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 associated with a verocytotoxin producing Escherichia coli infection. Endothelial cell damage of glomeruli and arterioles of the kidney plays a central role in the pathogenesis of HUS. A number of observations in vivo and in vitro indicate that inflammatory mediators contribute to this process. In this study we investigated the binding of 125I-verocytotoxin-1 (VT-1) to freshly isolated human nonadherent monocytes as well 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 binding sites for 125I-VT-1 per cell. Preincubation of nonadherent monocytes with bacterial lipopolysaccharide (LPS) caused a 23- to 30-fold increase of specific binding sites for VT-1 as shown by Scatchard plot analysis. Thin-layer chromatography of extracted neutral glycolipids of the cells and subsequent binding of 125I-VT-1 showed that human monocytes bind VT-1 to a globotriaosylceramide (Gb3) species that is different from that found on endothelial cells, probably a short-chain fatty acyl Gb3 or an alpha-OH-Gb3. In addition, we evaluated the functional 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 VT-1 did not inhibit overall protein synthesis, nor under basal conditions, neither after stimulation with LPS, in contrast to previous observations with endothelial cells. Furthermore, we found that VT-1 induces 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 increase in the production of cytokines was parallelled by an increase in mRNA, as was demonstrated for IL-6 by reverse transcription-polymerase chain reaction. These data suggest that inflammatory mediators locally produced by VT-1-stimulated monocytes may contribute to the pathogenic mechanism of the HUS.
van Setten
P A
PA
Department of Paediatrics, University Hospital, Nijmegen, The Netherlands.
Monnens
L A
LA
Verstraten
R G
RG
van den Heuvel
L P
LP
van Hinsbergh
V W
VW
D001427:Bacterial Toxins / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites; D018929:Cell Culture Techniques / Q000295:instrumentation; D002470:Cell Survival; D002478:Cells, Cultured; D004926:Escherichia coli / Q000378:metabolism / Q000472:pathogenicity; D005786:Gene Expression Regulation / Q000187:drug effects*; D006463:Hemolytic-Uremic Syndrome / Q000378:metabolism; D006801:Humans; D007375:Interleukin-1 / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D007378:Interleukins / Q000378:metabolism*; D008024:Ligands; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D009000:Monocytes / Q000187:drug effects* / Q000378:metabolism; D011138:Polytetrafluoroethylene; D014176:Protein Biosynthesis; D022622:Shiga Toxin 1; D014281:Trihexosylceramides / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D001427:Bacterial Toxins; D007375:Interleukin-1; D015850:Interleukin-6; D016209:Interleukin-8; D007378:Interleukins; D008024:Ligands; D008070:Lipopolysaccharides; D022622:Shiga Toxin 1; D014281:Trihexosylceramides; D014409:Tumor Necrosis Factor-alpha; C018549:globotriaosylceramide; D011138:Polytetrafluoroethylene