REGULATION OF FAS MEDIATED APOPTOSIS IN EOSINOPHILS

建议进行研究以检查Fas-1的机制和后果。 介导人嗜酸性粒细胞的凋亡。核心的、基本的假设 对于这些研究来说，Fas诱导的嗜酸性粒细胞杀伤代表着一种 炎症分解的自然机制，通常是受调节的 通过特定的细胞外和细胞内机制。为了测试这一点 假设，我们提出了三个具体的研究领域：(1)Fas诱导 嗜酸性粒细胞中的细胞凋亡。A)Fas和Fas-2的表达与调控 肿瘤坏死因子-α和干扰素-γ诱导人嗜酸性粒细胞凋亡 用RT-PCR和流式细胞仪分析研究和特异性分析 嗜酸性粒细胞凋亡；b)Fas表达的调节 氨基肽酶N/CD13将用特异性抗CD13封闭进行检测 抗体和氨基肽酶及其他抑制物；c)细胞内 Bcl2相关基因和ICE家族基因及蛋白在食管癌组织中的表达 易感和抗性嗜酸性粒细胞群体，使用RT-PCR，Northern， 免疫印迹和流式细胞仪检测细胞内特异性 Fas诱导细胞凋亡的调控机制。(2)Fas-Fas配体 嗜酸性粒细胞与呼吸道上皮细胞的相互作用。A) Fas配体的表达类似于研究Fas的表达 嗜酸性粒细胞的逆转录聚合酶链式反应和特异性相互作用的Norther分析 将在嗜酸性粒细胞和上皮细胞的“共同培养”中进行检测 单层；c)将测试金属蛋白酶抑制剂对 FasL的表达。(3)Fas-Fas配体相互作用与嗜酸性粒细胞 生物活性。A)在Fas易感和Fas-2易感人群中核因子-kappaB的激活 抗性细胞将使用电迁移率漂移分析和 核抽提物的超移位分析；b)表达 Fas-α对核因子-kappaB诱导的细胞因子基因及其蛋白产物的影响 诱导的嗜酸性粒细胞将用Northern、Western和ELISA法研究 分析以确定自分泌、旁分泌的潜力 和/或旁分泌与肺组织和细胞的相互作用；c) 细胞活性颗粒蛋白MBP和EPO的释放 坏死性和脱颗粒性嗜酸性粒细胞将通过蛋白质进行评估- 特定的化验。这些研究应该阐明关键机制 调节RAS介导的细胞凋亡及其在体内的潜在作用 药物或生理上诱导的这种细胞凋亡 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.