Suppression of gap junctional intercellular communication in HUVEC by neutrophil adhesion

Interaction between leukocytes and endothelium is believed to be important in many pathophysiological states. But little is known about the influence of leukocytes on gap junctional intercellular communication (GJIC) between endothelial cells. The activity of GJIC was measured by the the method of fluorescence recovery after photobleaching using an interactive lase cytometer. For adhesion assay, human umbilical vein endothelial cells (HUVEC) grown to confluency were stimulated for 5 h with LPS and then exposed to a LPS-stimulated neutrophils suspension. After incubation for 1 h, nonadherent neutrophils were removed by washing. Then the GJIC was measured. GJIC of HUVEC which had adherent neutrophils was suppressed. This suppression was abolished by inhibiting neutrophils adhesion to HUVEC with antihuman ICAM-1 antibody. Assay using intercell chambers which prevented the direct contact of neutrophils with HUVEC, showed that no suppression of GJIC was observed. The suppression of HUVEC GJIC by adherent neutrophils was not abolished by protease inhibitors. This suppression was abolished by pretreatment of HUVEC with tyrosine kinase inhibitors. To characterize the nature of this suppression, we examined the GJIC by anti-ICAM-1 antibody cross-linking. Cross-linking of ICAM-1 in HUVEC suppressed the GJIC. These results indicate that: 1) the suppression of GJIC by adherent neutrophils is not affected by proteases, and 2) this suppression is attributed to tyrosine phosphorylation of the gap junction protein, caused by adhesion to HUVEC through integrin and ICAM-1 binding.