Cytokines, Oxidants, Neutrophils and Lung Injury

本次续期申请中拟议研究的总体目标（年）。13-17）是为了解决促炎细胞因子如TNF α介导粘附分子ICAM-1在内皮细胞中的表达并由此诱导牢固的嗜中性粒细胞（PMN）粘附的关键信号传导途径。我们已经证明了内皮细胞中氧化剂信号在激活ICAM-1转录中的重要功能，以及NADPH氧化酶复合物在ICAM-1表达信号中的关键作用。此外，我们已经确定了涉及PKC zeta和PI 3激酶/Akt的信号通路，它们可能通过NADPH氧化酶激活氧化剂信号。由于稳定和坚定的ICAM-1依赖性粘附的中性粒细胞的内皮细胞将需要快速启动的蛋白质合成独立的ICAM-1的表达，以及延迟的蛋白质合成依赖性ICAM-1的表达，我们将探讨其表达的早期过程涉及细胞表面的变化，在内皮细胞中的组成型ICAM-1，以及延迟的表达需要从头蛋白质合成。在拟议的研究中，我们将确定（1）gp 91/phox和p41/phox NADPH氧化酶亚基在介导ICAM-1表达和PMN粘附至内皮细胞中的氧化剂信号传导，（2）PKCzeta在激活氧化剂信号传导中的作用，从而在NF-κ B激活和ICAM-1表达中的作用，（4）GTPases在氧化信号传导中的作用，介导ICAM-1表达和PMN粘附中的早发性蛋白质合成非依赖性成分。研究将利用分子方法来剖析信号通路以及肺中PMN隔离和迁移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TNF α诱导内皮细胞ICAM-1表达，从而介导不适当的PMN粘附和迁移通过肺微血管屏障的机制。我们希望确定氧化剂信号传导和信号传导途径在介导ICAM-1表达中的作用，从而促进内皮细胞对PMN的粘附性及其穿过血管壁的迁移。这些研究将是重要的，在提供一个更好地了解炎症性疾病状态的基础上，如成人呼吸窘迫综合征（ARDS）与增加中性粒细胞隔离和迁移，使代理商可以开发阻断特定的信号通路。

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.