Eosinophil-Airway Epithelial Fas-FasL Interactions

本研究拟探讨气道上皮细胞Fas配体（FasL）的功能表达及其诱导嗜酸性粒细胞细胞死亡的作用。中心假设是上皮FasL的功能有助于限制或消除炎性细胞浸润，如嗜酸性粒细胞，并且在哮喘中，该功能受损导致持续的肺嗜酸性粒细胞增多。这些研究将从三个方面探讨调节FasL在气道上皮细胞上功能表达的分子机制：(1)确定NF-kappaB在人气道上皮细胞中Fas配体（FasL）的组成性和诱导性表达中的作用，以及基质（MMP）和崩解素（ADAM）金属蛋白酶在这些细胞中可溶性FasL （sFasL）释放中的作用。实验将确定(a)细胞质和膜结合FasL的组成表达；(b)细胞因子在nf - kappab调控的气道上皮细胞FasL表达中的作用；(c)可溶性FasL的释放以及MMP-3或-7、ADAM-17或其他上皮表达的MMP/ADAMS在该释放中的作用。(2)评估fas介导的气道上皮FasL对人和鼠嗜酸性粒细胞和亲酸性细胞系的杀伤作用，以及icam -1介导的嗜酸性粒细胞粘附上皮细胞在这些相互作用中的作用。实验将(a)评估上皮细胞介导的人或鼠嗜酸性粒细胞杀伤和细胞间接触的必要性；(b)评估sFasL对上皮细胞或激动性抗fas诱导嗜酸性粒细胞凋亡的影响；和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)通过小鼠肺嗜酸性炎症模型，评估上皮FasL在体内炎症消退中的生理作用。实验将(a)确定分别使用fasl突变（gld）和fasl缺陷（lpr）小鼠的上皮细胞和嗜酸性粒细胞对嗜酸性细胞-上皮细胞相互作用的体外影响；(b)确定MMP和NF-kappaB敲除小鼠细胞对这些相互作用和膜(m)FasL和sFasL调控的体外影响；(c)在lpr、gold、MMP和NF-kappaB敲除小鼠和骨髓嵌合小鼠的体内模型中，与正常小鼠相比，确定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理解fas介导的嗜酸性粒细胞清除机制，并针对哮喘的慢性嗜酸性粒细胞炎症性质提供更具体的治疗。

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.