Control of neutrophil migration via Ly6 family members

描述（由申请人提供）：中性粒细胞是免疫防御的核心，但也参与免疫介导的病理学。我们最近发现鼠蛋白Ly 6 G与β2整联蛋白相互作用，Ly 6 G的抗体连接阻断了中性粒细胞向炎症部位的整联蛋白依赖性募集。相比之下，在感染性肺炎和腹膜炎中，募集没有类似的受损，这可能反映了β2整合素在无菌性和脓毒性炎症中的不同作用。重要的是，我们发现人类嗜中性粒细胞特异性Ly 6家族成员CD 177具有相似的活性，这两种蛋白质都参与了调节整合素依赖性中性粒细胞迁移的新机制。这项提议的目的是了解这种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。在目的I中，我们使用具有可操纵Ly 6 G表达的新生成的模型中性粒细胞来测试该蛋白质的天然功能是促进还是抑制迁移。使用体外和体内模型系统的无菌和脓毒性炎症，以及 由于细胞来自野生型和β2整联蛋白缺陷小鼠，我们将测试Ly 6 G阻断是否对中性粒细胞募集的β2整联蛋白表现出差异依赖性。在目的II中，我们询问Ly 6 G控制原代中性粒细胞中β2整合素的机制。使用先进的FLIM分子成像，我们将测试Ly 6 G与β2整合素的物理结合如何随活化状态而变化。我们将在体外和组织中确定Ly 6 G、整合素和其他相关分子在中性粒细胞与内皮相互作用上的空间关系。我们将测试Ly 6 G调节整合素内化和/或蛋白水解的假设。在目的III中，我们检验了CD 177是人中性粒细胞中整合素介导的迁移的Ly 6 G样调节剂的假设。认识到潜在的操纵需要更好地了解CD 177 pos和CD 177 neg中性粒细胞亚群，我们将免疫表型这些细胞在健康和关节炎的捐助者，并研究他们如何在血液中变化，关节炎滑液和感染性渗出液。我们将使用一种新的表达CD 177的中性粒细胞系来确定这种蛋白质在迁移中的功能，并在原代人中性粒细胞中使用FLIM来测试CD 177和β2整合素之间的相互作用如何在炎症性疾病中靶向。这些研究开始于偶然的观察，即即使在没有中性粒细胞耗竭的情况下，抗Ly 6 G抗体也能阻断小鼠实验性关节炎（1）。拟议的研究将采用新的工具来定义体外和体内这种效应的分子机制，同时将这些观察结果转化为与人类疾病相关的新机制的定义，重点是炎症性关节炎。

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.