S-nitrosoglutathione inhibits TNF-alpha-induced NFkappaB activation in neutrophils.
OBJECTIVE
Cytokine expression is controlled by transcription factors including NFkappaB, which has recently been found to exist in human neutrophils. We previously showed that exogenous nitric oxide (NO) induces neutrophil apoptosis and hypothesized that this NO effect could be mediated by inhibition of NFkappaB activation.


METHODS
Isolated human neutrophils were incubated with or without S-nitrosoglutathione (GSNO 0.1 mM-5 mM; Sigma) for 2 h. Neutrophils were either unstimulated or stimulated with TNFalphalpha or n-formyl methionyl leucine phenylalanine (fMLP). Viability was assessed by vital dye cytotoxicity assay. After nuclear extraction and measurement of protein concentration, NFkappaB binding was determined by electrophoretic mobility shift assay. Effects of GSNO on activation of IkappaB alpha, which inhibits intranuclear translocation of NFkappaB, were measured by Western immunoblot technique. For comparison, experiments were also performed in the presence of the NFkappaB inhibitor PDTC.


RESULTS
TNFalpha increased nuclear NFkappaB activity compared to unstimulated neutrophils (p < 0.001, n = 5). GSNO (500 microM) decreased TNFalpha-induced NFkappaB activity (p<0.05) and inhibited NFkappaB activity whether given prior to or during TNFalpha exposure. IkappaB alpha was significantly degraded at 30 and 120 min of TNFalpha exposure compared to control neutrophils (p < 0.05). GSNO exposure (500 microM) inhibited IkappaB alpha degradation in the presence of TNFalpha. PDTC enhanced neutrophil cell death and DNA fragmentation, in association with decreased NFkappaB activity, similar to GSNO effects.


CONCLUSIONS
Neutrophils possess NFkappaB activity that is increased by stimulation with TNFalpha. GSNO inhibits NFkappaB activity in association with inhibiting TNFalpha-induced degradation of IkappaB alpha. GSNO effects are similar to those seen with NFkappaB inhibition by PDTC. Inhibition of NF kappaB could represent a potential anti-inflammatory effect of GSNO.
Fortenberry
J D
JD
Critical Care Division, Children's Healthcare of Atlanta at Egleston, GA 30322, USA. James.Fortenberry@choa.org
Owens
M L
ML
Chen
N X
NX
Brown
L A
LA
D000328:Adult; D002467:Cell Nucleus / Q000378:metabolism; D053938:DNA Fragmentation; D004268:DNA-Binding Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005978:Glutathione / Q000031:analogs & derivatives* / Q000494:pharmacology*; D006801:Humans; D020672:I-kappa B Proteins; D020287:In Situ Nick-End Labeling; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009504:Neutrophils / Q000187:drug effects* / Q000378:metabolism* / Q000648:ultrastructure; D009603:Nitroso Compounds / Q000494:pharmacology*; D011392:Proline / Q000031:analogs & derivatives / Q000494:pharmacology; D026422:S-Nitrosoglutathione; D013859:Thiocarbamates / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D009603:Nitroso Compounds; D013859:Thiocarbamates; D014409:Tumor Necrosis Factor-alpha; C066229:prolinedithiocarbamate; D000072000:NF-KappaB Inhibitor alpha; D026422:S-Nitrosoglutathione; D009240:N-Formylmethionine Leucyl-Phenylalanine; D011392:Proline; D005978:Glutathione