Role of endogenous interferon-beta in lipopolysaccharide-triggered activation of the inducible nitric-oxide synthase gene in a mouse macrophage cell line, J774.
The role of endogenous tumor necrosis factor alpha (TNF-alpha) and interferon-beta (IFN-beta) in lipopolysaccharide (LPS)-induced activation of the inducible nitric-oxide synthase (i-NOS) gene was investigated. By Northern analysis or reverse-transcription polymerase chain reaction, the mouse macrophage cell line  (J774) was found to respond to LPS treatment by increased expression of mRNAs specific for TNF-alpha, IFN-beta, and i-NOS with the kinetics unique for each gene. Bioassay of the culture supernatants showed that TNF-alpha and IFN-beta secreted by J774 cells increased from an undetectable level to about 300 and 340 units/ml, respectively, 3-6 h after LPS stimulation. Nitrite concentration was found to increase from 0 to 7.8 and 28.5 microM by 12 and 24 h, respectively, in the culture supernatant of LPS-treated J774 cells. The presence of a neutralizing dose of antibodies against IFN-beta, but not against TNF-alpha, during treatment with either 10 ng or 1 microgram of LPS/ml significantly, but not completely decreased the level of i-NOS-specific mRNA expression and NO production. The incubation of J774 cells with mouse natural IFN-beta itself (up to the level of 1,200 units/ml) did not induce i-NOS-specific mRNA and therefore did not stimulate J774 cells to produce NO. However, natural IFN-beta synergistically augmented the expression of i-NOS mRNA and the production of NO by J774 cells triggered by suboptimal concentrations of LPS (1 to 5 ng/ml). These data thus suggest that endogenous IFN-beta, but not TNF-alpha, produced by LPS-stimulated J774 cells specifically contributes, probably in an auto/paracrine fashion, to the activation of the i-NOS gene expression by LPS.
Fujihara
M
M
Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City 66160-7420.
Ito
N
N
Pace
J L
JL
Watanabe
Y
Y
Russell
S W
SW
Suzuki
T
T
D000594:Amino Acid Oxidoreductases / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D004790:Enzyme Induction / Q000235:genetics; D005260:Female; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology*; D016899:Interferon-beta / Q000502:physiology*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000201:enzymology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics
D017931:DNA Primers; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D016899:Interferon-beta; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases