Neutrophil-platelet interactions during vascular inflammation

摘要 实时荧光活体显微镜提供了令人信服的证据表明，血小板-中性粒细胞 血管内皮细胞激活的相互作用是微血管闭塞的主要决定因素 发炎。以往的研究表明，中性粒细胞Mac-1(αMβ2整合素)在 炎症条件下的血小板-中性粒细胞相互作用。此外，我们发现中性粒细胞AKT2是一种关键的 细胞激活过程中Mac-1膜转位和激活的调节因子。然而，详细的 AKT2调节Mac-1功能的机制尚不清楚。我们的初步研究表明 中性粒细胞AKT2使基质相互作用分子1(STIM1)磷酸化，STIM1是一种钙离子感受器，对储存- 操作钙离子内流(SOCE)。重要的是，模拟STIM1上AKT2磷酸化位点的多肽显著 抑制中性粒细胞活化过程中的SOCE，提示AKT2磷酸化的STIM1在 诱导SOCE。另一个关键结果是，中性粒细胞NADPH氧化酶2(NOX2)在介导Mac-2的表达中起重要作用。 1激活而不是膜转位。在这项提议中，我们将检验中性粒细胞 Akt2诱导的SOCE刺激巨噬细胞产生ROS和配体结合活性 1，从而促进有害的中性粒细胞-血小板相互作用和微血管闭塞。 炎症状态。我们提出了以下具体目标来检验这一假设： 目的1：确定中性粒细胞AKT2在刺激STIM1和Mac-1功能中的作用。我们将定义 AKT2在STIM1磷酸化介导SOCE中的作用我们还将讨论AKT2是如何诱导 SOCE刺激Mac-1的配体结合活性。 目的2：明确中性粒细胞AKT2诱导的SOCE在激活NOX2和硫醇二硫键中的作用 暴露在Mac-1中。我们将确定AKT2诱导的SOCE在激活NOX2中的作用并研究 AKT2-NOX2信号是否诱导Mac-1胞外区的硫醇交换并刺激其 配体结合功能是通过细胞表面PDI活性实现的。我们还将鉴定变构二硫化物中的半胱氨酸残基 Mac-1债券。 目的3：探讨AKT2诱导的SOCE在中性粒细胞-血小板调节中的病理生理作用 相互作用和微血管闭塞是血管炎症的中心机制。我们会 确定AKT2诱导的SOCE在中性粒细胞募集、中性粒细胞-血小板相互作用以及 炎症状态下的血管闭塞。此外，我们还将研究AKT2诱导的SOCE的作用 镰状细胞病血管闭塞事件中异型细胞-细胞相互作用和聚集的信号转导 (SCD)患者和小鼠。

ABSTRACT Real-time fluorescence intravital microscopy has provided compelling evidence that platelet-neutrophil interactions on the activated endothelium are a major determinant of microvascular occlusion during vascular inflammation. Previous studies demonstrated that neutrophil Mac-1 (αMβ2 integrin) plays a critical role in platelet-neutrophil interactions under inflammatory conditions. Further, we found that neutrophil AKT2 is a critical regulator for the membrane translocation and activation of Mac-1 during cell activation. However, the detailed mechanisms by which AKT2 regulates Mac-1 function remain unclear. Our preliminary studies showed that neutrophil AKT2 phosphorylates stromal interaction molecule 1 (STIM1), a Ca2+ sensor essential for store- operated Ca2+ entry (SOCE). Importantly, peptides mimicking AKT2 phosphorylation sites on STIM1 significantly inhibited SOCE during neutrophil activation, suggesting the important role of AKT2-phosphorylated STIM1 in inducing SOCE. Another key result is that neutrophil NADPH oxidase 2 (NOX2) is important for mediating Mac- 1 activation but not membrane translocation. In this proposal, we will test the novel hypothesis that neutrophil AKT2-induced SOCE stimulates reactive oxygen species (ROS) generation and ligand-binding activity of Mac- 1, thereby promoting the deleterious neutrophil-platelet interaction and microvascular occlusion under inflammatory conditions. We propose the following specific aims to test this hypothesis: AIM 1: Determine the role of neutrophil AKT2 in stimulating STIM1 and Mac-1 function. We will define the role of AKT2 in mediating SOCE through STIM1 phosphorylation. We will also address how AKT2-induced SOCE stimulates ligand-binding activity of Mac-1. AIM 2: Define the role of neutrophil AKT2-induced SOCE in activating NOX2 and thiol-disulfide exposure in Mac-1. We will determine the role of AKT2-induced SOCE in activating NOX2 and investigate whether AKT2-NOX2 signaling induces thiol exchange in the extracellular domain of Mac-1 and stimulates its ligand-binding function through cell surface PDI activity. We will also identify Cys residues in allosteric disulfide bonds of Mac-1. AIM 3: Investigate the pathophysiologic role of AKT2-induced SOCE in mediating neutrophil-platelet interaction and microvascular occlusion as a central mechanism of vascular inflammation. We will determine the role of AKT2-induced SOCE in neutrophil recruitment, neutrophil-platelet interactions, and vascular occlusion under inflammatory conditions. Further, we will also examine the role of AKT2-induced SOCE signaling in heterotypic cell-cell interaction and aggregation during vaso-occlusive events in sickle cell disease (SCD) patients and mice.