Immunopathogenetic features characterize the autoimmune inflammatory myopathies - polymyositis, dermatomyositis, and related diseases: lymphocytic destruction of muscle cells, and humoral autoimmunity distinguished by a striking set of disease-specific autoantibodies. Although the muscle cell destruction is mediated by lymphocytes, the autoantibodies, particularly those directed against the family of functionally related but structurally diverse aminoacyl-tRNA synthetases, seem to offer a useful window on the disease and have been the focus of much of this group's research for a number of years. The major efforts in this project are currently directed towards developing a useful animal model of inflammatory myopathy; towards understanding the mode of death of muscle cells in human myositis; and towards understanding the development of myositis-specific autoantibodies.To achieve the first of these goals, we have made two approaches. First, we have forced the muscle-specific expression of MHC Class I, a molecule that is normally absent from muscle cells but that is up-regulated early in myositis. We achieved this by using transgenic mice that express the tetracycline transactivator under the control of a muscle-specific promoter, creatine kinase, and mating them to mice transgenic for the MHC Class I of the target strain whose transcription has been placed under the control of a tetracycline responsive protein, the product of the transactivator. The doubly transgenic progeny - but not mice bearing only one of the transgenes - will express MHC I in muscle when tetracycline is withdrawn. These doubly transgenic mice develop a myopathy with many features of human myositis. Most strikingly, the disease becomes self-perpetuating even if the transgenes are turned off, and autoantibodies that are myositis-specific in the human disease are made in some of the sick animals. These experiments suggest that the stimulus to making autoantibodies is the product of the inflammatory conditions within a particular tissue rather than any specific outside stimulus. We have begun, and will continue in the coming year, experiments to characterize the disease further by mating to animals lacking some parts of the inflammatory pathway. Furthermore, we plan to test the generality of the mechanism by analogous experiments involving other tissues. Second, we have worked with the Rosen/Casciola-Rosen lab at Johns Hopkins to test the hypothesis - which is consonant with the findings in the MHC Class I transgenic mice - that autoimmunity arises when endogenous proteins liberated by cell death pathways are proteolytically cleaved to yield novel fragments that are antigenic. We have immunized mice with muscle cells exposed to granzymes and sought evidence of autoimmunity. Although a clinical myopathy does not develop, novel autoantibodies do. They are currently being characterized.In the past year, we have completed a series of studies on the role of apoptosis in myositis. The absence of apoptosis in myositis muscle biopsies has been shown to correlate with the up-regulation of the anti-apoptotic molecule, FLIP. FLIP up-regulation has been demonstrated in muscle biopsy specimens at the level of individual muscle cells using laser capture microscopy and in cultured human muscle cells exposed to pro-inflammatory stimuli. These experiments have been published in the past year.In experiments stimulated by recently published observations on the chemotactic activity of tyrosyl-tRNA synthetase, we have joined with the Oppenheim lab in NCI at Frederick and with the Rosen lab at Hopkins to explore possible similar activity in other aminoacyl-tRNA synthetases, especially the principal myositis autoantigenic target, histidyl-tRNA synthetase and fragments derived from it. These experiments have shown promising results Their relevance to the human or to the mouse model disease will be explored.We have collaborated with the group of Eric Hoffman at Children's Hospital to explore a potential role for AAV (adeno-associated virus, a non-pathogenic parvovirus) in protecting muscle against developing an inflammatory myopathy. In contrast to muscle biopsies from normal individuals, in which the genome of AAV is often found, biopsies from patients do not have detectable AAV. These results have been accepted for publication, and follow-up studies are under discussion.
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