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.

The Trojan Horse of the microbiological arms race: phage‐encoded toxins as a defence against eukaryotic predators

“…In mammalian cells, the import of cytolethal quantities of Shiga toxin requires the binding of toxin to a plasma membrane receptor (Sandvig et al, 2010). Mammalian leukocytes, which lack the Stx receptor, are not susceptible to killing either by cell-free Shiga toxin or toxin produced by bacteria internalized within these cells (Tesh et al, 1994;Ramegowda and Tesh, 1996;van Setten et al, 1996). Similar to Shiga toxin, import of cytolethal diphtheria toxin into mammalian cells is also mediated by a plasma membrane receptor.…”

“…Cell-free Stx or Stx produced by bacteria encapsulated within food vacuoles are both cytotoxic to Tetrahymena (Stolfa and Koudelka, 2013). Cytotoxic Stx can only access the cytoplasm of susceptible mammalian cells by receptor mediated endocytosis; Stx released from bacteria internalized by leukocytes is not lethal to these cells (Tesh et al, 1994;Ramegowda and Tesh, 1996;van Setten et al, 1996). Given these possible modes of entry, we wished to determine the requirements for entry of cytotoxic Stx into Acanthamoeba.…”

Interactions between Shiga toxins and human polymorphonuclear leukocytes

“…Eppendorf tubes were precoated with PBS containing 1% BSA to avoid nonspecific loss of toxins [32]. Endotoxin-free neutrophils (2ϫ10 6 /ml) or HL-60 were incubated with different concentrations of unlabeled or radiolabeled Stx (0.5-75 nM) in PBS containing 1% BSA for 90 min at 37°C, with occasional stirring by gentle inversion of the tube.…”

Shiga toxin and its use in targeted cancer therapy and imaging

“…Although not fully characterized, Gb3 appears to show a relatively restricted expression in normal human tissues, being mostly found in kidney epithelium and endothelium (Lingwood, 1994;Khan et al, 2009), in microvascular endothelial cells in intestinal lamina propria (Miyamoto et al, 2006;Schuller et al, 2007), in platelets (Cooling et al, 1998), and in subsets of germinal centre B lymphocytes (Murray et al, 1985;Gregory et al, 1988;Mangeney et al, 1991). A low level of Gb3 expression has been reported in monocytes (van Setten et al, 1996), and in monocyte-derived macrophages and dendritic cells (Falguières et al, 2001). Recent reports indicate that Gb3 is also expressed by intestinal pericryptal myofibroblasts (Schuller et al, 2007), neurons (Obata et al, 2008) and endothelial cells in the central nervous system (Johansson et al, 2006;Obata et al, 2008), and the possible implications of this needs to be further clarified.…”