Cell Signaling of Human Neutrophil Peptides in Mediating Inflammatory Responses in Human Lung Epithelium
CCCF ePoster library. Han B. Oct 26, 2015; 117371; P11 Disclosure(s): This project was supported by CIHR grant
Dr. Bing Han
Dr. Bing Han
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Abstract
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P11


Topic: Basic/Translational Science


Cell Signaling of Human Neutrophil Peptides in Mediating Inflammatory Responses in Human Lung Epithelium



Bing Han, A. Khine, A. Slutsky, H. Zhnag

Interdepartmental Division of Critical Care Medicine, Departments of Anesthesia and Physiology, University of Toronto, St. Michael's Hospital, Toronto, Canada | Interdepartmental Division of Critical Care Medicine, Departments of Anesthesia and Physiology, University of Toronto, St. Michael's Hospital, Toronto, Canada | Interdepartmental Division of Critical Care Medicine, Departments of Anesthesia and Physiology, University of Toronto, St. Michael's Hospital, Toronto, Canada | Interdepartmental Division of Critical Care Medicine, Departments of Anesthesia and Physiology, University of Toronto, St. Michael's Hospital, Toronto, Canada

Introduction:

Human neutrophil peptides (HNP) are most abundant antimicrobial proteins in neutrophild, and play important role in innate immunity with a wide range of antimicrobial spectrum. Increasing evidence suggests that HNP also exert pro-inflammatory properties in animal models of acute respiratory distress syndrome (ARDS) and high concentrations of HNP have been observed in patients with pneumonia, sepsis andARDS . This evidence suggests that HNP may play a key role in neutrophil-mediated lung injury. However, the mechanisms by which HNP contribute to inflammatory responses remain unknown. Based on our preliminary data, we hypothesized that HNP act on cells through interacting with surface receptor(s).



Objectives:

To examine the signaling pathway for HNP-mediated inflammatory responses.



Methods:

HNP binding proteins in membrane fraction of human lung epithelial cells were detected by mass spectrometry using HNP as bait, and subsequent receptor complex interaction and functional assays were performed using cell and molecular biology techniques.



Results:

The mass spectrometry showed that activated leukocyte cell adhesion molecule (ALCAM/CD166), basal cell adhesion molecule (BCAM/CD239) and P2Y purinoceptor 6 (P2Y6) are the putative binding partners for HNP in human lung epithelial (BEAS-2B) -cells. Immunofluorescence staining assay showed that HNP are well co-localized with the three receptors on both BEAS-2B and human primary small airway epithelial (SAEC) cells. Co-immunoprecipitation assay demonstrated interactions between HNP and ALCAM, BCAM or P2Y6 in both BEAS-2B and SAEC cells. Direct binding of HNP to the receptors was further verified with protein-protein interaction assay by pulling down HNP with the recombinant receptor proteins. The pull-down assay with recombinant proteins also showed direct interactions among the three receptors. Using specific gene knock down, antagonists or blocking antibodies targeting the receptors revealed that the HNP-induced IL-8 production is largely modulated by P2Y6 receptor, while ALCAM and BCAM are mainly responsible for HNP-enhanced cell adhesion.



Conclusion:

P2Y6, ALCAM and BCAM are the receptors, which can interact each other to form receptor complex in mediating HNP-induce inflammatory response and adhesion in human lung epithelial cells, may serve as therapeutic target for neutrophil-induced lung injury.



References: Blood 2006,107:2936-42
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