Neutrophils are central to immune defense but also participate in immune-mediated pathology. We have recently shown that the murine protein Ly6G interacts with ?2 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 ?2 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 ?2 integrin-deficient mice, we will test whether Ly6G blockade exhibits differential dependence on ?2 integrins for neutrophil recruitment.
In Aim II, we interrogate mechanisms by which the Ly6G controls ?2 integrins in primary neutrophils. Using cutting-edge FLIM molecular imaging, we will test how the physical association of Ly6G with ?2 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 ?2 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.

Public Health Relevance

Neutrophils are the most abundant inflammatory cells in circulating blood, and their entry into inflamed sites can contribute to tissue injury in immune-mediated diseases such as arthritis. We have identified related proteins in mouse and human neutrophils that abrogate 2-integrin mediated migration when targeted by specific antibodies. Using a combination of in vitro and in vivo approaches, we seek to understand how these proteins function in order to identify novel targets for intervention in human inflammatory disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
1R01AR065538-01A1
Application #
8759409
Study Section
Arthritis, Connective Tissue and Skin Study Section (ACTS)
Program Officer
Mao, Su-Yau
Project Start
2014-07-08
Project End
2019-06-30
Budget Start
2014-07-08
Budget End
2015-06-30
Support Year
1
Fiscal Year
2014
Total Cost
$398,008
Indirect Cost
$145,448
Name
Brigham and Women's Hospital
Department
Type
DUNS #
030811269
City
Boston
State
MA
Country
United States
Zip Code
02115