Function of secreted PLA2 group X in eosinophil biology

描述（由申请人提供）：嗜酸性粒细胞合成半胱氨酰白三烯（CysLT）在哮喘发病机制中起重要作用。我们发现分泌型PLA 2 X组（sPLA 2-X）参与嗜酸性粒细胞CysLT的合成，这与长期以来认为胞浆型PLA 2 <$（cPLA 2 <$）是嗜酸性粒细胞中参与CysLT形成的唯一主要PLA 2的观点相矛盾。我们的总体假设是sPLA 2-X在气道炎症过程中在嗜酸性粒细胞中上调，并在嗜酸性粒细胞形成CysLT中起重要作用。我们最近证明，外源性sPLA 2-X通过依赖于sPLA 2-X的酶功能的机制启动人嗜酸性粒细胞合成CysLT，但也涉及通过p38和c-Jun末端激酶（JNK）激活cPLA 2？。在初步的数据中，我们证明sPLA 2-X是内源性表达的人嗜酸性粒细胞和sPLA 2-X的高度选择性抑制剂减弱CysLT形成fMLP刺激的嗜酸性粒细胞。在第一个具体的目标，我们将研究sPLA 2-X在人类嗜酸性粒细胞使用共聚焦显微镜的细胞内位置。我们将确定sPLA 2-X在嗜酸性粒细胞中的表达是否被IL-3、IL-5、GMCSF和其他细胞因子增加，这些细胞因子在嗜酸性粒细胞进入气道时“引发”嗜酸性粒细胞活化。在本研究的翻译部分，我们将确定sPLA 2-X表达是否增加，从哮喘患者的嗜酸性粒细胞。在第二个具体目标中，我们将首先通过sPLA 2-X和cPLA 2 <$的选择性抑制剂，然后通过在嗜酸性粒细胞样细胞系HL-60克隆-15中慢病毒介导的sPLA 2-X的shRNA敲低，来检查sPLA 2-X在嗜酸性粒细胞中的功能。由于sPLA 2-X可以在细胞内和分泌后起作用，我们将确定sPLA 2-X在活化过程中是否与5-脂氧合酶（5-LO）共定位，以及它是否在逐段脱粒过程中释放，从而使酶作用于细胞外膜。最后，我们将研究sPLA 2-X在无细胞嗜酸性粒细胞颗粒中的潜在功能，这些颗粒作为具有分泌能力的细胞器浸润哮喘患者的气道组织。确定sPLA 2-X在嗜酸性粒细胞CysLT形成中的重要功能将为这种酶作为哮喘治疗靶点提供强有力的理论基础。

DESCRIPTION (provided by applicant): The synthesis of cysteinyl leukotrienes (CysLT)s by eosinophils plays an important role in the pathogenesis of asthma. We have discovered that secreted PLA2 group X (sPLA2-X) is involved in eosinophil CysLT synthesis, contradicting the long-held view that cytosolic PLA2¿ (cPLA2¿) is the only major PLA2 involved in CysLT formation in eosinophils. Our overall hypothesis is that sPLA2-X is upregulated in eosinophils during inflammation of the airways and plays an important role in CysLT formation by eosinophils. We have recently demonstrated that exogenous sPLA2-X initiates CysLT synthesis by human eosinophils through a mechanism that is dependent upon the enzymatic function of sPLA2-X, but also involves the activation of cPLA2¿, through p38 and c-Jun-terminal kinase (JNK). In preliminary data, we demonstrate that sPLA2-X is endogenously expressed in human eosinophils and that a highly selective inhibitor of sPLA2-X attenuates CysLT formation by fMLP-stimulated eosinophils. In the first specific aim, we will examine the intracellular location of sPLA2-X in human eosinophils using confocal microscopy. We will determine if the expression of sPLA2-X in eosinophils is increased by IL-3, IL-5, GMCSF and other cytokines that "prime" eosinophils for activation as they enter the airways. In a translational portion of this study, we will determine if sPLA2-X expression is increased in eosinophils from patients with asthma. In the second specific aim, we will examine the function of sPLA2-X in eosinophils first through selective inhibitors of sPLA2-X and cPLA2¿, and then through lentiviral-mediated shRNA knockdown of sPLA2-X in the eosinophil-like cell line HL-60 clone-15. Since sPLA2-X can act within cells and after secretion, we will determine if sPLA2-X co-localizes with 5-lipoxygenase (5-LO) during activation, and if it is released during piecemeal degranulation, allowing the enzyme to act on the outer cell membrane. Finally, we will examine the potential function of sPLA2-X in cell-free eosinophil granules that act as secretion competent organelles infiltrating the airway tissues of patients with asthma. Establishing the important function of sPLA2-X in eosinophil CysLT formation will provide a strong rationale for this enzyme as a therapeutic target in asthma.