Eosinophil-Airway Epithelial Fas-FasL Interactions

嗜酸性粒细胞-气道上皮 Fas-FasL 相互作用

• 批准号: 6383514
• 负责人: KIMM J HAMANN
• 金额: $ 32.87万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-25 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6383514
• 关键词: CD95 molecule; RNase protection assay; apoptosis; asthma; atopy; cell adhesion; cell line; chemotaxis; cord blood; enzyme linked immunosorbent assay; eosinophil; flow cytometry; gene targeting; genetically modified animals; human tissue; inflammation; interleukin 9; laboratory mouse; ligands; metalloendopeptidases; nuclear factor kappa beta; protein protein interaction; receptor expression; respiratory epithelium

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.

本研究拟探讨气道上皮细胞表达Fas配体（FasL）的功能及其对人嗜酸性粒细胞死亡的诱导作用。中心假设是上皮细胞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，在哮喘中，这种功能受损，导致持续性肺嗜酸性粒细胞增多。 这些研究将从三个方面阐明调节气道上皮细胞FasL功能表达的分子机制：（1）确定NF-κ B在人气道上皮细胞Fas配体（FasL）组成性和诱导性表达中的作用，以及基质（MMP）和去整合素（ADAM）金属蛋白酶在这些细胞释放可溶性FasL（sFasL）中的作用。 实验将确定（a）胞质和膜结合的FasL的组成性表达;（B）细胞因子对气道上皮细胞上NF-κ B调节的FasL表达的作用;和（c）可溶性FasL的释放和MMP-3或-7、ADAM-17或其他上皮表达的MMP/亚当斯在该释放中的作用。(2)评估气道上皮FasL对人和鼠嗜酸性粒细胞和嗜酸性粒细胞系的Fas介导（凋亡）杀伤作用，以及ICAM-1介导的嗜酸性粒细胞与上皮细胞粘附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的杀伤以及细胞-细胞接触的必要性;（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作用;以及sFasL的趋化活性，以及（c）确定sFasL在细胞-细胞接触中的作用。（NF-κ B调节的）ICAM-1表达在促进细胞相互作用中的作用;（3）使用肺嗜酸性粒细胞炎症的鼠模型评估上皮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-上皮细胞相互作用的体外作用;（B）确定使用来自MMP和NF-κ B敲除小鼠的细胞对这些相互作用和膜（m）FasL和sFasL调节的体外作用;和（c）确定与正常小鼠相比，lpr、gld和MMP和NF-κ B敲除和骨髓嵌合小鼠体内模型中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导致更好地了解贡献Fas介导的嗜酸性粒细胞清除机制和更具体的治疗慢性嗜酸性粒细胞性哮喘的性质。