Endothelial Regulation of Leukocyte Transmigration

描述（申请人提供）：中性粒细胞和单核白细胞与内皮的相互作用在急慢性炎症、免疫反应、血管损伤和动脉粥样硬化的白细胞募集中起着关键作用。在上一个供资期间，取得了四项主要成就。(1)我们在T细胞亚群中发现了一种新的依赖e -选择素的粘附途径。(2)我们发现T -bet T细胞在体内和体外运输中存在缺陷，这与PSGL-1功能受损和CXCR4表达失败有关。(3)我们建立了一种活细胞荧光法来分析LFA-1和ICAM-1在白细胞迁移过程中的时空行为。(4)我们开发了一种非连接性中性粒细胞迁移的体外检测方法。这一建议建立在这一科学进展的基础上，将包括三个相互关联的具体目标，将重点放在内皮细胞ICAM-1尾部在白细胞转运中的作用。我们的工作假设是，在白细胞粘附过程中，LFA-1结合ICAM-1，触发其与细胞骨架的关联，这一步骤在“外向内”信号传导中起作用，促进ICAM-1在迁移的白细胞周围聚集。特异性目的1将阐明ICAM-1细胞质尾在白细胞转运中的作用，使用生化和生物物理技术，以及新开发的活细胞荧光成像系统和血管内皮，支持连接和非连接转运。特异性Aim 2将在体内炎症模型中测试ICAM-1尾部在化学引诱剂驱动的白细胞迁移中的作用。共聚焦活体荧光成像将利用细胞-细胞连接的荧光标记物可视化白细胞迁移的位置（连接或非连接路线），并同时确定迁移路线是否对血管通透性产生不利影响。特异性Aim 3将研究ICAM-1尾部在体外动脉内皮细胞炎症模型中的作用。动脉或静脉表型的内皮单层将在体外通过使用最近描述的锥形板装置在动脉或静脉波形中施加脉动剪切应力来创建。血液单核细胞和记忆T细胞与上述条件内皮的粘附相互作用和迁移位点将被检查。从这些研究中获得的信息将有助于描述白细胞转运的ICAM-1依赖机制，并可能因此确定免疫和炎症性疾病治疗干预的新靶点。

DESCRIPTION (provided by applicant): The interaction of neutrophils and mononuclear leukocytes with endothelium plays a pivotal role in leukocyte recruitment in acute and chronic inflammation, immune reactions, vascular injury and atherosclerosis. In the previous funding period, four major accomplishments occurred. (1) We identified a novel E-selectin dependent adhesion pathway in T cells subsets. (2) We determined that t-bet T cells have a defect in trafficking in vivo and in vitro related to impaired PSGL-1 function and failure to express CXCR4. (3) We developed a live cell fluorescence assay to resolve the temporal and spatial behavior of LFA-1 and ICAM-1 during leukocyte transmigration. (4) We developed an in vitro assay for non-junctional neutrophil transmigration. This proposal builds on this scientific progress and will consist of three interrelated specific aims that will focus on the role of endothelial cell ICAM-1 tail in leukocyte transmigration. Our working hypothesis is that during leukocyte adhesion, LFA-1 binds ICAM-1, triggering its association with the cytoskeleton, and this step functions in "outside in" signaling to promote ICAM-1 clustering around transmigrating leukocytes. Specific Aim 1 will elucidate the role of ICAM-1 cytoplasmic tail in leukocyte transmigration using biochemical and biophysical techniques as well as a newly developed live cell fluorescence imaging system and vascular endothelium that support both junctional and non-junctional transmigration. Specific Aim 2 will test the role of the ICAM-1 tail in chemoattractant driven leukocyte transmigration in an in vivo model of inflammation. Confocal intravital fluorescence imaging will visualize the sites of leukocyte transmigration (junctional or non-junctional route) using fluorescent markers of cell-cell junctions, and in parallel, determine whether the route of transmigration adversely effects vascular permeability. Specific Aim 3 will study the role of the ICAM-1 tail in an arterial endothelial cell model of inflammation in vitro. Endothelial monolayers of an arterial or venous phenotype will be created in vitro by applying a pulsatile shear stress in an arterial or venous waveform using a recently described cone-plate apparatus. The adhesive interactions and sites of transmigration of blood monocytes and memory T cells with the above conditioned endothelium will be examined. The information gained from these studies will help delineate the ICAM-1 dependent mechanisms underlying leukocyte transmigration and may thus define new targets for therapeutic intervention in immune and inflammatory diseases.