REGULATION OF FAS MEDIATED APOPTOSIS IN EOSINOPHILS

建议研究Fas的机制和后果， 介导的人嗜酸性粒细胞凋亡。核心的，基础的假设 Fas诱导的嗜酸性粒细胞的杀伤代表了一种 炎症消退的天然机制，通常是受调节的 通过特定的细胞外和细胞内机制。为了验证这一 假设，我们提出了三个具体的研究领域：（1）Fas诱导的 嗜酸性粒细胞凋亡。a）Fas和Fas的表达和调节- TNF-α和IFN-γ诱导的人嗜酸性粒细胞凋亡将是 使用RT-PCR和流式细胞术分析以及 B）Fas表达的调节 将使用特异性抗CD 13阻断剂检测氨肽酶N/CD 13 抗体和氨肽酶及其他抑制剂; c）细胞内 Bcl-2相关基因和ICE家族mRNA及蛋白表达 使用RT-PCR，北方， Western和流式细胞术分析，以澄清特定的细胞内 Fas诱导细胞凋亡的调控机制。(2)Fas-Fas配体 嗜酸性粒细胞和气道上皮细胞之间的相互作用。（一） Fas配体的表达类似于研究Fas表达的那些。 嗜酸性粒细胞，包括特异性相互作用的RT-PCR和Norther分析 将在嗜酸性粒细胞和上皮细胞的“共培养物”中进行检查 c）将测试金属蛋白酶抑制剂对细胞单层的影响。 FasL表达。(3)Fas-Fas配体相互作用与嗜酸性粒细胞 生物活性a）Fas-敏感和Fas-不敏感细胞中NF-κ B的活化。 将使用电迁移率变动测定来评估抗性细胞， 来自这些细胞的核提取物的超移位测定; B）表达 NF-κ B诱导的细胞因子基因及其蛋白产物的Fas- 将通过北方、西方和ELISA研究诱导的嗜酸性粒细胞 分析，以确定自分泌的可能性， 和/或与肺组织和细胞的旁分泌相互作用; 细胞凋亡与细胞凋亡引起的细胞活性颗粒蛋白MBP和EPO的释放， 将通过蛋白质- 具体分析。这些研究应阐明关键机制 调节Ras介导的细胞凋亡，并确定潜在的体内效应 药理学或生理学诱导这种细胞凋亡， 嗜酸性粒细胞

Studies are proposed to examine the mechanisms and consequences of Fas- mediated apoptosis of human eosinophils. The central, underling hypothesis for these studies is that Fas-induced killing of eosinophils represents a natural mechanism for inflammatory resolution which normally is regulated by specific extracellular and intracellular mechanisms. To test this hypothesis, we propose three specific areas of study: (1) Fas-induced apoptosis in eosinophils. a) The expression and regulation of Fas and Fas- induced apoptosis of human eosinophils by TNF-alpha and IFN-gamma will be studied using RT-PCR and flow cytometric assays and specific analyses of eosinophil apoptosis; b) The regulation of Fas expression by aminopeptidase N/CD13 will be examined using specific anti-CD13 blocking antibodies and aminopeptidase and other inhibitors; c) Intracellular expression of Bcl-2 related and ICE family mRNA and proteins in susceptible and resistant eosinophil populations, using RT-PCR, Northern, Western and flow cytometric assays to clarify specific intracellular mechanisms of regulation of Fas-induced apoptosis. (2) Fas-Fas ligand interactions between eosinophils and airway epithelial cells. a) The expression of Fas ligand similar to those for studying Fas expression of eosinophils including RT-PCR and Norther analyses of specific interaction will be examined in "co-cultures" of eosinophils and epithelial monolayers; c) Metalloprotease inhibitors will be tested for effects on FasL expression. (3) Fas-Fas ligand interactions and eosinophil bioactivity. a) The activation of NF-kappaB in Fas-susceptible and Fas- resistant cells will be assessed using electromobility shift assays and supershift assays of nuclear extracts from these cells; b) The expression of NF-kappaB inducible cytokine genes and their protein products by Fas- induced eosinophils will be studied by Northern, Western and ELISA analyses in order to determine the potential for autocrine, juxtacrine and/or paracrine interactions with pulmonary tissues and cells; c) The release of cytoactive granule proteins, MBP and EPO, by apoptotic versus necrotic versus de-granulating eosinophils will be assessed by protein- specific assays. These studies should elucidate critical mechanisms regulated Ras-mediated apoptosis and determine potential in vivo effects of pharmacologic or physiologic induction of this apoptosis of eosinophils.