Function of secreted PLA2 group X in eosinophil biology

描述(申请人提供)：嗜酸性粒细胞合成半胱氨酰白三烯(CysLT)S在哮喘发病机制中起重要作用。我们发现分泌型PLA2组X(sPLA2-X)参与了嗜酸性粒细胞CysLT的合成，这与长期以来认为胞浆PLA2(CPLA2)是嗜酸性粒细胞CysLT形成的唯一主要PLA2的观点相矛盾。我们的总体假设是，在呼吸道炎症过程中，sPLA2-X在嗜酸性粒细胞中表达上调，并在嗜酸性粒细胞形成CysLT过程中发挥重要作用。我们最近证实，外源sPLA2-X通过一种依赖于sPLA2-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cPLA2。在初步数据中，我们证明了sPLA2-X在人嗜酸性粒细胞中内源性表达，并且sPLA2-X的高选择性抑制剂可以减少fMLP刺激的嗜酸性粒细胞形成CysLT。在第一个具体目标中，我们将使用共聚焦显微镜检查sPLA2-X在人嗜酸性粒细胞中的细胞内定位。我们将确定IL-3、IL-5、GMCSF和其他细胞因子是否会增加嗜酸性粒细胞中sPLA2-X的表达，这些细胞因子可以在嗜酸性粒细胞进入呼吸道时为其激活做好准备。在这项研究的翻译部分，我们将确定哮喘患者的嗜酸性粒细胞中sPLA2-X的表达是否增加。在第二个特定目标中，我们将首先通过选择性抑制sPLA2-X和cPLA2β，然后通过慢病毒介导的shRNA在嗜酸性粒细胞样细胞系HL-60 Clone-15中下调sPLA2-X，来检测sPLA2-X在嗜酸性粒细胞中的功能。由于sPLA2-X可以在细胞内和分泌后发挥作用，我们将确定sPLA2-X在激活过程中是否与5-脂氧合酶(5-LO)共定位，以及是否在分段脱颗粒过程中释放，允许酶作用于细胞外膜。最后，我们将检测sPLA2-X在无细胞嗜酸性粒细胞中的潜在功能，该颗粒作为分泌活性细胞器渗透到哮喘患者的呼吸道组织中。确定sPLA2-X在嗜酸性粒细胞CysLT形成中的重要作用将为该酶作为哮喘的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.