Control of neutrophil migration via Ly6 family members

通过 Ly6 家族成员控制中性粒细胞迁移

• 批准号: 8886943
• 负责人: Peter A Nigrovic
• 金额: $ 38.19万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-08 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8886943
• 关键词: Adhesions; Adoptive Transfer; Antibodies; Arthritis; Attenuated; Biological Models; Biology; Blocking Antibodies; Blood; Cell Line; Cell model; Cells; Chimeric Proteins; Dependence; Disease; Endothelium; Ensure; Escherichia coli; Exhibits; Exudate; Family member; Fluorescence; Goals; Health; Histology; Homologous Gene; Human; ITGB2 gene; Image; Immigration; Immune; Immunophenotyping; In Situ; In Vitro; Infection; Inflammation; Inflammatory; Inflammatory Arthritis; Injury; Integrins; Intervention; Joints; Laboratories; Ligands; Ligation; Mediating; Microscopy; Modeling; Molecular; Mus; Neutrophil Infiltration; Opportunistic Infections; Oranges; Outcome; Pathology; Pathway interactions; Patients; Peritonitis; Pneumococcal Pneumonia; Pneumonia; Positioning Attribute; Protein Dynamics; Protein Family; Proteins; Proteolysis; Pulmonary Inflammation; Recruitment Activity; Research Personnel; Risk; Role; Site; Sterility; Synovial Fluid; Testing; Thioglycolates; Tissues; Translating; Work; biobank; human disease; in vivo; in vivo Model; migration; molecular imaging; neutrophil; novel; protein function; protein protein interaction; research study; septic; spatial relationship; tool

DESCRIPTION (provided by applicant): Neutrophils are central to immune defense but also participate in immune-mediated pathology. We have recently shown that the murine protein Ly6G interacts with β2 integrins, and that antibody ligation of Ly6G blocks integrin-dependent recruitment of neutrophils to sites of inflammation. By contrast, in infectious pneumonia and peritonitis, recruitment is not similarly impaired, potentially reflecting the differential role of β2 integrins in sterile and septic inflammation. Critically, we find that the human neutrophil-specific Ly6 family member CD177 exhibits similar activity, implicating both proteins in a novel mechanism regulating integrin-dependent neutrophil migration. The goal of this proposal is gain understanding of this new and potentially targetable pathway governing the egress of neutrophils into inflamed tissues. To accomplish this important goal, we propose three independent aims. In Aim I, we employ newly- generated model neutrophils with manipulable Ly6G expression to test whether the native function of this protein is to promote or inhibit migration. Using in vitro and in in vivo models systems of sterile and septic inflammation, as well as cells developed from wild-type and β2 integrin-deficient mice, we will test whether Ly6G blockade exhibits differential dependence on β2 integrins for neutrophil recruitment. In Aim II, we interrogate mechanisms by which the Ly6G controls β2 integrins in primary neutrophils. Using cutting-edge FLIM molecular imaging, we will test how the physical association of Ly6G with β2 integrins varies with activation state. We will determine the spatial relationship of Ly6G, integrins and other relevant molecules on neutrophils interacting with endothelium in vitro and in tissue. We will test the hypothesis that Ly6G modulates integrin internalization and/or proteolysis. In Aim III, we test the hypothesis that CD177 is a Ly6G-like modulator of integrin-mediated migration in human neutrophils. Recognizing that potential manipulation demands better understanding of CD177pos and CD177neg neutrophil subsets, we will immunophenotype these cells in healthy and arthritic donors and study how they vary in blood, arthritic synovial fluid and infectious exudates. We will employ a new CD177-expressing neutrophil cell line to define the function of this protein on migration, and employ FLIM in primary human neutrophils to test how the interaction between CD177 and β2 integrins could be targeted in inflammatory disease. These studies began with the serendipitous observation that antibodies against Ly6G blocked experiment arthritis in mice, even in the absence of neutrophil depletion (1). The proposed studies will employ new tools to define the molecular mechanism of this effect, in vitro and in vivo, while translating these observations into the definition of new mechanisms relevant for human disease, with a focus on inflammatory arthritis.

描述(由申请人提供)：中性粒细胞是免疫防御的中心，但也参与免疫介导的病理。我们最近发现，小鼠蛋白Ly6G与β2整合素相互作用，抗体连接Ly6G可阻止依赖整合素的中性粒细胞重新聚集到炎症部位。相比之下，在感染性肺炎和腹膜炎中，募集没有类似的损害，潜在地反映了β2整合素在无菌和败血症炎症中的不同作用。关键的是，我们发现人中性粒细胞特异的Ly6家族成员CD177具有相似的活性，这意味着这两种蛋白都参与了一种新的调控整合素依赖的中性粒细胞迁移的机制。这项建议的目标是了解这一新的和潜在的靶向途径，控制中性粒细胞进入炎症组织。为了实现这一重要目标，我们提出了三个独立的目标。在目标I中，我们使用新产生的具有可操纵的Ly6G表达的中性粒细胞来测试该蛋白的天然功能是促进还是抑制迁移。使用体外和体内无菌和脓毒症炎症模型系统 随着野生型和β2整合素缺陷小鼠的细胞发育，我们将测试Ly6G阻断是否表现出对中性粒细胞募集β2整合素的不同依赖。在AIM II中，我们询问了Ly6G控制原代中性粒细胞中β2整合素的机制。利用尖端的薄膜分子成像技术，我们将测试Ly6G与β2整合素的物理结合如何随着激活状态的变化而变化。我们将确定Ly6G、整合素和其他相关分子在中性粒细胞与血管内皮细胞体外和组织内相互作用的空间关系。我们将检验Ly6G调节整合素内化和/或蛋白分解的假设。在目标III中，我们验证了CD177是人中性粒细胞中整合素介导的迁移的Ly6G样调制子的假设。认识到潜在的操作需要更好地了解CD177pos和CD177neg中性粒细胞亚群，我们将在健康和关节炎捐赠者中对这些细胞进行免疫表型，并研究它们在血液、关节炎滑液和感染性渗出液中的变化。我们将使用一种新的表达CD177的中性粒细胞株来确定该蛋白在迁移中的功能，并在原代人类中性粒细胞中使用FLIM来测试CD177和β2整合素之间的相互作用如何在炎症性疾病中发挥作用。这些研究始于一项偶然的观察，即抗Ly6G的抗体阻止了小鼠的实验性关节炎，即使在没有中性粒细胞耗尽的情况下也是如此。拟议的研究将使用新的工具来定义这种效应的分子机制，无论是在体外还是在体内，同时将这些观察转化为与人类疾病相关的新机制的定义，重点是炎性关节炎。