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，类二十烷酸的产生受其生物合成的单个酶的时间和空间定位的调节，最有可能在核膜上。这是通过它们在静止细胞中的定位，它们在特定亚细胞位置的诱导表达，以及它们在细胞激活时向底物来源的易位来实现的。小鼠骨髓源性肥大细胞（mBMMC）可在类二十烷生成的三个阶段被激活。在构成期，mBMMC被c-kit （KL）配体激活，或被IgE和抗原与FepsilonRI交联激活，快速生成白三烯（LT） C4和前列腺素（PG） D2，该过程在10分钟内完成。在延迟期，KL和白细胞介素（IL）10，无论是IL-1 β还是IgE和抗原，在2至10小时内诱导PGD2的产生。在12小时后开始的第三阶段，称为即刻启动，KL与辅助细胞因子一起启动mBMMC以增强PGD2的产生。即刻生成（无论是构成型还是启动型）和延迟生成的PGD2分别依赖于前列腺素内过氧化物合成酶(PGHS)-1和PGHS-2。二十烷类生物合成的第一步是通过磷脂酶A2 （PLA2）的作用从细胞膜磷脂释放游离花生四烯酸，这是一个不断增长的酶家族。花生四烯酸供给PGHS-1和PGHS-2的分离可能是由不同PLA2酶在每个阶段的参与介导的。IV组和V组酶可能在构成期以合作的方式起作用，而未识别的肝素敏感PLA2在延迟期起作用。我们使用了具有特异性的兔抗肽抗体，以显示其在mBMMC激活的直接阶段向核膜的易位。我们还鉴定了一种富含半胱氨酸的PLA2，称为XI PLA2组，优先表达于T辅助（Th） 2亚群的T细胞中。我们建议将这些新的观察结果扩展到研究PLA2在类二十烷细胞产生的每个阶段的表达、亚细胞定位和易位。我们将通过反义DNA抑制、药物和基因破坏来确认他们的参与。我们将使用位点定向诱变来确定对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，检查其在发育和激活的小鼠肥大细胞中的表达，并验证其在Th2细胞的分化和/或效应功能中起作用的假设。