Distinct mechanisms for N-acetylcysteine inhibition of cytokine-induced E-selectin and VCAM-1 expression

We have examined the effects of N-acetyl-L-cysteine (NAG), a well-characterized, thiol-containing antioxidant, on agonist-induced monocytic cell adhesion to endothelial cells (EC). NAC inhibited interleukin-1 (IL-1 beta)-induced, but not basal, adhesion with 50% inhibition at similar to 20 mM. Monocytic cell adhesion to EC in response to tumor necrosis factor-alpha (TNF alpha), lipopolysaccharide (LPS), alpha-thrombin, or phorbol 12-myristate 13-acetate (PMA) was similarly inhibited by NAG. Unlike published studies with pyrrolidinedithiocarbamate, which specifically inhibited vascular cell adhesion molecule 1 (VCAM-1), NAC inhibited IL-1 beta-induced mRNA and cell surface expression of both E-selectin and VCAM-1. NAC had no effect on the half-life of E-selectin or VCAM-1 mRNA. Although NAC reduced nuclear factor-kappa B (NF-kappa B) activation in EC as measured by gel-shift assays using an oligonucleotide probe corresponding to the consensus NF-kappa B binding sites of the VCAM-1 gene (VCAM-NF-kappa B), the antioxidant had no appreciable effect when an oligomer corresponding to the consensus NF-kappa B binding site of the E-selectin gene (E-selectin-NF-kappa B) was used. Because NF-kappa B has been reported to be redox sensitive, we studied the effects of NAC on the EC redox environment. NAC caused an expected dramatic increase in the reduced glutathione (GSH) levels in EC. In vitro studies demonstrated that whereas the binding affinity of NF-kappa B to the VCAM-NF-kappa B oligomer peaked at a GSH-to-oxidized glutathione (GSSG) ratio of similar to 200 and decreased at higher ratios, the binding to the E-selectin-NF-kappa B oligomer appeared relatively unaffected even at ratios >400, i.e., those achieved in EC treated with 40 mM NAC. These results suggest that NF-kappa B binding to its consensus sequences in the VCAM-1 and E-selectin gene exhibits marked differences in redox sensitivity, allowing for differential gene expression regulated by the same transcription factor. Our data also demonstrate that NAC increases the GSH-to-GSSG ratio within the EC, suggesting one possible mechanism through which this antioxidant inhibits agonist-induced monocyte adhesion to EC.