Molecular and Force dynamics of leukocyte adhesion molecules

描述（由申请人提供）： 从循环到炎症的血管部位的神经元募集是一个精细协调的多步骤过程，其通过上调白细胞和内皮细胞粘附分子（CAM）（包括选择、整联蛋白和ICAM）的膜表达和功能活化而启动。其中编码选择素或β 2-整联蛋白的单个或多个基因的转基因小鼠模型已经揭示了这些分子在支持细胞-细胞和细胞-基质粘附中的独特和重叠功能，以及不同功能组的细胞内信号传导。尽管通过选择素和整联蛋白破译白细胞募集的分子解剖学方面取得了进展，但缺乏指导白细胞并可靶向治疗作用的结合和信号传导事件的序列。在我们之前的FIRST和R 01奖项的任期内，已经发现了三个重要的参与规则，这些规则指导了本提案的具体目标：1）选择素在中性粒细胞募集中作为粘附和信号转导受体发挥作用; 2）选择素和β 2-整联蛋白的膜聚集对其功能至关重要，并通过涉及MAP激酶的主动转运发生; 3）β 2-整合素构象的改变决定了亲和力和膜聚集，这两者都调节中性粒细胞在我们表示为炎症突触的界面处的内皮上的停滞。主要假设是中性粒细胞通过精确调节粘附键的寿命和强度来导航它们从血流到发炎组织的旅程。本项目的目的1是确定选择素如何参与信号稳定的粘附和内皮细胞迁移。在目标2中，我们将研究白细胞如何通过β 2-整合素调节粘附强度和寿命。目的3将验证和靶向中性粒细胞信号传导和粘附通过CD 18和选择素在小鼠皮肤创伤模型中的募集机制。我们的策略需要使用新鲜分离的人中性粒细胞和常规微循环的活体显微镜检查，目的是确定更有效的预后和治疗炎症性疾病的调控途径和分子靶点。

DESCRIPTION (provided by applicant): Neutrophil recruitment from the circulation to vascular sites of inflammation is a finely orchestrated multistep process initiated by upregulation of membrane expression and functional activation of leukocyte and endothelial cell adhesion molecules (CAMs) including selections, integrins, and ICAMs. Transgenic mouse models in which a single or multiple genes encoding selectins or beta2-integrins have revealed both unique and overlapping functions of these molecules in supporting cell-cell and cell-matrix adhesion, as well as intracellular signaling of a diverse set of functions. Despite the progress in deciphering the molecular anatomy of leukocyte recruitment via selectins and integrins the sequence of binding and signaling events that direct leukocytes and that may be targeted for therapeutic action is lacking. Over the tenure of our preceding FIRST and R01 awards three important rules of engagement have been discovered and these guide the specific aims of this proposal: l) Selectins function as both adhesive and signal transduction receptors in neutrophil recruitment; 2) membrane clustering of selectins and [32-integrins is critical to their function and occurs via active transport involving MAP kinases; 3) a shift in [32-integfin conformation determines affinity and membrane clustering both of which regulate neutrophil arrest on endothelium at an interface we denote the inflammatory synapse. The primary hypothesis is that neutrophils navigate their journey from the bloodstream to inflamed tissue by precise regulation of the lifetime and strength of adhesive bonds. Aim 1 of this project is to determine how selectin engagement signals stable adhesion and endothelial transmigration. In Aim 2, we will examine how leukocytes regulate adhesion strength and lifetime through beta2-integrins. Aim 3 will validate and target mechanisms of neutrophil signaling and adhesion via CD18 and selectins in recruitment to a cutaneous skin wound model in mouse. Our strategy entails the use of freshly isolated human neutrophils and intravital microscopy of routine microcirculation with the objective of identifying regulatory pathways and molecular targets for more effective prognosis and treatment of inflammatory diseases.