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Effects of verocytotoxin-1 on nonadherent human monocytes: binding
characteristics, protein synthesis, and induction of cytokine release
PA van Setten, LA Monnens, RG Verstraten, LP van den Heuvel and VW van Hinsbergh
Department of Paediatrics, University Hospital, Nijmegen, The Netherlands.
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.
Volume 88,
Issue 1,
pp. 174-183,
07/01/1996
Copyright © 1996 by The American Society of Hematology
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