PHOSPHOLIPASE A IN ALLERGIC INFLAMMATION

磷脂酶 A 在过敏性炎症中的作用

• 批准号: 6353056
• 负责人: Jonathan Peter Arm
• 金额: $ 32.71万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6353056
• 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，类二十烷酸的生成受到其生物合成中各个酶的时间和空间定位的调节，最有可能在核膜上。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易位至底物源来实现的。小鼠骨髓来源的肥大细胞（mBMMC）可被激活以产生类二十烷酸的三个阶段。在组成型立即阶段，mBMMC 被 c-kit (KL) 配体激活，或通过 IgE 和抗原交联 FepsilonRI 来激活，以快速生成白三烯 (LT) C4 和前列腺素 (PG) D2，即在延迟阶段，KL 和白细胞介素 (IL)10 与 IL-1beta 或 IgE 和抗原一起诱导 PGD2 生成。 2 至 10 小时后开始，称为立即启动阶段，KL 与辅助细胞因子启动 mBMMC，以增强 PGD2 的生成（无论是组成型还是启动型）和延迟的 PGD2 生成，这取决于前列腺素内过氧化物合酶。 (PGHS)-1 和 PGHS-2 类二十烷酸生物合成的第一步是释放游离花生四烯酸。花生四烯酸通过磷脂酶 A2 (PLA2) 的作用从细胞膜磷脂中分离出来，花生四烯酸向 PGHS-1 和 PGHS-2 的供应分离可能是由不同 PLA2 酶在每个阶段的参与介导的。 IV 组和 V 组酶可能在组成立即相中以协作方式起作用，而未识别的肝素敏感 PLA2 在延迟相中起作用。我们使用了对 V 组酶具有特异性的兔抗肽抗体，以显示其在 mBMMC 激活的立即阶段易位至核膜。我们还鉴定了富含半胱氨酸的 PLA2，称为 XI PLA2 组，优先在 T 辅助细胞 (Th) 2 子集的 T 细胞中表达。我们建议将这些新的观察结果扩展到对类二十烷酸生成的每个阶段中各个 PLA2 物种的表达、亚细胞定位和易位的研究。我们将通过反义 DNA、药物制剂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 族酶的静止位置和易位至关重要的基序。我们将进一步表征 XI PLA2 组，检查其在发育和活化的小鼠肥大细胞中的表达，并检验其在 Th2 细胞的分化和/或效应功能中发挥作用的假设。