Mechanisms of Neutrophil Activation

描述（由申请人提供）：“储存操作钙进入”（SOCE）进入细胞是诱导中性粒细胞激活的主要细胞内信号反应之一。最近，通过鉴定细胞内钙传感器蛋白STIM1和STIM2以及质膜钙通道蛋白ORAI1、2和3，已经确定了SOCE的分子机制。作为对免疫刺激的反应，钙从内质网的细胞内“储存”中释放出来，这导致STIM分子的构象改变，使它们能够与质膜上的ORAI通道蛋白物理结合，导致通道打开，允许细胞外钙进入。在淋巴细胞中，SOCE的缺失导致细胞对各种刺激的增殖反应和细胞因子产生不良。中性粒细胞中STIM/ORAI信号的研究尚未见报道。使用stim1-/-骨髓嵌合小鼠，我们发现SOCE的缺失导致中性粒细胞激活的严重阻断。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的目标。由于中性粒细胞功能的缺陷，stim1-/-嵌合体在急性腹膜炎酶酶酶模型中免受组织损伤，在肝缺血再灌注模型中表现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导致中性粒细胞活化的分子机制，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活化中最重要，3)开发针对Orai蛋白的新型单链单抗阻断试剂，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是否会限制组织损伤。我们将通过生化、遗传和化学遗传的方法来检验PKCs是中性粒细胞钙靶的假设。在小鼠的Stim和Orai蛋白中，尚不清楚哪一种在中性粒细胞的SOCE中起主导作用。我们将通过在小鼠中培养中性粒细胞谱系特异性突变体stim1、stim2、orai1和orai2来确定哪个Stim和Orai分子在中性粒细胞中最重要。最后，我们将利用UCSF与辉瑞公司的合作开发新的单链单克隆抗体，以Orai1为靶点，来验证阻断SOCE将逆转持续炎症性疾病的假设。我们的目标是确定中性粒细胞中SOCE的机制和相关蛋白，然后询问靶向这些蛋白是否会逆转炎症性疾病。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.