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A role of Hsp 60 in autoimmune diabetes : Analysis in a transgenic model ( transgenic mice y nonobese diabetic mouse )

A pathogenic role for self-reactive cells against the stress protein Hsp60 has been proposed as one of the events leading to autoimmune destruction of pancreatic b cells in the diabetes of nonobese diabetic (NOD) mice. To examine this hypothesis, we generated transgenic NOD mice carrying a murine Hsp60 transgene driven by the H-2Ea class II promoter. This would be expected to direct expression of the transgene to antigen-presenting cells including those in the thymus and so induce immunological tolerance by deletion. Detailed analysis of Hsp60 expression revealed that the endogenous gene is itself expressed strongly in thymic medullary epithelium (and weakly in cortex) yet fails to induce tolerance. Transgenic mice with retargeted Hsp60 showed overexpression of the gene in thymic cortical epithelium and in bone marrow-derived cells. Analysis of spontaneous T-cell responses to a panel of self and heterologous Hsp60 antigens showed that tolerance to the protein had not been induced, although responses to an immunodominant 437–460 epitope implicated in disease were suppressed, probably indicating an epitope shift. This correlated with changes in disease susceptibility: insulitis in transgenic mice was substantially reduced so that pathology rarely progressed beyond periislet infiltration. This was ref lected in a substantial reduction in hyperglycemia and disease. These data indicate that T cells specific for some epitopes of murine Hsp60 are likely to be involved in the islet-cell destruction that occurs in NOD mice. For the majority of spontaneous autoimmune diseases, the primary target autoantigens are not known. Attention in various diseases has focused on the stress protein Hsp60y65 as a possible candidate for T-cell recognition. Hsp60 molecules are highly immunogenic components in the response to infection yet they are highly conserved in evolution: mycobacterial Hsp65 is 48% homologous with human Hsp60 (1). This conservation is presumed to reflect a vital chaperone role in the correct folding, assembly, and compartmentalization of intracellular proteins (2). Responses to Hsp60 have been investigated in several autoimmune diseases. For example, adjuvant arthritis of Lewis rats is transferable by T-cell clones specific for the 180–188 epitope of mycobacterial Hsp65, which is apparently cross-reactive with self tissues in the rat (3). The search for the primary target autoantigen in insulindependent diabetes mellitus has focused on T-cell responses to islet components in patients and on anti-self T cells that spontaneously develop early in pathogenesis andyor have the ability to transfer disease in the nonobese diabetic (NOD) mouse disease model. Glutamic acid decarboxylase (GAD) meets many of the criteria for a candidate autoantigen since it is expressed in pancreatic b cells (as well as other sites), insulin-dependent diabetes mellitus patients respond to disease-specific GAD epitopes, NOD mice spontaneously develop responses to GAD epitopes early in the pathogenic process, and induction of tolerance to GAD prevents disease (4–6). Another candidate is a b-cell granule antigen, the target of a T-cell clone capable of transferring diabetes to prediabetic NOD mice. Expressing the T-cell receptor of this clone as a transgene greatly accelerated diabetes (7, 8). Elias and colleagues (9, 10) proposed Hsp60 as an early antigen in the triggering of insulin-dependent diabetes mellitus: antibody and T-cell responses to Hsp60 precede disease, Hsp60-reactive T cells can transfer disease to prediabetic NOD mice, and vaccination with Hsp65 in PBS blocks disease induction. Murine Hsp60 was cloned and the expressed cDNA clone was used in conjunction with a panel of synthetic peptides to show that the dominant epitope is within amino acid residues 437–460 (a peptide termed p277) (10). Vaccination with p277 blocks the development of disease and can halt ongoing disease (11, 12). Moreover, immunization to peptide p277 conjugated covalently to an immunogenic protein carrier could induce hyperglycemia and insulitis in some standard strains of mice not prone to spontaneous diabetes (13). Thus, the p277 peptide of Hsp60 is functionally involved in murine diabetes. The target antigen in any tissue-specific autoimmune disease should presumably have distribution limited largely to that tissue, whereas Hsp60 is thought to be ubiquitously inducible. However, heat shock proteins show different levels of constitutive expression and can be locally induced in vivo, for example, after tissue injury or fever (14, 15). Furthermore, there may be tissue-specific differences in the Hsp60 epitopes generated for presentation to T cells. Experiments show that expression of a transgene in professional thymic antigen-presenting cells leads to clonal deletion of T cells specific for that transgene product, the archetypal promoter for this analysis being the mouse class II Ea promoter (16). For example, thymic expression of self HyK ATPase, a candidate autoantigen in autoimmune gastritis, induces immunological tolerance and abrogates the disease (17). We have investigated the role of Hsp60 as a candidate autoantigen by overexpressing the gene in the thymus to manipulate self recognition by T cells and so alter pathogenesis. Transgenic expression of murine Hsp60 has allowed us to approach its role as a target in autoimmunity, analyzing various diseases to assess differences in pathology. MATERIALS AND METHODS Hsp60 Transgenic Mice. A construct consisting of the Xba I–Nru I linear fragment from the pDOI-5 vector (supplied by D. Mathis, Institut National de la Santé et de la Recherche The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviations: NOD, nonobese diabetic; GAD, glutamic acid decarboxylase; r, recombinant.