TUMOR-NECROSIS-FACTOR-ALPHA INDUCES ENDOTHELIAL GALACTOSYL TRANSFERASE-ACTIVITY AND VEROCYTOTOXIN RECEPTORS - ROLE OF SPECIFIC TUMOR-NECROSIS-FACTOR RECEPTORS AND PROTEIN-KINASE-C

Infections with verocytotoxin (VT) producing Escherichia coli have bee n strongly implicated in the epidemic form of hemolytic uremic syndrom e (HUS). Endothelial damage plays a central role in the pathogenesis o f HUS. In vitro studies have shown that VT can damage endothelial cell s after interaction with its cellular receptor globotriaosylceramide ( GbOse(3)cer). Cytokines, such as tumor necrosis factor alpha (TNF alph a) and interleukin-1 (IL-1) can potentiate the toxic effect of VT by i nducing a protein-synthesis dependent increase in VT receptors on endo thelial cells. In this study, the mechanisms underlying the increase i n endothelial VT receptors induced by TNF alpha were studied in more d etail. To investigate which proteins were involved in this induction, endothelial cells were incubated with and without TNF alpha in the pre sence of C-14-galactose or C-14-glucose. Thin-layer chromatography (TL C) analysis of the glycolipid extracts of these cells demonstrated a m arkedly enhanced incorporation of C-14-galactose in GbOse(3)cer and ot her galactose-containing glycolipids, suggesting that TNF alpha enhanc ed galactosyl-transferase activity. To examine the role of the two rec ently cloned TNF-receptors (TNFR-p75 and TNFR-p55) in the TNF alpha-in duced increase in GbOse(3)cer in human endothelial cells, cells were i ncubated with TNF alpha, the TNFR-p55 selective R32W-S86T-TNF alpha-mu tant, or the TNFR-p75 selective D143N-A145R-TNF alpha-mutant. The effe ct of TNF alpha activation, determined by binding-experiments with I-1 25-VT-1, could be largely, but not completely mimicked by R32W-S86T=TM F alpha. Although incubation of cells with D143N-A145R-TNF alpha did n ot show an increase in VT-1 binding, the monoclonal antibody utr-1, wh ich prevents binding to TNFR-p75, decreased the TNF alpha-induced VT-1 binding. Activation of protein kinase C (PKC) by phorbol ester increa ses the expression of VT-1 receptors; this effect was prevented by the PKC inhibitor Ro31-8220 and by homologous desensitization by pretreat ment with phorbol ester. In contrast, the presence of the protein kina se inhibitor Ro31-8220 or desensitization of PKC activity reduced the TNF alpha-induced increase in VT-1 receptors maximally by 50% and 24%, respectively. Comparable reductions in overall protein synthesis and the synthesis of E-selectin and plasminogen activator inhibitor-1 (PAI -1) were observed. This suggests an effect on general protein synthesi s rather than a specific effect of PKC in the signal transduction path way, by which TNF alpha induces VT-1 receptors. Our results indicate t hat TNF alpha can increase the VT-1 receptors on endothelial cells by inducing galactosyl-transferase activity, that this action of TNF alph a mainly occurs via the TNFR-p55; and that PKC activation increases ex pression of VT-1 receptors by a separate mechanism that acts additivel y to the TNF alpha-induced increase in VT-1 receptors. (C) 1995 by The American Society of Hematology.