Endothelial cell damage mediated by human monocytes. Effects of Shiga toxin1 (Stx1)

RAMOS, MV; FERNANDEZ, GC; NEGROTTO, S; LANDONI, V; D´ATRI, P; BENTANCOR, L; SHATTNER, M; ISTURIZ, M; PALERMO, M

Cordoba, Argentina

Congreso; VII Congreso Latinoamericano de Inmunología; 2005

ALAI

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. These factors up regulate the expression of the chemokine fractalkine (FKN) on endothelial cells. FKN receptor, CX3CR1, is present in NK, T CD8 cells and Mo and is involved in cell traffic during inflammation. Objectives: a) Investigate whether Stx1(500ng/ml) and LPS(100 ng/ml) modulate CX3CR1 expression on purified Mo by flow cytometry. b) Determine the capacity of Stx1treated Mo (Mo(Stx1)) to damage endothelial cells (HUVEC). Methods and Results a) There was a significant decrease in the CX3CR1 expression in Mo incubated 4h with Stx1 or LPS, % MoCX3CR1+: basal= 69±5, LPS=45±7*,Stx1=54±7*;*p<0.01. b) HUVEC, sensitized with TNF a (10 ng/ml) were incubated with Mo(Stx1).After 6h cells were stained and supernatant were collected for measuring LDH activity to evaluate endothelial damage. The relative percentages of alive cells, considering basal as 100 were TNF= 87±7, Stx1=75±10, TNF+Stx1=9±4*, TNF+Mo(Stx1)=18±8*; *p<0.005. LDH activity shows similar results (U/L:basal=54.7±13,TNF= 65.7±15, TNF+Stx1=125.6±9*, TNF+Mo(Stx1)=145.7±9; *p<0.05) We demonstrate that a) inflammatory factors and Stx downregulate MoCX3CR1expression and b) TNF preincubation of HUVEC allows cytotoxicity by Stx1 and Mo (Stx1). We conclude that Mo contribute to endothelial damage not only through secretion of  inflammatory cytokines, but also through the binding of Stx, activation, migration and increased interaction with endothelium.