Purpose
The aim of this study was to investigate the participation of nitric oxide (NO) in the apoptosis of rat peritoneal macrophages induced by culture supernatants and shiga toxin from Escherichia coli.
Material and methods
Shiga toxin (Stx) was purified from a clinically isolated E. coli O157:H7 stain using a receptor-mediated affinity chromatography. Purity of toxin was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining. Rat peritoneal macrophages, obtained by Percoll gradient, were exposed to different dilutions of E. coli cell free supernatants or Stx for 24 hs at 37°C in a 5% CO2 humidified atmosphere. After incubation, apoptotic cells (propidium iodide staining) and NO production (Griess reaction) were evaluated. In some experiments, the cultures were performed in the presence of inducible nitric oxide synthase (iNOS) inhibitor aminoguanidine (AG, 1mM).
Results
Peritoneal macrophages incubated in presence of E. coli supernatants showed an increment in apoptosis levels as well as in the NO production. Furthermore, the inhibition of NO synthesis, induced by the addition of AG at cultures, was correlated with a diminution in the percentage of apoptotic cells in these cultures, indicating the participation of this metabolite in the apoptotic process. On the other hand, in order to identify one of the probable products involved in these phenomena, presents in the culture supernatants, rat peritoneal macrophages were cultivated in presence of different concentrations of Stx. In similar form at observed with the culture supernatants, the treatment of cells with Stx induced an increment in the NO production and apoptotic levels. In addition, these parameters also were reverted by the aggregated of AG at cultures.
Conclusion
Different E. coli-related products have been shown to induce apoptosis in a variety of cells types. However, the precise relationship between NO synthesis and induction of apoptosis has not been intensely investigated. In this study, we demonstrated that the treatment with E. coli supernatants, or with the major virulence factors of this pathogen, Stx, induces nitric oxide-mediated apoptosis of rat peritoneal macrophages. These results could contribute to better understand of the immunopathology of E. coli.
