Src Phosphorylation of Endothelial Cell Surface ICAM-1 Mediates Neutrophil Adhesion and Contributes to the Mechanism of Lung Inflammation
Vogel, Stephen M.
Danilov, Sergei M.
Malik, Asrar B.
Minshall, Richard D.
PublisherAmerican Heart Association
MetadataShow full item record
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.