Cytokine regulation of a rodent model of mercuric chloride-induced autoimmunity.

Experimental models of chemically induced autoimmunity have contributed to our understanding of the development of autoimmune diseases in humans. Heavy metals such as mercury induce a dramatic activation of the immune system and autoantibody production in genetically susceptible rats and mice. This autoimmune syndrome is dependent on T cells, which are important for B-cell activation and cytokine secretion. Several studies have focused on the roles of T-helper (Th)1 and Th2 cells and their respective cytokines in the pathogenesis of mercury-induced disease. This article reviews recent studies that have examined the patterns of cytokine gene expression and where investigators have manipulated the Th1 and Th2 responses that occur during mercury-induced autoimmunity. Finally, we will discuss some biochemical/molecular mechanisms by which heavy metals may induce cytokine gene expression.

In most autoimmune patients, the underlying causes of disease are unknown and the initial steps leading to loss of tolerance, activation of autoreactive T cells, and production of autoantibodies are poorly understood. Additionally, many endogenous and exogenous factors contribute to the development of autoimmune diseases, including sex hormones, genetic predisposition, infectious agents, and exposure to occupational or environmental pollutants (1,2). Although few animal models of chemically induced autoimmunity have been extensively studied, mercuric chloride (HgC12)-induced autoimmunity in rodents presents a well-established but poorly understood phenomenon.
The fact that mercury induces autoimmunity in rats and mice is especially relevant, since mercury is also an abundant environmental pollutant (3). Moreover, mercury has been used for thousands of years in mining and manufacturing and can still be found today in dental amalgam, skin creams, and fluorescent light tubes (3). Finally, the specificity of the autoantibody response elicited in mercury-treated mice is virtually identical to that of autoantibodies present in a subset of patients with scleroderma (or systemic sclerosis), the human autoimmune disease most frequently associated with exposure to environmental agents (4).
The most fascinating and compelling reason for studying this model is that it remains unknown how such a simple molecule can rapidly induce a loss of tolerance to specific self-antigens and a dramatic, self-limiting activation of the immune system. Recent investigations have examined the roles of cytokine-producing T cells in the induction and regulation of this syndrome and have provided a better understanding of mercuryinduced autoimmunity.

Description of the Model
In genetically susceptible mice and rats, subtoxic doses of HgCl2 (1.0 mg/kg weight) induce an autoimmune syndrome characterized by the production of specific autoantibodies, polyclonal activation of B and T cells, serum increases in IgGI and IgE, and glomerulonephritis with renal immune complex deposits (5). These manifestations appear 7-10 days after the beginning of HgCl2 injections and a single injection may be sufficient to induce the syndrome. Although most investigators administer HgC12 as a subcutaneous injection, the route of HgCl2 exposure is not critical and HgC12 can be given intraperitoneally, orally, in drinking water, or aerosolized (5). Additionally, methyl mercury or pharmaceutical ointments and solutions containing organic mercury are equally effective in inducing immune-type glomerulonephritis in rats, even when these products are applied on wounds or on normal skin (6). In susceptible strains, the effects of HgC12 are dose dependent, with increases in serum IgE positively correlated with dosage of HgCl2 in rats (3) and increases in serum IgG antinucleolar antibodies (ANoA) and IgG immune complexes correlated with HgC12 dosage in mice (7).
Rats treated with mercury develop autoantibodies of various specificities, including antiglomular basement membrane, anti-double-stranded DNA, antithyroglobulin, antiphospholipid, and antilaminin P1 (8,9). Whether these autoantibodies are simply a byproduct of the polyclonal activation seen in these animals or represent an antigenspecific response remains to be elucidated. In mice, however, mercury elicits a focused autoantibody response characterized by antibodies that target specific nucleolar antigens. Several studies have revealed that many of these ANoA are directed against fibrillarin, a 34-kDa basic protein that is a constituent of several nucleolar ribonucleoprotein complexes involved in preribosomal RNA processing (10)(11)(12). Mercury-induced disease is self-limiting and most of the manifestations resolve spontaneously after 4-5 weeks during a regulation phase, even if HgCl2 injections are continued. In rats this regulation phase is followed by a resistance phase, where rats are now resistant to further HgCl2 injections (5). The resistance phase in the rat is mediated by CD8+ T cells and IL-2-producing CD4+ T cells (5,13,14). It is unclear whether mice undergo a resistance phase (15).
Major histocompatibility complex (MHC) class II genes play a major role in determining susceptibility to mercury-induced disease. Inbred Brown Norway (BN) rats of the RT-1 n haplotype are susceptible to development of mercury-induced autoimmunity, whereas inbred Lewis (LEW) rats of the RT-11 haplotype are resistant to disease. In mice, expression of I-AS alleles confers a susceptible phenotype and expression of I-Ab or I-Ad confers resistance (16). Additionally, non-MHC genes may also contribute in determining the nature of glomerulopathy in rats (17), and the magnitude, specificity, or persistence of the ANoA response in mice (18). Although murine mercury-induced disease has been most often studied in H-2S mice such as A.SW, SJL, and B1O.S, other strains develop ANoA, including A.CA (H-2f), DBA/1 (H-2q), and P/J (H-2P) (19). Th1/Th2 Cells T cells participate in all phases of mercuryinduced autoimmunity (5). Following activation, CD4+ T cells can become polarized to become either T-helper (Th)1 or Th2type cells. This polarization is dependent on a variety of factors, including antigen dose (20), costimulatory molecules (21), type of antigen-presenting cell (22), and perhaps most importantly, the presence of critical cytokines (23). Interleukin (IL)-12, IL-18, and interferon (IFN)-y promote the appearance of Thl cells (24)(25)(26), which in turn secrete IL-2, IFN-y, and mediate cellular immunity (27). In contrast, IL-4 biases T cells toward a Th2 phenotype (23). These Th2 cells can secrete IL-4, IL-5, IL-6, IL-10, and IL-13 and participate in humoral, allergic, and helminthic immunity (28).

Cytokine Gene Expression in the Model
Although HgC12 induces a profound activation of the immune system marked by changes in the production of various cytokines in susceptible animals, a clear and comprehensive pattern of cytokine gene expression during the syndrome has yet to be determined. Several studies have used various approaches including semiquantitative reverse transriptase polymerase chain reaction, Northern blot analysis, and intracellular staining techniques to obtain a limited picture of cytokine expression in this model. Among these, investigators have reported increases of IL-4 and IFN-y mRNA in splenocytes of mercury-treated BN rats (29) as well as slight increases in IL-12 mRNA levels in spleen and lymph nodes from these animals (30). In these studies, HgCl2 failed to induce increases of cytokines in resistant LEW rats (29,30). mRNA analysis revealed higher baseline levels of IFN-y (29) and   (30) in resistant LEW rats as compared to those of BN rats, which may possibly account for this strain's resistance. Further analysis has demonstrated that treatment of BN rats with HgC12 downregulates the ex vivo generation of IFN-y-producing cells in concanavalin A (ConA)-stimulated splenocytes, and that this downregulation may be mediated by nitric oxide (31).
Additionally, several studies in the rat have indicated that HgCl2 can directly trigger naive cells to produce cytokines in vitro. Unfractionated splenocytes and purified T cells from BN rats express high levels of IL-4 mRNA after exposure to HgCl2 in vitro (32). Similar to in vivo findings, mercury induces IFN-y expression in cells of both LEW and BN rats but does not induce increases in IL-4 mRNA in cells from LEW rats (32). Mast cells of BN rats also respond to in vitro mercury exposure with induction of IL-4 mRNA and enhanced release of serotonin in response to IgE cross-linking agents (33). In contrast, mast cells from resistant LEW rats are less sensitive to mediator release and do not express IL-4 mRNA in response to exposure to HgCl2 (33). Thus, cytokine production in response to HgCl2 in rats differs according to genotype, and resistance may be due in part to higher baseline levels of Thl cytokines or a failure to upregulate Th2 cytokines.
Considerably less is known about the pattern of cytokine gene expression that occurs in mercury-treated mice. Earlier studies demonstrated that B1O.S mice (H-2S) respond to HgCl2 with a massive proliferation and activation of their B cells, increased IL-4 mRNA levels, and class switching to IgE, IgGl, and IgG2a (34). Additionally, mercury induces dramatic IL-4-dependent increases in B-cell major histocompatability complex II expression in susceptible H-2s mice and moderate increases in resistant H-2d mice (34). Recently, Johansson and colleagues (35) examined cytokine-producing cells in lymph nodes of susceptible and resistant mouse strains receiving HgC12. Susceptible A.SW mice (H-2s) respond to HgC12 with an early activation of T cells, indicated by increases in IL-2-producing cells and upregulation of the CD25, CD122, and CD71 activation markers. This activation was followed by increased numbers of CD4+ T cells. A.SW mice also show modest increases in tumor necrosis factor-a, IFN-y, and IL-4-producing cells at 8-10 days after HgCl2 injections. In contrast, genetically resistant A.TL mice (H~2tl) have minimal increases in T cells and no increases in cytokine-producing cells (35).
Mercury can also activate murine cells in vitro. In one study by Hu and colleagues, in vitro exposure to HgCl2 induced high proliferative responses in splenocytes from susceptible SJL mice and low proliferative responses in splenocytes of resistant C57B1/6 mice (H-2b) (36). Additionally, the continuous presence of HgCl2 in vitro induced IL-2 and IFN-y production, but no IL-4 production, from cells of both H~2S and H-2b mice. In contrast, pretreatment of cells with mercury followed by washing resulted in IL-4 production in cells of susceptible and resistant mice (36).

Cytokine Manipulations of the Model
Several studies have employed various cytokine manipulations or interventions to further elucidate the mechanisms of disease (Table 1). These observations indicate a greater complexity to the disease than was originally ascribed and suggest independent regulation of the various manifestations.
Early studies, noting the mercury-induced increases in IgG 1 and IgE (37,38), suggested a role for Th2-type T cells in the pathogenesis of disease, as IL-4 produced by Th2 cells promotes dass switching to these isotypes (2,5). Ochel and colleagues (39) addressed the role for this cytokine in mercury-induced autoimmunity by treating susceptible A.SW mice (H-2s) with an anti-IL-4 monodonal antibody (mAb) prior to HgC12 injections. Although the mercury-induced increases in IgGI ANoA and total IgE were partially or completely abrogated, respectively, levels of IgG2a, IgG2b, and IgG3 dass ANoA were enhanced (39). These results suggested that IL-4 was required for dass switching to the IgGI and IgE isotypes in this disease but was not essential for the loss of tolerance to nucleolar antigens.
Recendy, several investigators used different approaches to block the Th2 component seen in this syndrome. IFN-y secreted by ThI cells is important in regulating cellular immune responses and downregulating Th2 activities (28). Further, HgCI2 induces IFN-y-producing splenocytes in mercury-resistant LEW rats (31). In one study by Doth and colleagues, the effect of recombinant interferon (rIFN)-y pretreatment prior to HgCI2 injections was assessed in susceptible B1O.S mice (H-2s). In resistant B1.D2 mice (H-2d), HgCl2 induces an IFN-yLdependent suppression of antibody formation to sheep red blood cells (40). To investigate whether this immunosuppression was responsible for resistance to HgCI2, rIFN-y was administered to B10.S mice prior to HgCI2 treatment. This pretreatment led to reductions in serum IgE levels and anti-SRBC antibody levels but filled to prevent ANoA production and immune glomerulonephritis in B1O.S mice (40). Conversely, mAb neutrlization of IFN-,y in resistant B10.D2 mice alleviated the mercury-induced immunosuppression but could not convert the mice to a mercury-susceptible phenotype (40).
Using an alternate approach, we have tried to modulate the response to HgC12 observed in A.SW mice to that of a ThI phenotype by administration of IL-12 prior to treatment with mercury. IL-12 is critical for the development of the Thl phenotype and for the initiation of inflammatory immune responses (27). IL-12 also contributes to certain Thl-mediated autoimmune disorders (41,42). Further, in mice susceptible to Leishmania major infection, IL-12 administration can bias toward a protective Thl response (43,44). Thus, IL-12 can serve as a potentially important immunoregulator of disease outcome. In A.SW mice, IL-12 treatment dramatically reduced ANoA titers of all dasses and partially inhibited the HgCl2-induced increase in IgG1 but had no effect on serum levels of IgE and renal Ig deposits (45). This last finding was somewhat unexpected, as induction of IFN-y by IL-12 can downregulate class switching to both IgGI and IgE. Nevertheless, IL-12 can enhance IgE synthesis under certain circumstances (46); its ability to influence Th2 cytokine-dependent IgE production varies according to the immunologic setting. We also observed that IL-12 further potentiated the HgCl2 induction of IL-4 (45). This may be related to our observation that IL-12 had no effect on serum IgE levels. Specifically, treatment of mice with both HgCl2 and IL-12 may have led to the development of Th cells with a mixed ThO phenotype. The balance of cytokines produced by these cells may thus explain differential effects of IL-12 on isotype switching to IgG1 and IgE (45).
Although these studies are important in their attempt to understand the complex regulatory nature of this syndrome, their findings are somewhat difficult to reconcile with a simple Thl/Th2 imbalance. Moreover, a somewhat artificial environment may be introduced in that administration of recombinant cytokines can result in kinetics and distribution that differ greatly from those of the proteins secreted in vivo. Similarly, the use of neutralizing antibodies can result in incomplete or short-term inactivation. Recently, we and others have utilized a targeted genedisruption or knockout ap5roach to more accurately assess the roles of cytokine genes in this model. These studies have examined mercury-induced disease in IL-4-deficient H-2S mice and confirmed findings made in IL-4 mAb-treated A.SW mice (47,48). Although mercury-treated IL-4-deficient mice lack the characteristic increases in serum IgGI ANoA and total IgGI and IgE, levels of ANoA of other IgG subclasses are comparable to mercury-treated wild-type H-2s mice (47,48). Using IFN-y-deficient H-2S mice, Kono et al. have observed that mercury treatment does not elicit ANoA or tissue lesions in these animals (47). If confirmed, this novel finding would suggest that IFN-y plays a hitherto unidentified role in the loss of tolerance to nucleolar antigens that takes place in this model.

How Does Mercuric Chloride Induce Cytokine Expression?
As indicated before, it is unclear how mercury triggers an activation of T cells and an upregulation of cytokine production in genetically susceptible animals. mercury and other heavy metals can have mitogenlike effects on cells and are strong activators of the immune system (32,49,50). However, most of the available evidence, including the presence of self-reactive and metal-specific cells (51)(52)(53) as well as the specificity of the autoantibody response (12), suggests that mercury induces a specific antigen-driven response .
Druet and colleagues recently examined HgCl2-induced cytokine expression in autoreactive T-cell lines derived from mercury-treated LEW rats. These cells can transfer resistance to naive (BN x LEW)F1 hybrids and produce IL-2, IFN-y, and transforming growth factor-n (14). In contrast, T-cell lines derived from mercury-and goldtreated BN rats passively transfer susceptibility to mercury-induced disease in CD8-depleted BN rats and produce IL-4 in vitro when cultured with HgC12 (51). This induction of IL-4 is mediated by protein kinase C-dependent Ca2+ influxes through Ltype channels (54). In these experiments, HgCl2 was able to activate protein kinase C directly and upregulate IL-4 mRNA expression without de novo protein synthesis.
Several recent lines of evidence suggest a role for intracellular thiol levels in determining Thl/Th2 cytokine expression. Sulfhydryl compounds have many important biologic functions, including maintenance of intracellular redox balances (55). GSH, the most abundant intracellular thiol, is required for ConA-mediated induction of IFN-y-producing cells in vitro (56). This induction is suppressed in BN rats, but not LEW rats, after exposure to HgCl2 in vivo (56). Furthermore, N-acetyl-L-cysteine, a GSH precursor, inhibits IL-4 and IgE production by human T cells in vitro (57), whereas in vivo depletion of GSH in mice results in decreased IFN-y production and increased IL-4 production in vitro (58). Taken together, these studies indicate that high thiol levels favor development of Thl responses, whereas low levels favor Th2 responses. A recent study has reported straindependent differences in thiol levels between LEW and BN rats (59). In particular, CD4+ T cells from LEW rats have higher levels of total thiols than those of BN rats (59). Because mercury has a strong affinity for thiol groups, it is possible that mercury may bind intracellular thiols, resulting in decreased thiol levels and a shift toward a Th2 profile in susceptible animals. Supporting this hypothesis, administration of thiol compounds in mice prevents development of mercuryinduced IgGI ANoA and IgG1 immune complexes and reduces serum IgE levels in SJL mice (60). Strain differences in intracellular thiol levels may contribute to the differential responses elicited by HgCl2 in either resistant or susceptible animals, thus explaining predominantly Thl or Th2 patterns of cytokine expression.
In summary, HgC12-induced autoimmunity in rodents is a useful model of chemically related autoimmunity in humans. Much is still unknown concerning the T-cell-dependent induction and resolution of this disease. The conventional view is that HgC12 triggers a Th2 immune response in susceptible strains, whereas resistant strains develop a Thl response after mercury treatment. Thus, in this scenario Th2 cells mediate induction of the syndrome, and Thl cells are important in the resolution of disease. Early studies demonstrating increases in both Th2 cytokine mRNA and Th2 cytokine-regulated immunoglobulins in susceptible animals supported this hypothesis. However, recent examination of the roles of these cytokines has demonstrated that IL-4 production is not required for HgC12-induced disease, and the question remains whether Thi cells participate in both the induction and regulation of this syndrome. Thl and Th2 cytokines indeed play important roles in the B-cell activation seen in this model, although it is likely that loss of tolerance to nudeolar antigens arises by mechanisms independent of conventional Th l /Th2 regulation. Thus, interventions such as administration of rIFN-y, rIL-12, or anti-IL-4 mAb affect isotype switching and total levels of serum immunoglobulins but cannot prevent the loss of tolerance seen in this model. Future studies on HgCl2-induced autoimmunity are most likely to focus on dissecting the biochemical and molecular pathways by which Hg interacts with selfantigen to yield an autoimmune response. Linked to these studies are questions such as: How does a metal render a self component immunogenic? and Why are nucleolar antigens (fibrillarin) specifically targeted? Recent works by several investigators have begun to address these issues (1,61,62). Further studies are also needed to clearly elucidate the pattern of cytokine expression in this syndrome and to determine their exact requirement for disease development. The knowledge gained from studying HgCl2induced autoimmunity in rodents will contribute to our understanding of the relationships between genetics, environment, and human autoimmunity.