ENDOTHELIAL LATERAL JUNCTIONS AND LEUKOCYTE TRAFFICKING

炎症反应是宿主防御的基本组成部分。这个 白细胞在血管中的牢固黏附和移行 炎症过程中的内皮细胞发生在毛细血管后水平和 收集小静脉。尽管白细胞已经引起了人们的极大关注- 内皮细胞的黏附相互作用，直到最近才有分子 白细胞通过内皮细胞的机制 侧向连接成为研究的热点。最近，我们已经证明了 中性粒细胞黏附触发黏附连接的破坏 内皮细胞侧缘，在转位事件之前(1)。 具体地说，VE-钙粘蛋白复合体由VE-钙粘蛋白、α-钙粘附素-α-钙粘蛋白组成。 连环蛋白、β-连环蛋白、γ-连环蛋白(也称为白蛋白)，以及 P120/p100非共价连接，在最初的3分钟内中断 中性粒细胞结合，并导致该复合体从外侧丢失 VE-钙粘附素和β-连环素的连接和切割。洗过了 由中性粒细胞制备的膜(经蛋白酶抑制剂处理)能够 来调节这些变化，这表明破坏的刺激因素 内皮VE-钙粘附素复合体位于中性粒细胞表面， 颗粒蛋白水解物似乎不是必需的。 本项目的重点是更全面地描述内皮细胞 依赖-参与调节侧向连接的机制 用免疫学、细胞学和细胞生物学方法研究白细胞迁移 体外和体内模型中的分子生物学策略。这个 拟议的研究将关键地检验观察到的变化是否 侧向连接的组成是白细胞所必需的 轮回。具体目标我将确定细胞黏附的步骤 在白细胞过程中触发VE-钙粘素复合体的解离 轮回，使用实时免疫荧光分析来监测 白血球黏附过程中VE-钙粘附素定位的变化 体外流动模型，其次，检验解离和分离的假设 VE-钙粘蛋白复合体的裂解是转体所必需的。 特殊目标二将试图确定其他尚未确定特征的 定位于细胞间连接的内皮分子 参与白细胞黏附-迁移，使用一种单抗 筛选方法。在《特定目标2》中提出的实验将 确定参与白细胞的细胞内信号通路- 用药理学方法诱导VE-钙粘附素解离 针对特定目标开发的活时间免疫荧光分析I.特异性 Aim IV将利用急性或慢性炎症的体内模型来 检测白细胞诱生的潜在病理生理相关性 粘连连接处的变化。 拟议的研究与方案的整体研究有关，并 将与其他项目协同工作，以获得更好的理解 血管内皮细胞衬里在炎症和免疫中的作用 反应。

The inflammatory response is a fundamental component in host defense. The firm adhesion and transmigration of leukocytes across the vascular endothelium during inflammation occurs at the level of post-capillary and collecting venules. Although much attention has been paid to leukocyte- endothelial adhesive interactions, only recently have the molecular mechanisms involved in leukocyte passage through the endothelial cell lateral junctions become a focus of study. Recently, we have shown that neutrophil adhesion triggers disruption of adherens junctions at endothelial lateral borders, prior to the transmigration event (1). Specifically, the VE-cadherin complex, consisting of VE-cadherin, alpha- catenin, beta-catenin, gamma-catenin (also termed plakoglobin), and p120/p100 non-covalently linked, was disrupted within 3 minutes of initial neutrophil binding, and results in loss of this complex from lateral junctions and cleavage of both VE-cadherin and beta-catenin. Washed membranes (protease inhibitor-treated) prepared from neutrophils were able to mediate these changes, suggesting that the stimulus for disruption of the endothelial VE-cadherin complex resides in neutrophil surface and that granule proteolytic components do not appear to be required. The focus of this project is to characterize more fully the endothelial dependent-mechanisms involved in regulation of lateral junctions during leukocyte transmigration using immunological, cell biological and molecular biological strategies in in vitro and in vivo models. The proposed studies will test critically whether the observed changes in the composition of lateral junctions are necessary for leukocyte transmigration. Specific Aim I will determine the step in cell adhesion that trigger dissociation of the VE-cadherin complex during leukocyte transmigration, using live time immunofluorescence assays to monitor changes in VE-cadherin localization during leukocyte adhesion in a in vitro flow model, and secondly, test the hypothesis that dissociation and cleavage of the VE-cadherin complex is necessary for transmigration. Specific Aim II will attempt to identify other, as yet uncharacterized, endothelial molecules localized to cell-to-cell junctions that are involved in leukocyte adhesion-transmigration, using a monoclonal antibody screening approach. The experiments proposed in Specific Aim II will determine the intracellular signaling pathways involved in leukocyte- induced VE-cadherin dissociation using pharmacological approaches and the live time immunofluorescence assays developed in Specific Aim I. Specific Aim IV will utilize in vivo models of acute or chronic inflammation to test the potential pathophysiologic relevance of leukocyte-induced alterations in adherens junctions. The proposed studies are relevant to the overall them of the program and will work in concert with the other project to gain a better understanding of the role of the vascular endothelial lining in inflammation and immune reactions.