Mechanisms and Consequences of Eosinophil Integrin Activation

本课题主要研究pi整合素对小鼠血液中嗜酸性粒细胞(EOS)的激活机制。 哮喘患者；这种激活在EOS选择性进入呼吸道中的作用；以及‘二 我们假设的现象对于EOS在呼吸道内的运动、足体的形成 Amp2整合素的激活。目标1解决了血液中EOS和P选择素相互作用的假说 与P-选择素的糖蛋白配体(PSGL)，通常隔离在血小板的α-颗粒中 和内皮细胞的韦贝尔-帕拉德小体，负责增强p1的激活。这一假说将 通过对不同严重程度的哮喘患者的血液样本进行相关研究进行测试 哮喘志愿者在全肺或节段性肺抗原攻击后进行研究。测量结果将是 由循环EOS上pi的N29激活敏感表位组成，与P-选择素相关 循环EOS、血小板表面P-选择素、血浆中可溶性P-选择素。在……里面 体外研究将比较不同形式的P-选择素如何增强N29表位的表达 当Eos被添加到血液中时，并确定对增加表达至关重要的信号通路。目标2 提出的假说是激活a4p1，这是EOS上的主要pi整合素，启动EOS在 血管细胞黏附分子(VCAM)表达哮喘肺血管，从而进入呼吸道。我们 将N29表位的表达与肺内嗜酸性炎症的各种指标相关联，并与 肺组织抗原攻击后N29表达的变化与P-选择素变化的时程关系。 平行的体外研究将测试P-选择素是否增强静态和流动状态下全血的EOS黏附 条件，并确定重要的信号通路，以增加黏附。足体是一种 介导与细胞外基质中黏附配体的相互作用和蛋白质分解的瞬时结构。 EOS的足体，当细胞在IL-5和/或肿瘤坏死因子-α刺激下通过a4p1黏附到 VCAM具有一组独特的相关细胞骨架和细胞膜蛋白 其他细胞类型的足体。包括在这一套是亚当斯膜金属蛋白酶。目标3 测试关于泊多体和亚当斯有助于粘附物强大的蛋白分解能力的假设 EOS；以及当EOS进入肺时会有一个“移交”，这样Amp2就会被激活 暴露在IL-5下，取代在足体中降解的a4p1，成为主要的粘附性整合素。这个 研究将有助于更好地了解哮喘患者中EOS的贩运以及贩运可能是如何发生的 从药理上调节的。

This project focuses on the mechanism of activation of pi integrins on eosinophils (EOS) in the blood of patients with asthma; the role of such activation in selective movement of EOS into the airway; and'two phenomena that we hypothesize are important for movement of EOS within the airway, formation of podosomes and activation of aMp2 integrin. Aim 1 addresses the hypothesis'that interaction of blood EOS Pselectin glycoprotein ligand (PSGL) with P-selectin, which is normally sequestered in a-granules of platelets and Weibel-Palade bodies of endothelial cells, is responsible for enhanced p1 activation. This hypothesis will be tested by correlative studies of blood samples obtained from patients with asthma of varying severity and asthmatic volunteers studied after whole lung or segmental lung antigen challenge. Measurements will be made of the amounts of the N29 activation-sensitive epitope of pi on circulating EOS, P-selectin associated with circulating EOS, P-selectin on the surface of circulating platelets, and soluble P-selectin in plasma. In vitro studies will compare how the various forms of P-selectin enhance expression of the N29 epitope on EOS when added to blood and identify signaling pathways important for the increased expression. Aim 2 addresses the hypothesis that activation of a4p1, the major pi integrin on EOS, primes the EOS to arrest in vascular cell adhesion molecule (VCAM)-expressing vessels of asthmatic lung and thus enter the airway. We will correlate N29 epitope expression with various measures of eosinophilic inflammation in lung and relate the time course of changes of N29 expression after lung Ag challenge vis-a-vis changes in P-selectin. Parallel in vitro studies will test if P-selectin enhances EOS adhesion from whole blood under static and flow conditions and identify signaling pathways important for the increased adhesion. The podosome is a transient structure that mediates interaction with and proteolysis of adhesive ligands in extracellular matrix. The podosome of EOS, which forms when cells stimulated with IL-5 and/or TNF-a adhere via a4p1 to VCAM, has a distinctive set of associated cytoskeletal and cell membrane proteins when compared to podosomes of other cell types. Included within this set is the ADAMS membrane metalloproteinase. Aim 3 tests the hypotheses that podosomes and ADAMS contribute to the robust proteolytic capacity of adherent EOS; and that there is a "hand-off1 when the EOS enter the lung such that aMp2, which is activated upon exposure to IL-5, replaces a4p1, which is degraded in podosomes, as the principal adhesive integrin. The research will lead to a better understanding of trafficking of EOS in asthma and how trafficking may be modulated pharmacologically.