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* Mario Negri Institute for Pharmacological Research, Bergamo, Italy;
Department of Microbiology and Immunology, School of Medicine, Vanderbilt University, Nashville, TN 37232;
Department of Pediatric Nephrology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; and
Unit of Nephrology and Dialysis, Azienda Ospedaliera, Ospedali Riuniti di Bergamo, Italy
Shiga toxins (Stx) are the virulence factors of enterohemorrhagic Escherichia coli O157:H7, a worldwide emerging diarrheal pathogen, which precipitates postdiarrheal hemolytic uremic syndrome, the leading cause of acute renal failure in children. In this study, we show that Stx2 triggered expression of fractalkine (FKN), a CX3C transmembrane chemokine, acting as both adhesion counterreceptor on endothelial cells and soluble chemoattractant. Stx2 caused in HUVEC expression of FKN mRNA and protein, which promoted leukocyte capture, ablated by Abs to either endothelial FKN or leukocyte CX3CR1 receptor. Exposure of human glomerular endothelial cells to Stx2 recapitulated its FKN-inducing activity and FKN-mediated leukocyte adhesion. Both processes required phosphorylation of Src-family protein tyrosine kinase and p38 MAPK in endothelial cells. Furthermore, they depended on nuclear import of NF-
B and other stress-responsive transcription factors. Inhibition of their nuclear import with the cell-penetrating SN50 peptide reduced FKN mRNA levels and FKN-mediated leukocyte capture by endothelial cells. Adenoviral overexpression of IB
inhibited FKN mRNA up-regulation. The FKN-mediated responses to Stx2 were also dependent on AP-1. In mice, both virulence factors of Stx-producing E. coli, Stx and LPS, are required to elicit hemolytic uremic syndrome. In this study, FKN was detected within glomeruli of C57BL/6 mice injected with Stx2, and further increased after Stx2 plus LPS coadministration. This was associated with recruitment of CX3CR1-positive cells. Thus, in response to Stx2, FKN is induced playing an essential role in the promotion of leukocyte-endothelial cell interaction thereby potentially contributing to the renal microvascular dysfunction and thrombotic microangiopathy that underlie hemolytic uremic syndrome due to enterohemorrhagic E. coli O157:H7 infection.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 P.M. was the recipient of a Fellowship from Fondazione Aiuti per la Ricerca sulle Malattie Rare, Bergamo, Italy and J.H. was supported by National Institutes of Health Grants HL69542 and HL68744.
2 C.Za. and C.Zo. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Carla Zoja, "Mario Negri" Institute for Pharmacological Research, Via Gavazzeni 11, 24125 Bergamo, Italy. E-mail address: zoja{at}marionegri.it
4 Abbreviations used in this paper: Stx, Shiga toxin; HUS, hemolytic uremic syndrome; D+HUS, diarrhea-associated HUS; Gb3, globotriaosyl ceramide; Tir, translocated intimin receptor; FKN, fractalkine; GEC, glomerular endothelial cell; PTK, protein tyrosine kinase; ODN, oligodeoxynucleotide; Ct, cycle threshold; BUN, blood urea nitrogen; vWF, von Willebrand factor; SRTF, stress responsive transcription factor; PMN, polymorphonuclear cell.
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