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（由应用提供）：嗜酸性粒细胞的胱烷基白细胞（CYSLT）S的合成在哮喘的发病机理中起着重要作用。我们已经发现，分泌的PLA2组X（SPLA2-X）参与嗜酸性粒细胞Cyslt合成，这与长期以来的观点相矛盾，即长期以来的观点是胞质Pla2¿（CPLA2¿）是唯一参与嗜酸性粒细胞质中CYSLT形成的主要PLA2。我们的总体假设是，在气道炎症期间，SpLA2-X在嗜酸性粒细胞中被上调，并且在嗜酸性粒细胞的CYSLT形成中起重要作用。我们最近证明，外源SPLA2-X通过一种依赖于SplA2-X的酶促功能的机制引发人嗜酸性粒细胞的CYSLT合成，但也涉及通过p38和C-Jun-terminal-terminal-terminal-terminal-listinal-listinal-listinal-listinal-lastinal-closigationcpla2¿在初步数据中，我们证明了SplA2-X在人嗜酸性粒细胞中的内源表达，并且高度选择性的spla2-X抑制剂可通过FMLP刺激的嗜酸菌抑制CYSLT的形成。在第一个特定目的中，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。我们将确定在嗜酸性粒细胞中SpLA2-X的表达是否增加了IL-3，IL-5，GMCSF和其他“素”嗜酸性粒细胞的细胞因子，以便在进入气道时激活。在这项研究的转化部分中，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。在第二个特定目的中，我们将首先通过SPLA2-X和CPLA2的选择性抑制剂检查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SPLA2-X的SPLA2-X的SHRNA敲低的嗜酸菌样细胞系HL-60克隆-15。由于SPLA2-X可以在细胞内作用和分泌后，因此我们将确定SplA2-X在激活过程中是否与5-脂氧酶（5-lo）共定位，以及是否在零碎的脱粒过程中释放出来，允许该酶在外部细胞膜上作用。最后，我们将研究SPLA2-X在无细胞的嗜酸性嗜酸性粒细胞颗粒中的潜在功能，这些粒细胞颗粒充当秘密主管的细胞器，渗入哮喘患者的气道组织。建立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的治疗靶标提供强大的理由。