Molecular and Force Dynamics: Leukocyte Adhesion Molecules

白细胞在急性炎症部位的募集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，其中包括选择素、整合素和Ig超家族配体。在R01的任期内，出现了一系列统一的主题，这些主题开始定义控制从白细胞滚动到牢固阻止和跨内皮细胞迁移的分子和力动力学：1)选择素具有机械和生化特性，使其能够作为粘附性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阻止和细胞迁移；3)β2-整合素亲和力和亲和力提供与跨内皮细胞迁移的内向外信号相关的把关机制。这些中性粒细胞参与的规则导致了最初的假设，即在急性炎症条件下，黏附和信号事件构成了一个系统，通过设计促进快速和不可逆转的迁移，同时维持基本的内皮屏障功能。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)确定中性粒细胞上的E-选择素配体如何作为机械转导，触发和放大整合素活化的炎症内皮细胞上的趋化因子信号。3)在皮肤创伤模型中确定内皮屏障功能和中性粒细胞募集是如何在从急性炎症向慢性炎症的转变中耦合的。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。

Recruitment of leukocytes to acute sites of 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 begin to define the molecular and force dynamics that govern the transition from leukocyte 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) β2-integrin affinity and avidity provide gatekeeper mechanisms associated with inside-out signaling of transendothelial migration. These rules of neutrophil engagement have lead to the primary hypothesis that under conditions of acute inflammation the adhesive and signaling events constitute a system that by design promotes rapid and irreversible transmigration while maintaining basal endothelial barrier function. 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 actvation on inflamed endothelium 3) Determine how endothelial barrier function and neutrophil recruitment are coupled in the transition from acute to chronic inflammation studied in a cutaneous wound model. 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.