Mechanisms of Neutrophil Activation

描述（由申请人提供）：“钙库操纵的钙进入”（SOCE）进入细胞是诱导嗜中性粒细胞活化的主要细胞内信号传导反应之一。SOCE的分子机制最近已经通过鉴定细胞内钙传感器蛋白STIM 1和STIM 2以及质膜钙通道蛋白ORAI 1、2和3而被定义。响应于免疫刺激，钙从内质网中的细胞内“储存”释放，这导致STIM分子的构象变化，使其与质膜中的奥赖通道蛋白物理缔合，导致通道开放，允许细胞外钙进入。在淋巴细胞中，SOCE的缺失导致响应于各种刺激的不良细胞增殖反应和细胞因子产生。目前还没有中性粒细胞中STIM/奥赖信号传导的研究。 使用stim 1-/-骨髓嵌合小鼠，我们发现SOCE的缺失导致中性粒细胞活化的深刻阻滞。我们的初步证据表明，PKC酶是中性粒细胞活化过程中细胞外钙离子的靶点。由于这种缺陷的中性粒细胞功能，stim 1-/-嵌合体在急性腹膜炎的酵母聚糖模型中被保护免受组织损伤，并且在肝缺血再灌注模型中显示出显著减少的组织损伤。 为了扩展这些观察结果，我们提出了一系列实验：1）确定SOCE导致嗜中性粒细胞活化的分子机制，2）产生缺乏单个Stim或奥赖分子的嗜中性粒细胞谱系特异性突变体，以确定哪种在嗜中性粒细胞活化中最重要，3）开发靶向奥赖蛋白的新型单链mAb阻断剂，这将使我们能够测试在持续的炎症反应期间中性粒细胞中SOCE的停止是否会限制组织损伤。我们将通过生物化学、遗传学和化学遗传学方法来检验PKC是中性粒细胞中钙的靶点这一假设。在小鼠的Stim和奥赖蛋白中，尚不清楚哪种蛋白在中性粒细胞的SOCE中起主导作用。我们将通过在小鼠中开发中性粒细胞谱系特异性的stim 1、stim 2、orai 1和orai 2突变体来确定哪种Stim和奥赖分子在中性粒细胞中最重要。最后，我们将利用UCSF / Pfizer公司的新合作开发新的单链mAb，靶向Orai 1，以验证阻断SOCE将逆转正在进行的炎症性疾病的假设。 我们的目标是确定中性粒细胞中参与SOCE的机制和蛋白质，然后询问靶向这些蛋白质是否会逆转炎症性疾病。鉴于我们使用stim 1-/-小鼠的初步发现的新奇，实现这些目标将是炎症研究的重大进展。

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.