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

通过Mac-1的调节控制中性粒细胞的上游迁移

• 批准号: 9756062
• 负责人: Daniel A Hammer
• 金额: $ 23.81万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9756062
• 关键词: Actins; Adhesions; Binding; Blocking Antibodies; Blood; Blood Vessels; CD34 gene; Cell Line; Cell Surface Receptors; Cell physiology; Cells; Confocal Microscopy; Endothelial Cells; Endothelium; Event; Expression Profiling; HL-60 Cells; Hematopoietic; Hematopoietic stem cells; Homing; Human; Immune; In Vitro; Infectious Agent; Inflammation; Inflammatory; Innate Immune Response; Integrin alpha4beta1; Integrins; Intercellular adhesion molecule 1; Leukocytes; Ligands; Lymphocyte Function-Associated Antigen-1; Lymphoid; Macrophage-1 Antigen; Mediating; Molecular; Myelogenous; Neutrophil Infiltration; Paper; Patient-Focused Outcomes; Phenotype; Population; RNA Interference; Recombinants; Reporting; Resolution; Secure; Signal Transduction; Site; Stimulus; Stream; Sum; Surface; Swimming; T-Lymphocyte; Testing; Therapeutic; Tissues; Umbilical vein; Vascular Cell Adhesion Molecule-1; cytokine; density; experimental study; first responder; improved; migration; mutant; neutrophil; peripheral blood; polymerization; postcapillary venule; preference; prevent; receptor; receptor binding; receptor expression; small hairpin RNA; trafficking

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.

摘要/摘要 免疫细胞从血液流到炎症部位的能力以找到 传染剂并将信息传输到其他免疫细胞是先天免疫反应的标志。 贩运受白细胞粘附级联的约束，这是一个特征良好的逐步事件序列 其中血液传播的免疫细胞束缚，首先逮捕，迁移，然后输入时间进行免疫 细胞功能。中性粒细胞是“第一反应者”，将立即访问炎症部位 感觉到炎症性侮辱。这些事件通常发生在毛细血管后静脉内，其中细胞 必须克服高剪切速率与内皮表面结合并最终移民。 我们和其他人已经确定了一种有趣的现象，其中某些造血存在细胞 - 尤其是T细胞和造血茎和祖细胞（HSPC） - 将在上游爬网 流动的方向，在包含配体间细胞间粘合分子1（ICAM-1）的表面上。这个上游 迁移是由β2整合素αLβ2（也称为淋巴细胞功能相关的抗原1）介导的（LFA-- 1）与ICAM-1结合。最初据报道，中性粒细胞无法在ICAM-1上游爬行，甚至 虽然中性粒细胞表达LFA-1。中性粒细胞表达了一种用于结合ICAM-1，MAC-1， 当激活中性粒细胞时，它会向上表达。我们假设中性粒细胞无法爬行 上游是因为MAC-1在中性粒细胞中占主导地位的ICAM-1，并且如果我们阻止或禁用MAC-1， 将回收上游迁移的表型。我们的初步结果表明，通过阻止Mac-1， 恢复中性粒细胞的上游迁移，从而为该应用程序设定了前提。 在此应用中，我们将表明中性粒细胞确实可以通过抑制相互作用 Mac-1，使用分化的HL-60细胞和原代嗜中性粒细胞。具体来说，在AIM 1中，我们将确定 中性粒细胞可以使用抗体依靠剪切流的方向爬行的条件 阻止以确定哪些整联蛋白抑制了分化的HL-60细胞和主要的上游迁移 人类嗜中性粒细胞，并确认阻断MAC-1会导致上游迁移。然后，在AIM 2中，我们将 使用RNA分析中性粒细胞缺乏中性粒细胞的定向迁移偏好 干扰使Mac-1和LFA-1缺陷的HL-60细胞系，并测试其方向 偏爱在纯化的分子表面以及受刺激的HUVEC上迁移。我们将验证 通过将CD34+造血干细胞转导的原代嗜中性粒细胞突变体的结果 shRNA在其细胞因子过程中针对MAC-1诱导嗜中性粒细胞的分化。这些研究会 最终确定指导或阻止上游负责的关键表面受体 中性粒细胞的迁移。这些信息将具有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并解决炎症性疾病。