Nitric oxide-mediated apoptosis in rat macrophages subjected to Shiga Toxin from Eschearichia coli

BARONETTI, JL; ANGEL VILLEGAS, N; AIASSA, V; SUÁREZ , ME; PARAJE, MG; ALBESA, I.

Buenos Aires

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

Asociación Argentina de Microbiología

PurposeThe aim of this study was to investigate the participation of nitric oxide (NO) in theapoptosis of rat peritoneal macrophages induced by culture supernatants and shiga toxinfrom Escherichia coli.Material and methodsShiga toxin (Stx) was purified from a clinically isolated E. coli O157:H7 stain using areceptor-mediated affinity chromatography. Purity of toxin was assessed by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining. Ratperitoneal macrophages, obtained by Percoll gradient, were exposed to different dilutionsof E. coli cell free supernatants or Stx for 24 hs at 37°C in a 5% CO2 humidifiedatmosphere. After incubation, apoptotic cells (propidium iodide staining) and NOproduction (Griess reaction) were evaluated. In some experiments, the cultures wereperformed in the presence of inducible nitric oxide synthase (iNOS) inhibitoraminoguanidine (AG, 1mM).ResultsPeritoneal macrophages incubated in presence of E. coli supernatants showed an incrementin apoptosis levels as well as in the NO production. Furthermore, the inhibition of NOsynthesis, induced by the addition of AG at cultures, was correlated with a diminution inthe percentage of apoptotic cells in these cultures, indicating the participation of thismetabolite in the apoptotic process. On the other hand, in order to identify one of theprobable products involved in these phenomena, presents in the culture supernatants, ratperitoneal macrophages were cultivated in presence of different concentrations of Stx. Insimilar form at observed with the culture supernatants, the treatment of cells with Stxinduced an increment in the NO production and apoptotic levels. In addition, theseparameters also were reverted by the aggregated of AG at cultures.ConclusionDifferent E. coli-related products have been shown to induce apoptosis in a variety of cellstypes. However, the precise relationship between NO synthesis and induction of apoptosishas not been intensely investigated. In this study, we demonstrated that the treatment withE. coli supernatants, or with the major virulence factors of this pathogen, Stx, induces nitricoxide-mediated apoptosis of rat peritoneal macrophages. These results could contribute tobetter understand of the immunopathology of E. coli.