RESUMEN
The inhibitory effect of interleukin-10 (IL-10), an anti-inflammatory cytokine, on lipopolysaccharide (LPS)-induced IL-6 production was characterized by simultaneous stimulation of RAW 264.7 cells with LPS and IL-10. The presence of IL-10 significantly inhibited LPS-induced IL-6 production at a transcriptional level. The expression of IkappaB-zeta, which promotes IL-6 production, was induced in response to LPS and it was definitely suppressed in the presence of IL-10. Further, IL-10 inhibited LPS-induced NF-kappaB activation. A pharmacological inhibitor of NF-kappaB prevented LPS-induced IkappaB-zeta expression, suggesting that IL-10 might inhibit LPS-induced IkappaB-zeta expression via the inactivation of NF-kappaB. In LPS- and IL-10-stimulated cells, the expression of Bcl-3 that inhibits NF-kappaB activation was significantly augmented. Introduction of Bcl-3 siRNA abolished IL-10-mediated IkappaB-zeta inhibition. In the presence of Bcl-3, siRNA IL-10 failed to inhibit LPS-induced IL-6 production. Therefore, it was suggested that Bcl-3 induced by IL-10 might reduce LPS-induced IkappaB-zeta activity via inactivation of NF-kappaB and that reduced IkappaB-zeta activity failed to promote LPS-induced IL-6 production.
Asunto(s)
Interleucina-10/farmacología , Interleucina-6/biosíntesis , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Recombinantes/farmacología , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Aspirina/farmacología , Proteínas del Linfoma 3 de Células B , Línea Celular , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Regulación hacia Abajo/inmunología , Interleucina-6/genética , Interleucina-6/metabolismo , Lipopolisacáridos/inmunología , Lipopolisacáridos/metabolismo , Activación de Macrófagos/efectos de los fármacos , Activación de Macrófagos/genética , Activación de Macrófagos/inmunología , Ratones , FN-kappa B/antagonistas & inhibidores , Proteínas Nucleares/genética , Proteínas Nucleares/inmunología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/inmunología , ARN Interferente Pequeño/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/inmunología , Factores de Transcripción/genética , Factores de Transcripción/inmunología , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/genética , Activación Transcripcional/inmunología , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The effect of Clostridium perfringens alpha-toxin on production of tumor necrosis factor (TNF)-alpha and nitric oxide (NO) in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells was studied. The pretreatment of wild type alpha-toxin, but not the inactive mutant, significantly decreased LPS-induced TNF-alpha and NO production. alpha-Toxin inhibited the expression of TNF-alpha and an inducible type of NO synthase protein and mRNA. Furthermore, it inhibited the phosphorylation of IkappaB-alpha and p65 NF-kappaB subunit, and the NF-kappaB luciferase reporter gene activity in LPS-stimulated cells. The pretreatment of alpha-toxin increased the level of intracellular ceramide. Taken together, Clostridium perfringens alpha-toxin pretreatment was suggested to inhibit LPS-induced TNF-alpha and NO production through the inhibition of NF-kappaB activation. The relationship between alpha-toxin-induced intracellular ceramide generation and the NF-kappaB inhibition is discussed.
Asunto(s)
Toxinas Bacterianas/inmunología , Toxinas Bacterianas/metabolismo , Proteínas de Unión al Calcio/inmunología , Proteínas de Unión al Calcio/metabolismo , Ceramidas/metabolismo , Lipopolisacáridos/inmunología , Óxido Nítrico/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Fosfolipasas de Tipo C/inmunología , Fosfolipasas de Tipo C/metabolismo , Animales , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Quinasa I-kappa B/metabolismo , Luciferasas/biosíntesis , Luciferasas/genética , Macrófagos/química , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , Fosforilación , Factor de Transcripción ReIA/metabolismoRESUMEN
Nystatin is known to deplete lipid rafts from mammalian cell membranes. Lipid rafts have been reported to be necessary for lipopolysaccharide signaling. In this study, it was unexpectedly found that lipopolysaccharide-induced nitric oxide production was not inhibited, but rather increased in the presence of a non-cytotoxic concentration of nystatin. Surprisingly, treatment with nystatin induced only NO production and iNOS expression in RAW264.7 cells. At the concentration used, no changes in the expression of GM1 ganglioside, a lipid raft marker on RAW264.7 cells, was seen. From studies using several kinds of inhibitors for signaling molecules, nystatin-induced NO production seems to occur via the ikappaB/NF-kappaB and the PI3 K/Akt pathway. Furthermore, because nystatin is known to activate the Na-K pump, we examined whether the Na-K pump inhibitor amiloride suppresses nystatin-induced NO production. It was found that amiloride significantly inhibited nystatin-induced NO production. The results suggest that a moderate concentration of nystatin induces NO production by Na-pump activation through the PI3 kinase/Akt/NF-kappaB pathway without affecting the condition of lipid rafts.