Endothelial and Epithelial Cell Damage Mediated By Human Monocytes and Shiga Toxin-1 (Stx1).

RAMOS M.V; FERN¨¢NDEZ G.C; LANDONI V.I.; PANEK C.A; D´ATRI P; FERNANDEZ-BRANDO R.J; SILBERSTEIN C; ISTURIZ M.A; PALERMO M.S

Buenos Aires

Simposio; 7th International Symposium on Shiga Toxin (Verocytotoxin)-producing Escherichia coli Infections; 2009

Sociedad Argentina de Microbiolog¨ªa

Infection with Stx-producing E.Coli leads to Hemolytic Uremic Syndrome (HUS). Cytotoxic effects on renal epithelial and endothelial cells caused by Stx, inflammatory factors and monocytes (Mo), are all involved in the development of HUS. In addition, inflammatory factors up regulate the expression of the chemokine fractalkine (CX3CL1) on endothelial cells and epithelial cells. CX3CL1 receptor, CX3CR1, is present on Mo and is involved in cell traffic during inflammation. The purpose of the study was: A) To determine the capacity of Stx1-treated Mo (Mo(Stx1)) to damage endothelial (Human umbilical vein endothelial cells, HUVEC) and epithelial cells (Human Renal Tubular epithelial cells, HRTEC). B) To investigate the role of CX3CL1 in Mo(Stx1)-mediated damage on endothelial and epithelial cells. Results: Human purified Mo (2x106 cell/ml) were incubated with medium alone (Mo) or with (5ng/ml) Stx1 for 1 h at 4¡ãC, then Mo were washed twice to eliminate Stx1 excess (Mo(Stx1)). Finally HUVEC or HRTEC cells, sensitized with TNF-¦Á (10 ng/ml), were coincubated with Mo, Mo(Stx1) alone or preincubated with anti-Stx1/Stx2 antibody (Ab, 2¦Ìg/ml) or soluble CX3CL1 (0.5¦Ìg/ml). After 20h HUVEC and HRTEC cells which remained viable were stained with May Gr¨¹nwald-Giemsa and counted by optic microscopy, to evaluate endothelial or epithelial damage. The relative percentages of living cells, considering cells without treatment as 100 were TNF= 100¡À7.6, TNF+Stx1=45.5¡À9.9*, TNF+Mo=74.0¡À11.1*; TNF+Mo(Stx1)=51.5¡À11.0*; TNF+Mo(Stx1)+Ab=67.2¡À12.0; TNF+Mo(Stx1)+CX3CL1s=78.1¡À14.5#; *p<0.005 compared to TNF; #p<0.05 compared to Mo(Stx1). In the case of HRTEC, the relative percentages of living cells were TNF=88.8¡À3.9; TNF+Stx1=49.5¡À3.9*; TNF+Mo=71.4¡À7.0; TNF+Mo(Stx1)=49.2¡À5.5*; TNF+Mo(Stx1)+Ab=95.5¡À7.9; TNF+Mo(Stx1)+CX3CL1s=70.1¡À15.1#; *p<0.05 compared to TNF; #p<0.05 compared to Mo(Stx1) We conclude that Mo contribute to endothelial and epithelial damage not only through secretion of inflammatory cytokines, but also through the binding of Stx, activation and increased interaction with endothelium, mediated at least in part by CX3CL1/CX3CR1. Even though we observed that MoStx1 mediated damage was partially counteracted by the presence of an anti-Stx antibody, it was also clear that CX3CL1 was involved in the interaction of Mo to endothelium/epithelium.