ACTIVATED MACROPHAGE/MONOCYTE TGFB1-INDUCED UPREGULATION OF COLLAGEN SYNTHESIS

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Systemic sclerosis/scleroderma is a chronic autoimmune disease of unknown etiology, characterized by the excessive deposition of collagen in viscerae and skin, altered cell-mediated immunity, and the production of autoantibodies. Existing murine models for scleroderma are limited in their usefulness for study of the complex immunologic abnormalities, and human disease is difficult to study because the early changes are subtle and diagnosis is often delayed. Evidence from in vitro and in vivo work on pulmonary lesions in human scleroderma suggest that TGFbeta1 produced by infiltrating monocytes is a potent stimulus for collagen gene upregulation by fibroblasts leading to fibrosis. A murine sclerodermatous graft-versus- host disease (GVHD) model (C57BL/6J to LP/J) in which animals develop GVHD, skin thickening and autoantibodies after bone marrow transplantation across minor histocompatibility loci (H-2b) provides the ideal opportunity to study these events in an intact organism. Control mice receiving the reciprocal bone marrow transplantation LP/6 to C57BL/6J develop GVHD and dermal mononuclear cell infiltrates, but do not develop the skin thickening. Aim I of this proposal will characterize fully the sclerodermatous GVHD mice and confirm their usefulness as a model for human scleroderma. Disease progression will be correlated with histologic, biochemical and immunologic parameters by measuring dermal thickness using ultrasonography and physical measurements of intact skin and histologic sections, assaying autoantibody production by antinuclear antibody tests, immunophenotyping of the dermal mononuclear infIltrating cells, and quantifying dermal collagen gene expression by northern blot analysis of total RNA prepared from dermis at time points after transplantation in sclerodermatous and control animals. Aim II tests the central role of TGFbeta1-producing monocytes in causing collagen gene upregulation leading to skin fibrosis. Immunophenotyping and in situ hybridization using TGFbeta1 and pro-alpha(I)collagen probes to demonstrate co-localization of TGFbeta1-producing monocytes and collagen-producing fibroblasts in early skin lesions is the goal of this proposed work. Finally, the model provides a system in which variables can be manipulated to test the hypothesis that monocyte TGFbeta1 production is critical to initiation and progression of fibrosis, a logical extension of the work proposed here. Developing the murine model, confirming its validity for scleroderma, and identifying major early immunologic events in scleroderma will provide a means to test innovative immunotherapies in vivo.