Lactosylceramide mediates tumor necrosis factor-alpha-induced intercellular adhesion molecule-1 (ICAM-1) expression and the adhesion of neutrophil inhuman umbilical vein endothelial cells

The endothelial expression of adhesion molecules by proinflammatory cytokin es such as tumor necrosis factor-alpha (TNF-alpha) has been suggested to co ntribute to the initiation of atherosclerotic plaque formation. Since lacto sylceramide (LacCer) accumulates in large quantities in human atherosclerot ic plaque, we have explored its role in TNF-alpha-induced expression of int ercellular adhesion molecule-1 (ICAM-1) in human umbilical vein endothelial cells and their consequent adhesion to polymorphonuclear leukocytes (PMNs) . We found that TNF-alpha increased LacCer synthesis by way of stimulating the activity of UDP-galactose:glucosylceramide beta(1-->4)galactosyltransfe rase in a time-dependent fashion. The TNF-alpha-induced expression of ICAM- 1 was abrogated by D-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PD MP), an inhibitor of UDP-galactose:glucosylceramide beta(1-->4)-galactosylt ransferase. However, the addition of LacCer reversed the D-PDMP effect on T MF-alpha-induced ICAM-1 expression in human umbilical vein endothelial cell s. Northern hybridization analysis of mRNA levels and enzyme-linked immunos orbent assays revealed that LacCer (5 mu M) specifically stimulated ICAM-1 at both the transcriptional and translational levels. This was accompanied by the adhesion of PMNs, which was visualized by confocal microscopy. Furth er studies revealed that LacCer stimulated the endogenous generation of sup eroxide radicals (O-2(radical anion)) about 5-fold compared with the contro l by specifically activating plasma membrane-associated NADPH-dependent oxi dase. This phenomenon was blocked by the antioxidant N-acetyl-L-cysteine, p yrrolidine dithiocarbamate, and the NADPM oxidase inhibitor, diphenylene io donium. Overexpression of endogeneous CuZn-superoxide dismutase via an aden oviral vector carrying cDNA for CuZn-superoxide dismutase, also inhibited L acCer-induced ICAM-1 expression in endothelial cells. In sum, our findings suggest that LacCer may play the role of a lipid second messenger in TNF-al pha-induced pathogenesis by activating an oxidant-sensitive transcriptional pathway that leads to the adhesion of PMNs to endothelial cells.