Mechanisms and Consequences of Eosinophil Integrin Activation

本项目的重点是在血液中的嗜酸性粒细胞（EOS）上活化pi整合素的机制， 哮喘患者;这种激活在EOS进入气道的选择性运动中的作用;以及 我们假设这些现象对于EOS在气道内的运动、足状体的形成和呼吸系统的重建是重要的。 和aMp 2整联蛋白的活化。目的1解决了“血液EOS P选择素相互作用”的假设 糖蛋白配体（PSGL）与P-选择素，这是正常情况下隔离在血小板的α-颗粒 和内皮细胞的韦伯-帕拉德体，负责增强p1激活。这一假设将 通过从不同严重程度的哮喘患者获得的血液样本的相关研究进行测试， 哮喘志愿者在全肺或肺段抗原激发后进行研究。测量将 由Pl在循环EOS上的N29活化敏感性表位的量组成，P-选择素相关 与循环EOS、循环血小板表面的P-选择素和血浆中的可溶性P-选择素有关。在 体外研究将比较各种形式的P-选择素如何增强N29表位在细胞上的表达。 EOS当加入血液中，并确定信号通路的重要性增加表达。目的2 解决了a4 p1（EOS上主要的pi整联蛋白）的激活引发EOS在 血管细胞粘附分子（VCAM）表达血管哮喘肺，从而进入气道。我们 将N29表位表达与肺中嗜酸性粒细胞炎症的各种测量相关， 肺组织Ag激发后N29表达变化的时间过程与P-选择素的变化。 平行的体外研究将测试P-选择素是否在静态和流动下增强来自全血的EOS粘附 条件，并确定信号通路的重要性增加的粘附。足体是一种 介导与细胞外基质中粘附配体相互作用和蛋白水解的瞬时结构。 当用IL-5和/或TNF-α刺激细胞时形成的EOS的足状体通过α 4 β 1粘附到 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podosomes。包括在这套是亚当斯膜金属蛋白酶。目标3 测试的假设，podosomes和亚当斯有助于强大的蛋白水解能力的粘附 EOS;并且当EOS进入肺时存在“切换1，使得aMp 2，其在EOS进入肺时被激活。 暴露于IL-5，取代在足体中降解的α 4 β 1，作为主要的粘附整联蛋白。的 研究将导致更好地了解EOS在哮喘中的贩运以及贩运可能是如何发生的。 调变频率

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.