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.

总结/摘要 免疫细胞从血流中运输到炎症部位的能力， 感染因子并将信息传递给其他免疫细胞是先天免疫应答的标志。 白细胞粘附级联反应是一种特征明确的逐步发生的事件序列， 其中血液携带免疫细胞系结、滚动、牢固地阻滞、迁移然后进入组织以进行免疫 细胞功能。中性粒细胞是“第一反应者”，会立即到达炎症部位 在感觉到炎症性损伤时。这些事件通常发生在毛细血管后小静脉内， 必须克服高剪切速率以结合并最终穿过内皮表面。 我们和其他人已经发现了一个有趣的现象， - 特别是T细胞和造血干细胞和祖细胞（HSPCs）-将向上游爬行， 流动方向，在含有配体细胞间粘附分子-1（ICAM-1）的表面上。该上游 迁移是由β2整合素αLβ2介导的，αLβ2也称为淋巴细胞功能相关抗原-1（LFA-1）。 1)与ICAM-1结合。最初有报道称，中性粒细胞不能在ICAM-1上向上游爬行，即使 但中性粒细胞表达LFA-1。中性粒细胞表达用于结合ICAM-1的另外的受体，Mac-1， 当嗜中性粒细胞被激活时其被上调表达。我们假设中性粒细胞无法爬行 因为Mac-1在中性粒细胞中主导结合ICAM-1，如果我们阻断或禁用Mac-1， 将恢复上游迁移的表型。我们的初步结果表明，通过阻断Mac-1， 恢复了中性粒细胞的上游迁移，从而为该应用奠定了前提。 在这个应用中，我们将证明中性粒细胞确实可以通过抑制 Mac-1，使用分化的HL-60细胞和原代中性粒细胞。具体而言，在目标1中，我们将确定 中性粒细胞可以利用抗体逆着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HL-60细胞中的上游迁移。 人中性粒细胞，并证实阻断Mac-1导致上游迁移。在目标2中，我们 使用RNA分析Mac-1缺陷的中性粒细胞改变的方向迁移偏好， 干扰制备Mac-1和LFA-1缺陷的HL-60细胞系，并检测其定向表达， 在纯化的分子表面上以及在刺激的HUVEC上的迁移的偏好。我们将验证 通过转导CD 34+造血干细胞， 针对Mac-1的shRNA在其细胞因子体外诱导分化为中性粒细胞期间。这些研究将 最终确定关键的表面受体，负责引导或阻止上游 中性粒细胞的迁移。这些信息将具有改善患者结局的治疗潜力， 更好地引导中性粒细胞到达炎症部位并解决炎症性疾病。