Lack of graft-versus-host-like pathology in mercury-induced autoimmunity of Brown Norway rats.

The repeated administration of mercury to Brown Norway (BN) rats induces the production of autoantibodies to laminin 1 and other autoantigens, accompanied by renal deposition of immunoglobulins and a membranous glomerulonephropathy. A graft-versus-host-like (GVHL) syndrome, characterized by widespread necrotizing leukocytoclastic vasculitis of the bowel, skin, and other tissues, has also been observed after mercury treatment of BN rats. These findings have suggested that the autoimmunity caused by the administration of mercury to BN rats may result as a xenobiotic-induced GVHL effect under the control of OX22+ T lymphocytes. However, previous studies of mercury-induced autoimmunity have never reported any evidence of GVHL lesions. Therefore, we have carefully examined various tissues from a large group of BN rats injected with HgCl(2) to identify possible areas of inflammatory reactions that may have been unnoticed in previous investigations. In addition, we have determined by flow cytometry whether exposure to mercury results in percentage and numerical alterations of OX22+ or other lymphocyte subpopulations in lymphoid organs of HgCl(2)-treated BN rats. The present article confirms that mercury induces autoimmune responses to laminin 1 but does not corroborate the hypothesis of a GVHL syndrome regulated by OX22+ lymphocytes. First, changes in OX22+ cells during treatment with HgCl(2) were infrequent and had no significant correlation with the kinetics of autoimmune responses to laminin 1. Second, we detected no GVHL lesions in skin and intestine of mercury-treated BN rats.

Metals and kidney autoimmunity.

The causes of autoimmune responses leading to human kidney pathology remain unknown. However, environmental agents such as microorganisms and/or xenobiotics are good candidates for that role. Metals, either present in the environment or administered for therapeutic reasons, are prototypical xenobiotics that cause decreases or enhancements of immune responses. In particular, exposure to gold and mercury may result in autoimmune responses to various self-antigens as well as autoimmune disease of the kidney and other tissues. Gold compounds, currently used in the treatment of patients with progressive polyarticular rheumatoid arthritis, can cause a nephrotic syndrome. Similarly, an immune-mediated membranous nephropathy frequently occurred when drugs containing mercury were commonly used. Recent epidemiologic studies have shown that occupational exposure to mercury does not usually result in autoimmunity. However, mercury induces antinuclear antibodies, sclerodermalike disease, lichen planus, or membranous nephropathy in some individuals. Laboratory investigations have confirmed that the administration of gold or mercury to experimental animals leads to autoimmune disease quite similar to that observed in human subjects exposed to these metals. In addition, studies of inbred mice and rats have revealed that a few strains are susceptible to the autoimmune effects of gold and mercury, whereas the majority of inbred strains are resistant. These findings have emphasized the importance of genetic (immunogenetic and pharmacogenetic) factors in the induction of metal-associated autoimmunity. (italic)In vitro(/italic) and (italic)in vivo(/italic) research of autoimmune disease caused by mercury and gold has already yielded valuable information and answered a number of important questions. At the same time it has raised new issues about possible immunostimulatory or immunosuppressive mechanisms of xenobiotic activity. Thus it is evident that investigations of metal-induced renal autoimmunity have the potential to produce new knowledge with relevance to autoimmune disease caused by xenobiotics in general as well as to idiopathic autoimmunity.

Mercury-induced autoimmunity in Brown Norway rats: kinetics of changes in RT6+ T lymphocytes correlated with IgG isotypes of circulating autoantibodies to laminin 1.

Repeated exposure to mercury causes various autoimmune effects in rats of the Brown Norway (BN) strain. Previous studies from our laboratory have shown that on day 15 of HgCl2 treatment BN rats exhibit a relative decrease in RT6.2+ T cells. At the same time, they produce high levels of autoantibodies to renal antigens and experience a membranous glomerulonephropathy. In contrast, Lewis (LEW) rats are resistant to autoimmunity caused by mercury and do not demonstrate a decrease in RT6+ cells after administration of HgCl2. In the present paper we provide novel information on the correlation between changes in RT6.2+ lymph node T cells and the production of autoantibodies to laminin 1, obtained by detailed kinetic studies of HgCl2-treated BN rats. We have confirmed a decrease in the percentage of RT6.2+ lymphocytes on day 15 of mercury treatment, despite a significant increase in the number of peripheral lymphocytes. No such changes were observed in LEW rats. We have determined that on day 15 the percentage decrease in RT6+ cells is evident in both RT6.2+CD4+ and RT6.2+CD8+ T cell subsets. Kinetic studies demonstrated that significant changes in the percentage of RT6.2+ cells are first observed by day 8 and continue through days 11 and 15. We have also observed a significant percent decrease in CD4+ T lymphocytes as well as an increase in CD4-CD8- cells. The dramatic increase in the percentage of these double negative cells at the level of peripheral lymphoid tissues does not appear to be due to higher thymic output, since there was a decrease in the percentage of TCR+Thy1+ cells, a phenotype that is associated with recent thymic emigrants. Finally, we have demonstrated that 100% of HgCl2-treated BN rats had circulating antibodies that reacted with both mouse and rat laminin 1, i.e. are autoantibodies to laminin 1. These autoantibodies were predominantly of the IgG1 and IgG2a isotype, possibly as the result of a polarized autoimmune response driven by Type 2 cytokines. A kinetic investigation showed that significant levels of IgG1 and IgG2a autoantibodies to laminin 1 were first presentin the circulation by day 11. The inverse correlation between levels of RT6.2+ T lymphocytes and autoantibodies to laminin 1 suggests that mercury may induce autoimmune responses in BN rats by its effects on these immunoregulatory cells.

Autoimmunity caused by xenobiotics.

A number of clinical reports and experimental studies have shown that autoimmune responses and/or autoimmune diseases are induced in humans and laboratory animals by chronic exposure to various chemicals. The present review is focused on the most frequent groups of chemically induced human disorders. Results obtained from studies of the available counterparts of these diseases, i.e. those models obtained from the exposure of laboratory animals to various chemicals are analyzed. Although significant progress has been achieved to the realization that environmental agents may cause profound changes in our immune system and result in autoimmune disease, epidemiological, clinical and experimental work is still needed to acquire a better understanding of the mechanisms involved.

Effects of HgCl2 on the expression of autoimmune responses and disease in diabetes-prone (DP) BB rats.

Repeated exposure of Brown Norway (BN) rats to relatively low doses of HgCl2 induces autoantibodies to renal antigens (e.g., laminin) and a membranous glomerulonephropathy characterized by proteinuria. In contrast, Lewis (LEW) rats are "resistant" to the autoimmune effects of mercury and, when exposed to this metal, are protected against experimental autoimmune encephalomyelitis (EAE) and Heymann's nephritis. To date, there is no information on "suppressive" effects of mercury in naturally occurring (so-called "spontaneous") rat models of autoimmune disease. Therefore, we have administered HgCl2 to diabetes-prone (DP) BB rats, animals that spontaneously develop both insulin-dependent diabetes mellitus (IDDM) and thyroiditis. We found that DP rats treated with mercury or water for a period of 40-125 days developed autoantibodies to thyroglobulin, with a higher incidence in HgCl2-injected animals (92% vs. 56% in H2O-injected controls). A novel finding of our study was the detection of autoantibodies to laminin in the same rats, again with an increased incidence after HgCl2 treatment (83% vs. 44%). IgG2a was the most frequently detected isotype of antibodies to laminin, followed by IgG1, IgG2b and IgG2c. The IgG isotype profile suggests that treatment with HgCl2 may activate both Th1 and Th2 lymphocytes in BB rats. In spite of these stimulatory effects on autoantibody responses, we found that there was no difference in the incidence of IDDM and thyroiditis between HgCl2-treated and control animals. We conclude that the suppressive effects of mercury previously observed in EAE and Heymann's nephritis of LEW rats do not occur in "spontaneous" autoimmune IDDM and thyroiditis of BB rats. Therefore, immune suppression caused by HgCl2 cannot be considered a common phenomenon, but may be a genetically determined characteristic of LEW rats, possibly related to a specific or unique cytokine profile of this particular rat strain. In contrast, while mercury does not seem to recruit, induce or rescue regulatory T cell function in DP rats, it does stimulate autoantibody responses in these animals.

Thymus atrophy and changes in thymocyte subpopulations of BN rats with mercury-induced renal autoimmune disease.

Administration of low doses of mercury induces autoantibodies to laminin and autoimmune glomerulonephropathy in BN, MAXX and DZB rats as well as in (BN x LEW)F1 hybrids. LEW strain rats are resistant to these immunotoxic effects. Susceptible rats also show lymphoid hyperplasia in spleen and lymph nodes and severe thymic atrophy. It is still uncertain whether these mercury-induced changes have any role in the induction of autoimmune responses to laminin. In the present study, we have examined the effects of mercury on the thymus of susceptible and resistant rats. Histological analysis of thymuses from BN rats revealed extensive disorganization within 15 days following mercury treatment, with loss of demarcation between cortex and medulla. Numbers of thymus cells were significantly decreased in both BN and (BN x LEW)F1 hybrid rats injected with HgCl2. There was no apparent increase in apoptotic cells in the thymus of these animals. By flow cytometry we detected a relative and absolute loss of double-positive CD4+ CD8+ thymocytes in BN (but not in LEW rats) within 15 days of mercury treatment. There was a corresponding increase in the relative proportion of single-positive (CD4+ or CD8+) and double-negative CD4- CD8- thymocytes in mercury-treated BN rats. Absolute increases in the number of CD4+ single-positive thymocytes were also observed. In contrast, mercury-treated LEW rats had no changes in thymus architecture or significant decreases in cell numbers. Since the thymus is important in both position and negative selection of developing thymocytes, immunotoxic effects of mercury on its structure and thymocyte subpopulations may have multiple consequences. Alternatively, we suggest the hypothesis that autoimmunity (and in particular autoantibodies to laminin) may be responsible for the changes observed in the thymus.

Autoimmunity and heavy metals.

This brief review is focused on those heavy metals (cadmium, gold and mercury) that have strong associations with autoimmunity. Cadmium treatment of rats and mice results in autoimmune responses that vary with species and inbred strain of animals. However, there is no solid evidence demonstrating that the renal pathology observed in humans exposed to cadmium has an autoimmune pathogenesis. More clear-cut are the autoimmune effects of preparations containing gold salts, that have been widely used in the treatment of rheumatoid arthritis. Gold may cause autoimmune thrombocytopenia, immune complex-mediated glomerulonephritis and other autoimmune disorders. Similarly, there is solid evidence that mercury can induce autoimmune disease both in humans and experimental animals. The lessons to be derived from metal-induced autoimmunity relate to structure-activity relationship, pathogenesis, etiology and genetics. They probably apply to xenobiotic-induced autoimmune disease in general.

Role of RT6+ T lymphocytes in mercury-induced renal autoimmunity: experimental manipulations of "susceptible" and "resistant" rats.

Brown Norway (BN) rats, "susceptible" to the autoimmune effects of mercury, experience a decrease of peripheral RT6.2+ T lymphocytes after the injection of relatively low doses of mercuric chloride. This change coincides with the appearance of circulating autoantibodies to renal antigens (e.g., laminin). Lewis (LEW) rats, "resistant" to the autoimmune effects of mercury, do not show significant decreases of RT6+ T cells. It is possible that BN rats are particularly sensitive to stress induced by mercury and that secretion of adrenocortical hormones decreases levels of RT6+ T cells in this rat strain. Alternatively, mercury may induce a graft-versus-host-like syndrome in BN rats, resulting in higher levels of corticosteroids capable of affecting RT6+ lymphocytes. To eliminate the possible influence of adrenocortical hormones, we have adrenalectomized BN rats prior to administration of mercury. Autoimmune responses to renal antigens were not affected by this experimental manipulation. Similarly, adrenalectomized rats exposed to mercury showed a significant decrease of RT6+ T lymphocytes in cervical lymph nodes. Overall, these observations do not support the hypothesis that increases in adrenocortical hormones play a major role in mercury-induced changes of RT6+ T cells. We have also explored whether experimental depletion of RT6+ T lymphocytes would result in autoimmunity. Gamma irradiation of BN rats led to a decrease of RT6+ T splenocytes, but by itself (i.e., without exposure to mercury) did not cause autoimmune responses to renal antigens. In addition, gamma-irradiated BN rats treated with mercury had autoimmune responses similar to those observed in mercury-treated nonirradiated controls. Depletion of RT6+ T cells in LEW rats through the use of a monoclonal antibody against the RT6.1 alloantigen did not by itself cause renal autoimmunity in this "resistant" strain. Depletion followed by administration of mercury also failed to induce renal autoimmunity. The lack of autoimmune effects in RT6-depleted BN and LEW rats suggests that a combination of several factors may be necessary to break self-tolerance and cause mercury-induced autoimmunity. Such factors likely comprise both environmental (mercury) and endogenous, genetically determined components. The latter include regulatory T cells (possibly RT6+), major histocompatibility complex (MHC), and T-cell receptors (TCR). Thus, BN rats with decreased percentages of immunoregulatory RT6+ T lymphocytes require additional immunotoxic and/or toxic effects of mercury for autoimmunity to occur. On the other hand, LEW rats depleted of regulatory T cells may still be unable to develop renal autoimmunity after exposure to mercury because they lack the appropriate MHC and TCR.

Mercury-induced renal autoimmunity in BN-->LEW.1N chimeric rats.

Repeated exposure to relatively low doses of mercuric chloride causes a variety of autoimmune responses in rats of the Brown Norway (BN) strain. These animals experience a membranous glomerulonephritis, characterized by the production of autoantibodies to renal antigens (e.g., laminin) and proteinuria. In contrast, Lewis (LEW) rats are "resistant" to the autoimmune effects of mercury. Despite extensive investigations, the mechanisms of immunoregulation in this animal model are still unknown. RT6+ T lymphocytes may have a regulatory role in both BN and LEW rats. This hypothesis is suggested by our finding of a mercury-associated decrease of RT6+ T cells in lymph nodes of BN rats exposed to mercury and the lack of such effect in similarly treated LEW rats. In the present report we show that congenic LEW.1N or BN.1L had no renal autoimmune disease after treatment with HgCl2. FCM analysis of mercury-treated LEW.1N revealed that RT6.1+ T lymphocytes were significantly decreased in both spleen and lymph nodes of these animals. Experimental depletion of RT6+ T cells (by monoclonal antibody treatment or gamma irradiation) in LEW.1N and BN.1L rats did not favor the induction of renal autoimmunity after exposure to mercury. On the other hand, BN-->LEW.1N chimeras (obtained by adoptive transfer of BN lymphocytes into gamma-irradiated LEW.1N rats) experienced autoimmune responses to kidney antigens when treated with HgCl2. They had autoantibodies to laminin and linear binding of immunoglobulins in their kidneys as well as a decreased percentage of RT6.2+ T lymphocytes in cervical lymph nodes. Therefore, the different components of this experimental model can now be dissected using various types of BN-->LEW.1N chimeras, obtained by the adoptive transfer of purified T cell subsets.

Immunomodulation by metals.

A symposium entitled Immunomodulation by Metals was held at the 32nd Annual Meeting of the Society of Toxicology (SOT) in New Orleans, Louisiana. The symposium was co-sponsored by the Immunotoxicology and Metals Specialty Sections of SOT and was designed to describe the types of adverse immunological reactions which occur in response to environmental and/or occupational exposure to metals. Epidemiological evidence and underlying mechanisms responsible for the observed alterations were also discussed. The following is a summary of each of the individual presentations.

Mercury-induced renal autoimmunity: changes in RT6+ T-lymphocytes of susceptible and resistant rats.

The repeated administration of mercury to rats of the Brown Norway (BN) inbred strain results in a self-limiting production of autoantibodies to renal antigens (e.g., laminin) and autoimmune glomerulonephritis. In contrast, rats of the Lewis (LEW) strain do not develop renal autoimmunity after mercury treatment. Suppressor T-cells and/or the idiotype-anti-idiotype network have been implicated in the control of autoimmunity in susceptible (BN) rats as well as the "resistant" state of nonsusceptible (LEW) animals. In our investigations of the immune regulation of mercury-induced autoimmune glomerulonephritis, we have performed a phenotypic analysis of lymphocyte subpopulation in the spleens and lymph nodes of mercury-treated and control LEW, BN, and (BN x LEW) F1 hybrid rats. Of particular interest were RT6+ T-cells, a subpopulation of lymphocytes that may have immunoregulatory properties and show a relative decrease in mercury-treated BN rats concomitantly with the development of autoimmune responses to renal autoantigens. LEW rats did not develop renal autoimmunity after mercury treatment and had no significant change in the ratio of RT6+ to RT6- T-lymphocytes. Interestingly, the administration of mercury to (BN x LEW) F1 hybrid rats caused effects similar to those observed in the BN strain. Auto-immune responses to antigens of the kidney coincided with a change in the balance within the RT6 cell population, which was altered in favor of T-lymphocytes that do not express the RT6 phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)

The in vitro effects of mercury on peritoneal leukocytes (PMN and macrophages) from inbred brown Norway and Lewis rats.

The present paper demonstrates that HgCl2 can affect rat peritoneal polymorphonuclear leukocyte (PMN) and macrophage (M phi) functions in vitro. In addition, we have noticed that these effects of mercury vary according to the rat strain: for example, HgCl2 stimulates H2O2 release from Lewis (LEW) but not Brown Norway (BN) PMN. Similarly, LEW M phi produce high levels of H2O2 when exposed to HgCl2 in vitro, whereas BN M phi do not. Finally, mercury inhibits erythrophagocytosis of both LEW and BN "resident" peritoneal M phi. Preliminary experiments using M phi from other rat strains have also shown that MAXX M phi are stimulated by HgCl2 to release H2O2 in vitro, whereas Yoshida M phi are inhibited. Differences in lymphocyte responses (e.g. delayed-type hypersensitivity reactions and mitogen stimulation) between rats of various strains are well known. To these examples one may now add variations in PMN and M phi responses to mercury and possibly other metals. Our results suggest that caution should be exercised in interpreting the outcome of immunotoxicity studies in experimental animals. In particular, outbred rats may not provide appropriate models, that might be better obtained by comparative investigations of rats from various inbred strains.

Acute optic neuritis associated with immunization with the CNS myelin proteolipid protein.

Optic nerve tissue for SJL/J mice immunized with the central nervous system (CNS) myelin-specific proteolipid protein (PLP) was examined for histopathologic evidence of optic neuritis. Optic nerves isolated 17 d after immunization with PLP revealed an interstitial and submeningeal inflammatory infiltrate consisting of neutrophils and monocytes. In all cases, histologic evidence of optic nerve involvement correlated serologically with the presence of circulating anti-PLP antibodies. Control animals had no histopathologic evidence of disease or anti-PLP antibody. In many respects, the observed histopathologic profile of PLP-induced optic neuritis is similar to that associated with human inflammatory demyelinating diseases such as multiple sclerosis (MS). Because optic neuritis frequently is associated with some of the earliest clinical symptoms of MS, the acute nature of optic nerve involvement in this animal model suggests that immune recognition of the myelin PLP may play a significant role in the pathophysiology of optic nerve damage associated with sensitization to CNS-specific antigens.

Reduction of the RT6.2+ subset of T lymphocytes in brown Norway rats with mercury-induced renal autoimmunity.

Chemically induced autoimmunity is a recently recognized environmental hazard that may affect individuals genetically predisposed to autoimmune disease and chronically exposed to certain chemicals. For example, moderate concentrations of mercury may lead to renal autoimmune disease in a small but significant percentage of the exposed population. Mercury also induces autoimmune glomerulonephritis in susceptible Brown Norway (BN) and MAXX inbred strain rats. Autoimmune responses, directed to epitopes of the renal glomerular basement membrane (GBM), are rapid in onset and have a self-limiting course in mercury-treated rats. Both regulatory T cells and idiotype-anti-idiotype network have been implicated in the resolution of this autoimmune process. In our investigations of immune regulation of mercury-induced autoimmune glomerulonephritis, we have used flow cytometry to quantitate lymphocyte subpopulations in the spleen and lymph nodes of mercury-treated and control BN rats. Of particular interest was the RT6+ T cell subset, that appears to have important immunoregulatory properties in a rat model of autoimmune insulin-dependent diabetes mellitus. Spleen and lymph nodes from control BN rats contained 22 and 52%, respectively, RT6+ cells. Spleens from mercury-treated animals contained 21% RT6+ cells on Day 10 of treatment, 13% on Day 17, 16% on Day 24 and 20% on Day 30. Lymph nodes from the same rats had 36% RT6+ cells on Day 10, 23% on Day 17, 29% on Day 24, and 28% on Day 30. The decrease in RT6+ cells correlated inversely with autoimmune responses to GBM, which peaked on Days 17-24 and declined by Day 30. Moreover, autoimmune responses were also associated with elevated RT6-:RT6+ T cell ratios. Similar results were obtained in two additional groups of BN rats, comprising both younger and older animals, sacrificed at Day 18 of mercury treatment. Analysis of other lymphocyte subpopulations demonstrated a decrease of CD4+ and CD5+ cells, whereas B cells as well as CD8+, IL-2 receptor+, and MHC class II+ subsets showed no consistent correlation with the onset or resolution of the autoimmune process. These findings suggest that mercury-induced changes in RT6+ T lymphocytes may be related to the development of renal autoimmune disease in genetically predisposed BN rats.