Molecular Mechanisms of Neutrophil-Endothelial Cell Adhesion Induced by Redox Imbalance

Abstract

Abstract —Previous studies have implicated a role for intracellular thiols in the activation of nuclear factor-κB and transcriptional regulation of endothelial cell adhesion molecules. This study was designed to determine whether changes in endothelial cell glutathione (GSH) or oxidized glutathione (GSSG) can alter neutrophil adhesivity and to define the molecular mechanism that underlies this GSSG/GSH-induced adhesion response. Treatment of human umbilical vein endothelial cell (HUVEC) monolayers for 6 hours with 0.2 mmol/L diamide and 1 mmol/L buthionine sulfoximine (BSO) decreased GSH levels and increased the ratio of GSSG to GSH without cell toxicity. These redox changes are similar to those observed with anoxia/reoxygenation. Diamide plus BSO–induced thiol/disulfide imbalance was associated with a biphasic increase in neutrophil adhesion to HUVECs with peak responses observed at 15 minutes (phase 1) and 240 minutes (phase 2). N -Acetylcysteine treatment attenuated neutrophil adhesion in both phases, which indicated a role for GSH in the adhesion responses. Interestingly, phase 1 adhesion was inversely correlated with GSH levels but not with the GSSG/GSH ratio, whereas phase 2 neutrophil adhesion was positively correlated with GSSG/GSH ratio but not with GSH levels. Intercellular adhesion molecule-1 and P-selectin–specific monoclonal antibodies attenuated the increased neutrophil adhesion during both phases, whereas an anti–E-selectin monoclonal antibody also attenuated the phase 2 response. Pretreatment with actinomycin D and cycloheximide or with competing ds -oligonucleotides that contained nuclear factor-κB or activator protein-1 cognate DNA sequences significantly attenuated the phase 2 response, which implicated a role for de novo protein synthesis. Surface expression of intercellular adhesion molecule-1, P-selectin, and E-selectin on HUVECs correlated with the phase 1 and 2 neutrophil adhesion responses. This study demonstrates that changes in endothelial cell GSSG/GSH cause transcription-independent and transcription-dependent surface expression of different endothelial cell adhesion molecules, which leads to a 2-phase neutrophil–endothelial adhesion response.