Src Phosphorylation of Endothelial Cell Surface Intercellular Adhesion Molecule-1 Mediates Neutrophil Adhesion and Contributes to the Mechanism of Lung Inflammation

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. Methods and Results—TNFα exposure of mouse lungs for 5 minutes produced a 3-fold increase in 125I-anti-ICAM-1 monoclonal antibody (mAb) binding and 111In 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 Bmax 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 effec...