Shiga toxin-2 triggers endothelial leukocyte adhesion and transmigration via NF-kappaB dependent up-regulation of IL-8 and MCP-1.
BACKGROUND
Shiga toxin (Stx)-producing E. coli is a causative agent of the epidemic form of hemolytic uremic syndrome (HUS), the most common cause of acute renal failure in children. Endothelial injury and leukocyte activation are instrumental to the development of microangiopathic lesions. To obtain more insight into the mechanisms favoring endothelium-leukocyte interaction, we studied (1) the effect of Stx-2 on leukocyte adhesion and transmigration in human endothelial cells under flow; (2) the effect of Stx-2 on endothelial expression of monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) and their functional role in the adhesive phenomena; and (3) the role of nuclear factor-kappaB (NF-kappaB) in endothelial chemokine expression.


METHODS
For adhesion experiments, human umbilical vein endothelial cells (HUVEC) and human glomerular endothelial cells (GEC) were incubated for 24 hours with Stx-2 (25 pmol/L), with or without anti-IL-8 or MCP-1 antibodies, and then exposed to leukocyte suspension under flow (1.5 dynes/cm2). IL-8 and MCP-1 expression was evaluated in Stx-2 treated endothelial cells (6 hours) by Northern blot. NF-kappaB activity was assessed by electrophoretic mobility shift assay. The role of NF-kappaB in Stx-induced chemokines was evaluated by transfecting HUVEC with an adenovirus coding for IkappaBalpha.


RESULTS
Stx-2 significantly enhanced the number of leukocytes that adhered and then migrated across the endothelium. Stx-2 increased the expression of IL-8 and MCP-1, which was preceded by NF-kappaB activation. Blocking of endothelial IL-8 and MCP-1 with corresponding antibodies significantly inhibited Stx-induced leukocyte adhesion and migration either in HUVEC or GEC. Adenovirus-mediated gene transfer of IkappaBalpha down-regulated IL-8 and MCP-1 mRNA and also inhibited the adhesion and transmigration of leukocytes in Stx-treated HUVEC.


CONCLUSIONS
Stx-2 via a transcriptional activation mechanism specifically mediated by NF-kappaB up-regulates endothelial MCP-1 and IL-8. Both chemokines are important modulators of leukocyte adhesion and transmigration under flow. These findings might be relevant to understand the nature of microvascular lesions in HUS and open future perspectives for better treatment of microvascular thrombosis.
Zoja
Carla
C
Mario Negri Institute for Pharmacological Research, Azienda Ospedaliera, Ospedali Riuniti di Bergamo, Via Gavazzeni 11, 24125 Bergamo, Italy. zoja@marionegri.it
Angioletti
Stefania
S
Donadelli
Roberta
R
Zanchi
Cristina
C
Tomasoni
Susanna
S
Binda
Elena
E
Imberti
Barbara
B
te Loo
Maroeska
M
Monnens
Leo
L
Remuzzi
Giuseppe
G
Morigi
Marina
M
D000256:Adenoviridae / Q000235:genetics; D002448:Cell Adhesion / Q000187:drug effects / Q000276:immunology; D002465:Cell Movement / Q000187:drug effects / Q000276:immunology; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000235:genetics*; D004730:Endothelium, Vascular / Q000166:cytology / Q000276:immunology; D015870:Gene Expression / Q000187:drug effects / Q000276:immunology; D006463:Hemolytic-Uremic Syndrome / Q000276:immunology; D006801:Humans; D020672:I-kappa B Proteins / Q000235:genetics; D016209:Interleukin-8 / Q000235:genetics*; D007678:Kidney Glomerulus / Q000166:cytology / Q000276:immunology*; D007962:Leukocytes / Q000166:cytology*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D022641:Shiga Toxin 2 / Q000633:toxicity*; D014162:Transfection; D014471:Umbilical Veins / Q000166:cytology; D015854:Up-Regulation / Q000187:drug effects / Q000276:immunology
D018932:Chemokine CCL2; D020672:I-kappa B Proteins; D016209:Interleukin-8; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D012333:RNA, Messenger; D022641:Shiga Toxin 2; D000072000:NF-KappaB Inhibitor alpha