K. Michael Pollard

Criteria for Environmentally Associated Autoimmune Diseases

Increasing evidence supports a role for the environment in the development of autoimmune diseases, as reviewed in the accompanying three papers from the National Institute of Environmental Health Sciences Expert Panel Workshop. An important unresolved issue, however, is the development of criteria for identifying autoimmune disease phenotypes for which the environment plays a causative role, herein referred to as environmentally associated autoimmune diseases. There are several different areas in which such criteria need to be developed, including: 1) identifying the necessary and sufficient data to define environmental risk factors for autoimmune diseases meeting current classification criteria; 2) establishing the existence of and criteria for new environmentally associated autoimmune disorders that do not meet current disease classification criteria; and 3) identifying in clinical practice specific environmental agents that induce autoimmune disease in individual patients. Here we discuss approaches that could be useful for developing criteria in these three areas, as well as factors that should be considered in evaluating the evidence for criteria that can distinguish individuals with such disorders from individuals without such disorders with high sensitivity and specificity. Current studies suggest that multiple lines of complementary evidence will be important and that in many cases there will be clinical, serologic, genetic, epigenetic, and/or other laboratory features that could be incorporated as criteria for environmentally associated autoimmune diseases to improve diagnosis and treatment and possibly allow for preventative strategies in the future.

Animal models used to examine the role of the environment in the development of autoimmune disease: Findings from an NIEHS Expert Panel Workshop

Autoimmunity is thought to result from a combination of genetics, environmental triggers, and stochastic events. Environmental factors, such as chemicals, drugs or infectious agents, have been implicated in the expression of autoimmune disease, yet human studies are extremely limited in their ability to test isolated exposures to demonstrate causation or to assess pathogenic mechanisms. In this review we examine the research literature on the ability of chemical, physical and biological agents to induce and/or exacerbate autoimmunity in a variety of animal models. There is no single animal model capable of mimicking the features of human autoimmune disease, particularly as related to environmental exposures. An objective, therefore, was to assess the types of information that can be gleaned from the use of animal models, and how well that information can be used to translate back to human health. Our review notes the importance of genetic background to the types and severity of the autoimmune response following exposure to environmental factors, and emphasizes literature where animal model studies have led to increased confidence about environmental factors that affect expression of autoimmunity. A high level of confidence was reached if there were multiple studies from different laboratories confirming the same findings. Examples include mercury, pristane, and infection with Streptococcus or Coxsackie B virus. A second level of consensus identified those exposures likely to influence autoimmunity but requiring further confirmation. To fit into this category, there needed to be significant supporting data, perhaps by multiple studies from a single laboratory, or repetition of some but not all findings in multiple laboratories. Examples include silica, gold, TCE, TCDD, UV radiation, and Theilers murine encephalomyelitis virus. With the caveat that researchers must keep in mind the limitations and appropriate applications of the various approaches, animal models are shown to be extremely valuable tools for studying the induction or exacerbation of autoimmunity by environmental conditions and exposures.

Gender differences in autoimmunity associated with exposure to environmental factors

Autoimmunity is thought to result from a combination of genetics, environmental triggers, and stochastic events. Gender is also a significant risk factor with many diseases exhibiting a female bias. Although the role of environmental triggers, especially medications, in eliciting autoimmunity is well established less is known about the interplay between gender, the environment and autoimmunity. This review examines the contribution of gender in autoimmunity induced by selected chemical, physical and biological agents in humans and animal models. Epidemiological studies reveal that environmental factors can be associated with a gender bias in human autoimmunity. However many studies show that the increased risk of autoimmunity is often influenced by occupational exposure or other gender biased activities. Animal studies, although often prejudiced by the exclusive use of female animals, reveal that gender bias can be strain specific suggesting an interaction between sex chromosome complement and background genes. This observation has important implications because it argues that within a gender biased disease there may be individuals in which gender does not contribute to autoimmunity. Exposure to environmental factors, which encompasses everything around us, adds an additional layer of complexity. Understanding how the environment influences the relationship between sex chromosome complement and innate and adaptive immune responses will be essential in determining the role of gender in environmentally-induced autoimmunity.

Toxicology of autoimmune diseases.

Susceptibility to most autoimmune diseases is dependent on polygenic inheritance, environmental factors, and poorly defined stochastic events. One of the significant challenges facing autoimmune disease research is in identifying the specific events that trigger loss of tolerance and autoimmunity. Although many intrinsic factors, including age, sex, and genetics, contribute to autoimmunity, extrinsic factors such as drugs, chemicals, microbes, or other environmental factors can also act as important initiators. This review explores how certain extrinsic factors, namely, drugs and chemicals, can promote the development of autoimmunity, focusing on a few better characterized agents that, in most instances, have been shown to produce autoimmune manifestations in human populations. Mechanisms of autoimmune disease induction are discussed in terms of research obtained using specific animal models. Although a number of different pathways have been delineated for drug/chemical-induced autoimmunity, some similarities do exist, and a working model is proposed.

Proteolytic Cleavage of a Self-Antigen Following Xenobiotic-Induced Cell Death Produces a Fragment with Novel Immunogenic Properties

The heavy metal mercury elicits a genetically restricted autoantibody response in mice that targets the nucleolar autoantigen fibrillarin. HgCl2-induced cell death of macrophages resulted in the proteolytic cleavage of fibrillarin. A prominent feature of mercury-induced cell death was the generation of a 19-kDa fragment of fibrillarin that was not found following apoptotic or nonapoptotic cell death induced by stimuli other than mercury. Proteolysis of fibrillarin lacking cysteines, and therefore unable to bind mercury, also produced the 19-kDa fragment, suggesting that a mercury-fibrillarin interaction was not necessary for the unique cleavage pattern of this self-Ag. In contrast to immunization with full-length fibrillarin, the 19-kDa fragment produced anti-fibrillarin Abs with some of the properties of the HgCl2-induced anti-fibrillarin response. We propose that cell death following exposure to an autoimmunity-inducing xenobiotic can lead to the generation of novel protein fragments that may serve as sources of antigenic determinants for self-reactive T lymphocytes.

Costimulation Requirements of Induced Murine Systemic Autoimmune Disease

Costimulation between T cells and APC is required for productive immune responses. A number of receptor/ligand pairs have been shown to mediate costimulation, including CD28/B7 molecules (CD80 and CD86), CD40/CD40 ligand (CD40L, CD154), and LFA-1 (CD18)/ICAM-1 (CD54). T-B cell costimulation also plays a significant role in autoimmune diseases such as systemic lupus erythematosus. Murine HgCl2-induced autoimmunity (mHgIA) is a T cell-dependent systemic autoimmune disease that shares a number of common pathogenic mechanisms with idiopathic lupus. In this report, the significance of costimulation in mHgIA is examined by attempting to induce disease in mice deficient in either CD40L, CD28, or ICAM-1. Unlike absence of ICAM-1, homozygous deficiencies in either CD40L or CD28 significantly reduced the development of mHgIA. CD40L displayed a gene dosage effect as heterozygous mice also showed reduction of autoantibody responses and immunopathology. Markers of T cell activation such as CD44 and CTLA-4 were associated with disease expression in wild-type and ICAM-1-deficient mice but not in CD40L- or CD28-deficient mice. Absence of CTLA-4 expression in CD40L−/− mice suggests that signaling via both CD28 and CD40L is important for T cell activation and subsequent autoimmunity in mHgIA. Attempts to circumvent the absence of CD40L by increasing CD28 signaling via agonistic Ab failed to elicit CTLA-4 expression. These findings indicate that breaking of self-tolerance in mHgIA requires signaling via both the CD28/B7 and CD40/CD40L pathways.

The in vitro proliferation of murine lymphocytes to mercuric chloride is restricted to mature T cells and is interleukin 1 dependent

The aims of this study were to compare the in vitro responses of murine lymphocytes to HgCl2 to determine the requirement for adherent cells, and the contribution that costimulation plays in T cell proliferation. The in vitro proliferative response of murine splenocytes to HgCl2 was found to be both cell concentration- and HgCl2 concentration-dependent with the greatest response occurring with 5 x 10(6) cells/ml in the presence of 10(-5) M HgCl2. Both CD4+ and CD8+ T cells proliferated in response to HgCl2, but B cells and immature T cells (thymocytes) did not. Proliferation required the presence of splenic adherent cells and was inhibited by addition of anti-IL-1 alpha antibodies. Antibodies to the other co-stimulatory molecules CD40 ligand, CD80 (B7-1), and CD86 (B7-2), although inhibitory, were less effective. Xenobiotics such as the heavy metal mercury can elicit a spectrum of immunological responses ranging from immunosuppression to autoimmunity. The most common response, in vivo and in vitro, is lymphoproliferation, which may be a prelude to immune activation. Although a number of the requirements for mercury-induced T cell proliferation in vitro have been described, the role that adherent cells play remains to be explained. The studies described here show that interaction between co-stimulatory molecules of adherent cells and mature T cells contributes to HgCl2-induced T cell proliferation. Among these co-stimulatory molecules, IL-1 appears to play an important role. The requirement for mature T cells, adherent cells, and co-stimulatory molecules argues that HgCl2-induced T cell proliferation possesses the properties of an antigen-induced response.

Molecular cloning and sequence analysis of U3 snoRNA-associated mouse fibrillarin.

We have isolated and determined the sequence of a 1.1-kb cDNA from a murine WEHI-3 macrophage library which encodes the highly conserved, nucleolar protein, fibrillarin. The murine fibrillarin protein sequence displays 94.2% identity with human fibrillarin, 82.9% identity with amphibian fibrillarin and 74.0% identity with the yeast fibrillarin homolog, NOP1. Immunoprecipitation showed that anti-fibrillarin autoantibodies from human scleroderma sera and the monoclonal autoantibody 72B9 recognize the approx. 34-36 kDa in vitro transcribed and translated protein. Mouse fibrillarin contains a N-terminal glycine- and arginine-rich (GAR) domain which although conserved among the fibrillarins is not as strongly conserved as several regions in the carboxy tail of the protein. Specific amino acid residues in yeast NOP1 thought to be associated with the synthesis and maturation of ribosomes show strong conservation between the mouse, human, amphibian and yeast protein sequences.

Using Single-Gene Deletions to Identify Checkpoints in the Progression of Systemic Autoimmunity

Abstract: Systemic lupus erythematosus is a multigenic disorder of unknown etiology. To investigate the roles that specific genes play in lupus, we have examined the disease profiles in mice with single‐gene deletions. In total, some 17 genes have been studied. Absence of certain genes, such as CD40L, CD28, or Igh6, abrogated induction of autoimmunity. Other genes, such as Igh5, IL‐4, or ICAM‐1, had little effect on the development of disease. Intermediate effects were observed in IL‐6‐deficient mice, while absence of β2‐microglobulin resulted in loss of hypergammaglobulinemia and IgG1 autoantibodies, but produced little change in anti‐chromatin antibodies or glomerular deposits. The most interesting observations were obtained with genes related to the expression or function of interferon‐γ (IFN‐γ). Reductions in IFN‐γ levels in murine lupus are associated with reductions in both autoantibody levels and immune‐complex‐ mediated pathology. Genes involved in up‐regulation of IFN‐γ expression, such as IL‐12, STAT‐4, or ICE, did not significantly influence autoimmunity, whereas absence of IFN‐γ or IFN‐γ receptor led to greatly reduced autoantibody response and immunopathology. Absence of IRF‐1, a gene ex‐pressed in response to IFN‐γ, resulted in selective retention of anti‐chromatin antibodies but little glomerular pathology. These studies suggest that the presence of a baseline level of IFN‐γ, rather than increased expression, is important for autoimmunity. Furthermore, as the IRF‐1 knockout demonstrates, specific defects in signaling pathways and gene expression subsequent to IFN‐γ/IFN‐γ receptor interaction may influence only certain disease parameters. It has not escaped our attention that IFN‐γ influences the expression and function of other immunologically relevant genes, such as IL‐4, IL‐6, and β2‐microglobulin. Thus, these genes may be part of the downstream events following IFN‐γ/IFN‐γ receptor interaction that promote the development of autoimmunity.

Role of Nucleic Acid–Sensing TLRs in Diverse Autoantibody Specificities and Anti-Nuclear Antibody–Producing B Cells

Nucleic acid (NA)–sensing TLRs (NA-TLRs) promote the induction of anti-nuclear Abs in systemic lupus erythematosus. However, the extent to which other nonnuclear pathogenic autoantibody specificities that occur in lupus and independently in other autoimmune diseases depend on NA-TLRs, and which immune cells require NA-TLRs in systemic autoimmunity, remains to be determined. Using Unc93b13d lupus-prone mice that lack NA-TLR signaling, we found that all pathogenic nonnuclear autoantibody specificities examined, even anti-RBC, required NA-TLRs. Furthermore, we document that NA-TLRs in B cells were required for the development of antichromatin and rheumatoid factor. These findings support a unifying NA-TLR–mediated mechanism of autoantibody production that has both pathophysiological and therapeutic implications for systemic lupus erythematosus and several other humoral-mediated autoimmune diseases. In particular, our findings suggest that targeting of NA-TLR signaling in B cells alone would be sufficient to specifically block production of a broad diversity of autoantibodies.