Molecular and Force Dynamics: Leukocyte Adhesion Molecules

描述(由申请人提供)：将白细胞招募到急性炎症部位是一个精心策划的过程，由白细胞和内皮细胞黏附分子(CAM)的膜表达和功能激活启动，包括选择素、整合素和免疫球蛋白超家族配体。在R01的任期内出现了一系列统一的主题，这些主题提供了分子尺度的洞察，以了解PMN如何在从滚动到牢固阻止和跨内皮迁移的转变中整合力量作为空间-机械线索：1)选择素被赋予机械和生化性质，使其能够作为粘附性受体和信号转导受体发挥作用；2)切应力和跨膜钙释放激活的钙通道(CRAC)调节细胞内钙通量，其功能是同步整合素介导的停滞和细胞迁移；3)22-整合素亲和力和结合ICAM-1的键机制通过调节跨内皮迁移的内外信号提供一种把关机制。在这一竞争更新中，我们应用我们创新的血管模拟微流体通道与实时免疫荧光成像相结合，以追求以下特定目标：1)检测钙释放激活的钙流量在中性粒细胞招募的多步骤过程中的协调作用。2)确定中性粒细胞表面的E-选择素配体如何作为机械转导分子触发和放大炎症内皮上整合素活化的趋化因子信号。3)确定LFA-1中性粒细胞形状极化由外向内信号的分子和力学机制。我们的策略需要使用新鲜分离的人中性粒细胞和小鼠炎症模型，首要目标是确定炎症性疾病的预后和治疗的调控途径和分子靶点。

DESCRIPTION (provided by applicant): Recruitment of leukocytes to sites of acute inflammation is a finely orchestrated process initiated by membrane expression and functional activation of leukocyte and endothelial cell adhesion molecules (CAMs) including selectins, integrins, and Ig-super family ligands. A set of unifying themes have emerged over the tenure of this R01 which provide molecular scale insight into how PMNs integrate force as a spatio-mechanical cue in the transition from rolling to firm arrest and transendothelial migration: 1) Selectins are endowed with mechanical and biochemical properties which allow them to function as both adhesive and signal transduction receptors; 2) Shear stress and transmembrane calcium release-activated Ca2+ (CRAC) channels regulate intracellular calcium flux which functions to synchronize integrin mediated arrest and cell migration; 3) 22-integrin affinity and bond mechanics in binding ICAM-1 provide a gatekeeper mechanism via regulation of outside-in signaling of transendothelial migration. In this competitive renewal we apply our innovative vascular mimetic microfluidic channels combined with real-time immunofluorescence imaging to pursue the following specific aims: 1) Examine the role of calcium release activated calcium flux in orchestration of the multistep process of neutrophil recruitment. 2) Determine how E-selectin ligands on neutrophils serve as mechano-transducers that trigger and amplify chemokine signaling of integrin activation on inflamed endothelium 3) define the molecular and mechanical mechanisms underlying LFA-1 outside-in signaling of neutrophil shape polarization. Our strategy entails the use of freshly isolated human neutrophils and murine models of inflammation with the overarching goal of identifying regulatory pathways and molecular targets for prognosis and treatment of inflammatory diseases.