Molecular and Force Dynamics: Leukocyte Adhesion Molecules

白细胞向急性炎症部位的募集是由白细胞和内皮细胞粘附分子（CAM）（包括选择素、整联蛋白和Ig超家族配体）的膜表达和功能活化引发的精细协调的过程。在R 01的任期内出现了一组统一的主题，其开始定义了控制从白细胞滚动到牢固停滞和跨内皮迁移的转变的分子和力动力学：1）选择素被赋予机械和生物化学性质，这使得它们能够充当粘附和信号转导受体; 2）剪切应力和跨膜钙释放激活的Ca ~（2+）（CRAC）通道调节细胞内钙流，其功能是同步整合素介导的阻滞和细胞迁移; 3）β2-整联蛋白亲和力和亲合力提供了与跨内皮迁移的由内而外信号传导相关的看门人机制。中性粒细胞参与的这些规则导致了主要假设，即在急性炎症条件下，粘附和信号传导事件构成了一个系统，该系统通过设计促进快速和不可逆的迁移，同时维持基础内皮屏障功能。在这次竞争性更新中，我们将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，以实现以下特定目标：1）检查钙释放激活的钙流量在中性粒细胞募集的多步骤过程中的作用。2)确定中性粒细胞上的E-选择素配体如何作为机械换能器，触发和放大炎症内皮上整联蛋白激活的趋化因子信号传导3）确定在皮肤伤口模型中研究的从急性到慢性炎症的转变中内皮屏障功能和中性粒细胞募集如何耦合。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，其总体目标是确定炎症疾病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.