Effects of verocytotoxin-1 on nonadherent human monocytes: binding characteristics, protein synthesis, and induction of cytokine release

Abstract

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.

Pathogenic role of inflammatory response during Shiga toxin-associated hemolytic uremic syndrome (HUS)

“…Alternatively, Stx appears to be routed into a degradative pathway in both macrophages and dendritic cells of human origin [25]. Associated with this pathway, Stx induces the expression of cytokines, such as IL-1β, IL-6, IL-8, and TNF-α in human monocytes [8,26] and murine peritoneal macrophages [27] in vitro. In contrast to primary cells, a human myeloid leukemia cell line, THP-1 cells, expresses abundant Gb3 on the cell membrane and is susceptible to apoptosis by Stx through the activation of caspases 2, 6, 8, and 9 [25].…”

Reduction of pathogenic bacteria in ground beef using a Lactobacillus-based biological intervention

“…In vitro studies revealed that stx can bind to human erythrocytes (15), platelets (41), and activated monocytes (160). Recent studies have found that stx immediately and more efficiently binds to polymorphonuclear leukocytes (PMNL).…”

Hemolytic uremic syndrome: pathogenesis and update of interventions

“…Although Louise et al suggested that Stx may enhance the procoagulant effect of LPS [62], in vivo studies have demonstrated that toxicity of Stx is potentiated by LPS [63][64][65]. In particular, LPS, chemokines and cytokines released by inflammatory cells or injured cells upregulate the expression of Stx receptors and sensitize microvascular endothelial cells (glomerular and others) to Stx-induced injury [17,[66][67][68].…”