Mechanisms of Neutrophil Activation

描述(申请人提供)：“储存操作的钙进入细胞”(SOCE)是诱导中性粒细胞激活的主要细胞内信号反应之一。最近通过鉴定细胞内钙离子感受器蛋白STIM1和STIM2以及质膜钙通道蛋白ORAI1、2和3来确定SOCE的分子机制。在免疫刺激的响应下，钙从内质网的细胞内“储藏物”中释放出来，导致STIM分子的构象变化，使它们与质膜上的ORAI通道蛋白物理结合，导致通道开放，允许细胞外钙进入。在淋巴细胞中，SOCE的丢失导致细胞增殖反应和细胞因子产生对各种刺激的反应较差。目前还没有关于中性粒细胞STIM/ORAI信号转导的研究。使用STIM1/-骨髓嵌合小鼠，我们发现SOCE的丢失导致中性粒细胞激活的深刻阻断。我们的初步证据表明，在中性粒细胞激活过程中，PKC酶是细胞外钙的靶标。由于中性粒细胞功能缺陷，在酵母多糖急性腹膜炎模型中，STIM1/-嵌合体免受组织损伤，而在肝脏缺血再灌注模型中，STIM1/-嵌合体的组织损伤显著减少。为了扩大这些观察，我们提出了一系列实验来：1)确定SOCE导致中性粒细胞激活的分子机制，2)产生缺乏单个STIM或Orai分子的中性粒细胞系特异性突变体，以确定哪些在中性粒细胞激活中最重要，3)开发针对Orai蛋白的新型单链mAb阻断试剂，这将使我们能够测试在持续的炎症反应中停止中性粒细胞中的SOCE是否将限制组织损伤。我们将通过生化、遗传学和化学遗传学的方法来检验PKCs是中性粒细胞钙的靶标这一假设。在小鼠的STim和Orai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。我们将通过开发中性粒细胞STIM1、STIM2、ORAI1和ORAI2的中性粒细胞系特异性突变体来确定哪些STIM和ORAI分子在中性粒细胞中最重要。最后，我们将利用加州大学旧金山分校和辉瑞公司的新合作开发针对Orai1的新型单链单抗，以测试阻断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SOCE中涉及的机制和蛋白质，然后询问靶向这些蛋白质是否会逆转炎症性疾病。考虑到我们在STIM1-/-小鼠身上的初步发现的新颖性，实现这些目标将是炎症研究的一大进步。

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.