Reduced endothelial basal nitric oxide induces leukocyte adhesion through Src‐dependent phosphorylation of constitutive intercellular adhesion molecule‐1

Abstract

Nitric oxide (NO), in addition to being a potent vasodilator, has also been indicated to play dual roles in inflammation. Excessive NO production induced by inflammatory mediators contributes to an increase in microvessel permeability and plays a pro‐inflammatory role. Basal NO, on the other hand, is a known anti‐adhesive molecule and plays anti‐inflammatory roles in preventing platelet and leukocyte adhesion. Reduced basal NO has been proposed to increase the adhesiveness of microvessel walls, but the underlying mechanisms remain unclear. Our previous study conducted in intact rat venules found that reduced basal NO by the application of caveolin‐1 scaffolding domain (AP‐CAV) promotes ICAM‐1‐mediated adhesion of leukocytes in 10–30 min. The firm adhesion of leukocytes in a classical pattern is mediated by increased expressions ICAM‐1 that requires protein synthesis and usually occurs hours after cytokine‐mediated activation. Our in vivo observation of AP‐CAV‐induced ICAM‐1‐mediated early phase leukocyte adhesion led us to hypothesize that reduced basal NO increases the adhesiveness of constitutive ICAM‐1 via conformational changes. We tested this hypothesis in cultured HUVECs. Cellular fractionation and western blot were used to determine the relative levels of ICAM‐1 at different regions of the cell and their activation status. The ICAM‐1 adhesiveness were examined by measuring its binding affinity to an inhibitory antibody using immunoprecipitation assay in non‐denatured proteins and immunofluorescence staining with confocal imaging in cultured ECs. The adhesion strength of cultured ECs were measured with atomic force microscopy (AFM). Results showed that the majority of the constitutive ICAM‐1 was located in the cell membrane. AP‐CAV treatment for 30 min caused a significant increase in phosphorylated ICAM‐1. The binding affinity to an inhibitory antibody of ICAM‐1 was also increased in the absence of ICAM‐1translocation and de novo synthesis. The AP‐CAV‐induced increases in ICAM‐1 phosphorylation was abolished by pre‐treating ECs with Src inhibitor, PP1. AFM conducted in cultured ECs showed an increase of the mean rupture force from 0.17 ± 0.002 (control) to 0.27 ± 0.002 nN after 30 min of AP‐CAV treatment. The application of an NOS inhibitor, L‐NMMA, for 30 min showed similar increases in ICAM‐1 binding affinity to that of AP‐CAV in both protein assay and confocal images from cultured ECs, supporting the effect of AP‐CAV to be the results of reduced basal NO. These results demonstrated that reduced basal NO activated ICAM‐1 via Src‐mediated phosphorylation that increased the adhesive capacity of ICAM‐1 without protein synthesis and translocation, suggesting a conformational change of constitutive membrane ICAM‐1. This study provides new insights into the mechanisms of how reduced basal NO in mediating leukocyte adhesion.Support or Funding InformationSupported by HL056237, HL084338 and DK097391.