Molecular Mechanisms of Leukocyte Activation

白细胞激活的分子机制

基本信息

批准号：
8074999
负责人：
RICHARD D YE
金额：
$ 38.86万
依托单位：
UNIVERSITY OF ILLINOIS AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
1993
资助国家：
美国
起止时间：
1993-01-01 至 2015-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8074999
关键词：
1,2-diacylglycerol 4-ethoxymethylene-2-phenyl-2-oxazoline-5-one Abbreviations Acute Bacteria Bacterial Proteins Binding Blood Vessels Bone Marrow Cells Chemotactic Factors Clinical Complex Cytoplasmic Granules Data Dependency Diglycerides Disease Endothelial Cells Enzymes Equilibrium Event Family Fluorescein Fluorescence Fluorescence Resonance Energy Transfer Generations Grant Guanine Nucleotide Exchange Factors Health Benefit Host Defense Host Defense Mechanism Immune Individual Inflammation Inflammatory Inflammatory Response Injection of therapeutic agent Injury Leukocytes MAP kinase phosphatase MKP-5 MAPK14 gene MEKs Maintenance Measures Mediating Membrane Microbe Modeling Molecular Molecular Conformation Mus Mutagenesis NADP Organ failure Output Oxidants Oxidases Peroxidases Phagocytes Phagocytosis Phosphatidylserines Phosphorylation Phosphotransferases Play Production Proline-Rich Domain Protein Isoforms Protein Kinase Proteins Reaction Reactive Oxygen Species Regulation Research Resistance Role Signal Transduction Site Superoxides System Testing Therapeutic Intervention Tissues Work bactericide base cytochrome b558 design extracellular fMet-Leu-Phe receptor fungus human CYBA protein in vivo insight leukocyte activation macrophage methionyl-leucyl-phenylalanine mouse model mutant myristoylation neutrophil neutrophil cytosol factor 67K novel prevent public health relevance research study vascular inflammation

项目摘要

DESCRIPTION (provided by applicant): Chemoattractant-induced phagocyte activation is an important mechanism of host defense. In phagocytes, induced activation of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase leads to robust production of reactive oxygen species (ROS), which is essential for the elimination of ingested bacteria and fungi. However, extracellular ROS production by phagocyte NADPH oxidase (also termed Nox2) can be harmful to the tissue and is a major cause of vascular injury in acute inflammation. This revised competing renewal application aims to understand how chemoattractant-induced neutrophil superoxide generation is regulated at the molecular and cellular levels. Building on the systems we developed and preliminary results obtained in the previous grant cycle, the application will focus on the critical roles for p47phox in the assembly of an NADPH oxidase. Experiments are proposed in 3 specific aims to challenge existing concept and identify novel regulatory mechanisms for NADPH oxidase activation. Aim 1 will characterize Akt isoforms in chemoattractant-induced ROS production. In neutrophil research, Akt has been taken as one class of protein kinases functionally, but our preliminary study has led to an unexpected finding that the two major Akt isoforms in neutrophils play different roles in NADPH oxidase activation. Experiments are proposed to test the hypothesis that membrane translocation of an Akt isoform dictates its ability to mediate NADPH oxidase activation, and to examine whether the two Akt isoforms phosphorylates p47phox differently. Aim 2 is based on our recent characterization of a mutant p47phox protein that mediates spontaneous superoxide production in the absence of physical interaction with p67phox. Experiments are designed to characterize a potentially novel mechanism for 47phox-mediated conformational change in cytochrome b558 that facilitates the assembly of a functional NADPH oxidase complex. Aim 3 will investigate an important regulatory mechanism for p47phox-dependent oxidant production. We will examine the negative regulation of NADPH oxidase by MAP kinase phosphatase 5 (MKP5). Using in vivo and ex vivo approaches, we will investigate how MKP5 protects against LPS-induced vascular injury through suppression of neutrophil ROS production in a mouse model of vascular inflammation. Collectively, these studies are expected to provide novel insights into the activation and inactivation mechanisms of phagocyte NADPH oxidase, thereby facilitating therapeutic intervention of ROS-mediated tissue injury. PUBLIC HEALTH RELEVANCE: Phagocytes (a class of white blood cells that engulf bacteria and fungi) produce large amounts of reactive oxygen species that are toxic to bacteria and to host tissues. A balanced act of phagocyte activation is important for the maintenance of host defense capability and for minimizing unwanted tissue injury. The proposed studies will investigate how these cells are regulated by intrinsic mechanisms that prevent over-production of reactive oxygen species during acute inflammation, and how the machinery for oxidant production is assembled and activated when phagocytes are stimulated. Information derived from these studies is expected to have health benefit by reducing inflammatory tissue injury and the resulting failure of organ functions.

描述（由申请人提供）：趋化剂诱导的吞噬细胞激活是宿主防御的重要机制。在吞噬细胞中，诱导的烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶的激活会导致活力的活性氧（ROS）的强大产生，这对于消除摄入的细菌和真菌是必不可少的。然而，吞噬细胞NADPH氧化酶（也称为NOX2）的细胞外ROS产生可能对组织有害，并且是急性炎症中血管损伤的主要原因。这项修订的竞争性更新应用旨在了解趋化因子诱导的中性粒细胞超氧化物的产生如何在分子和细胞水平下调节。在我们开发的系统和在上一个赠款周期中获得的初步结果的基础上，该应用将重点关注P47phox在NADPH氧化酶组装中的关键作用。在3个特定目的中提出了实验，以挑战现有概念并确定NADPH氧化酶激活的新型调节机制。 AIM 1将表征化学吸引剂诱导的ROS产生中Akt同工型。在中性粒细胞研究中，AKT在功能上被视为一类蛋白激酶，但是我们的初步研究导致了一个意外的发现，即中性粒细胞中的两个主要AKT同工型在NADPH氧化酶激活中起着不同的作用。提出了实验来检验Akt同工型的膜易位决定其介导NADPH氧化酶活性的能力的假设，并检查两个AKT同工型是否磷酸化P47Phox的磷酸化。 AIM 2基于我们最近对突变体P47Phox蛋白的表征，该突变体P47Phox蛋白在没有与P67Phox的物理相互作用的情况下介导自发性超氧化物的产生。实验旨在表征细胞色素B558中47phox介导的构象变化的潜在新机制，该机制促进了功能性NADPH氧化酶复合物的组装。 AIM 3将研究P47Phox依赖性氧化剂产生的重要调节机制。我们将通过MAP激酶磷酸酶5（MKP5）检查NADPH氧化酶的阴性调节。使用体内和离体方法，我们将研究MKP5在血管炎症的小鼠模型中如何通过抑制中性粒细胞ROS的抑制来防止LPS诱导的血管损伤。总的来说，这些研究预计将提供有关吞噬细胞NADPH氧化酶的激活和失活机制的新见解，从而促进ROS介导的组织损伤的治疗干预。公共卫生相关性：吞噬细胞（一类吞噬细菌和真菌的白细胞）产生大量对细菌有毒并宿主组织有毒的活性氧。吞噬细胞激活的平衡行为对于维持宿主防御能力和最大程度地减少不良组织损伤至关重要。拟议的研究将研究这些细胞如何受到固有机制的调节，这些机制可以防止在急性炎症过程中过量产生活性氧，以及当刺激吞噬细胞时如何组装和激活氧化剂的机械。预计从这些研究中得出的信息将通过减少炎症组织损伤和器官功能失败来具有健康益处。