Eosinophil-Airway Epithelial Fas-FasL Interactions

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Studies are proposed to examine the functional expression of Fas ligand (FasL) by airway epithelial cells and the induction of cell death in human eosinophils by this FasL. The central hypothesis is that epithelial FasL functions to help limit or resolve inflammatory cellular infiltrates such as eosinophils, and that in asthma this function is impaired contributing to persistent pulmonary eosinophilia. These studies will address molecular mechanisms which regulate the functional expression of FasL on airway epithelial cells in three specific aims: (1) Determine the role of NF-kappaB in the constitutive and inducible expression of Fas ligand (FasL) in human airway epithelial cells and the role of matrix (MMP) and disintegrin (ADAM) metalloproteases in the release of soluble FasL (sFasL) from these cells. Experiments will determine (a) constitutive expression of cytosolic and membrane-bound FasL; (b) the role of cytokines on the NF-kappaB-regulated expression of FasL on airway epithelial cells; and (c) the release of soluble (s)FasL and the roles of MMP-3 or -7, ADAM-17 or other epithelial-expressed MMP/ADAMS in this release. (2) Assess the Fas-mediated (apoptotic) killing of human and murine eosinophils and eosinophilic cell lines by airway epithelial FasL and the role of ICAM-1-mediated adherence of eosinophils to epithelial cells in these interactions. Experiments will (a) assess the epithelial cell-mediated killing of human or murine eosinophils and the necessity of cell-cell contact; (b) assess the effects of sFasL on induction of apoptosis of eosinophils by epithelial cells or agonistic anti-Fas; and the chemotactic activity of sFasL, and (c) determine the role of (NF-kappaB-regulated) ICAM-1 expression in promoting cell interactions; (3) Assess the physiological role of epithelial FasL in resolution of inflammation in vivo using a murine model of pulmonary eosinophilic inflammation. Experiments will (a) determine the in vitro effects of using epithelial cells and eosinophils from FasL-mutant (gld) and Fas-deficient (lpr) mice, respectively on eosinophil-epithelial cell interactions; (b) determine the in vitro effects of using cells from MMP and NF-kappaB knockout mice on these interactions and regulation of membrane(m)FasL and sFasL; and (c) determine the resolution of pulmonary eosinophilic inflammation in an in vivo model in lpr, gld, and MMP and NF-kappaB knockout and bone marrow chimeric mice compared to normal mice. Elucidation of the interactions of these interactions will lead to a better understanding of contributory Fas-mediated mechanisms of eosinophil clearance and to more specific therapies tailored to the chronic eosinophilic inflammatory nature of asthma.