Shiga toxin-1 effect on lipopolysaccharide-sensitized astrocytes induces toxicity and modulation of endothelial cells properties

LANDONI V,; C CALATAYUD; CAMPOS-NEBEL M; SCHIERLOH LP; FERNÁNDEZ GC; PALERMO MS; ISTURIZ MA

Buenos Aires

Simposio; 7th International Symposium on Shiga -Toxin (Verocytotoxin)- Producing Eschericchia coli (VTEC2009); 2009

Asociación Argentina de Microbiología

Although Stx is necessary for HUS development, it is known that the inflammatory response is able to potentiate Stx toxicity. In fact, bacterial lipopolysacharide (LPS) play a key role for the development of HUS.In severe cases central nervous system (CNS) complication are observed.In fact , brain damage is one of the most common causes of death. Although the mechanisms of neurological symptoms are not known, damage of brain endothelial cells (BEC), a component of the blood-brain barrier (BBB), is clear.Several in vivo studies have demonstrated that Stx not only is able to impair the BBB function but  also to cross the endothelial barrier. Astrocytes (AST), the most abundant glial cells in CNS, are in close contact with BEC and their interaction help to maintain the correct BBB function and morphology. However, in response to brain injury, AST release inflammatory mediators that modulate the permeability of BBB and  stimulate immune system attracting leukocytes to the site of injury. Given the anatomical association between AST and BEC, as well as the inflammatory role of AST on neuronal dysfunction, we hypothesize that LPS and Stx effects on AST may be involve in the brain damage observed in severe cases of HUS. Then, one of the the aims of this study was to evaluate if Stx1 alone, or in combination with LPS, is capable of inducing an inflammatory response on AST. In addition, we evaluated the effect of soluble factor released by Stx/LPS-treated AST on endothelial cells.Rat AST were treated with LPS; Stx1 or LPS+Stx1. We demonstrated an AST activation induced by Stx1+LPS since an increase of glial fibrillary acidic protein (GFAP) expression (p<0.05) and a maximal TNF-alpha secretion (p<0.05) was observed. Moreover, AST apoptosis was evaluated and significant induction of apoptosis was observed on LPS+Stx1-treated AST (p<0.05).In addition, taking into account that soluble factor released by AST have been shown to induce BBB endothelial cell phenotype in non-neural endothelial cells,human umbilical vein endothelial cell (HUVEC) monolayers were cultured with LPS/Stx-treated astrocyte SN (SN-AST) and the adhesion molecules expression were analized by flow cytometry. A significant increase of ICAM-1 and E-selectine expression was observed on HUVEC treated with SN-ASTLPS+Stx1 (p<0.001 vs SN-ASTcontrol and p<0.05 vs SN-AST LPS).Other authors have shown that conditioned media from AST is able to induce tubulogenesis in HUVEC cultures. However we found that this ability was dramatically reduced when LPS sensitized-AST were treated with Stx-1(p<0.05 vs SN-ASTcontrol,SN-AST LPS and SN-AST Stx1).Finally SN-AST-induced endothelial cell toxicity was evaluated. In this sense, higher cell death was observed when HUVEC were incubated with SN-ASTLPS+Stx1 (p<0.05 vs SN-ASTStx1 and SN-ASTLPS). Our results suggest that the response of AST to Stx1+LPS may contribute to inflammation leading to endothelial injury and disturbance of the BBB function.