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称为白三烯的脂质介质家族。这些类二十烷酸的生物活性包括中性粒细胞趋化因子（白三烯B4）以及支气管平滑肌收缩物质白三烯C4（LTC 4）。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的宿主防御反应中所起的作用的许多细节是已知的，但关于化学反应性中间体白三烯A4（LTA 4）在人中性粒细胞（合成LTA 4的主要细胞）内稳定的精确事件以及它如何离开细胞知之甚少。LTA 4具有小于3秒的化学半衰期，并且靠近细胞的核周区域，但其在称为“跨细胞生物合成”的过程中大部分转运到嗜中性粒细胞外，并在次级细胞内加工。虽然相当大的重点放在理解激活5-LO和酶释放花生四烯酸，cPLA 2a，很少有人知道的酶参与花生四烯酸转化回细胞磷脂。我们已经发现，花生四烯酸再酰化的抑制使人嗜中性粒细胞中白三烯的生物合成增加50- 100倍，因此将增加跨细胞生物合成。溶血磷脂酰基转移酶和脂肪酰辅酶A连接酶的研究，特别是花生四烯酸再酯化的建议，包括鉴定溶血磷脂酰酰基转移酶。拟议的工作的第二个重点涉及中性粒细胞胞质溶胶中的蛋白质的表征，其稳定LTA 4，允许其参与跨细胞生物合成。在某种程度上，这些研究将集中注意力在S100 A8/A9上，使用S100 A9（MRP-14 -/-）缺陷的遗传小鼠作为可能的蛋白质复合物，其可以稳定LTA 4，因为已知LTA 4是中性粒细胞中的主要花生四烯酸结合蛋白。将使用质谱和蛋白质组学技术鉴定LTA 4的稳定因子（称为嗜中性粒细胞稳定因子）。第三个研究领域将使用嵌合小鼠研究白三烯的跨细胞生物合成，所述嵌合小鼠来源于从LTA 4水解酶缺失或LTC 4合酶缺失小鼠骨髓移植到5-LO缺陷的受体小鼠中。在这些研究中，将使用LC/MS/MS脂质组学方法定量在腹膜炎炎症模型和急性肺损伤模型（LPS）中产生的花生四烯酸的所有代谢物。将刺激缺乏白三烯级联酶的巨噬细胞或嗜中性粒细胞的混合物，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.