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 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.