PHOSPHOLIPASE A IN ALLERGIC INFLAMMATION

磷脂酶 A 在过敏性炎症中的作用

• 批准号: 6496747
• 负责人: Jonathan Peter Arm
• 金额: $ 3.04万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-01 至 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6496747
• 关键词: antibody receptor; atopy; bone marrow; cell differentiation; eicosanoid metabolism; enzyme activity; enzyme mechanism; immunoglobulin E; inflammation; laboratory mouse; laboratory rabbit; leukotrienes; mast cell; phospholipase A2; prostaglandin endoperoxide synthase; prostaglandins; protein localization; site directed mutagenesis

There is growing evidence that eicosanoid generation is regulated by the temporal and spatial localization of the individual enzymes of their biosynthesis, most likely at the nuclear envelope. This is achieved through their localization in the resting cell, their induced expression at specific subcellular locations, and their translocation to a source of substrate when a cell is activated. The mouse bone marrow derived mast cell (mBMMC) may be activated for three phases of eicosanoid generation. In the constitutive-immediate phase, mBMMC are activated by the ligand for c-kit ((KL) or by cross-linking of FepsilonRI by IgE and antigen for the rapid generation of leukotriene (LT) C4 and prostaglandin (PG) D2 that is complete within 10 minutes. In the delayed phase KL and interleukin (IL)10, with either IL-1beta or IgE and antigen, elicits PGD2 generation from 2 to 10 hours. In the third phase, which begins after 12 hours and is termed prime-immediate, KL with accessory cytokines primes mBMMC for augmented PGD2 generation. Immediate (whether constitutive or primed) and delayed PGD2 generation re dependent upon prostaglandin endoperoxide synthase (PGHS)-1 and PGHS-2, respectively. The initial step in eicosanoid biosynthesis is the release of free arachidonic acid from cell membrane phospholipids by the action of phospholipase A2 (PLA2), a growing family of enzymes. The segregation of the supply of arachidonic acid to PGHS-1 and PGHS-2 is likely mediated by the participation of different PLA2 enzymes in each phase. The group IV and group V enzymes likely act in a co-operative fashion in the constitutive-immediate phase, and a unidentified heparin- sensitive PLA2 acts in the delayed phase. We have used rabbit anti-peptide antibodies with specificity for the group V enzyme to show its translocation to the nuclear envelope during the immediate phase of mBMMC activation. We have also identified a cysteine-rich PLA2, termed group XI PLA2, preferentially expressed in T cells of the T helper (Th) 2 subset. We propose to extend these novel observations to a study of the expression, subcellular localization, and translocation of individual PLA2 species during each phase of eicosanoid generation. We will confirm their participation through inhibition by antisense DNA, pharmacological agents, and gene disruption. We will use site-directed mutagenesis to determine the motifs critical to the resting location and translocation of the group V enzyme. We will further characterize the group XI PLA2, examin3e its expression in developing and activated mouse mast cells, and test the hypothesis that it has a role in the differentiation and/or effector function of Th2 cells.

越来越多的证据表明，类花生酸的产生是由其生物合成的各个酶的时间和空间定位，最有可能在核膜。这是通过它们在静息细胞中的定位、它们在特定亚细胞位置的诱导表达以及当细胞被激活时它们移位到底物来源来实现的。小鼠骨髓源性肥大细胞（mBMMC）在类花生酸生成过程中可分为三个阶段。在组成性即刻阶段，mBMMC被c-kit（KL）的配体或通过IgE和抗原交联FepsilonRI激活，以快速产生白三烯（LT）C4和前列腺素（PG）D2，在10分钟内完成。在延迟相KL和白细胞介素（IL）10，与IL-1 β或IgE和抗原，eliminating PGD 2生成从2到10小时。在第三阶段，12小时后开始，称为初免-立即，KL与辅助细胞因子初免mBMMC用于增强的PGD 2生成。PGD 2的即时生成（无论是组成型还是引发型）和延迟生成分别依赖于前列腺素内过氧化物合酶（PGHS）-1和PGHS-2。类花生酸生物合成的第一步是通过磷脂酶A2（PLA 2）（一种不断增长的酶家族）的作用从细胞膜磷脂中释放游离花生四烯酸。花生四烯酸向PGHS-1和PGHS-2的供应的分离可能是由每个阶段中不同的PLA 2酶的参与介导的。IV组和V组酶可能在组成性立即阶段以合作方式起作用，并且未鉴定的肝素敏感性PLA 2在延迟阶段起作用。我们已经使用了兔抗肽抗体与组V酶的特异性，以显示其易位到核膜在mBMMC激活的即时阶段。我们还确定了富含半胱氨酸的PLA 2，称为组XI PLA 2，优先表达在T细胞的辅助性T细胞（Th）2亚群。我们建议将这些新的观察扩展到研究类花生酸生成的每个阶段中单个PLA 2物种的表达、亚细胞定位和易位。我们将通过反义DNA、药物和基因破坏的抑制来确认它们的参与。我们将使用定点诱变来确定对V组酶的静止位置和易位至关重要的基序。我们将进一步表征组XI PLA 2，检查其在发育和活化的小鼠肥大细胞中的表达，并测试其在Th 2细胞的分化和/或效应子功能中起作用的假设。