We are studying a murine model for scleroderma, murine sclerodermatous graft versus host disease (Scl GVHD). In Scl GVHD, irradiated BALB/c (H-2d) mice transplanted with Bl0.D2 (H-2d ) bone marrow and spleen cells develop autoimmune disease, skin thickening and lung fibrosis; control animals transplanted with syngeneic cells do not develop disease. In Scl GVHD, increased cutaneous collagen production is driven by TGF-beta l, produced by activated CD11 b+ monocyte/macrophages and CD3+ T cells. We have previously shown that inhibiting TGF-beta with anti-TGF-beta antibodies administered intravenously abrogates skin thickening and lung fibrosis at day 21 after bone marrow transplantation (BMT). Mice with Scl GVHD develop cutaneous mononuclear cell infiltrates preceding upregulation of collagen synthesis, dense dermal thickening and lung fibrosis. We used RT/PCR analysis of total skin RNA and flow cytometry of single skin cell preparations and found that mRNAs for chemokines such as macrophage chemoattractant protein (MCP-1/JE), macrophage inhibitory protein (MIP-1alpha) and RANTES are upregulated in Scl GVHD before accumulation of inflammatory cells and skin thickening. Hypothesis: Chemokines that attract monocytes, macrophages (MCP-1, MIP-1 a) and T cells (RANTES) are critical in the pathogenesis of fibrosis, and may be targets for interventions in scleroderma.
Specific Aim I : 1st and 2nd years) To investigate the role of C-C chemokines MCP-1, MIP- 1alpha, and RANTES in early critical events leading to skin fibrosis in animals with Scl GVHD. What is the chemokine environment and time course of chemokine upregulation in skin of animals developing Scl GVHD? In animals developing Scl GVHD, what cells infiltrating skin secrete chemokines? Are they T cells or monocyte/macrophages, or both? Do fibroblasts and endothelial cells secrete chemokines in Scl GVHD? Is chemokine receptor upregulation on immune cells also present? Is a cytokine milieu (Th1or Th2) associated with the chemokine upregulation? We propose to characterize the cytokine and chemokine environments and effects of intervention in those pathways by immunostaining, flow cytometry of single skin cell suspensions, RT/PCR and RNase protection assays. To evaluate skin thickening, we use routine histology and image analysis.
Specific Aim II : (2nd and 3rd years) To use in vivo interventions to inhibit chemokines in Scl GVHD. Can antibodies to chemokines inhibit and reverse skin fibrosis? Neutralizing polyclonal hamster antibodies for MCP-1 (macrophages) and rat antimouse mAb for RANTES (T cells) will be administered in separate experiments to animals with Scl GVHD to determine the relative importance of T cell versus macrophage chemoattraction in Scl GVHD. The murine Scl GVHD model provides an ideal vehicle to test novel interventions for scleroderma. These pilot studies have high relevance to scleroderma and fibrosing diseases in general, and may lead to effective treatments for graft versus host disease as well.
