ENDOTHELIAL LATERAL JUNCTIONS AND LEUKOCYTE TRAFFICKING

炎症反应是宿主防御的基本组成部分。的 白细胞在血管中的牢固粘附和迁移 炎症过程中的内皮细胞发生在毛细血管后水平， 集合小静脉虽然白细胞已经受到了很大的关注- 内皮粘附相互作用，直到最近才有分子 白细胞通过内皮细胞的机制 横向连接成为研究的焦点。最近，我们已经证明， 中性粒细胞粘附触发粘附连接的破坏， 迁移事件之前的内皮外侧边界（1）。 具体地，VE-钙粘蛋白复合物，由VE-钙粘蛋白、α-钙粘蛋白和α-钙粘蛋白组成。 连环蛋白，β-连环蛋白，γ-连环蛋白（也称为斑珠蛋白），和 p120/p100非共价连接，在初始3分钟内被破坏 中性粒细胞结合，并导致该复合物从外侧丢失 VE-钙粘蛋白和β-连环蛋白的连接和裂解。洗涤 从嗜中性粒细胞制备的膜（蛋白酶促反应器处理的）能够 来调节这些变化，这表明， 内皮VE-钙粘蛋白复合物存在于中性粒细胞表面， 似乎不需要颗粒蛋白水解组分。 该项目的重点是更充分地表征内皮细胞， 参与调节侧连接的依赖机制 使用免疫学、细胞生物学和 在体外和体内模型中的分子生物学策略。的 拟议的研究将严格测试观察到的变化， 侧连接的组成是白细胞 轮回具体目标I将确定细胞粘附的步骤 触发VE-钙粘蛋白复合物的解离， 使用实时免疫荧光测定来监测 白细胞粘附过程中VE-钙粘蛋白定位的变化 体外流动模型，其次，测试假设，解离和 VE-钙粘蛋白复合物的裂解是迁移所必需的。 具体目标II将试图确定其他，尚未定性， 内皮分子定位于细胞与细胞连接处， 参与白细胞粘附-迁移，使用单克隆抗体 筛选方法。具体目标II中提出的实验将 确定参与白细胞的细胞内信号通路- 使用药理学方法诱导VE-钙粘蛋白解离， 在Specific Aim I中开发的实时免疫荧光测定。具体 目的IV将利用急性或慢性炎症的体内模型， 检测白细胞诱导的 粘附连接的改变。 拟议的研究与该计划的总体目标相关， 我将与其他项目合作，以更好地了解 血管内皮细胞在炎症和免疫反应中的作用 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.