REGULATION OF FAS MEDIATED APOPTOSIS IN EOSINOPHILS

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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.