Function of secreted PLA2 group X in eosinophil biology

分泌型 PLA2 X 组在嗜酸性粒细胞生物学中的功能

• 批准号: 8522225
• 负责人: TEAL S HALLSTRAND
• 金额: $ 18.39万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-15 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8522225
• 关键词: Abbreviations; Antibodies; Arachidonate 5-Lipoxygenase; Arachidonic Acids; Asthma; Attenuated; Biology; Cell Line; Cell membrane; Cells; Confocal Microscopy; Cytolysis; Cytoplasmic Granules; Data; Eicosanoids; Enzymes; Figs - dietary; G Protein-Coupled Receptor Genes; G-Protein-Coupled Receptors; Generations; HL60; Human; Inflammation; Inflammation Mediators; Inflammatory; Interleukin-3; Interleukin-5; JUN gene; Label; Life; Location; MAPK14 gene; Mediating; Mediator of activation protein; Modeling; Organelles; Pathogenesis; Patients; Phospholipase; Phospholipase A2; Phospholipids; Phosphotransferases; Play; Production; Public Health; Role; Signal Transduction; Site; Source; Stimulus; Subcellular structure; TNF gene; TSLP gene; Tissues; United States; airway inflammation; cysteinyl-leukotriene; cytokine; eosinophil; extracellular; human TSLP protein; in vivo; inhibitor/antagonist; lentiviral-mediated; lipid mediator; novel; overexpression; peripheral blood; small hairpin RNA; therapeutic target; translational study; young adult

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.

描述（由申请人提供）：嗜酸性粒细胞合成半胱氨酰白三烯（CysLT）在哮喘的发病机制中起着重要作用。我们发现分泌型 PLA2 X 基团 (sPLA2-X) 参与嗜酸性粒细胞 CysLT 合成，这与长期以来认为胞质 PLA2¿ (cPLA2¿) 是参与嗜酸性粒细胞 CysLT 形成的唯一主要 PLA2 的观点相矛盾。我们的总体假设是，气道炎症期间，sPLA2-X 在嗜酸性粒细胞中上调，并在嗜酸性粒细胞形成 CysLT 中发挥重要作用。我们最近证明，外源性 sPLA2-X 通过依赖于 sPLA2-X 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 sPLA2-X 在人嗜酸性粒细胞中内源表达，并且 sPLA2-X 的高选择性抑制剂可减弱 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人嗜酸性粒细胞中 sPLA2-X 的细胞内位置。我们将确定嗜酸性粒细胞中 sPLA2-X 的表达是否会因 IL-3、IL-5、GMCSF 和其他细胞因子的增加而增加，这些细胞因子在嗜酸性粒细胞进入气道时“启动”嗜酸性粒细胞的激活。在本研究的转化部分中，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。在第二个具体目标中，我们将首先通过 sPLA2-X 和 cPLA2¿ 的选择性抑制剂来检查 sPLA2-X 在嗜酸性粒细胞中的功能，然后通过慢病毒介导的 shRNA 敲低嗜酸性粒细胞样细胞系 HL-60 clone-15 中的 sPLA2-X。由于 sPLA2-X 可以在细胞内和分泌后发挥作用，因此我们将确定 sPLA2-X 在激活过程中是否与 5-脂氧合酶 (5-LO) 共定位，以及它是否在分段脱颗粒过程中释放，从而允许酶作用于外细胞膜。最后，我们将检查 sPLA2-X 在无细胞嗜酸性粒细胞颗粒中的潜在功能，这些颗粒充当浸润哮喘患者气道组织的分泌细胞器。确定 sPLA2-X 在嗜酸性粒细胞 CysLT 形成中的重要功能将为该酶作为哮喘治疗靶点提供强有力的理论依据。