Mechanisms of Neutrophil Activation

中性粒细胞激活机制

基本信息

批准号：
8991703
负责人：
Clifford A Lowell
金额：
$ 39.63万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-07-01 至 2018-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8991703
关键词：
Acute Adult Animals Arthritis Autoimmune Process Binding Biochemical Genetics Bone Marrow Calcium Calcium Channel Calcium Signaling Calcium-Binding Proteins Cell membrane Cells Chimera organism Cloning Collaborations Complex Cytoplasmic Protein Data Defect Development Disease Disease Progression Disease model Embryo Endoplasmic Reticulum Enzymes Fetal Liver Generations Genes Genetic Goals Health Immune Immunologic Deficiency Syndromes Individual Infection Inflammation Inflammatory Inflammatory Response Injury Ischemia Knockout Mice Ligands Liver Stem Cell LoxP-flanked allele Lymphocyte Mediating Membrane Methods Modeling Molecular Molecular Target Mus Mutant Strains Mice NADPH Oxidase Neutrophil Activation Peptide Hydrolases Peritonitis Phagocytes Play Process Production Protein Isoforms Protein Kinase C Proteins Reagent Reperfusion Injury Reperfusion Therapy Research Respiratory Burst Rheumatoid Arthritis Role S100A8 gene STIM1 gene Second Messenger Systems Series Signal Pathway Signal Transduction Stimulus TYRP1 gene Testing Therapeutic Tissues Vasculitis Work Zymosan animal tissue chemical genetics citrate carrier cytokine design extracellular genetic approach immunopathology killings liver ischemia man mouse model mutant neutrophil new therapeutic target novel novel strategies pathogen receptor release of sequestered calcium ion into cytoplasm research study response second messenger sensor

项目摘要

DESCRIPTION (provided by applicant): "Store-operated calcium entry" (SOCE) into cells is one of the major intracellular signaling responses that induce neutrophil activation. The molecular mechanism of SOCE has recently been defined through the identification of the intracellular calcium sensor proteins, STIM1 and STIM2, and the plasma membrane calcium channel proteins ORAI1, 2 and 3. In response to immune stimuli, calcium is released from intracellular "stores" in the endoplasmic reticulum, which leads to a conformational change in the STIM molecules, allowing them to physically associate with ORAI channel proteins in the plasma membrane, leading to channel opening allowing entry of extracellular calcium. In lymphocytes, the loss of SOCE results in poor cellular proliferative responses and cytokine production in response to a variety of stimuli. There have been no studies of STIM/ORAI signaling in neutrophils. Using stim1-/- bone marrow chimeric mice, we have found that loss of SOCE leads to a profound block in neutrophil activation. Our preliminary evidence suggests that PKC enzymes are the target of extracellular calcium during neutrophil activation. As a result of this defective neutrophil function, stim1-/- chimeras are protected from tissue injury in the zymosan model of acute peritonitis and show significantly reduced tissue injury in a hepatic ischemia reperfusion model. To expand on these observations, we propose a series of experiments to: 1) determine the molecular mechanisms by which SOCE leads to neutrophil activation, 2) generate neutrophil lineage specific mutants lacking individual Stim or Orai molecules, to determine which are most important in neutrophil activation, 3) develop novel single chain mAb blocking reagents, targeting Orai proteins, that will allow us to test whether cessation of SOCE in neutrophils during an ongoing inflammatory response will limit tissue injury. We will test the hypothesis that PKCs are the target of calcium in neutrophils through biochemical, genetic and chemical genetic approaches. Of the Stim and Orai proteins in mice, it is unclear which play the dominant role in SOCE in neutrophils. We will determine which of the Stim and Orai molecules are most important in neutrophils by development of neutrophil lineage specific mutants of stim1, stim2, orai1 and orai2 in mice. Finally, we will take advantage of a new UCSF / Pfizer Corp collaboration to develop novel single chain mAbs that will target Orai1, to test the hypothesis that blockade of SOCE will reverse ongoing inflammatory disease. Our goal is to determine the mechanisms and proteins involved in SOCE in neutrophils, then ask whether targeting these proteins will reverse inflammatory disease. Given the novelty of our initial findings using stim1-/- mice, achieving these goals will be a major advance in inflammation research.

描述（由申请人提供）：“商店经营的钙进入”（SOCE）进入细胞是诱导嗜中性粒细胞激活的主要细胞内信号传导反应之一。最近，通过鉴定细胞内钙传感器蛋白，stim1和stim2以及质膜钙通道蛋白ORAI1、2和3。钙在对内质的刺激性变化中允许刺激性变化，从而，SOCE的分子机制是通过鉴定钙蛋白ORAI1、2和3的质膜钙通道蛋白ORAI1、2和3定义的定义。与质膜中的Orai通道蛋白相结合，导致通道打开，允许进入细胞外钙。在淋巴细胞中，SOCE的丧失导致细胞增殖反应不良和响应各种刺激的细胞因子产生。中性粒细胞中没有刺激/ORAI信号传导的研究。使用Stim1 - / - 骨髓嵌合小鼠，我们发现SOCE丧失会导致中性粒细胞激活的深刻障碍。我们的初步证据表明，PKC酶是嗜中性粒细胞活化过程中细胞外钙的靶标。由于这种缺陷的中性粒细胞功能，刺激性嵌合体在急性腹膜炎的Zymosan模型中受到了组织损伤的保护，并在肝缺血再灌注模型中显示出显着降低的组织损伤。 To expand on these observations, we propose a series of experiments to: 1) determine the molecular mechanisms by which SOCE leads to neutrophil activation, 2) generate neutrophil lineage specific mutants lacking individual Stim or Orai molecules, to determine which are most important in neutrophil activation, 3) develop novel single chain mAb blocking reagents, targeting Orai proteins, that will allow us to test whether cessation of SOCE在持续的炎症反应过程中，中性粒细胞将限制组织损伤。我们将通过生化，遗传和化学遗传学方法来检验假说，即PKC是中性粒细胞中钙的靶标。在小鼠中的刺激蛋白和ORAI蛋白质中，尚不清楚哪个在中性粒细胞中发挥了主要作用。我们将通过在小鼠中的STIM1，STIM2，ORAI1和ORAI2的中性粒细胞特异性突变体的发展来确定哪种刺激和ORAI分子在中性粒细胞中最重要。最后，我们将利用新的UCSF / Pfizer Corp协作来开发靶向Orai1的新型单链MAB，以检验以下假设：SOCE的封锁将逆转持续的炎症性疾病。我们的目标是确定中性粒细胞中SOCE的机制和蛋白质，然后询问靶向这些蛋白质是否会反向炎症性疾病。鉴于我们使用STIM1 - / - 小鼠的初步发现的新颖性，实现这些目标将是炎症研究的重大进步。