PHOSPHOLIPASE A IN ALLERGIC INFLAMMATION

越来越多的证据表明，二十烷类化合物的产生受到其生物合成的单个酶的时间和空间局部化的调节，最有可能的是在核膜上。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转移到底物来源来实现的。小鼠骨髓来源的肥大细胞(MBMMC)可在类二十烷生成的三个阶段被激活。在构成-即刻阶段，MBMMC被c-kit的配体((KL)激活，或通过IgE和抗原使FepsilonRI交联来快速产生白三烯(LT)C4和前列腺素(PG)D2，在10分钟内完成。在延迟期，KL和IL-10与IL-1β或IgE和抗原共同作用，可在2~10小时内诱导PGD2的产生。在第三阶段，在12小时后开始，称为即刻素数，KL和辅助细胞因子素数MBMMC用于增强PGD2的生成。PGD2的即时生成(无论是组成性的还是启动的)和延迟的生成分别依赖于前列腺素过氧化物合成酶(PGHS)-1和PGHS-2。二十烷基类化合物生物合成的第一步是在磷脂酶A2(PLA2)的作用下从细胞膜磷脂中释放出游离的花生四烯酸，PLA2是一个不断增长的酶家族。花生四烯酸向PGHS-1和PGHS-2的供应分离可能是由不同的PLA2酶参与每个阶段所介导的。IV组和V组酶在构成-即时阶段可能以协同方式发挥作用，而不明原因的肝素敏感的PLA2在延迟阶段发挥作用。我们使用了对V组酶具有特异性的兔抗肽抗体来显示其在MBMMC激活的即刻阶段移位到核膜上。我们还鉴定了一种富含半胱氨酸的PLA2，称为XI PLA2组，优先在T辅助分子(Th)2亚群的T细胞中表达。我们建议将这些新的观察扩展到研究单个PLA2物种在二十烷类化合物产生的每个阶段的表达、亚细胞定位和移位。我们将通过反义DNA抑制、药理学药物和基因破坏来确认它们的参与。我们将使用定点突变来确定对V组酶的静止位置和易位至关重要的基序。我们将进一步确定XI PLA2组的特征，检测其在发育中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/或效应功能中具有作用的假设。

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.