drug use, the distinction between drug-induced and idiopathic lupus has been made. However, some clinicians have challenged the notion that lupus not related to pharmaceutical exposure is idiopathic. Occupational exposure to the organic solvent hydrazine causes SLE in slow acetylators with the HLA-DR3 phenotype (Reidenberg et al., 1983). Tartrazine, a yellow dye used as an artificial coloring agent in food and drugs, has been associated with SLE-like illness (Pereyo, 1986). Ingestion of alfalfa sprout causes SLE-like illnesses in monkeys (Malinow et al., 1982).

Some patients with a genetic predisposition do not develop autoimmunity, whereas patients without a family history of the associated HLA haplotype can. Attention has focused increasingly on environmental influences. Ingestion of dietary iodine can lead to the expression of autoimmune thyroiditis. Pollutants such as heavy metals have been implicated in some cases of immune-complex glomerulonephritis. Infections and hormonal balance also can play a role in the induction of an autoimmune state.

In some cases, a combination of diet, chemical exposure, genetic susceptibility, infection, and hormonal balance can act synergistically to induce an autoimmune state; in others, an overwhelming environmental

exposure or some other single factor could be the cause. Individuals with a genetic predisposition could be at increased risk from a given environmental exposure, as was seen in the Spanish toxic oil syndrome.

The Spanish toxic oil syndrome, an epidemic of disease in Madrid and a surrounding region that began in May 1981, was probably related to ingestion of anilinedenatured rapeseed oil, although the actual toxicant has never been identified definitively. A disease developed in approximately 20,000 people beginning about one week after they had ingested the adulterated cooking oil. Their signs and symptoms were fever, rash, pruritus, interstitial pneumonitis, dyspnea, eosinophilia, thrombocytopenia, arthralgias, myalgias, malaise, intrahepatic cholestasis, gastrointestinal symptoms, intrahepatic cholestasis, and sometimes lymphadenopathy (Noriega et al., 1982). Most of the victims made an uneventful recovery, but approximately 15% developed features suggestive of autoimmune disease, with a scleroderma-like illness, Sjögren's syndrome; pulmonary hypertension; Raynaud's phenomenon; and dysphagia (Fernández-Segoviano et al., 1983; Alonso-Ruiz et al., 1986). There was an increased incidence of the HLA-DR3 and HLA-DR4 phenotypes among those who developed chronic disease (Vicario et al., 1982; Kammüller et al., 1988). Another epidemic occurred in the Netherlands in 1960 when 20,000 of 600,000 individuals who ate a new brand of margarine developed erythema multiforme (Mali and Malten, 1966). These examples illustrate the implications of environmental xenobiotics in a variety of autoimmune disorders.

A somewhat similar picture of progressive systemic sclerosis has been associated with exposure to trichloroethylene (Lockey et al., 1987). An outbreak of disease with strikingly similar signs has been described in individuals ingesting preparations of L-tryptophan (Belongia et al., 1990). There are case reports from Japan (Kumagai et al., 1979, 1984), Europe (Byron et al., 1984), and the United States (Varga et al., 1989, and references therein) of women who developed features of autoimmune disease, such as scleroderma, after receiving mammary injections or implants of silicone for cosmetic reasons. There is controversy about this association, because the calculated incidence in the United States among women with augmentation mammoplasty is no greater than that found in the population at large. Studies using animal models have suggested that silicone can trigger persistent fibrosis and inflammation, but have provided no evidence of autoimmunity (Ballantyne et al., 1965).

In most cases of autoimmunity associated with xenobiotic exposure, the precise mechanism by which the xenobiotic substance induces an autoimmune process is unknown. In some areas, however, the pathogenesis of these disorders is coming into focus. Drug-induced immune cytopenias are among the best studied examples of these conditions, partly because of the accessibility to the target tissue and the ability to monitor changes over time. For example, four mechanisms have been identified for drug-induced immune hemolytic disorders (Petz and Garratty, 1980). The drug can attach to the cell membrane, where it interacts with a drug-specific antibody. Alternatively, the drug could modify the cell membrane so that the patient's immune system regards the cell as foreign. Immune complexes formed between the drug and its respective antibody could adhere to the membrane to produce injury. A fourth mechanism involves red-cell sensitization due to production of red-cell autoantibody and is, in that sense, the only true autoimmune form of these reactions. Procainamide (Kleinman et al., 1984), α-methyldopa (Worlledge et al., 1966), and nomifensine

(Salama et al., 1989) are all examples of drugs involving red-cell sensitization. Procainamide also is associated with other autoimmune phenomena (Blomgren et al., 1972; Henningsen et al., 1975).

The immunopathy is thought to arise by blocking of the normal immunosuppression against self-antigens. Consistent with this is the observation that low doses of cyclophosphamide, also known to suppress suppressorcell activity differentially, can reliably induce red-cell autoantibodies in mice (Hutchings et al., 1985). Exogenous chemicals can induce the production of novel adducts that might serve as triggers of an immune response. For example, halothane-exposed guinea pigs and rabbits develop trifluoroacetylated (TFA) proteins, which evoke anti-TFA antibodies. Some of these animals show liver injury that resembles the hepatitis seen in some patients after halothane anesthesia (Hubbard et al., 1989).

Several recent observations suggest that immune-mediated damage occurs through a combination of mechanisms. Sera from patients with hemolytic anemia caused by drug-specific antibodies have occasionally been shown to react with red blood cells alone, suggesting the presence of true autoantibodies (Florendo et al., 1980; Habibi, 1985), which are produced by a host to its own tissues. Autoantibodies are found along with drug-dependent antibodies in patients with drug-induced immune neutropenia (Salama et al., 1989) and thrombocytopenia (Lerner et al., 1985).

The administration of high doses of a cephalosporin, one of a class of drugs thought to act principally as haptens, has recently been shown to induce red-cell, neutrophil, and platelet antibodies in dogs that can be demonstrated ex vivo in the absence of the drug (Bloom et al., 1988). These observations serve to further blur the line between hypersensitivity and xenobiotic-induced autoimmune disorders. The above mechanisms have been well studied, and similar mechanisms are known to play a role in xenobiotic-induced immune damage involving other tissues. In addition, cell-mediated immunity is thought to be important in some diseases with features of autoimmunity. For example, lymphocytes and macrophages infiltrate the myelin sheaths of patients with multiple sclerosis (Adams, 1983).

The many animal models of autoimmune diseases that have been studied fall into three groups.

1. Spontaneous autoimmunity. A good model is the well-studied New Zealand Black mouse, in which both sexes spontaneously develop autoimmune hemolytic anemia, B-and T-cell defects, hepatosplenomegaly, and glomerulonephritis because of a genetic predisposition (Milich and Gersjwon, 1980).

2. Experimental autoimmunization. In these models, animals receive injections of autoantigens and an appropriate adjuvant. An example is experimental allergic encephalomyelitis (EAE), in which demyelinating lesions accompanied by neurologic deficits suggestive of multiple sclerosis are produced by injection of myelin basic protein accompanied by an appropriate antigen. Adoptive transfer of EAE can be performed by infusions of lymphocytes (Paterson, 1960).

3. Chemically induced autoimmune disease. The animal is exposed to a chemical that induces an autoimmune disease. Pharmaceutical agents thought to induce autoimmunity in humans have been studied in animals, and species variability and strain variability have been observed. For example, hydralazine and procainamide given to BALB/c strain mice and A strain mice induce antinuclear antibodies in both strains; the A strain mice, but not the BALB/c strain mice, develop glomerulonephritis (Ten Veen and Feltkamp-Vroom, 1973). Canavanine, the arginine analogue, induces double-stranded

DNA antibodies in mice (Prete, 1985). Canavanine occurs in alfalfa sprouts and induces an autoimmune disease similar to lupus in primates (Malinow et al., 1982). The lesson learned from animal models of autoimmunity is that genetic influences, environmental exposure, or in some cases a combination of the two can lead to autoimmune disease. Our knowledge of autoimmune phenomena in humans suggests a similar situation.

Physical signs and symptoms can support the validation of biologic markers of autoimmune diseases. For example, characteristic joint deformities are indicative of rheumatoid arthritis. Diagnoses are ultimately made on clinical grounds, with constellations of signs and symptoms forming the patterns from which diseases are diagnosed. Likewise, historical data can be used to confirm both a marker of susceptibility to autoimmunity and a marker of effect. For example, the validity of the biologic marker is supported in cases in which a family history of an autoimmune disease suggests that family members are at increased risk or when historical data for a given region or group of people show a consistently high incidence of a given disease.

The HLA system can provide a marker of susceptibility to certain immune-system-mediated diseases, ranging from ankylosing spondylitis (associated with HLA-B27) to pemphigus vulgaris (associated with HLA-Rw4). These associations are not absolute; large numbers of healthy persons have susceptibility markers, and many affected persons do not. The relative risk of these diseases could vary with geography, sex, and race. Nevertheless, these markers are important research tools that have value in elucidating the epidemiology of the diseases, and they can, in selected cases, be an aid to diagnosis. Their clinical utility in autoimmune diseases is limited, and HLA typing should not be done routinely on all patients with autoimmune phenomena.

A closer association of HLA genes with autoimmune disease has been reported when alleles are identified by means of oligonucleotide sequences or individual amino acid products, instead of depending on the usual serologic reagents (Kwok et al., 1988; Nepom et al., 1989).

The genes of the major histocompatibility complex (MHC) and the genes that control the constant region of the immunoglobulin heavy chain regulate the immune response in experimental animals and humans. In humans, IgG molecules are polymorphic at the Gm locus, and a number of associations between Gm allotypes and autoimmune diseases have been described (Nakao and Kozma, 1988). Farid et al. (1977) reported an association between Gm phenotype f and autoimmune thyroiditis. Field et al. (1984) found a correlation between Glm(2) and increased susceptibility to insulin-dependent diabetes in individuals who had the HLA-DR4, but not the HLA-DR3, haplotype. Thus, there are interactions between MHC and Ig determinants. In the presence of a particular MHC haplotype, a particular Ig allotype will increase the risk of autoimmune disease.

The presence of thyroid-specific autoantibodies is frequently an indicator of autoimmune thyroid disease. Burek et al. (1984) studied the association of a number of genetic

markers with thyroid autoantibodies. They found that four genetic traits—HLA, Gm, ABO blood group, and Rh blood group—showed weak but significant associations with autoantibodies, but that in the aggregate these markers were quite predictive. The predisposition to autoimmunity is polygenic, and the greatest susceptibility is the result of inheritance of a number of genetic traits, which often act through different pathways (Rose and Burek, 1985).

The liver enzyme acetyl transferase metabolizes many drugs, such as isoniazid and hydralazine. The genes that control the level of this enzyme are polymorphic and separate the population into two groups: slow and fast acetylators. Among patients who ingested low total amounts of hydralazine, 60% of the slow acetylators developed the antinuclear antibodies characteristic of lupus, whereas none of the fast acetylators did (Perry et al., 1970). Persons who are slow acetylators of some drugs have an increased incidence of drug-induced lupus. Hence, slow hepatic acetylation of a drug also is a marker of susceptibility to drug-induced lupus.

Antinuclear antibodies are detected by incubating a tissue section with the subject's serum and then with a fluorescein-labeled antihuman antibody. If the subject is producing antibody to a nuclear constituent, that constituent will fluoresce in ultraviolet microscopy. Antinuclear antibodies are markers for a number of autoimmune diseases, the most notable of which is systemic lupus erythematosus (Ferrell and Tan, 1985). Antibodies to specific nuclear constituents are high specific for certain collagen vascular diseases. These tests are extremely valuable in making specific diagnoses and play a major role in clinical medicine. They can also be important in epidemiologic surveys of xenobiotic-exposed populations.

Serum antibodies to specific tissue antigens detected by immunofluorescence, radioimmunoassay, enzyme-linkedimmunosorbent assay (ELISA), immunoperoxidase, and other highly sensitive techniques have been useful in the diagnosis of certain autoimmune diseases. For example, the antibodies for antigens in the kidney basement membrane found in Goodpasture's syndrome are both diagnostic and pathogenic. Many other tissue-specific autoantibodies are valuable in diagnosis but do not necessarily play a role in pathogenesis. Autoantibodies of thyroglobulin and thyroid peroxidase, for example, are characteristic of chronic thyroiditis; autoantibodies to islet cells of the pancreas are found in many patients with insulin-dependent diabetes; and autoantibodies that react with actin of smooth muscle are prominent in chronic active hepatitis (Bigazzi et al., 1986). Similar autoantibodies, however, are common in healthy individuals, and their incidence increases with age. Recent studies suggest that patients with disease, as well as individuals at risk of developing disease, produce autoantibodies to distinct antigenic determinants on the thyroglobulin molecule (Bresler et al., 1990). Tests for specific tissue antibodies are important research tools that provide information about the pathogenesis of these diseases; they also have predictive value in the diagnosis of many autoimmune disorders.

The microscopic examination of specimens from tissue biopsies, appropriately fixed and stained, can be valuable in diagnosing autoimmune diseases and can elucidate the pathogenesis and mechanism of

immune-tissue damage. Direct immunofluorescence can detect antibody and complement deposition in tissues. For example, in bullous pemphigoid, an autoimmune disorder that produces bullous skin lesions, heavy depositions of IgG can be detected along the basement membrane between the epidermis and dermis of patients (Diaz et al., 1985).

Aggregates of antibody and antigen can be detected in serum and tissues in several disorders, including infectious diseases, autoimmune diseases, malignancies, and serum sickness secondary to administration of drugs. This lack of specificity makes them of little use as markers of autoimmunity, but they are markers of disease activity. Systemic lupus erythematosus is the prototype of an autoimmune disease for which the pathologic changes are due to the deposition of immune complexes in various tissues, especially the kidney. These complexes involve DNA-anti-DNA. Measurement of immune complexes and demonstration of antibodies to native DNA and determination of levels of hemolytic complement in serum are diagnostically useful (Toth et al., 1986).

Inflammatory disorders can deplete the immune complement, and an abnormally low level of complement (C3 consumption), although not a specific marker of autoimmunity, can be a biologic marker of disease activity in autoimmune disorders, most notably systemic lupus erythematosus (Toth et al., 1986).

Autoimmune diseases in humans are common and cause a great deal of morbidity and mortality. Xenobiotic exposures can induce autoimmune disorders, which sometimes require a genetic predisposition, in both humans and animals. A great deal is known about drug-induced autoimmunity, but our knowledge of autoimmune diseases arising from environmental exposure is in its infancy, and we do not know the extent to which apparently spontaneous autoimmune disorders are influenced by environmental factors.

Clinical research should determine the extent to which non-drug-induced autoimmune disorders are environmentally induced. If it is found that significant percentages of human autoimmune disorders are environmentally related and that these diseases can go into remission by removing the incriminated chemical or by reducing exposure to it, techniques must be developed to allow clinicians to identify environmental factors in autoimmunity.

Inexpensive and practical tests to screen populations for susceptibility to autoimmune disease should be developed. The best current predictor of susceptibility to autoimmune disease is the HLA class II haplotype. Determinations based on nucleotide base (or amino acid) sequence are more discriminating than are conventional serologic assays, and they are more predictive of autoimmune disease (Nepom, 1989).

Populations exposed to toxic substances because of spills of hazardous materials, proximity to toxic-waste sites, and occupation could then be easily screened for autoimmune phenomena, including subclinical or asymptomatic disease. An example of such a test is the indirect immunofluorescence assay for a battery of autoantigens.

Better data on the epidemiology of autoimmune diseases should be collected continuously. It is important to document changes in the incidence of these diseases and the geographic clustering of cases in acquiring an understanding of environmental factors. Autoimmune diseases do not require reports to local health departments, so

existing epidemiologic data are a patchwork of areas of controversy. This mission would be ideal for the Agency for Toxic Substances and Disease Registry.

The association between autoimmune disease and some pharmaceuticals is well established. Efforts should be made to investigate the prevalence of autoimmune responses and markers among pharmaceutical-manufacture workers. Perhaps health-care workers routinely exposed to these drugs could also be monitored for the prevalence of drug-induced autoimmune disease.

The environmental control unit discussed in Chapter 3 could be an effective tool for investigating the role of xenobiotic substances in autoimmunity. It allows isolation of the patient from the ubiquitous chemical environment. If improvement can be objectively quantitated, chemicals from the patient's daily life can be reintroduced to see which, if any, produce disease. This simple concept is well grounded in common sense, and examination of its utility in the study of autoimmune diseases is merited.