ENDOTHELIAL CELL AND MONOCYTE ACTIVATION IN A MURINE GVHD SCLERODERMA MODEL

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The early events in systemic sclerosis/scleroderma, a chronic fibrosing autoimmune disease of unknown etiology, are poorly understood and have been difficult to study in patients. The disease is characterized by insidious clinical progression, altered cell-mediated immunity, production of autoantibodies, and excessive deposition of collagen by fibroblasts in viscerae and skin. Cutaneous fibrosis appears to occur first around dermal vessels, suggesting triggering events localized to the perivasculature in human scleroderma. Hypothesis: activation of recruited monocytes in dermal perivascular compartments is a critical early event in scleroderma which can be modeled in murine sclerodermatous graft versus host disease (GVHD). When bone marrow and spleen cells from B10.D2 (H-2k) mice are transplanted into lethally irradiated Balb/c mice (H-2k), the animals develop mild GVHD, autoimmune disease, and skin thickening resembling that of human scleroderma. Control animals receiving syngeneic bone marrow and spleen cells (Balb/c transplanted to Balb/c) show no such changes. Specific Aim I: To determine the sequence of early critical events in monocyte activation in the cutaneous microvascular compartment of animals with sclerodermatous GVHD in order to better understand the pathophysiology of sclerodermatous disease. Immunostaining for endothelial cell (ICAM- 1, VCAM-1, E-selectin, and von Willebrand factor) and monocyte (Mac- 1,Ia, VLA-4, CD16, and iNOS) activation markers at time points before detectable collagen mRNA upregulation and skin will identify these early events. Image analysis is used to quantitate density and phenotypes of infiltrating cells. Specific Aim II: Using flow cytometry, correlate immunostaining results from Aim I to identify and quantify phenotypes of cell populations infiltrating skin of animals with sclerodermatous GVHD at early time points. Intracytoplasmic staining for TGFbeta1 and surface Mac-1 will address the question: does infiltration by TGFbeta-producing monocytes precede dermal fibrosis? Correlating these early events in sclerodermatous disease will point to interventions testing novel monocyte-directed immunotherapies in vivo, then translated into clinical trials for human scleroderma.