Protein disulfide isomerase in neutrophil recruitment and vascular inflammation

说明(申请人提供)：中性粒细胞是血管炎症和损伤过程中先天免疫反应所必需的。中性粒细胞重新聚集到血管损伤部位是一个多步骤的过程，包括中性粒细胞滚动、牢固黏附和迁移。选择素与其配体的相互作用介导中性粒细胞在炎症的内皮细胞上滚动，并进一步调节整合素的功能。随后，活化的LFA-1和Mac-1在中性粒细胞与内皮细胞的黏附和迁移中发挥关键作用。然而，整合素在中性粒细胞募集过程中被激活的分子机制仍不清楚。有人认为整合素中氧化还原敏感的硫醇-二硫键交换可能是其激活的重要因素。蛋白质二硫键异构酶(PDI)是一种定位于细胞表面的硫醇异构酶，在b3整合素的硫醇交换中发挥重要作用，从而调节b3整合素介导的血小板和内皮细胞功能。我们的假设是，中性粒细胞表面PDI通过促进氧化还原敏感的硫醇交换，将b2整合素转变为其在炎症过程中b2整合素介导的中性粒细胞募集所需的活性构象，来调节b2整合素的功能。基于我们的初步数据，在目标1中，我们将确定细胞表面PDI在b2整合素与其对偶受体ICAM-1相互作用中的作用，以及在b2整合素介导的中性粒细胞黏附和迁移中的作用。在目标2中，我们将确定PDI与b2整合素相互作用的分子机制，以调节它们的功能。在目标3中，我们将利用实时荧光活体显微镜研究炎症条件下细胞表面PDI在b2整合素介导的中性粒细胞黏附和迁移中的病理生理作用。更好地了解表面PDI如何调节b2整合素介导的中性粒细胞重新聚集到炎症部位，可能会导致新疗法的发展，以预防和治疗炎症和组织损伤。

DESCRIPTION (provided by applicant): Neutrophils are essential for the innate immune response during vascular inflammation and injury. Neutrophil recruitment into the site of vascular injury is a multi-step process consisting of neutrophil rolling, firm adhesion, and transmigration. Interaction of selectins with their ligands mediates neutrophil rolling over the inflamed endothelium and further regulates integrin function. Subsequently, activated LFA-1 and Mac-1 play critical roles in neutrophil adhesion to and transmigration across the endothelium. However, the molecular mechanism by which the integrins are activated during neutrophil recruitment remains unclear. It has been proposed that redox-sensitive thiol-disulfide exchange in integrins may be important for their activation. Protein disulfide isomerase (PDI), a cell surface localized thiol isomerase, plays an important role in thiol exchange in b3 integrins, thereby regulating b3 integrin-mediated platelet and endothelial cell function. It is our hypothesis that neutrophil surface PDI regulates b2 integrin function by facilitating redox-sensitive thiol exchange, shifting b2 integrin to its active conformation required for b2 integrin-mediated neutrophil recruitment during inflammation. Based on our preliminary data which strongly support the hypothesis, in Aim 1, we will define the role of cell surface PDI in the interaction of b2 integrin with its counter-receptor, ICAM-1 and in b2 integrin-mediated neutrophil adhesion and transmigration. In Aim 2, we will identify the molecular mechanisms by which PDI interacts with b2 integrins to regulate their function. In Aim 3, we will investigate the pathophysiological role of cell surface PDI in b2 integrin-mediated neutrophil adhesion and transmigration under inflammatory conditions using real-time fluorescence intravital microscopy in vivo. A better understanding of how surface PDI regulates b2 integrin-mediated neutrophil recruitment into the site of inflammation may lead to the development of novel therapeutics to prevent and treat inflammation and tissue injury.