Shiga Toxin 2 and Lipopolysaccharide Induce Human Microvascular Endothelial Cells To Release Chemokines and Factors That Stimulate Platelet Function

Abstract

Shiga toxins (Stxs) produced by Shigella dysenteriae type 1 and enterohemorrhagic Escherichia coli are the most common cause of hemolytic-uremic syndrome (HUS). It is well established that vascular endothelial cells, mainly those located in the renal microvasculature, are targets for Stxs. The aim of the present research was to evaluate whether E. coli-derived Shiga toxin 2 (Stx2) incubated with human microvascular endothelial cells (HMEC-1) induces release of chemokines and other factors that might stimulate platelet function. HMEC-1 were exposed for 24 h in vitro to Stx2, lipopolysaccharide (LPS), or the Stx2-LPS combination, and chemokine production was assessed by immunoassay. More interleukin-8 was released than stromal cell-derived factor 1␣ (SDF-1␣) or SDF-1␤ and RANTES. The Stx2-LPS combination potentiated chemokine release, but Stx2 alone caused more release of SDF-1␣ at 24 h than LPS or Stx2-LPS did. In the presence of low ADP levels, HMEC-1 supernatants activated platelet function assessed by classical aggregometry, single-particle counting, granule secretion, P-selectin exposure, and the formation of platelet-monocyte aggregates. Supernatants from HMEC-1 exposed only to Stx2 exhibited enhanced exposure of platelet P-selectin and platelet-THP-1 cell interactions. Blockade of platelet cyclooxygenase by indomethacin prevented functional activation. The chemokine RANTES enhanced platelet aggregation induced by SDF-1␣, macrophage-derived chemokine, or thymus and activationregulated chemokine in the presence of very low ADP levels. These data support the hypothesis that microvascular endothelial cells exposed to E. coli O157:H7-derived Stx2 and LPS release chemokines and other factors, which when combined with low levels of primary agonists, such as ADP, cause platelet activation and promote the renal thrombosis associated with HUS.

The CXCR4/CXCR7/SDF-1 pathway contributes to the pathogenesis of Shiga toxin–associated hemolytic uremic syndrome in humans and mice

“…Increased endothelial cell surface expression of CXCR4 was also observed by flow cytometry (Supplemental Figure 4A). The observed translational effects of Stx on SDF1 were less pronounced than those on the SDF-1 receptors ( Figure 4C) but were consistent with a previous report of enhanced SDF-1 release from microvascular endothelial cells exposed to Stx (39). The translational changes observed for these Stx target genes are specific and clearly different from other genes, such as cyclophilin A, which appears to fall off the ribosomes in the presence of only 10 fM Stx ( Figure 4D).…”

Platelet activation in patients with sickle disease, hemolysis-associated pulmonary hypertension, and nitric oxide scavenging by cell-free hemoglobin

“…Hemoglobinemia, disordered platelet function, and thrombosis are features of paroxysmal nocturnal hemoglobinuria, thrombotic thrombocytopenic purpura, hemolytic-uremic syndrome, and disseminated intravascular coagulation. There is evidence of platelet activation in these conditions, [52][53][54][55][56][57][58][59] but the potential contribution of NO scavenging by plasma hemoglobin to platelet activation and thrombosis has not yet been investigated. This mechanism is supported by evidence of NO scavenging by cell-free cross-linked hemoglobin artificial blood substitutes in rats, promoting platelet deposition at sites of subintimal injury.…”

Alternative Pathway Activation of Complement by Shiga Toxin Promotes Exuberant C3a Formation That Triggers Microvascular Thrombosis

“…After STEC ingestion, Stx is transported in the circulation to the capillary bed of target organs, including the kidney (5,6). Glomerular endothelium that expresses the receptor globotriaosyl ceramide (Gb3)/CD77 (10,11) is the main target of the toxic effects of Stx1 and 2 (12), which activate a cascade of signals contributing to vascular dysfunction, leukocyte recruitment, and thrombus formation (13)(14)(15)(16)(17)(18)(19). We previously reported that Stx promoted von Willebrand factor (VWF)-dependent thrombus growth on microvascular endothelium under flow via upregulation of P-selectin (17).…”