AIASSA VIRGINIA
Congresos y reuniones científicas
Título:
Nitric oxide-mediated apoptosis in rat macrophages subjected to Shiga Toxin from Eschearichia coli
Autor/es:
BARONETTI, JL; ANGEL VILLEGAS, N; AIASSA, V; SUÁREZ , ME; PARAJE, MG; ALBESA, I.
Lugar:
Buenos Aires
Reunión:
Simposio; 7th International Symposium on Shiga Toxin (Verocytotoxin)-Producing Eschearichia coli infections; 2009
Institución organizadora:
Asociación Argentina de Microbiología
Resumen:
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.
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.
