Cytokines, Oxidants, Neutrophils and Lung Injury

本次更新申请（13-17年）中提出的研究的总体目标是解决促炎细胞因子如TNFalpha介导内皮细胞中粘附分子ICAM-1表达的关键信号通路，从而诱导强中性粒细胞（PMN）粘附。我们已经证明了内皮细胞中氧化信号在激活ICAM-1转录中的重要功能，以及NADPH氧化酶复合物在ICAM-1表达信号传导中的关键作用。此外，我们已经确定了涉及PKC zeta和PI3激酶/Akt的信号通路，这些信号通路可能通过NADPH氧化酶激活氧化信号。由于PMN与内皮细胞的稳定和牢固的ICAM-1依赖性粘附需要独立于ICAM-1表达的快速蛋白合成，以及蛋白质合成依赖性ICAM-1表达的延迟，我们将探索其表达的早期过程，包括内皮细胞中构成ICAM-1的细胞表面改变，以及需要从头合成蛋白质的延迟表达。在即将提出的研究中，我们将确定(1)gp91/phox和p41/phox NADPH氧化酶亚基在介导ICAM-1表达和PMN粘附内皮细胞中的氧化信号，(2)PKCzeta在激活氧化信号，从而在NF-kappaB激活和ICAM-1表达中的作用，最后(4)gtpase在氧化信号传导和介导ICAM-1表达和PMN粘附的早发蛋白合成独立成分中的作用。研究将利用分子方法来剖析PMN在肺部的隔离和迁移的信号通路，以及肺微血管通透性和水肿形成的生理评估。随着这些研究的完成，我们将进一步了解TNFalpha诱导内皮细胞ICAM-1表达的机制，从而介导PMN不适当的粘附和跨肺微血管屏障的迁移。我们希望确定氧化信号和信号通路在介导ICAM-1表达中的作用，从而促进内皮细胞对PMN的粘附及其在血管壁上的迁移。这些研究将有助于更好地了解炎症性疾病状态的基础，如与PMN隔离和迁移增加相关的成人呼吸窘迫综合征（ARDS），从而开发出阻断特定信号通路的药物。

The overall objective of the proposed studies in this renewal application (yr. 13-17) is to address the critical signaling pathways by which pro- inflammatory cytokines such as TNFalpha mediate the expression of adhesion molecule, ICAM-1, in endothelial cells and thereby induce firm neutrophil (PMN) adhesion. We have shown an important function of oxidant signaling in endothelial cells in activating transcription of ICAM-1 and also a critical role of the NADPH oxidase complex in signaling ICAM-1 expression. In addition, we have identified signaling pathways involving PKC zeta and PI3 kinase/Akt that may activate oxidant signaling via NADPH oxidase. As stable and firm ICAM-1 dependent adhesion of PMN to endothelial cells will require rapid-onset protein synthesis independent of ICAM-1 expression as well as delayed protein synthesis-dependent ICAM-1 expression, we will explore both the early course of its expression involving cell surface alterations in the constitutive ICAM-1 in endothelial cells as well as delayed expression requiring de novo protein synthesis. In the proposed studies, we will determine (1) oxidant signaling of the gp91/phox and p41/phox NADPH oxidase subunits in mediating ICAM-1 expression and PMN adhesion to endothelial cells, (2) role of PKCzeta in activating oxidant signaling, and thereby in NF-kappaB activation and ICAM-1 expression, and finally (4) role of GTPases in oxidant signaling and mediating the early-onset protein synthesis-independent component of ICAM-1 expression and PMN adhesion. Studies will utilize molecular approaches to dissect the signaling pathways as well as physiological assessments of PMN sequestration and migration in lungs as well as pulmonary microvascular permeability and edema formation. With the completion of these studies, we will advance the understanding of the mechanisms by which TNFalpha induces endothelial cell ICAM-1 expression, and thereby mediates inappropriate PMN adhesion and migration across the pulmonary microvessel barrier. We hope to define the role of oxidant signaling and the signaling pathways in mediating ICAM-1 expression, and in thereby promoting endothelial adhesivity to PMN and their migration across the vessel wall. These studies will be important in providing a better understanding of the basis of inflammatory disease states such as the adult respiratory distress syndrome (ARDS) associated with increased PMN sequestration and migration so that agents can be developed to block specific signaling pathways.