ARACHIDONATE METABOLISM BY ACTIVATED MAST CELLS

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The cysteinyl leukotrienes (LTC4, and its derivatives, LTD4 and LTE4), and prostaglandin (PG) D2 are potent lipid mediators with distinct biologic properties, which are derived from the metabolism of arachidonic acid by the 5-lipoxygenase (5-LO) and cyclooxygenase pathways, respectively. They have been implicated in the pathogenesis of allergic disease, in particular bronchial asthma. Rat and mouse serosal mast cells preferentially generate PGD2 compared to LTC4, whereas rat mucosal mast cells of apparent maturity preferentially generate LTC4 relative to PGD2. The mouse, IL-3 dependent bone marrow-derived mast cell (mBMMC), which preferentially generates LTC4 is a pluripotent progenitor capable of differentiation into mature mast cells of either phenotype depending upon the influence of the local microenvironment. The first aim of this project seeks to identify tissue-based elements, whether soluble cytokines or cell-associated, that lead to the differentiation and/or maturation of BALB/c mBMMC toward a phenotype that preferentially generates PGD2, whilst downregulating LTC4 generation. Analysis of RNA transcription and degradation, and SDS-PAGE immunoblot analysis, will be used to investigate the cellular biologic mechanisms of these events. Although we can assess the differential regulation of the 5-LO and cyclooxygenase pathways of mBMMC with molecular and immunochemical probes for cPLA2, 5-LO, FLAP, and cyclooxygenase I and II, we lack the probes for the terminal enzymes in each sequence. The second aim is therefore to clone the cDNA for LTC4 synthase from the human KG-1 cell line, and to obtain the cDNA for the mouse enzyme by cross-hybridization. Specific antibody to PGD2 synthase will be used to obtain the cDNA for this enzyme from a mouse mast cell cDNA library in lambda-ZAP. The availability of cDNAs for LTC4 synthase and mast cell PGD2 synthase will provide a consensus amino acid sequence suitable for identifying peptides to which antiserum can be raised and used for SDS-PAGE immunoblot analysis and perhaps immunohistochemistry and ultrastructural localization of these enzymes. A parallel aim of this project is to delineate those regulatory events directed only at the genes which encode LTC4 synthase and PGD2 synthase. To this end the cDNAs will be used as probes to clone the genes for these enzymes. The genomic organization will be characterized, the exons and 5' flanking region will be sequenced, and the cis-acting regulatory elements will be defined.