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Regulation of Murine Mast Cell Homeostasis by TGF-β1 and CD4+CD25+C Regulatory T Cells

REGULATION OF MURINE MAST CELLS HOMEOSTASIS BY TGF-P1 AND C D ~ + C D ~ ~ + REGULATORY T CELLS By Mohit Kashyap A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University Virginia Commonwealth University, 2006 Director: John J. Ryan, Ph.D., Associate Professor, Department of Biology Understanding mast cell development is central to allergic disease pathophysiology. Our laboratory has previously shown that cytokines such as IL-4 and IL-10 inhibit mast cell development from bone marrow progenitors. These studies encouraged our interest in other regulatory cytokines, including transforming growth factor+ 1 (TGF-P 1). TGF-P 1 has many cellular sources, one of which is C D ~ + C D ~ ~ + regulatory T cells (Tregs). We wanted to determine the effects of Transforming Growth Factor (TGF)Pl on mast cell development. We find that TGFPl decreased FccRI, c-Kit, TllST2 and FcyR expression, and inhibited granule formation in developing mast cells. Accessory cells were not required for this inhibition. Smad3-deficiency did not alter the response of bone marrow cells to TGFP1. TGFPl inhibited expression of the FccRI a subunit protein, without decreasing P or y proteins. Mast cells derived in the presence of TGFPl were functionally impaired, as IgE-mediated cytokine secretion was greatly reduced. The changes in granule formation and surface antigen expression were long-standing, as they were not reversed by transfer to WIWV mice. The TGF-P 1 dependent transcriptional regulation of bone marrow cells from which mast cells develop was examined through DNA microarray analysis. Wild type (WT) bone marrow cells were stimulated with IL3+SCF+vehicle or IL-3+SCF+TGF-PI for 10 days and their transcriptomes* analyzed. The results identified which components of transcriptional regulation were regulated by TGF-P 1. Of particular interest was the upregulation of the P subunit of the FccRI, inspite of no receptor surface expression and the differential regulation of various mast cell proteases (MCPs). This initial survey provides a potential starting point for further analysis of the role of TGF-P 1 -dependent signaling in developing mast cells. Because they produce TGF-P1 and/or IL-10, regulatory T cell-dependent murine mast cell inhibition was examined. Co-culture of mast cells with regulatory T cells for 6 days downregulated mast cell number, high affinity IgE receptor and c-Kit surface expression. This led to a decrease in TNFa release making mast cells functionally impaired. By using Tregs from IL-10 KO mice, this effect was proven to be IL-10 dependent. Mast cells are mediators of inflammatory disease. TGFPl and IL-10 may contribute to mast cell homeostasis by inhibiting maturation from bone marrow precursors. The effects of TGFP 1 and regulatory T cell derived IL-10 result in greatly diminished expression of cell surface markers, reduced granulation, and lack of responsiveness to IgE-mediated activation. Thus TGFPl and/or C D ~ + C D ~ ~ + T cells can serve as potent and multifunctional regulators of mast cell maturation and/or function. * A set of genes that are expressed in a cell at any given time.