Controlling the Upstream Migration of Neutrophils through the Modulation of Mac-1

摘要/摘要 免疫细胞从血流到炎症部位的运输能力，以便发现 感染病原体并将信息传递给其他免疫细胞是先天免疫反应的一个标志。 贩运是由白细胞粘附级联反应控制的，这是一个特征明确的、循序渐进的事件序列 在这个过程中，血液传播的免疫细胞系住、滚动、牢固地阻止、迁移，然后进入组织进行免疫 细胞功能。中性粒细胞是“第一反应者”，它会立即进入炎症部位。 在感觉到这种煽动性的侮辱之后。这些事件通常发生在毛细血管后小静脉内，在那里细胞 必须克服高剪切率才能结合到内皮细胞表面并最终通过内皮细胞表面迁移。 我们和其他人发现了一种有趣的现象，在这种现象中，某些起源于造血的细胞 -特别是T细胞和造血干细胞和祖细胞(HSPC)-将向上爬行，对抗 流动方向，在含有配体细胞间黏附分子-1(ICAM-1)的表面上。这是上游的 β2整合素，αLβ2，又称淋巴细胞功能相关抗原-1(LfA-1)介导了细胞的迁移。 1)与ICAM-1结合。最初有报道称，中性粒细胞无法在ICAM-1上上游爬行，甚至 尽管中性粒细胞表达LFA-1。中性粒细胞表达一种额外的结合ICAM-1、Mac-1、 当中性粒细胞被激活时，它会上调表达。我们假设中性粒细胞不能爬行 上游是因为Mac-1在中性粒细胞中主导结合ICAM-1，如果我们阻止或禁用Mac-1， 上游迁移的表型将恢复。我们的初步结果表明，通过阻止Mac-1， 中性粒细胞的上游迁移被恢复，从而为这一应用奠定了前提。 在这个应用中，我们将展示中性粒细胞确实可以通过抑制相互作用而向上爬行 Mac-1，使用分化的HL-60细胞和原代中性粒细胞。具体来说，在目标1中，我们将确定 利用抗体，中性粒细胞可以逆剪切流方向爬行的条件 阻断以确定哪些整合素抑制分化的HL-60细胞和原代细胞的上游迁移 人类中性粒细胞，并证实阻断Mac-1会导致上游迁移。然后，在《目标2》中，我们将 用RNA分析Mac-1缺陷的中性粒细胞的定向迁移偏好改变 干扰使缺失Mac-1和LFA-1的HL-60细胞株，并检测其定向 在纯化的分子表面以及刺激的人脐静脉内皮细胞上偏爱迁移。我们将验证 通过转导CD34+造血干细胞获得原代中性粒细胞突变体的结果 针对Mac-1的shRNA在其细胞因子体外诱导分化为中性粒细胞。这些研究将 最终确定关键表面受体负责引导或阻止上游 中性粒细胞的迁移。这些信息将通过以下方式保持改善患者预后的治疗潜力 更好地将中性粒细胞引导到炎症部位，化解炎症性疾病。

Summary/Abstract The ability of immune cells to traffic from the blood stream to the sites of inflammation in order to find infectious agents and transmit information to other immune cells is a hallmark of the innate immune response. Trafficking is governed by the leukocyte adhesion cascade, a well-characterized, step-wise sequence of events in which blood borne immune cells tether, roll, firmly arrest, migrate and then enter tissues to perform immune cell functions. Neutrophils are the “first responders” and will traffic to the sites of inflammation immediately upon sensing the inflammatory insult. These events normally occur within post-capillary venules, where cells must overcome high shear rates to bind to and eventually transmigrate through the endothelial surface. We and others have identified an interesting phenomenon wherein certain cells of hematopoietic origin - notably T-cells and hematopoietic stem and progenitor cells (HSPCs) – will crawl upstream, against the direction of flow, on surfaces that contain the ligand intercellular adhesion molecule-1 (ICAM-1). This upstream migration is mediated by the β2 integrin, αLβ2, also known as Lymphocyte Function-associated Antigen-1 (LFA- 1) binding to ICAM-1. Originally it was reported that neutrophils are unable to crawl upstream on ICAM-1, even though neutrophils express LFA-1. Neutrophils express an additional receptor for binding ICAM-1, Mac-1, which is up expressed when neutrophils are activated. We hypothesized that neutrophils are unable to crawl upstream because Mac-1 dominates binding ICAM-1 in neutrophils, and that if we block or disable Mac-1, the phenotype for upstream migration would be recovered. Our preliminary results show that by blocking Mac-1, upstream migration of neutrophils is recovered, thus setting the premise for this application. In this application we will show that neutrophils can indeed crawl upstream by inhibiting interactions of Mac-1, using both differentiated HL-60 cells and primary neutrophils. Specifically, in Aim 1, we will Determine the conditions in which neutrophils can crawl against the direction of shear flow, using antibody blocking to determine which integrins inhibit upstream migration in differentiated HL-60 cells and primary human neutrophils, and confirm that blocking Mac-1 leads to upstream migration. Then, in Aim 2, we will analyzing the altered directional migration preferences of neutrophils deficient in Mac-1, using RNA interference to make HL-60 cell lines that are deficient in Mac-1 and LFA-1, and test their directional preference for migration on purified molecular surfaces, as well as on stimulated HUVECs. We will validate the results of this aim with mutants of primary neutrophils by transducing CD34+ Hematopoietic Stem cells with shRNA against Mac-1 during their cytokine induced differentiation into neutrophils in-vitro. These studies will ultimately identify the critical surface receptors responsible in either guiding or preventing the upstream migration of neutrophils. This information will hold therapeutic potential for improving patient outcomes by better guiding neutrophils to the sites of inflammation and resolving inflammatory ailments.