The proposed investigations on the cells, mast cells and eosinophils, and the mediators, preformed secretory granule constituents and newly derived membrane lipid metabolites, of immediate hypersensitivity reactions continue a long term commitment. With the culture/coculture of mast cells and eosinophils, the regulation of phenotype of non-transformed cells by fibroblast factors operating in concert with defined hemopoietic cytokines can be examined so that possible tissue effects on the chemical composition and/or function of these effector cell types can be identified. Kirsten sarcoma virus immortalized murine mast cells (KiSV-MC) provide a homogeneous and substantial source of connective tissue-like murine mast cells (CTMC) for isolation of specific proteins and cloning of their specific cDNA from the KiSV-MC cDNA library; the cDNA will be used to examine transcription of these proteins during phenotypic changes in interleukin 3 (IL-3)-dependent bone marrow- derived murine mast cells (BMMC) elicited by fibroblasts or during regulation of proliferation and/or phenotype of KiSV-MC by IL-3 and/or IL-4. These same cDNA probes will be used for genomic cloning of mouse secretory granule carboxypeptidase A and to initiate cloning of the cDNA that encodes the analogous human mast cell neutral proteases. With regard to the analysis of the 5-lipoxygenase pathway to leukotriene C4 generation (LTC4), the techniques for culturing eosinophils ex vivo and for retrovirus immortalization of murine mast cells, KiSV-MC, provide new approaches. The ability to culture peripheral blood human eosinophils ex vivo will permit the mechanism and regulation of the distinct step for LTC4 export to be fully characterized. The KiSV-MC provide a homogeneous source of cells with adequate amounts of LTC4 synthase for isolation and amino acid sequence analysis of selected portions so as to allow synthesis of oligonucleotides and initiation of cloning from the KiSV-MC library. Taken together, these studies will yield new insights into the following: the extracellular regulation of mast cell and eosinophil phenotypes; transcription, translation, and degradation of secretory granule constituents of mast cells; the protein and/or nucleotide sequence for microsomal LTC4 synthase and mechanistic information on LTC4 export; and the presence in selected human cDNA libraries of nucleotide sequences analogous to those that encode the murine mast cell proteases.
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