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

Fadila Guessous,¹ Marek Marcinkiewicz,¹ Renata Polanowska-Grabowska,¹ Sudawadee Kongkhum,^1, Daniel Heatherly,^2, Tom Obrig,² and Adrian R. L. Gear^1*

Department of Biochemistry & Molecular Genetics,¹ Division of Nephrology, Department of Medicine, University of Virginia School of Medicine, Charlottesville, Virginia 22908²

Received 2 July 2005/ Returned for modification 17 August 2005/ Accepted 7 September 2005

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-1ß 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 activation-regulated 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.

Editor: J. T. Barbieri

Present address: Department of Medical Technology, Faculty of Allied Health Sciences, Thammasat University, Rangsit Campus, 99 Mhu 18 Klongneung, KlongLuang, Pathumthani 12120, Thailand.

Present address: RD 1 Box 158, Glen Easton, WV 26039.

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