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

Objective: To determine whether TNFα-induced Src activation and ICAM-1 phosphorylation rapidly increases endothelial cell adhesivity and PMN sequestration independent of de novo ICAM-1 synthesis. Methods and Results: TNFα exposure of mouse lungs for 5 min produced a 3-fold increase in 125I-anti-ICAM-1 mAb binding and 111In oxine-labeled PMN sequestration as well as Src activation, ICAM-1 Tyr518 phosphorylation, and pTyr518-ICAM-1 co-immunoprecipitation with actin. The response was absent in Nox2-/- lungs or following Src inhibition. In COS-7 cells transfected with wild-type (WT), phospho-defective (Y518F), or phospho-mimicking (Y518D) 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 Y518F-ICAM-1 indicating increased binding avidity secondary to ICAM-1 phosphorylation. This effect was mimicked by expression of the Y518DICAM- 1 mutant. TNFα also increased the staining intensity and cell surface clustering of YN1/1.7.4 mAb-labeled WT-ICAM-1 that co-localized with F-actin which was not observed with Y518F-ICAM-1 but was recapitulated with Y518D-ICAM-1. Finally, overexpression of ICAM-1 in mouse lungs significantly increased LPS-induced transvascular albumin leakage and bronchoalveolar lavage PMN counts at 2 and 24 hrs after LPS inhalation compared to lungs expressing Y518F 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.