Role And Mechanism Of Lipid Raft In PSGL-1 Mediated Neutrophil Adhesion

PMN(Polymorphonuclear neutrophils)plays an important rolein innate immune system.Under numerous pathological or physiological conditions,Neutrophils will transmigrate through the endothelium clefts to the underlying tissue.The recruitment of neutrophils to a site of infection or tissue injury entails acascade of cellular adhesive events,including tethering,rolling,adhesion,transmigration.Adhesion molecules play an important role in this physiological process,Selectins mediate leukocyte tethering and rolling,and integrins mediate adhesion of leukocytes.Early in inflammation,PSGL-1 binds to P-or E-selectin expressed on activated endothelium,and mediates leukocyte rolling on endothelium.Subsequently,integrins are activated and mediate the cell arrest and stable adhesion to endothelium under shear flow conditions.In addition to its direct role in the capture of leukocytes from the bloodstream,PSGL-1 also functions as a signal transduction receptor and initiates a series of intracellular signal events during leukocytes activation,including secretion of cytokines,transcriptional activation,activation of kinases,especially the activation of integrins.The activation of integrin is a prerequisite for leukocyte adhesion.In the present study,we adopted Cell adhesion assay under static conditions and Time-lapse microscopy of adhesion confirmed that PSGL-1 ligation promoted neutrophil adhesion on ICAM-1.Immunofluorescence labeling experiments found that PSGL-1 ligation induced ?2 integrin clustering.Lipid raft,a liquid ordered plasma membrane microdomain,is related to cell surface receptor function.We adopted Cell adhesion assay under flow and static conditions found that lipid rafts involve in the PSGL-1-mediated neutrophil adhesion.Immunofluorescence double-labeling experiments confirmed that lipid raft were essential for adhesion receptor PSGL-1 and ?2 integrin clustering.Immunoprecipitation experiments and lipid raft fraction extraction showed that Syk kinase is involved in the regulation of ?2 integrin clustering,and then play a role in PSGL-1 mediated ?2 integrin-dependent neutrophil adhesion.In the present study,we used shotgun proteomic technique to analyze lipid raft proteome quantitatively in PSGL-1 ligated or unligated HL-60 cells.We successfully screen the interest protein ?-adducin.By immunofluorescence double-labeling experiments,Membrane fractionation,Detergent-resistant cell lysate fractions found that ?-adducin relocation in PSGL-1 ligated HL-60 cells.And immunoprecipitation experiments showed that PSGL-1 ligation induced Src activation,and the Src activation is very important for ?-adducin relocation.By using in vitro expression system GST-pull down experiments,we found that he C-teeminal domain and proline-rich region of ?-adducin play a pivotal role for its efficient interaction with Src kinase.We used Site-directed mutagenesis techniques found that Tyr-568 of ?-adducin was very important for its activation.This study reveals for the first time that lipid raft and raft-associated protein play crucial parts in leukocyte adhesion by regulating PSGL-1 clustering.