EFFECTS OF VEROCYTOTOXIN-1 ON NONADHERENT HUMAN MONOCYTES - BINDING CHARACTERISTICS, PROTEIN-SYNTHESIS, AND INDUCTION OF CYTOKINE RELEASE

The epidemic form of the hemolytic uremic syndrome (HUS) has been asso ciated with a verocytotoxin producing Escherichia coli infection. Endo thelial cell damage of glomeruli and arterioles of the kidney plays a central role in the pathogenesis of HUS. A number of observations in v ivo and in vitro indicate that inflammatory mediators contribute to th is process. In this study we investigated the binding of I-125-verocyt otoxin-1 (VT-1) to freshly isolated human nonadherent monocytes as wel l as the nature of the ligand to which VT-1 binds on monocytes. On the average, freshly isolated monocytes have 0.07 x 10(5) specific bindin g sites for I-125-VT-1 per cell. Preincubation of nonadherent monocyte s with bacterial lipopolysaccharide (LPS) caused a 23- to 30-fold incr ease of specific binding sites for VT-1 as shown by Scatchard plot ana lysis. Thin-layer chromatography of extracted neutral glycolipids of t he cells and subsequent binding of I-125-VT-1 showed that human monocy tes bind VT-1 to a globotriaosylceramide (Gb(3)) species that is diffe rent from that found on endothelial cells, probably a short-chain fatt y acyl Gb(3) or an alpha-OH-Gb(3). In addition, we evaluated the funct ional consequences of VT-1 binding to human monocytes by investigating the effects of VT-1 on the total protein synthesis and, specifically, the production of the cytokines interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), IL-6, and IL-8. We observed that V T-1 did not inhibit overall protein synthesis, nor under basal conditi ons, neither after stimulation with LPS, in contrast to previous obser vations with endothelial cells. Furthermore, we found that VT-1 induce s the synthesis of the cytokines IL-1 beta, TNF-alpha, IL-6, and IL-8 in nonstimulated monocytes by a LPS-independent cell activation. The i ncrease in the production of cytokines was parallelled by an increase in mRNA, as was demonstrated for IL6 by reverse transcription-polymera se chain reaction. These data suggest that inflammatory mediators loca lly produced by VT-1-stimulated monocytes may contribute to the pathog enic mechanism of the HUS. (C) 1996 by The American Society of Hematol ogy.