title: Src phosphorylation of endothelial cell surface intercellular adhesion molecule-1 mediates neutrophil adhesion and contributes to the mechanism of lung inflammation.
abstract: OBJECTIVE
  The goal of this study was to determine whether tumor necrosis factor α (TNFα)-induced Src activation and intercellular adhesion molecule-1 (ICAM-1) phosphorylation rapidly increase endothelial cell adhesivity and polymorphonuclear leukocyte (PMN) sequestration independently of de novo ICAM-1 synthesis.
  
  
  RESULTS
  TNFα exposure of mouse lungs for 5 minutes produced a 3-fold increase in (125)I-anti-ICAM-1 monoclonal antibody (mAb) binding and (111)In oxine-labeled PMN sequestration, as well as Src activation, ICAM-1 Tyr518 phosphorylation, and phospho- Tyr518-ICAM-1 coimmunoprecipitation with actin. The response was absent in Nox2(-/-) lungs or following Src inhibition. In COS-7 cells transfected with wild-type (WT), phospho-defective (Tyr518Phe), or phospho-mimicking (Tyr518Asp) mouse ICAM-1 cDNA constructs, TNFα increased the B(max) of YN1/1.7.4 anti-ICAM-1 mAb binding to WT-ICAM-1 but not to Tyr518Phe-ICAM-1, indicating increased binding avidity secondary to ICAM-1 phosphorylation. This effect was mimicked by expression of the Tyr518Asp-ICAM-1 mutant. TNFα also increased the staining intensity and cell surface clustering of YN1/1.7.4 mAb-labeled WT-ICAM-1 that colocalized with F-actin, which was not observed with Tyr518Phe-ICAM-1 but was recapitulated with Tyr518Asp-ICAM-1. Finally, overexpression of ICAM-1 in mouse lungs significantly increased lipopolysaccharide-induced transvascular albumin leakage and bronchoalveolar lavage PMN counts at 2 and 24 hours after lipopolysaccharide inhalation compared with lungs expressing the Tyr518Phe ICAM-1 mutant.
  
  
  CONCLUSIONS
  Src-dependent phosphorylation of endothelial cell ICAM-1 Tyr518 induces PMN adhesion by promoting ICAM-1 clustering, which we propose mediates rapid-phase lung vascular accumulation of PMNs during inflammation.
authors.affiliation: Department of Pharmacology, University of Illinois, Chicago, IL, USA.
authors.forename: Guoquan
authors.initials: G
authors.lastname: Liu
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Vogel
authors.forename: Xiaopei
authors.initials: X
authors.lastname: Gao
authors.forename: Kamran
authors.initials: K
authors.lastname: Javaid
authors.forename: Guochang
authors.initials: G
authors.lastname: Hu
authors.forename: Sergei M
authors.initials: SM
authors.lastname: Danilov
authors.forename: Asrar B
authors.initials: AB
authors.lastname: Malik
authors.forename: Richard D
authors.initials: RD
authors.lastname: Minshall
mesh_terms: D000818:Animals; D019556:COS Cells; D002448:Cell Adhesion; D002522:Chlorocebus aethiops; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000633:toxicity; D008297:Male; D008562:Membrane Glycoproteins / Q000502:physiology; D051379:Mice; D008810:Mice, Inbred C57BL; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000502:physiology; D009504:Neutrophils / Q000166:cytology / Q000502:physiology*; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D010766:Phosphorylation; D011014:Pneumonia / Q000209:etiology*; D011493:Protein Kinase C / Q000502:physiology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D019061:src-Family Kinases / Q000378:metabolism*; D000099221:Protein Kinase C zeta
chemical_list: D018799:Intercellular Adhesion Molecule-1; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D019869:Phosphatidylinositol 3-Kinases; D011493:Protein Kinase C; D014409:Tumor Necrosis Factor-alpha; D019061:src-Family Kinases; D000099221:Protein Kinase C zeta; C056558:CYBB protein, human