Leukotrienes and Slow Reacting Substance of Anaphylaxis

描述（由申请人提供）：人中性粒细胞在宿主防御反应中起重要作用，部分原因是5-脂氧合酶（5-LO）的活化和花生四烯酸氧化成一类称为白三烯的脂质介质。这些类二十烷类化合物的生物活性包括中性粒细胞趋化因子（白三烯B4）和支气管平滑肌收缩物质白三烯C4 （LTC4）。尽管关于这些脂质介质的生物合成以及这些分子在肺系统中宿主防御反应中所起作用的许多细节已经知道，但对于化学反应中间体白三烯A4 （LTA4）在人类中性粒细胞（合成LTA4的主要细胞）内稳定的确切事件以及它如何退出细胞知之甚少。LTA4的化学半衰期小于3秒，在细胞的核周区域附近制造，但它在被称为“跨细胞生物合成”的过程中大部分被运送到中性粒细胞外，并在次级细胞内加工。虽然相当多的重点放在了解5-LO和释放花生四烯酸的酶cPLA2a的激活上，但对将花生四烯酸转化回细胞磷脂的酶知之甚少。我们发现，抑制花生四烯酸再酰化可使人中性粒细胞中白三烯的生物合成增加50- 100倍，从而增加跨细胞生物合成。提出了用于花生四烯酸再酯化的溶血磷脂酰基转移酶和脂肪酰基辅酶a连接酶的研究，包括溶血磷脂酰基转移酶的鉴定。所提出的工作的第二个重点涉及中性粒细胞细胞质中蛋白质的表征，该蛋白质稳定LTA4，使其能够参与跨细胞生物合成。在某种程度上，这些研究将把注意力集中在S100A8/A9上，利用S100A9基因缺陷小鼠（MRP-14 -/-）作为一种可能的蛋白质复合物，该蛋白复合物可以稳定LTA4，因为已知LTA4是中性粒细胞中主要的花生四烯酸结合蛋白。LTA4的中性粒细胞稳定因子的鉴定将使用质谱法和蛋白质组学技术进行。第三个研究领域将研究白三烯的跨细胞生物合成，使用从LTA4水解酶缺失或LTC4合酶缺失小鼠骨髓移植获得的嵌合小鼠到缺乏5-LO的受体小鼠中。在这些研究中，花生四烯酸酯在腹膜炎炎症模型和急性肺损伤模型（LPS）中产生的所有代谢物将采用LC/MS/MS脂质组学方法进行定量。缺乏白三烯级联酶的巨噬细胞或中性粒细胞的混合物将被刺激，并在特定细胞类型的跨细胞生物合成研究中研究其产物。

DESCRIPTION (provided by applicant): 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.