Eosinophil-Airway Epithelial Fas-FasL Interactions

本研究旨在探讨呼吸道上皮细胞Fas配体(FasL)的功能性表达及其诱导嗜酸性粒细胞死亡的作用。中心假说是，上皮性FasL功能有助于限制或化解炎性细胞浸润，如嗜酸性粒细胞，在哮喘中，这一功能受损，导致持续性肺嗜酸性粒细胞增多症。这些研究将从三个方面探讨调节呼吸道上皮细胞FasL功能表达的分子机制：(1)确定核因子-kappaB在人呼吸道上皮细胞Fas配体(FasL)结构性和诱导性表达中的作用，以及基质(MMP)和去整合素(ADAM)金属蛋白水解酶在这些细胞释放可溶性FasL(SFasL)中的作用。实验将确定(A)胞浆和膜结合的FasL的构成表达；(B)细胞因子在NF-kappaB调节的呼吸道上皮细胞FasL表达中的作用；(C)可溶性(S)FasL的释放以及基质金属蛋白酶-3或-7、ADAM-17或其他上皮表达的基质金属蛋白酶/亚当斯在这种释放中的作用。(2)探讨Fas介导的FasL对人和小鼠嗜酸性粒细胞及嗜酸性粒细胞系的杀伤作用，以及ICAM-1介导的嗜酸性粒细胞与上皮细胞的黏附在这种相互作用中的作用。实验将(A)评估上皮细胞介导的对人或小鼠嗜酸性粒细胞的杀伤以及细胞与细胞接触的必要性；(B)评估sFasL在上皮细胞或激发型抗Fas诱导嗜酸性粒细胞凋亡中的作用；以及sFasL的趋化活性，以及(C)确定(NF-kappaB调节的)ICAM-1表达在促进细胞相互作用中的作用；(3)利用小鼠肺嗜酸性粒细胞炎症模型，评估上皮FasL在体内消退炎症中的生理作用。实验将(A)确定分别来自FasL突变(GLD)和Fas缺陷(LPR)小鼠的上皮细胞和嗜酸性粒细胞对嗜酸性粒细胞与上皮细胞相互作用的体外影响；(B)确定来自MMP和NF-kappaB基因敲除小鼠的细胞在体外对这些相互作用及膜(M)FasL和sFasL的调节的影响；(C)在与正常小鼠相比的体内模型中，确定LPR、GLD和MMP以及NF-kappaB基因敲除小鼠和骨髓嵌合小鼠肺嗜酸性粒细胞炎症的消退。阐明这些相互作用的相互作用将有助于更好地了解Fas介导的嗜酸性粒细胞清除机制，并为哮喘的慢性嗜酸性粒细胞炎症特性量身定做更具体的治疗方法。

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.