Leukotrienes and Slow Reacting Substance of Anaphylaxis

描述(申请人提供)：人中性粒细胞在宿主防御反应中发挥重要作用，部分原因是5-脂氧合酶(5-LO)的激活和花生四烯酸被氧化成一类称为白三烯的脂质介质。这些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体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已经知道了很多细节，但关于化学反应中间体白三烯A4(LTA4)在人中性粒细胞内稳定的确切事件，人类中性粒细胞是合成LTA4的主要细胞，以及它是如何离开细胞的，人们知之甚少。LTA4的化学半衰期不到3秒，靠近细胞的核周区域，但它主要在中性粒细胞外运输，这一过程被称为“跨细胞生物合成”，并在次级细胞内进行处理。虽然人们相当重视了解5-LO和释放花生四烯酸的酶cPLA2a的激活，但对参与将花生四烯酸转化为细胞磷脂的酶知之甚少。我们发现，抑制花生四烯酸反应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。提出了花生四烯酸再酯化所特有的溶血磷脂酰基转移酶和脂肪酰辅酶A连接酶的研究，包括溶血磷脂酰基转移酶的鉴定。拟议工作的第二个重点涉及中性粒细胞胞浆中蛋白质的表征，该蛋白质稳定了LTA4，使其能够参与跨细胞生物合成。在一定程度上，这些研究将集中关注S100A8/A9，利用S100A9基因缺陷的小鼠(MRP-14-/-)作为一种可能的蛋白质复合体，可以稳定LTA4，因为它是已知的中性粒细胞中主要的花生四烯酸结合蛋白。LTA4的稳定因子称为中性粒细胞稳定因子，将使用质谱学和蛋白质组学技术进行鉴定。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合成酶缺失的小鼠骨髓移植到缺乏5-LO的受体小鼠的嵌合小鼠。在这些研究中，将使用LC/MS/MS脂质组学方法对腹膜炎炎症模型和急性肺损伤(LPS)模型中产生的所有花生四烯酸代谢产物进行定量。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产品。

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.