The human neutrophil plays an important role in host defense reactions in part due to activation of the enzyme 5-lipoxygenase (5-LO) and the oxidation of arachidonic acid into a family of lipid mediators called leukotrienes. The biological activities of these eicosanoids include the neutrophil chemotactic factor (leukotriene B4) as well as the bronchial smooth muscle contraction substance, leukotriene C4 (LTC4). Even though many details are known concerning the biosynthesis of these lipid mediators as well as the role these molecules play in host defense reactions in the pulmonary system, little is known about the precise events by which the chemically reactive intermediate, leukotriene A4 (LTA4) is stabilized within the human neutrophil, the major cell synthesizing LTA4 and how it exits the cell. LTA4 has a chemical half-life of less than 3 sec, and is made close to the perinuclear region of the cell, but it is largely transported outside of the neutrophil in a process termed """"""""transcellular biosynthesis"""""""" and processed within a secondary cell. While considerable emphasis has been placed on understanding activation of both 5-LO and the enzyme which releases arachidonic acid, cPLA2a, little is known about the enzymes involved in converting arachidonic acid back into cellular phospholipids. We have found that inhibition of arachidonate reacylation increases 50- to 100-fold the biosynthesis of leukotrienes in the human neutrophil and will thus increase transcellular biosynthesis. Studies of lysophospholipid acyltransferase and fatty acyl-CoA ligase specific for arachidonic acid reesterification are proposed including identification of lysophosphatidyl acyltransferase. A second focus of the proposed work involves characterization of the protein in the neutrophil cytosol which stabilizes LTA4 that permits it to participate in transcellular biosynthesis. In part, these investigations will focus attention on S100A8/A9 using genetic mice deficient in S100A9 (MRP-14 -/-) as a possible protein complex which can stabilize LTA4 since it is known to be the major arachidonate binding protein in the neutrophil. Identification of the stabilizing factor called the neutrophil stabilizing factor for LTA4 will be carried out using mass spectrometry and techniques in proteomics. A third area of investigation will study transcellular biosynthesis of leukotrienes using chimeric mice derived from bone marrow transplantation from either LTA4 hydrolase null or LTC4 synthase null mice into a recipient mouse deficient in 5-LO. In these studies, all metabolites of arachidonate generated in a peritonitis inflammation model and acute lung injury model (LPS) will be quantitated using LC/MS/MS lipidomics approach. Mixture of macrophage or neutrophils deficient in leukotriene cascade enzymes will be stimulated and products studied in studies of transcellular biosynthesis in specific cell types.
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