Abstract 5851: Neutrophil Binding Induces Src-dependent ICAM-1 Phosphorylation and eNOS Activation in Endothelial Cells
The interaction of β2 integrins on neutrophils with intercellular adhesion molecule (ICAM-1) on endothelial cells induces ICAM-1 clustering and activation of outside-in signaling culminating in transendothelial neutrophil migration. ICAM-1 activation is known to stimulate sustained Src kinase activity (as measured by the increase in Src Tyr418 autophosphorylation and dephosphorylation of Src inhibitory phospho-Tyr529). Moreover, the phosphatase SHP2 is known to dephosphorylate Src Tyr529 and prolong Src activation. We therefore hypothesized that prolonged Src signaling downstream of ICAM-1 phosphorylation (Tyr485) and clustering is required for efficient neutrophil transmigration. We also predicted that prolonged Src activation would produce long lasting eNOS phosphorylation and NO production by the well characterized PI3-kinase-Akt pathway. In the present study, we showed that ICAM-1 cross-linking induced the activation of Src in endothelial cells that was dependent on SHP2 since the phosphatase-dead mutant (Cys463Ser) blocked Src activation and therefore ICAM-1 Tyr485 phosphorylation. In addition, ICAM-1 cross-linking promoted the phosphorylation of Akt at Ser473 and eNOS at Ser1177 leading to prolonged NO production. Both Akt and eNOS phosphorylation and resultant NO production were sensitive to Src inhibitor PP2, PI3-kinase inhibitor wortmannin, and ICAM-1 phospho-defective mutant (Tyr485Phe) suggesting that ICAM-1 activation stimulates the Src-PI3K-Akt-eNOS pathway in endothelial cells. In vitro, neutrophil transmigration was dependent on Src activation, ICAM-1 phosphorylation, and NO production. Importantly, in a model of carrageenan-induced acute lung injury, neutrophil infiltration into the lung was also significantly reduced in ICAM-1−/− and eNOS−/− mice. In conclusion, activation of endothelial cell ICAM-1 leads to sustained Src activity accompanied by enhanced NO production and increased neutrophil transmigration.