Kenneth Michael Pollard

Resistance to Xenobiotic-Induced Autoimmunity Maps to Chromosome 1

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.

Silica, Silicosis, and Autoimmunity.

Inhalation of dust containing crystalline silica is associated with a number of acute and chronic diseases including systemic autoimmune diseases. Evidence for the link with autoimmune disease comes from epidemiological studies linking occupational exposure to crystalline silica dust with the systemic autoimmune diseases systemic lupus erythematosus, systemic sclerosis, and rheumatoid arthritis. Although little is known regarding the mechanism by which silica exposure leads to systemic autoimmune disease, there is a voluminous literature on silica exposure and silicosis that may help identify immune processes that precede development of autoimmunity. The pathophysiology of silicosis consists of deposition of silica particles in the alveoli of the lung. Ingestion of these particles by macrophages initiates an inflammatory response, which stimulates fibroblasts to proliferate and produce collagen. Silica particles are encased by collagen leading to fibrosis and the nodular lesions characteristic of the disease. The steps in the development of silicosis, including acute and chronic inflammation and fibrosis, have different molecular and cellular requirements, suggesting that silica-induced inflammation and fibrosis may be mechanistically separate. Significantly, it is unclear whether silica-induced inflammation and fibrosis contribute similarly to the development of autoimmunity. Nonetheless, the findings from human and animal model studies are consistent with an autoimmune pathogenesis that begins with activation of the innate immune system leading to proinflammatory cytokine production, pulmonary inflammation leading to activation of adaptive immunity, breaking of tolerance, and autoantibodies and tissue damage. The variable frequency of these immunological features following silica exposure suggests substantial genetic involvement and gene/environment interaction in silica-induced autoimmunity. However, numerous questions remain unanswered.

Selective induction of anti‐fibrillarin autoantibodies by silver nitrate in mice

Female SJL (H‐2s) mice developed serum IgG anti‐nucleolar antibodies (ANoA) after 5 weeks treatment with 0·05% or 0·01% silver nitrate (AgNO3) in drinking water. Five more weeks of treatment increased the ANoA titre to 3410 ± 853 and 640±175 (reciprocal mean ± s.e.m.), respectively. Controls receiving ordinary tap water and mice given 0·002% AgNO3, showed no antinucleolar antibodies. The high‐titre ANoA targeted a 34‐kD nucleolar protein identified as fibrillarin, the major autoantigen in murine mercury‐induced autoimmunity and in a fraction of patients with systemic scleroderma. Serum autoantibodies to chromatin or histones, kidney, spleen, stomach, thyroid, or skin antigens (except the nucleolus) were not found in any of the mice. There was no consistent significant increase of serum IgG1 igG2a, IgG2b, or IgG3 concentrations after AgNO3 treatment compared with controls. Mice treated with 0·05% AgNO3 for 10 weeks showed a slight decrease in serum IgG1, IgG2b and lgG3 concentrations. These mice also showed a small but statistically significant increase in renal, mesangial IgM deposits, which was not accompanied by any increase in C3c deposits, whereas mice given lower doses of silver nitrate showed no significant increase in mesangial immunoglobulin immune deposits. Systemic vessel wall immune deposits were not found in any of the mice. In mice given 0·05% silver nitrate, the kidney showed the highest concentration of silver (12·2±0·09 μg Ag/g wet weight; mean ± s.e.m.). followed by the spleen (8·7±1·3), and the liver (3·9±0·4). Treatment with 0·01% silver nitrate caused a different distribution of silver, with the highest concentration in the spleen (2·1±0·16 μg Ag/g), followed by the kidney (0·63±0·037), and the liver (< 0·29 μg Ag/g; mean). Silver seems to be a more specific inducer of antinucleolar/anti‐fibrillarin autoantibodies than mercury and gold, lacks the general immune stimulating potential of mercury, and has only a weak tendency to induce renal immune deposits. These observations suggest that the autoimmune sequelae induced in mice by metals is dependent, not only upon the genetic haplotype of the murine strain, but also on the metal under investigation.

Gold- and silver-induced murine autoimmunity – requirement for cytokines and CD28 in murine heavy metal-induced autoimmunity

Treatment with gold in the form of aurothiomaleate, silver or mercury (Hg) in genetically susceptible mouse strains (H‐2s) induces a systemic autoimmune condition characterized by anti‐nuclear antibodies targeting the 34‐kDa nucleolar protein fibrillarin, as well as lymphoproliferation and systemic immune‐complex (IC) deposits. In this study we have examined the effect of single‐gene deletions for interferon (IFN)‐γ, interleukin (IL)‐4, IL‐6 or CD28 in B10.S (H‐2s) mice on heavy metal‐induced autoimmunity. Targeting of the genes for IFN‐γ, IL‐6 or CD28 abrogated the development of both anti‐fibrillarin antibodies (AFA) and IC deposits using a modest dose of Hg (130 µg Hg/kg body weight/day). Deletion of IL‐4 severely reduced the IgG1 AFA induced by all three metals, left the total IgG AFA response intact, but abrogated the Hg‐induced systemic IC deposits. In conclusion, intact IFN‐γ and CD28 genes are necessary for induction of AFA with all three metals and systemic IC deposits using Hg, while lack of IL‐4 distinctly skews the metal‐induced AFA response towards T helper type 1. In a previous study using a higher dose of Hg (415 µg Hg/kg body weight/day), IC deposits were preserved in IL‐4−/− and IL‐6−/− mice, and also AFA in the latter mice. Therefore, the attenuated autoimmunity following loss of IL‐4 and IL‐6 is dose‐dependent, as higher doses of Hg are able to override the attenuation observed using lower doses.

Expert Panel Workshop Consensus Statement on the Role of the Environment in the Development of Autoimmune Disease

Autoimmune diseases include 80 or more complex disorders characterized by self-reactive, pathologic immune responses in which genetic susceptibility is largely insufficient to determine disease onset. In September 2010, the National Institute of Environmental Health Sciences (NIEHS) organized an expert panel workshop to evaluate the role of environmental factors in autoimmune diseases, and the state of the science regarding relevant mechanisms, animal models, and human studies. The objective of the workshop was to analyze the existing data to identify conclusions that could be drawn regarding environmental exposures and autoimmunity and to identify critical knowledge gaps and areas of uncertainty for future study. This consensus document summarizes key findings from published workshop monographs on areas in which “confident” and “likely” assessments were made, with recommendations for further research. Transcribed notes and slides were reviewed to synthesize an overview on exposure assessment and questions addressed by interdisciplinary panels. Critical advances in the field of autoimmune disease research have been made in the past decade. Collaborative translational and interdisciplinary research is needed to elucidate the role of environmental factors in autoimmune diseases. A focus on exposure assessment methodology is needed to improve the effectiveness of human studies, and more experimental studies are needed to focus on causal mechanisms underlying observed associations of environmental factors with autoimmune disease in humans.

The effect of xenobiotic exposure on spontaneous autoimmunity in (SWR x SJL)F1 hybrid mice.

F1 hybrids of SWR (H-2 q ) and SJL (H-2 s ) mice spontaneously develop a lupuslike condition in an age-dependent manner, and these two H-2 haplotypes also confer susceptibility to induction of systemic autoimmunity by heavy metals such as mercury, silver, and gold with anti-fibrillarin antibodies (AFA) as marker. The aim of this study was to determine how the mixing of two susceptible genomes might influence expression of idiopathic and induced autoimmunity over a period of 14 mo of exposure to mercury and silver. Spontaneous autoimmunity first appeared as antinuclear antibodies (ANA) in females at 10 wk of age and in males at 10 mo of age, and was followed by development of anti-chromatin antibodies. Antibodies to double-stranded DNA developed in 60% of males and 20% of females. Thirty percent of males and 10% of females developed a coarsely speckled ANA pattern associated with high titers of anti-Sm antibodies. Glomerular immune complex (IC) deposits and a proliferative glomerulonephritis were seen at 17 mo of age. The F1 hybrids treated with metals showed no exaggeration of spontaneous autoimmunity. However, the metals suppressed the spontaneous development of anti-Sm and antichromatin antibodies. The metal-induced AFA, linked to the H-2 s and H-2 q haplotype, reached a maximum after 3–4 mo of treatment and then declined; 33% of the silver-treated hybrids finally became AFA-negative, despite continuous treatment. The decline in ANoA during metal treatment is contrary to the situation in metal-treated SJL mice. This indicates that dominant SWR background genes suppressed induction of certain autoimmune traits in the (SWR × SJL)F1 hybrid mice. This work was supported by a grant from the Swedish Research Council, Branch of Medicine (project 9453), to P.Hultman, and by NIH grants ES09802, ES08080, ES08666, and ES07511. We thank Robert L. Rubin, The Scripps Research Institute, for supplying chromatin preparations, and Elham Nikookhesal for technical assistance. Yngve Åberg, PhD, provided statistical assistance.

Environment, autoantibodies, and autoimmunity

Susceptibility to autoimmune disease is multifactorial and includes genetic predisposition, gender, ethnicity, age, and environment. While no single factor has been identified as preeminent, the role of the environment has garnered increasing interest. This reflects the ubiquitous nature of the environment, which encompasses everything around us including the air we breathe, the water we drink, the food we eat, synthetic and natural chemicals, microorganisms, industrial by-products, and physical factors such as radiation (1). The most convincing evidence for a role of exogenous factors in autoimmunity comes from studies implicating numerous medications in the induction of autoimmune disease, particularly the association of drug-induced systemic lupus erythematosus (SLE) with procainamide and hydralazine (2). Identification of the causal role of medications in the induction of autoimmune disease is due in large part to the fact that medications are taken under medical supervision where drug exposure and possible side effects can be closely monitored. This is not the case with non-therapeutic exposure to environmental factors where contact may include numerous exogenous factors at any particular time. Nonetheless, evidence for the association of (non-therapeutic) environmental exposure with autoimmunity has come from two well documented exposures. In 1981, in Spain, the ingestion of analine adulterated rapeseed oil was linked to a previously unknown disease, subsequently called toxic oil syndrome (TOS), which was characterized by myalgias, peripheral eosinophilia, and pulmonary infiltrates (3). The adulterated oil was sold as “olive oil” by street vendors and subsequently used for cooking. The determination that the adulterated oil was the cause of TOS was based on robust epidemiological evidence. More than 20,000 people were affected and some 2,000 perished. Chronic conditions, including scleroderma and neurologic changes, have been described in the survivors. A clinically similar, though epidemiologically distinct, syndrome was identified in United States in 1989 (3, 4). Eosinophilia myalgia syndrome (EMS) affecting approximately 1,500 individuals was suggested to be due to ingestion of certain lots of l-tryptophan from a single manufacturer. Akin to TOS, EMS is a scleroderma-like syndrome found more frequently in women but unlike TOS was not restricted to a geographical area. The acute phase of the syndrome was characterized by myalgia and eosinophilia, followed by chronic cutaneous lesions, progressive neuropathy, and myopathy. These causative exposures are rare examples in a field hampered by the difficulty of linking putative environmental risk factors with autoimmune disease in humans. Recently, the National Institute of Environmental Health Sciences (NIEHS) convened an expert panel in a workshop setting to review the role of the environment in the development of autoimmune disease. The meeting addressed specific areas of mechanisms, animal models, epidemiology, diagnostic criteria, and exposure assessment focusing, in particular, on the contribution of chemical, physical, and biological agent exposures; medications were not considered. A series of papers were published summarizing the workshop findings (5–8), and a consensus statement was recently published (9). Together these publications constitute the most recent summary of the state of knowledge on the role of environmental exposures in autoimmune disease. In this opinion piece, I will expand upon some of the findings of the NIEHS workshop and our own studies to examine how environmental exposure can contribute to our understanding of autoimmunity and autoimmune diseases.

Gold causes genetically determined autoimmune and immunostimulatory responses in mice

Natrium aurothiomaleate (GSTM) is a useful disease‐modifying anti‐rheumatic drug, but causes a variety of immune‐mediated adverse effects in many patients. A murine model was used to study further the interaction of GSTM with the immune system, including induction of systemic autoimmunity. Mice were given weekly intramuscular injections of GSTM and controls equimolar amounts of sodium thiomaleate. The effects of gold on lymphocyte subpopulations were determined by flow cytometry. Humoral autoimmunity was measured by indirect immunofluorescence and immunoblotting, and deposition of immunoglobulin and C3 used to assess immunopathology. Gold, in the form of GSTM, stimulated the murine immune system causing strain‐dependent lymphoproliferation and autoimmunity, including a major histocompatibility complex (MHC)‐restricted autoantibody response against the nucleolar protein fibrillarin. GSTM did not cause glomerular or vessel wall IgG deposits. However, it did elicit a strong B cell‐stimulating effect, including both T helper 1 (Th1)‐ and Th2‐dependent isotypes. All these effects on the immune system were dependent on the MHC genotype, emphasizing the clinical observations of a strong genetic linkage for the major adverse immune reactions seen with GSTM treatment.

Decay-accelerating factor 1 (Daf1) deficiency exacerbates xenobiotic-induced autoimmunity

Absence of decay‐accelerating factor 1 (Daf1) has been shown to enhance T‐cell responses and autoimmunity via increased expression of specific cytokines, most notably interferon (IFN)‐γ. To determine if Daf1 deficiency can exacerbate IFN‐γ‐dependent murine mercury‐induced autoimmunity (mHgIA), C57/BL6 Daf1+/+ and Daf1−/− mice were exposed to mercuric chloride (HgCl2) and examined for differences in cytokine expression, T‐cell activation and features of humoral autoimmunity. In the absence of Daf1, mHgIA was exacerbated, with increased serum immunoglobulin G (IgG), anti‐nuclear autoantibodies (ANAs) and anti‐chromatin autoantibodies. This aggravated response could not be explained by increased T‐cell activation but was associated with increased levels of IFN‐γ, interleukin (IL)‐2, IL‐4 and IL‐10 but not IL‐17 in Daf1‐deficient mice. Anti‐CD3/anti‐CD28 costimulation of Daf1−/− CD4+ T cells in vitro was also found to increase cytokine expression, but the profile was different from that of mHgIA, suggesting that the cytokine changes observed in Daf1 deficiency reflect a response to mercury. The role of Daf1 in influencing cytokine expression was further examined by stimulation of CD4+ T cells in the presence of anti‐CD3 and CD97, a molecular partner for Daf1. This resulted in increased IL‐10, decreased IL‐17 and IL‐21 and decreased IFN‐γ. These findings demonstrate that the absence of Daf1 exacerbates mHgIA, with changes in the profile of expressed cytokines. Interaction between Daf1 and its molecular partner CD97 was found to modify expression of mHgIA‐promoting cytokines, suggesting a possible approach for the suppression of overaggressive cytokine production in autoimmunity.

The Role of Decay Accelerating Factor in Environmentally Induced and Idiopathic Systemic Autoimmune Disease

Decay accelerating factor (DAF) plays a complex role in the immune system through complement-dependent and -independent regulation of innate and adaptive immunity. Over the past five years there has been accumulating evidence for a significant role of DAF in negatively regulating adaptive T-cell responses and autoimmunity in both humans and experimental models. This review discusses the relationship between DAF and the complement system and highlights major advances in our understanding of the biology of DAF in human disease, particularly systemic lupus erythematosus. The role of DAF in regulation of idiopathic and environmentally induced systemic autoimmunity is discussed including studies showing that reduction or absence of DAF is associated with autoimmunity. In contrast, DAF-mediated T cell activation leads to cytokine expression consistent with T regulatory cells. This is supported by studies showing that interaction between DAF and its molecular partner, CD97, modifies expression of autoimmunity promoting cytokines. These observations are used to develop a hypothetical model to explain how DAF expression may impact T cell differentiation via interaction with CD97 leading to T regulatory cells, increased production of IL-10, and immune tolerance.