Control of neutrophil migration via Ly6 family members

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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.