Molecular & Force dynamics:leukocyte adhesion molecules

描述(由申请人提供)： 中性粒细胞从循环到炎症血管部位的募集是一个精心策划的多步骤过程，由白细胞和内皮细胞黏附分子(包括选择素、整合素和ICAM)的膜表达上调和功能激活启动。在转基因小鼠模型中，编码选择素或β2整合素的单个或多个基因揭示了这些分子在支持细胞-细胞和细胞-基质黏附方面的独特和重叠功能，以及一系列不同功能的细胞内信号。尽管在破译通过选择素和整合素的白细胞募集的分子解剖学方面取得了进展，但指导白细胞和可能作为治疗作用靶点的结合和信号事件的序列仍然缺乏。在我们之前的FIRST和R01奖项的任期内，我们发现了三条重要的参与规则，这些规则指导了本提案的具体目标：L)选择素在中性粒细胞招募中既作为黏附受体又作为信号转导受体；2)选择素和[32-整合素]的膜聚集对它们的功能至关重要，并通过涉及MAP激酶的主动转运发生；3)[32-整合素构象的变化决定了亲和力和膜聚集，这两者都调节中性粒细胞在内皮上的结合界面，我们将其称为炎性突触。主要的假设是，中性粒细胞通过精确调节粘附键的寿命和强度，从血流到炎症组织。该项目的目标1是确定选择素参与如何信号稳定的黏附和内皮迁移。在目标2中，我们将研究白细胞如何通过β2整合素调节黏附强度和寿命。目的研究CD18和选择素在小鼠皮肤创伤模型中对中性粒细胞信号转导和黏附的作用机制。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，目的是确定调控途径和分子靶点，以便更有效地预测和治疗炎症性疾病。

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.