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Deficiency in Daf1, a complement regulatory protein, has been shown to exacerbate development of various autoimmune diseases and recent studies have suggested that this may be explained by Daf1 acting to limit T-cell hyper-responsiveness. It has been suggested that the absence of Daf1 aggravates autoimmune disease in a complement-dependent manner, but others have shown that activation of T cells in the absence of Daf1 can be complement independent. However, the relationship between Daf1, complement components, lymphocyte activation, cytokine expression and antibody production remains to be determined in mice that are not Daf1 deficient. We have recently demonstrated, in murine mercury-induced autoimmunity (mHgIA), that an accumulation of CD44(high) Daf(low) CD4(+) T cells is associated with the development of autoimmunity. In this study we observed that complement depletion does not affect the accumulation of activated CD4(+) T cells, elevation of splenic interleukin-4 expression and autoantibody production in mHgIA. In addition, neither the accumulation of CD44(high) Daf(low) CD4(+) T cells nor the down-regulation of Daf1 expression on CD4(+) T cells was influenced by a lack of complement. In conclusion, these studies show that initiating events in xenobiotic-induced autoimmunity, including lymphocyte activation, cytokine expression and autoantibody production, are not dependent on complement., (© 2011 The Authors. Immunology © 2011 Blackwell Publishing Ltd.)

Biological impairments due to mercury discharge into the environment are now an issue of global concern. From the three forms of mercury found in aquatic ecosystems, the immunotoxic effects of mercury chloride were examined in the model animal, the blue mussel. In order to investigate the toxic potency of this chemical, three exposure regimes were carried out: chronic exposure of groups of individuals, a new protocol "in tubo" designed for sub-acute exposures of individuals, and acute exposures of target cells. Chronic exposure revealed significant immunotoxic effects after 7 days at 10(-6)M, while acute exposures showed significant inhibition of phagocytosis at 10(-4)M and 10(-3)M. In sub-acute exposures both circulating haemocytes and haemocyte mortality increased at 10(-4)M and 10(-3)M while phagocytosis and the clearance rate drew hormetic toxic effects on healthy individuals. These results suggest the use of the "in tubo" design for bivalve toxicological individual studies.

Inorganic mercury (mercuric chloride--HgCl(2)) induces in mice an autoimmune syndrome (HgIA) with T cell-dependent polyclonal B cell activation and hypergammaglobulinemia, dose- and H-2-dependent production of autoantibodies targeting the 34 kDa nucleolar protein fibrillarin (AFA), and systemic immune-complex deposits. The organic mercury species methylmercury (MeHg) and ethylmercury (EtHg--in the form of thimerosal) induce AFA, while the other manifestations of HgIA seen after treatment with HgCl(2) are present to varying extent. Since these organic Hg species are converted to the autoimmunogen Hg(2+) in the body, their primary autoimmunogen potential is uncertain and the subject of this study. A moderate dose of HgCl(2) (8 mg/L drinking water--internal dose 148 micro gHg/kg body weight [bw]/day) caused the fastest AFA response, while the induction was delayed after higher (25 mg/L) and lower (1.5 and 3 mg/L) doses. The lowest dose of HgCl(2) inducing AFA was 1.5 mg/L drinking water which corresponded to a renal Hg(2+) concentration of 0.53 micro g/g. Using a dose of 8 mg HgCl(2)/L this threshold concentration was reached within 24 h, and a consistent AFA response developed after 8-10 days. The time lag for the immunological part of the reaction leading to a consistent AFA response was therefore 7-9 days. A dose of thimerosal close to the threshold dose for induction of AFA (2 mg/L drinking water--internal dose 118 micro gHg/kg bw per day), caused a renal Hg(2+) concentration of 1.8 micro g/g. The autoimmunogen effect of EtHg might therefore be entirely due to Hg(2+) formed from EtHg in the body. The effect of organic and inorganic Hg species on T-helper type 1 and type 2 cells during induction of AFA was assessed as the presence and titre of AFA of the IgG1 and IgG2a isotype, respectively. EtHg induced a persistent Th1-skewed response irrespectively of the dose and time used. A low daily dose of HgCl(2) (1.5-3 mg/L) caused a Th1-skewed AFA response, while a moderate dose (8 mg/L) after 2 weeks resulted in a balanced or even Th2-skewed response. Higher daily doses of HgCl(2) (25 mg/L) caused a balanced Th2-Th1 response already from onset. In conclusion, while metabolically formed Hg(2+) might be the main AFA-inducing factor also after treatment with EtHg, the quality of the Hg-induced AFA response is modified by the species of Hg as well as the dose.

Mercuric chloride (HgCl2)-induced autoimmunity in Brown Norway rats is a spontaneously resolving autoimmune response driven by the activation of T helper type 2 lymphocytes (Th2 cells). Treatment with antibody to OX40-ligand (OX40-L) from the time of the first HgCl2 injection for 12 days had little effect. Delayed treatment commenced 8 days after the first HgCl2 injection significantly suppressed immunoglobulin E production, splenomegaly, weight loss and mortality. This makes OX40/OX40-L signalling an attractive therapeutic target for Th2-driven autoimmune diseases. Intravenous administration of the murine antibody to OX-40-L elicited a vigorous anti-mouse immunoglobulin antibody response that was significantly enhanced compared to the response to control immunoglobulin. It is likely that this response significantly reduced the plasma half-life of the anti-OX40-L antibody and this observation has clear implications for the interpretation of data from experiments where anti-OX40-L is used in vivo.

The initiation and severity of systemic autoimmune diseases are influenced by a variety of genetic and environmental factors, in particular bacterial infections and products. Here, we have employed bacterial lipopolysaccharide (LPS), which non-specifically activates the immune system, to explore the involvement of innate immunity in mercury-induced autoimmunity in mice. Following treatment of mouse strains resistant [DBA/2 (H-2(d))] or susceptible [SJL(H-2(s))] to such autoimmunity with mercuric chloride and/or LPS or with physiological saline alone (control), their immune/autoimmune responses were monitored. Resistant DBA/2 mice were rendered susceptible to mercury-induced autoimmunity by co-administration of LPS, exhibiting pronounced increases in the synthesis of IgG1 and IgE, high titres of IgG1 deposits in the kidneys and elevated circulating levels of IgG1 antibodies of different specificities. Furthermore, the percentages of the T cells isolated from the spleens of DBA/2 mice exposed to both mercury and LPS that produced pro-inflammatory cytokines were markedly increased by in vitro stimulation with phorbol myristate acetate (PMA) and ionomycin, which was not the case for splenic T cells isolated from mice receiving mercuric chloride, LPS or saline alone. In addition, exposure of susceptible SJL mice to mercury in combination with LPS aggravated the characteristic features of mercury-induced autoimmunity, including increased synthesis of IgG1 and IgE, the production of IgG1 anti-nucleolar antibodies (ANolA) and the formation of renal deposits of IgG1. In summary, our findings indicate that activation of the innate immune system plays a key role in both the induction and severity of chemically induced autoimmunity.

In susceptible mice, mercuric chloride induces a systemic autoimmune response that is characterized by elevated serum levels of immunoglobulin G1 (IgG1) and immunoglobulin E (IgE), production of anti-nucleolar antibodies (ANolAs) and the formation of renal IgG deposits. We have previously shown that mercury can also enhance immune/autoimmune responses in mouse strains genetically prone to develop spontaneous autoimmune disease. Here, we investigated whether mercury can enhance the severity of murine collagen-induced arthritis (CIA), an inducible (acquired) autoimmune disease that cannot be induced by mercury itself. While mercury administered prior to the induction phase of CIA exerted little, if any, influence, administration of mercury during the induction phase and following onset aggravated the symptoms of this disease and increased the serum levels of IgE and IgG2a antibodies directed against collagen type II (CII). Furthermore, while animals injected with mercury alone exhibited a significant decrease in the ratio of the levels of interferon-gamma (IFN-gamma) to interleukin-4 (IL-4) mRNA in their spleens, this ratio was increased in mice with CIA, with or without administration of mercury. Finally, the production of anti-nuclear antibodies, a hallmark of autoimmunity in response to mercury, was observed in all mice with CIA treated with this heavy metal. Our findings suggest that exposure to mercury during the development of CIA may influence immunological factors in such a way as to synergistically promote disease development.

Administration of mercuric chloride to Brown Norway rats results in T helper type 2 (Th2)- dominated autoimmunity characterized by high immunoglobulin E (IgE) concentrations, the production of multiple IgG autoantibodies, including those to glomerular basement membrane (GBM), arthritis and caecal vasculitis. After 14 days animals immunoregulate and auto-immunity resolves even if mercuric chloride injections are continued. In a third phase, if animals are re-challenged with mercuric chloride 6 weeks later, they show only attenuated autoimmunity with lower anti-GBM antibody concentrations and arthritis scores. Resistance to the induction of anti-GBM antibodies can also be achieved following an initial challenge with low-dose (one-tenth standard dose) mercuric chloride. We have now studied this resistant phase in more detail. We have shown, first, that following an initial full-dose mercuric chloride challenge, resistance also affects susceptibility to caecal vasculitis. Second, following an initial full-dose mercuric chloride challenge, the IgE response upon re-challenge is initially accelerated but subsequently enters a resistant phase and third, following an initial challenge with low-dose mercuric chloride, resistance is also seen to the induction of caecal vasculitis but is not seen in IgE serology (where results suggest competing effector and regulatory cell populations). Studying such regulatory phases in animal models of autoimmunity may be of benefit in the future in designing new therapies for human vasculitis.

In susceptible mice, mercuric chloride induces a systemic autoimmune disease characterized by increased serum levels of immunoglobulin (Ig) G1 and IgE, production of anti-nucleolar autoantibodies (ANolA) and formation of renal IgG deposits. We have previously hypothesized that mercury confers more adverse immunological effects on those mouse strains, which are genetically prone to develop spontaneous autoimmune diseases than on normal strains. In this study, we tested our hypothesis in tight skin 1 (Tsk1/+) mice, a murine model for human scleroderma. As a support for our hypothesis, we observed that in Tsk1/+ mice, B cells were spontaneously hyperactive and that treatment with mercury induced a strong immune/autoimmune response in these mice, but not in their non-Tsk (+/+) littermates. This response was characterized by the formation of high numbers of splenic IgG1, IgG2b and IgG3 antibody-secreting cells, increased serum levels of IgE, production of IgG1 antibodies against single-stranded DNA (ssDNA), trinitrophenol (TNP) as well as thyroglobulin and the development of renal IgG1 deposits. Neither Tsk1/+ mice nor F1 hybrid crosses between this strain, and mercury susceptible B10.S (H-2(s)) were able to produce IgG1-ANolA in response to mercury. Moreover, mercury-induced immune activation in Tsk1/+ was not able to potentiate the progression of skin fibrosis in this strain. Thus, exposure to mercury accelerates the immune dysregulation, but not the development of skin fibrosis in Tsk1/+ mice.

A rise in interleukin (IL) 4-dependent immunoglobulin E (IgE) is a hallmark of the mercuric chloride (HgCl2)-induced Th2-mediated autoimmune syndrome in the Brown Norway (BN) rat, and one of the mediators in allergic asthma in human. Oxidative stress, a potential factor related to the pathogenesis of allergy and asthma, has been shown to up-regulate IL-4 in mast cells and predispose to degranulation in vitro. However, it remains unknown whether oxidative/antioxidative imbalance plays a role in this Th2-driven model of autoimmunity in the rat. Here we show that administration of the non-sulphydryl-containing antioxidant desferrioxamine i.p. and s.c. to BN rats reduces HgCl2-enhanced IL-4 gene expression and inhibits HgCl2-induced Th2-mediated autoimmunity. Desferrioxamine treatment suppresses significantly IgE production and lymphoproliferation, and reduces tissue injury in the form of caecal vasculitis in the HgCl2-induced autoimmune syndrome. These results support a role for oxidative stress in the pathogenesis of the HgCl2-induced Th2-dominated autoimmune syndrome. This finding might have implications for understanding the mechanisms involved in Th2 cell responses as seen in allergy and asthma and thereby aid the development of new therapeutic strategies for these diseases.

T-helper cells type 1 (Th1) and type 2 (Th2) play an important role in the pathogenesis of autoimmune diseases. In many Th1-dependent autoimmune models, treatment with recombinant interleukin-12 (rIL-12) accelerates the autoimmune response. Mercury-induced autoimmunity (HgIA) in mice is an H-2 regulated condition with antinucleolar antibodies targeting fibrillarin (ANoA), systemic immune-complex (IC) deposits and transient polyclonal B-cell activation (PBA). HgIA has many characteristics of a Th2 type of reaction, including a strong increase of IgE, but disease induction is critically dependent on the Th1 cytokine IFN-gamma. The aim of this study was to investigate if a strong deviation of the immune response in HgIA towards Th1 would aggravate HgIA. Injections of both rIL-12 and anti-IL-4 monoclonal antibody (alpha-IL-4) reduced the HgCl2-(Hg-)induced concentration of the Th2-dependent serum IgE and IgG1, but increased the Th1-dependent serum IgG2a. The IgG-ANoA developed earlier and attained a higher titre after combined treatment, and the ANoA titre of the IgG1 isotype decreased while the ANoA titre of the Th1-associated IgG2a, IgG2b and IgG3-ANoA isotypes increased. Treatment with rIL-12 alone increased the Hg-induced IgG2a and IgG3 ANoA titres, the PBA, and the IC deposits in renal and splenic vessel walls, while treatment with alpha-IL-4 + Hg inhibited renal but not splenic vessel wall IC deposits. We conclude that manipulating the cytokine status, by altering the Th1/Th2 balance, will influence autoimmune disease manifestations. This might be an important way of modulating human autoimmune diseases.

Immunological tolerance can be achieved in animals by exposure of newborn to a foreign antigen. Depending on the dose and timing of the antigenic challenge, tolerance has been reported to result in clonal deletion, anergy or active suppression. In this latter case, regulatory T cells prevent autoimmunity by suppressing the reactivity of pathogenic self-reactive T cells. We have previously reported the generation of a neonatal, mercury-specific, and dominant tolerance to autoimmunity induced by mercury salts in rats. Chronic exposure to mercury salts can lead to SLE-like autoimmune responses, mediated by autoreactive CD4+ Th2 cells, that regulate and are followed by a resistant state mediated by protective CD8+ T cells. The aim of the study was to compare the resistance to the neonatal tolerance to mercury disease, and to further characterize the CD8+ T cells endowed with regulatory capacity in the neonatal tolerance model. We report here that resistance to mercury disease is long lasting and not mercury-specific, suggesting that different CD8+ T cells are involved in resistance and neonatal tolerance, and that regulatory CD8+ Tc1 cells generated in tolerance are required to control the CD8- cell population from developing Th2-mediated autoimmunity. Upon mercury recall, CD8+ CD45RC(high) T cells, that represent the Tc1 subset in the rat, expanded and were polarized towards IFNgamma production. Interestingly, identical results were obtained with the CD8+ CD25+T cell population. Substantial amounts of FasL gene expression were detected in CD8+ T lymphocytes upon recall with the tolerogen. AICD may be one of the regulatory mechanisms used by these regulatory CD8+ Tc1 cells that control neonatal tolerance to a Th2-mediated autoimmune disorder.

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.

After exposure to subtoxic doses of heavy metals such as mercury, H-2(s) mice develop an autoimmune syndrome consisting of the rapid production of IgG autoantibodies that are highly specific for nucleolar autoantigens and a polyclonal increase in serum IgG1 and IgE. In this study, we explore the role of two inhibitory immunoreceptors, CTLA-4 and FcgammaRIIB, in the regulation of mercury-induced autoimmunity. In susceptible mice treated with mercuric chloride (HgCl(2)), administration of a blocking anti-CTLA-4 Ab resulted in a further increase in anti-nucleolar autoantibodies and in total serum IgG1 levels. Furthermore, in some DBA/2 mice, which are normally resistant to heavy metal-induced autoimmunity, anti-CTLA-4 treatment leads to the production of anti-nucleolar Abs, thereby overcoming the genetic restriction of the disease. In mice deficient for the FcgammaRIIB, HgCl(2) administration did not trigger autoantibody production, but resulted in an increase in IgE serum levels. Taken together, these results indicate that different inhibitory mechanisms regulate various manifestations of this autoimmune syndrome.

Background: In 97% of all patients with oral lichenoid reactions (OLR) associated with dental amalgam a removal of the fillings leads to a decline of the lesions, as a minimum., Objectives: The aim of this study was to determine if contact allergic or local toxic effects or both may contribute to OLR using an animal model with mercury-sensitive and non-sensitive rats., Methods: Twenty Brown Norway rats, which have a genetic predisposition for an autoimmune syndrome after exposure to mercury and 20 Lewis rats, not mercury sensitive, were treated as follows: 10 animals of each group were sensitized with a low dose of mercuric chloride. Half of all animals received local exposure of the right buccal mucosa to amalgam (left: control), the others to amalgam alloy free of mercury. All rats were patch tested with an amalgam series., Results: After 20 days of exposure 96% of all animals showed white mucosal lesions restricted to the contact zone of the alloy on the treated side, but only up to 25% had a positive patch test reaction to amalgam or inorganic mercury (INM). The lesions showed no relation to species, alloy, sensitization or patch test reaction., Conclusions: While allergic mechanisms may contribute to mucosal contact lesions in Brown Norway rats, this is less probable in Lewis rats. Mercury in general appears to be irrelevant in the development of ORL in this study. If this holds true for humans as well, patch testing with an amalgam series may be helpful in a minor fraction of all patients with OLR.

Exposure of newborn animals to a foreign Ag may result in immunological tolerance to that specific Ag, a phenomenon called neonatal tolerance. We have previously reported that neonatal administration to Brown-Norway rats of mercury, a heavy metal toxicant, induces a dominant tolerance, specific for the chemical otherwise responsible for Th2 cell-mediated autoimmune responses in this susceptible strain of rats. Neonatal exposure to Ags can prime immunity, rather than inactivate or delete responses, and sustain regulatory functions effective against autoreactive T cells. Here, we address whether such a tolerant response is due to the generation of regulatory cells. The results suggest that the CD8(+) T cell subset is involved in neonatal tolerance to mercuric salt-induced Th2 autoimmune disease. Thus, we demonstrate that in vivo CD8 depletion breaks tolerance following mercury recall in animals under a neonatal tolerance protocol. Furthermore, adoptive cotransfer of splenocytes from naive and tolerant rats as well as transfer of CD8(+) T cells from tolerant animals prevent naive syngeneic rats from developing pathologic Th2 immune responses. These observations indicate that CD8(+) T cells are endowed with regulatory functions in neonatal tolerance and mediate active suppression. Moreover, neonatal tolerance induced the expansion of CD8(+)CD45RC(high) T cells and the emergence of a high percentage of IFN-gamma-synthesizing CD8(+) T cells, which probably reflects the implication of regulatory Tc1 cells. Thus, in vivo induction of neonatal tolerance suppresses Th2 autoimmune responses via generation of a CD8(+) cell-mediated regulatory response.

It is clear that functional heterogeneity of T cells may be explained by differential cytokine production. The aim of this paper was to review evidence for regulatory cells, generated after HgCl(2)-exposure. They differ from classical Th1 and Th2 cells, produce transforming growth factor-beta and interleukin-10 and exert their regulatory functions in a Th1/Th2-unrestricted fashion.

Although evidence indicates that environmental factors play a major role in precipitating systemic autoimmunity in genetically susceptible individuals, little is known about the mechanisms involved. Certain heavy metals, such as mercury, are potent environmental immunostimulants that produce a number of immunopathologic sequelae, including lymphoproliferation, hypergammaglobulinemia, and overt systemic autoimmunity. Predisposition to such metal-induced immunopathology has been shown to be influenced by both MHC and non-MHC genes, as well as susceptibility to spontaneous lupus, in mice and other experimental animals. Among the various mouse strains examined to date, the DBA/2 appears to uniquely lack susceptibility to mercury-induced autoimmunity (HgIA), despite expressing a susceptible H-2 haplotype (H-2d). To define the genetic basis for this trait, two genome-wide scans were conducted using F2 intercrosses of the DBA/2 strain with either the SJL or NZB strains, both of which are highly susceptible to HgIA. A single major quantitative trait locus on chromosome 1, designated Hmr1, was shown to be common to both crosses and encompassed a region containing several lupus susceptibility loci. Hmr1 was linked to glomerular immune complex deposits and not autoantibody production, suggesting that DBA/2 resistance to HgIA may primarily involve the later stages of disease pathogenesis. Identification and characterization of susceptibility/resistance genes and mechanisms relevant to the immunopathogenesis of mercury-induced autoimmunity should provide important insights into the pathogenesis of autoimmunity and may reveal novel targets for intervention.

Previous studies have shown that genetic factors control the susceptibility to mercury-induced immunoglobulin (Ig)G1 antibody formation, IgE synthesis, renal IgG deposits and antinucleolar autoantibodies (ANolA) production in the susceptible mice. In this study, we examined the genetic control of resistance to these characteristics after HgCl2 injection in F1 hybrid crosses between the highly mercury resistant DBA/2 and mercury susceptible NZB (H-2d), SJL (H-2 s), A.CA (H-2f) and DBA/1 (H-2q) mice and also in backcross hybrids between (DBA/2 x SJL)F1 and SJL mice. We observed that mercury-induced immune/autoimmune manifestations were profoundly downregulated in most (if not all) of the F1 hybrids, indicating that the resistance to mercury was a dominant trait. Analysis of mercury-induced immune/autoimmune responses in the (DBA/2 x SJL) x SJL backcross hybrids suggested that only one gene or a cluster of genes determined the resistance to the ANolA production, whereas the resistance to other characteristics was controlled by two and/or three gene loci. By H-2 genotyping the backcross mice, it was found that H-2d haplotype per se could confer resistance to ANolA production. However, we did not find any significant association between the H-2d haplotype and the resistance to increase of IgG1 and IgE synthesis and the development of renal IgG1 deposits. Thus, while in DBA/2 mice, gene(s) in the H-2 loci strictly contribute to the inheritance of resistance to ANolA production; non-H-2 genes mainly govern the inheritance of unresponsiveness regarding other characteristics.

Autoreactive T cells exist in healthy individuals and represent a potential reservoir of pathogenic effectors which, when stimulated by microbial adjuvants, could trigger an autoimmune disease. Experimental studies have indicated that xenobiotics, well defined from a chemical point of view, could promote the differentiation of autoreactive T cells towards a pathogenic pathway. It is therefore theoretically possible that compounds present in vaccines such as thiomersal or aluminium hydroxyde can trigger autoimmune reactions through bystander effects. Mercury and gold in rodents can induce immunological disorders with autoimmune reactions. In vitro, both activate signal transduction pathways that result in the expression of cytokines, particularly of IL-4 and IFNgamma. In a suitable microenvironment heavy metals could therefore favour the activation of autoreactive T cells. In that respect, genetic background is of major importance. Genome-wide searches in the rat have shown that overlapping chromosomal regions control the immunological disorders induced by gold salt treatment, the development of experimental autoimmune encephalomyelitis and the CD45RC(high)/CD45RC(low)CD4(+)T cells balance. The identification and functional characterization of genes controlling these phenotypes may shed light on key regulatory mechanisms of immune responses. This should help to improve efficacy and safety of vaccines., (Copyright 2001 Academic Press.)

Suppression of IgE responses is a major goal for immunotherapy, especially in the field of allergy. The Th2 subset of helper T cells plays a vital role in class switching of B cells to IgE production by releasing IL-4. In susceptible rat strains, mercuric chloride (HgCl2) induces activation of Th2 cells, with enhanced expression of IL-4, polyclonal B cell activation and very high levels of circulating IgE. We have previously shown that spontaneous regulation of this response coincides with enhanced expression of Th1/type-1 cytokines, including interferon-gamma (IFN-gamma) and IL-12. We now report the effects of administration of exogenous type-1 cytokines on HgCl2-induced Th2 responses. At high doses, recombinant rat IFN-gamma markedly reduced serum IgE levels. Recombinant mouse IL-12 was less effective at suppressing the IgE response following HgCl2, although it caused marked up-regulation of IFN-gamma gene expression in the spleen. In Lewis rats, which are resistant to HgCl2-induced autoimmunity, a rise in serum IFN-gamma was observed after HgCl2, but administration of polyclonal anti-IFN-gamma antibodies did not render them susceptible to induction of a Th2 response by HgCl2. Our data show that individual type-1 cytokines are capable of suppressing the dramatic Th2 response induced by HgCl2 in the rat, even when they are not given until after starting HgCl2 administration. IFN-gamma is a pivotal cytokine in ameliorating the Th2 response and measures aimed at selective up-regulation of this cytokine may be of therapeutic value in suppression of unwanted IgE responses.

Hg2+ induces lymphocyte proliferation when added to cell cultures from both healthy and mercury-allergic subjects. Consequently, when measuring DNA synthesis a possible Hg2+-specific response, resulting from proliferating memory cells, cannot be discriminated from a non-allergic response. The mechanism behind this non-allergic response is unknown but a superantigenic effect of Hg2+ has been suggested. In this study, five mercury-allergic patients, with oral lichen planus (OLP) lesions adjacent to dental amalgam and a positive patch test to Hg0, and five healthy subjects without amalgam were examined. The immunophenotype and the T cell receptor Vbeta (TCR Vbeta) repertoire of Hg2+-induced lymphoblasts as well as the expression of the lymphocyte activation markers CD23 and CD134 were analysed for possible differences between healthy and allergic subjects. The mechanism of Hg2+-induced proliferation was examined by comparing the TCR Vbeta expression of Hg- and staphylococcal enterotoxin B (SEB)-activated lymphoblasts, the latter used as a positive superantigen control. It was not possible to discriminate between mercury-allergic and healthy subjects using the immunophenotype or the TCR Vbeta profile of the Hg2+-induced lymphoblasts or the expression of CD23 and CD134. However, Hg2+-induced CD4+ lymphoblasts showed a skewing towards Vbeta2. This relative increase in Vbeta2 was only detected in the CD4+ but not in the CD8+ lymphoblast population. In conclusion, Hg2+ induced a proliferation-dependent skewing towards CD4+ but not CD8+ lymphocytes expressing Vbeta2. In this respect Hg2+ differs from the classical bacterial superantigen SEB, which also stimulates unique TCR Vbeta families among CD8+ cells.

Blood lymphocytes from 20 patients with oral contact lesions to dental amalgam and 10 healthy individuals were analyzed for HgCl2-induced proliferation in vitro, using both a modified assay and a conventional assay. The release of interferon-gamma (IFN-gamma) was measured in cell supernatants. Six patients displayed positive reactions in patch tests to mercuric compounds. No significant differences were recorded in HgCl2-induced proliferation in cells from patients and controls, since only few in the whole material responded to submitogenic concentrations. IFN-gamma was detectable in cell supernatants from some patients but also from controls and is not predictive of mercury allergy. Neither the phenotypes of peripheral lymphocyte subsets, the frequency of circulating cells expressing the interleukin-2 (IL-2) receptor, spontaneous lymphocyte proliferation nor concentrations of serum interleukin-6 differed between patient and control samples. In contrast to what has been claimed before, we did not find any evidence for specific in vitro lymphocyte reactivity in patients with oral contact lesions.

Mercuric chloride induces autoimmunity in Brown Norway rats with polyclonal B-cell activation, hyper-IgE and multiple autoantibodies. Pre-treatment with low-dose HgCl2 (one-tenth of the standard dose) induces resistance to later full-dose HgCl2; we have studied the mechanism of this resistance. Brown Norway rats given low-dose HgCl2 showed only a modest increase in serum IgE level, three logs lower than rats given standard-dose HgCl2, and no up-regulation of splenic interleukin (IL)-4 mRNA. There was up-regulation of splenic interferon (IFN)-gamma gene expression and a progressive rise in serum IFN-gamma. Neither IL-12 nor IL-18 were induced, but there was up-regulation of IL-12 receptor beta2-chain (IL-12Rbeta2) expression. IL-10 and transforming growth factor (TGF)-beta expression did not change. Serum IgE and splenic IL-4 mRNA expression remained static when these rats were rechallenged, confirming resistance. Thereafter IFN-gamma expression gradually fell, after which IL-4 expression and serum IgE rose slightly. Our observations suggest that low-dose HgCl2 confers protection in Brown Norway rats to further HgCl2 by up-regulation of IFN-gamma, associated with enhanced IL-12Rbeta2 expression. The immunological response to HgCl2 in susceptible rat strains is more complex than previously appreciated and is dose dependent, with low doses inducing a T helper '(Th)1' type of response in contrast to the 'Th2' type response associated with standard doses.

Exposure to low doses of mercury chloride induces autoantibodies to the nucleolar protein fibrillarin in H-2s, but not in H-2b, mice. Surprisingly, F1 crosses between resistant and sensitive haplotypes are resistant. Previously, we have shown that the resistance in these F1 mice was due to coexpression of the resistant class II allele. Using adoptive transfer techniques we have examined several mechanisms by which the resistant haplotype could be down-regulating the antifibrillarin response in F1 (s/b) mice. Similar to other autoimmune models, mercury-induced autoimmunity requires cognate MHC-restricted T cell help. The absence of autoantibody production in F1 mice was not due to a difference in thymic education or to the absence of antifibrillarin-specific T cell help. These results suggest that the resistance is due to an intrinsic property of the haplotype-heterozygous B cells.

Imbalances of Th1- and Th2-type responses have been postulated to be a predisposing factor for both humoral and cellular mediated autoimmune diseases. To further define their roles in systemic autoimmunity, IL-4 and IFN-gamma gene knockout mice were studied for susceptibility to the prototypic Th2-mediated mercury-induced autoimmunity. A predominant Th2-type response following HgCl2 treatment of wild-type B10.S mice was confirmed by the findings of a significant increase in splenic IL-4 and hypergammaglobulinemia primarily of the IgG1 isotype, without an increase in IFN-gamma levels. Paradoxically, IL-4-deficient mice developed the characteristic anti-nucleolar autoantibodies and tissue deposition of immune complexes, while IFN-gamma-deficient mice had very low autoantibody levels and essentially normal immunohistology. Studies to define defects in Ab responses of IFN-gamma-deficient mice, using the T-dependent Ag (4-hydroxy-3-nitrophenyl)acetyl, revealed an attenuated IgG response to low and to a lesser extent high doses of (4-hydroxy-3-nitrophenyl)acetyl-hemocyanin, but maintenance of affinity maturation. These results indicate that Th1/Th2 imbalance does not directly play a role in susceptibility to mercury-induced autoimmunity, and suggest that the dependence on Th1-type responses in certain autoimmune diseases is due to the requirement for IFN-gamma for Ab production to weakly antigenic self molecules.

Exposure to certain drugs and environmental chemicals can provoke the onset of autoimmune disease in susceptible individuals by releasing (self) epitopes for which tolerance has not been established, while simultaneously providing the necessary adjuvant activity. The resulting response type is influenced by the genotype of exposed individuals and relates to susceptibility to the adverse immune effects of the chemicals. Here, we assessed the modulatory role of the chemical compounds themselves. A single injection of streptozotocin (STZ) increased the number of CD8+ cells, macrophages, apoptotic cells, and IFN-gamma-producing T helper and T cytotoxic cells, whereas the number of CD4+ cells and B cells was reduced in the draining lymph node. Coinjection with the reporter antigen TNP-OVA resulted in primary and secondary production of TNP-specific antibodies that were predominantly of IgG2a and IgG2b isotype, whereas STZ did not enhance priming for delayed-type hypersensitivity (DTH) responses to TNP-OVA. Injection of HgCl2 on the other hand, reduced the number of IFN-gamma-producing cells, induced accumulation of B cells and CD4+ and CD8+ T cells, enhanced IgG1 and IgE production to TNP-OVA, and primed for secondary IgG1 and IgE production as well as for DTH reactions. Together these results indicate that a single injection of STZ stimulates type-1 responses, whereas HgCl2 enhanced mixed type-1 and -2 responses in BALB/c mice. These response types match the (auto)immune effects elicited to unknown (auto)antigens following multiple injections of these chemicals.

Susceptibility to induction of both T helper 1- (Th1) and Th2-mediated autoimmunity is multifactorial and involves genetic linkage to the major histocompatibility complex (MHC) class II haplotype. Brown Norway (BN) rats exposed to mercuric chloride develop a Th2-dependent systemic autoimmunity, whereas Lewis rats, which are highly susceptible to Th1-mediated autoimmunity, develop immune suppression after mercuric chloride exposure. Exposure to mercuric chloride is known to enhance B-lymphocyte expression of the MHC class II molecule RT1.B, predominantly in BN rats. We demonstrate that, in contrast, expression of RT1.D was unmodified on these B cells, whereas both RT1.B and RT1.D were up-regulated on epithelial cells. Regulation of B-cell MHC class II isotype expression was further studied in vitro, using BN rat lymph node (LN) cells. Interleukin-4 (IL-4) strongly enhanced B-cell expression of RT1.B (2.8-fold), whereas RT1.D expression was only slightly, although significantly, modified (1.2-fold). B cells from Lewis rats showed a similar IL-4-induced enhancement of RT1.B expression (2.5-fold), whereas, in contrast, RT1.D expression was unmodified. Exposure of LN cells from BN rats to interferon-gamma induced a moderate increase of B-cell MHC class II expression, predominantly of RT1.B. Strong and rapid enhancement of B-cell RT1.D expression was observed after stimulation by phorbol 12-myristate 13-acetate and ionomycin. Rat IL-13 did not modify B-cell MHC class II expression; however, it induced typical morphological changes in peritoneal macrophages. These experiments demonstrate isotype-specific and strain-dependent regulation of MHC class II expression on rat B lymphocytes, which may be of pathophysiological relevance for the strain-dependent susceptibility for Th1- or Th2-mediated autoimmunity.

Upon treatment with HgCl2, H-2s mice, such as B10.S, develop an activation of B lymphocytes that depends, at least partially, on activation of T helper type 2 (Th2) cells and results in increased serum levels of IgG1 and IgE, appearance of IgG autoantibodies, and development of immune glomerulonephritis and vasculitis. Results of previous studies and of experiments presented here indicate that the B cell activation and systemic autoimmune disease fail to develop in MHC-congenic B10.D2 (H-2d) and B10.BR (H-2k) mice treated with HgCl2, although B10.D2 T cells showed signs of activation by and specificity for HgCl2 comparable to those seen in strain B10.S. Here, we report that following HgCl2 injections the antibody response to sheep erythrocytes is normal in B10.S, but suppressed in B10.D2 mice. This suppression was prevented by MoAb to mouse IFN-gamma. Conversely, treatment of B10.D2 mice with murine recombinant IFN-gamma (rIFN-gamma) was able to reproduce the immunosuppression seen after HgCl2 treatment. In B10.S mice, it took administration of both rIFN-gamma and HgCl2 to suppress the anti-sheep erythrocyte response. Although rIFN-gamma diminished the increase in IgE serum levels of HgCl2-treated B10.S mice, it failed to prevent their autoantibody production and immune glomerulonephritis. These findings further strengthen the concept that B10.S mice react to HgCl2 by preferential activation of their Th2 cells producing IL-4, whereas B10.D2 mice react to HgCl2 by preferential activation of their Th1 cells, which produce IFN-gamma and thus suppress antibody responses.

Mercuric chloride (HgCl2) induces the production of antinucleolar antibodies (ANucA) in susceptible strains of mice. Responder strains bearing the H-2(5) haplotype as well as several ANucA resistant strains have been shown to develop renal immune complex deposits after HgCl2 treatment. Sera obtained throughout 12 to 16 weeks of HgCl2 treatment from mice of four ANucA responder strains (A.SW/SnJ, A.CA/SnJ, DBA/1J, and P/J) and one ANucA-resistant strain (C57BL/10SnJ) were examined for ANucA production. Terminal sera were also tested for the presence of anti-glomerular basement membrane antibodies, and the kidneys were examined for the deposition of IgG and C3. Only one strain, A.SW, developed significant deposits of IgG in the renal glomeruli, although all four responder strains exhibited similar ANucA induction/production profiles. The differences seen by direct immunofluorescence assay (IFA) in renal immune complex deposition between the A.SW and histocompatibility congenic A.CA mice were corroborated by individually eluting and then quantitating the deposited IgG from renal tissues of Hg-treated A.SW and A.CA mice as well as control A.SW mice. The average amount of IgG eluted from A.SW renal tissue was significantly greater than that eluted from either A.CA or control A.SW renal tissues. All eluates from Hg-treated animals gave only a nucleolar fluorescence pattern when assayed by indirect IFA against a panel of rat organ tissues. In summary, no correlation was found between ANucA production and renal IgG deposition in response to treatment with HgCl2.

The heavy metal mercury elicits a genetically restricted, anti-nucleolar autoantibody response that targets fibrillarin, a 34-kDa protein component of many small nucleolar ribonucleoprotein particles. The mechanisms by which a toxin such as mercury elicits an autoantibody response that predominantly targets a single intracellular protein autoantigen remain uncertain, but may be prefaced by mercury gaining access to the intracellular environment. Mercury-induced cell death was associated with loss of fibrillarin antigenicity and modification of the molecular properties of fibrillarin as revealed by aberrant migration under nonreducing conditions in SDS-PAGE. Addition of mercury to isolated nuclei also resulted in aberrant migration of fibrillarin, but not other nuclear autoantigens. The sensitivity of the HgCl2-induced modification of fibrillarin to 2-ME, iodoacetamide, and hydrogen peroxide suggested interaction of mercury with the two cysteines in the fibrillarin sequence. This was confirmed by mutation of the cysteines to alanines, which abolished the aberrant migration of fibrillarin in the presence of HgCl2. The modification of the molecular structure of fibrillarin by mercury reduced immunoprecipitation by anti-fibrillarin autoantibodies, pointing to unmodified fibrillarin as the B cell Ag and implicating mercury-modified fibrillarin as the source of T cell antigenicity. These observations demonstrate for the first time that an environmental toxin can alter the physicochemical properties of an autoantigen and may help to explain the antigenic specificity of mercury-induced murine autoimmunity.

Repeated exposure of subtoxic doses of mercuric chloride (HgCl2) can induce antinucleolar autoantibodies in susceptible mice. To study the immunopathological mechanism(s), sodium selenite (Na2SeO3), which is known to reduce the toxicity of mercury, is injected simultaneously with HgCl2 into mice. Equimolar Na2SeO3 nor the same amount of Se in Na2SeO3 as Hg in HgCl2 could not reduce antinucleolar autoantibody induction by HgCl2. These results indicate that the mechanism of autoimmunity induction by HgCl2 might be independent of its toxicity.

HgCl2 treatment of B10.S mice induces IgG autoantibodies to fibrillarin, a component of small nuclear ribonucleoprotein particles, and histone. Here, we demonstrate the activation by HgCl2 of autoreactive T cells specific for these nuclear proteins. Of nine CD4+ T cell hybridoma clones obtained from HgCl2-treated B10.S mice, one clone reacted to histone Hl and eight clones to fibrillarin. One of the fibrillarin-specific clones only recognized fibrillarin pretreated with HgCl2 (Hg2+ fibrillarin), suggesting that Hg2+ can induce the presentation of a novel fibrillarin epitope. Four fibrillarin-specific hybridoma clones studied for cytokine production were shown to produce interleukin (IL)-2, and three of them also produced IL-4. For stimulation of fibrillarin-specific T cell hybridomas, exogenous murine fibrillarin had to be added when antigen-presenting cells (APCs) came from untreated mice, but not when the APCs were obtained from HgCl2-treated animals. Apparently, HgCl2 treatment induces the presentation by APCs of a novel Hg2+ fibrillarin epitope and up-regulates the presentation of unaltered fibrillarin epitopes, thus activating 'Hg(2+)-specific' as well as autoreactive CD4+ T cells.

Brown Norway (BN) rats given mercuric chloride (HgCl2), gold (Au) salts or D-penicillamine develop a T helper 2 (Th2) cell-mediated autoimmune syndrome. The recent observation of tissue injury within 24 h of HgCl2 treatment suggested the involvement of a non-T cell. We therefore examined the effect of these compounds on rat mast cells in vitro. Incubation of BN rat peritoneal mast cells with HgCl2 enhanced the release of serotonin in response to IgE cross-linking agents. Mast cells from Lewis rats, a strain not susceptible to the autoimmune syndrome in vivo, were affected to a lesser extent. The effect was observed with purified BN mast cells, suggesting a direct action. Similar effects were seen with D-penicillamine in the presence of copper ions, a combination that produces hydrogen peroxide, and Au. HgCl2 caused significant induction of interleukin (IL)-4 mRNA in mast cells from BN, but not Lewis rats. The data demonstrate a novel enhancing effect of a number of compounds on mast cell mediator release, and an inducing effect of HgCl2 on mast cell IL-4, expression. These findings are consistent with our hypotheses that mast cells may contribute to early tissue injury, and also, via production of IL-4, may initiate and/or augment, the Th2 response in the BN rat model of chemical-induced autoimmunity.

We studied characteristics of the anti-glomerular basement membrane (GBM) antibody response in three animal models of Goodpasture's disease: treatment of Brown Norway (BN) rats with the polyclonal activator mercuric chloride (HgCl2), and immunization of BN and Wistar Kyoto (WKY) rats with rat GBM. Serial serum samples were obtained over the time course of the models, and anti-GBM antibodies eluted from the kidneys. Functional affinity of the anti-GBM antibodies was measured in a solid-phase ELISA incorporating the mild chaotropic agent diethylamine. Evidence for shared epitope specificity with human anti-GBM antibodies was sought using competition ELISA. As with recent studies in human anti-GBM disease, there was no evidence for affinity maturation of the anti-GBM response in the serum of any of the animal models. Antibodies eluted from the kidneys were of higher functional affinity than serum antibodies only in the HgCl2-treated BN rat. There was no obvious correlation between the functional affinity of the antibodies and the severity of nephritis in the three models. Competition studies between eluted anti-GBM antibodies from the rat models and human anti-GBM antibodies did not provide any evidence for shared epitope recognition. This study provides further information on the extent to which these models reflect the human disease.

Light microscopy and immunocytochemistry have been used to study the tissue reaction to non-irritant concentrations of mercury painted onto the oral mucosa of genetically mercury-sensitive BN rats. Low-dose skin injections of HgCl2 in BN rats result in an autoimmune syndrome, including also a spontaneous migration of T lymphocytes into the oral mucosa. Our results show that such infiltrates confer an increased degree of reactivity (contact stomatitis) to HgCl2 painted onto the BN (Hg) rat oral mucosa. In contrast, results were negative in the LEW rat strain, which is also resistant to development of autoimmunity to skin-injected mercury. The possible involvement of mucosal mercury-loaded macrophages is discussed. The results are also discussed with respect to possible etiologic and pathogenetic mechanisms involved in the development of dental material (amalgam)-associated lichenoid lesions of human oral mucosa.

Mercuric chloride (HgCl2) induces autoimmunity in susceptible rat strains, with hyper-IgE, appearance of a number of autoantibodies, and widespread tissue injury, including necrotizing vasculitis in the gut. In the early phase of tissue injury there is granulocyte infiltration; later there is immunoglobulin deposition along basement membranes in vessels. We have analysed the role of complement in this model using cobra venom factor (CVF), which causes decomplementation lasting around 5 days. The characteristic time course when HgCl2 is given over 10 days is that tissue injury and autoantibody levels reach a peak at around day 15 (start of HgCl2 = day 0). We therefore gave CVF either early (day 0), intermediate (day 5) or late (day 10); a fourth group (controls) received HgCl2 but no CVF. At each time point, CVF caused complete decomplementation which lasted for at least 5 days. Serum IgE and autoantibody levels were similar in all four experimental groups. Tissue injury in the 'early' CVF group and in the 'late' CVF group was not significantly different from controls, but in the intermediate group tissue injury was significantly more severe than in controls. These data indicate that the complement system does not play a major role in the induction of autoantibodies by HgCl2, nor in the effector phase of tissue injury. We speculate that the exacerbation of tissue injury by CVF in the group given this agent at an intermediate stage of the model is explained by the presence of products of C3 activation which have proinflammatory effects during the phase of active granulocyte-mediated tissue injury.

Monoclonal antibodies (mAbs) that react with soluble mercuric ions have been produced by injection of BALB/c mice with a hapten-carrier complex designed to maximize exposure of the metal to the immune system. Three hybridomas producing antibodies that reacted with bovine serum albumin (BSA)-glutathione-HgCl, but not with BSA-glutathione, were isolated from the spleen of a mouse given multiple injections with glutathione-HgCl conjugated to keyhole limpet hemocyanin. Stable subclones were established from two of these antibodies, designated mAb 4A10 and mAb 1F10. The binding of both antibodies to immobilized BSA-glutathione-HgCl was inhibited by soluble HgCl2, and dissociation constants for mercuric chloride binding were 2.3 and 3.7 nM for mAbs 4A10 and 1F10, respectively. Both antibodies bound mercuric acetate with similar affinities, demonstrating that the antibodies were capable of binding to mercuric ions in the presence of a different counterion than the one used in the immunogen. Reactions were not observed with other metal cations by either antibody. These data demonstrate the successful induction of antibodies that react very specifically with mercuric ions in solution regardless of the presence of a carrier.

Mercuric chloride (HgCl2) induces in Brown-Norway (BN) and (Lewis x Brown-Norway) F1 hybrid rats a transient autoimmune disease characterized by the production of various antibodies to self and non-self antigens and by a dramatic increase of serum IgE. Experimental autoimmune uveoretinitis (EAU) can be induced in Lewis (LEW) and (LEW x BN) F1 hybrid rats by a single immunization with retinal S-antigen (S-Ag). Besides uveoretinitis, animals immunized with S-Ag develop an autoimmune pinealitis (EAP). We demonstrate in this study that (LEW x BN) F1 hybrid rats, injected with HgCl2 7 days before S-Ag immunization, are quite efficiently protected against EAU and EAP. We also show that HgCl2-induced protection is neither due to a cytotoxic effect of HgCl2 nor to CD8+ T-cell dependent mechanisms nor to the HgCl2-induced increase of serum IgE concentration. The role of other hypothetical mechanisms, such as anti-S-Ag anti-idiotypic antibodies and/or HgCl2-induced unbalance between T-helper cell subsets, is discussed.

It has previously been shown that HgCl2 injected subcutaneously in Brown Norway (BN) rats (RT1n) induces an autoimmune syndrome with glomerulonephritis and epiphenomena in various tissues. We have, with monoclonal antibodies, investigated cellular infiltrates of the oral mucosa in Hg-treated BN rats. Our results show an infiltration of CD45RB- T cells and ED1+ monocytes. In comparison with experimental contact hypersensitivity to 2,4-dinitrofluorobenzene, we found a low expression of RT1B and RT1D. No B cells were found. SD rats (RT1(1/u], treated identically, did not respond. Intense reactions with dense mononuclear cell infiltrates were seen in BN rats injected with HgCl2 and subsequently challenged in the oral mucosa with low doses of HgCl2 in acetone/olive oil. In contrast, no significant infiltrates appeared in BN rats epicutaneously painted with HgCl2 followed by oral challenge. Thus, the route by which Hg is introduced to the immune system seems to be decisive for the appearance of oral lesions in BN rats. The findings indicate that low-dose exposure to Hg can bring about epiphenomena resulting in an increased T cell dependent reactivity of the oral mucosa to Hg in genetically predisposed individuals.

Female Balb/c and SJL mice exposed to HgCl2 by subcutaneous injection or via drinking water for up to 6 months showed immunostimulation with increased concentrations of immunoglobulins (Ig) in the serum. The Ig isotype pattern was dependent both on the strain and the use of immunopotentiation by addition of Freund's complete adjuvant (FCA). Balb/c mice showed a T cell dependent pattern, whereas the SJL mice developed an increase also in T cell independent isotypes. This latter pattern shifted to show T cell dependency after an initial addition of FCA. FCA also converted the lack of stimulation in C57BL/6J mice to a low response with a T cell dependent isotype pattern. No correlation emerged between the body burden of mercury, as assessed by the concentration in the kidneys, and the degree of immunostimulation by mercury. Mice showing a stimulation of the immune system developed mesangial immune complex (IC) glomerulonephritis and, later on, IC deposits in renal, splenic, and hepatic vessel walls with an isotype pattern corresponding to that seen in the serum.

Female SJL and Balb/c mice were given subcutaneous injections of 1.6 mg HgCl2/kg body weight every third day for 2, 4, 8, or 12 weeks. Indirect immunofluorescence using HEp-2 cells as substrate showed that SJL mice developed antinuclear antibodies (ANA) with a predominantly nucleolar and a weaker, homogeneous nuclear pattern after 4 weeks treatment. The nucleolar antigen was sensitive to treatment with trypsin and RNAse, but the antibody was not absorbed by calf liver RNA. The antigen responsible for the homogeneous nuclear pattern was sensitive to treatment with trypsin, DNAse, and acid solution, but reconstitution with histones on acid treated substrate did not restore the fluorescence. The corresponding antibody was not absorbed by double-stranded or single-stranded DNA, and the Crithidia luciliae assay was negative. This suggests that the antigen responsible for the homogeneous ANA pattern is a non-histone chromatin protein. No autoantibodies were found in Balb/c mice. Electron dense immune deposits containing IgG and C3 in a mesangial-vascular pattern developed after 4 weeks mercury treatment in SJL and Balb/c mice. Acid eluate from kidneys of SJL mice with immune deposits contained tissue-bound ANA with a strictly anti-nucleolar pattern, showing that such antibodies make up part of the renal immune deposits. No autoantibodies were found in the eluate from Balb/c mice. The findings demonstrate that mercury induces a polyclonal autoantibody response in SJL mice, and suggests a restricted antibody response with unknown specificity in Balb/c mice, in both cases leading to immune complex deposits in the kidneys.