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Double-stranded RNA-induced inducible nitric-oxide synthase expression and interleukin-1 release by murine
M R Heitmeier1, A L Scarim, J A Corbett
1Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, Missouri 63104, USA.
Abstract:
The effects of double-stranded RNA (synthetic polyinosinic-polycytidylic acid; poly(I-C)) on macrophage expression of inducible nitric-oxide synthase (iNOS), production of nitric oxide, and release of interleukin-1 (IL-1) were investigated. Individually, poly(I-C), interferon-gamma (IFN-gamma), and lipopolysaccharide (LPS) stimulate nitrite production and iNOS expression by RAW 264.7 cells. In combination, the effects of poly(I-C) + IFN-gamma are additive, while poly(I-C) does not further potentiate LPS-induced nitrite production. These results suggest that poly(I-C) and LPS may stimulate iNOS expression by similar signaling pathways, which may be independent of pathways activated by IFN-gamma. LPS-induced iNOS expression is associated with the activation of NF-kappaB. We show that inhibition of NF-kappaB by pyrrolidinedithiocarbamate prevents poly(I-C) + IFN-gamma-, poly(I-C) + LPS-, and LPS-induced iNOS expression, nitrite production and IkappaB degradation by RAW 264.7 cells. The effects of poly(I-C) on iNOS expression appear to be cell-type specific. Poly(I-C), alone or in combination with IFN-gamma, does not stimulate, nor does poly(I-C) potentiate, IL-1-induced nitrite production by rat insulinoma RINm5F cells. In addition, we show that the combination of poly(I-C) + IFN-gamma stimulates iNOS expression, nitrite production, IkappaB degradation, and the release of IL-1 by primary mouse macrophages, and these effects are prevented by pyrrolidinedithiocarbamate. These findings indicate that double-stranded RNA, in the presence of IFN-gamma, is a potent activator of macrophages, stimulating iNOS expression, nitrite production, and IL-1 release by a mechanism which requires the activation of NF-kappaB.
Insights
Double-stranded RNA (poly(I-C)) and interferon-gamma (IFN-gamma) together potently activate macrophages. This activation stimulates inducible nitric-oxide synthase (iNOS) expression, nitric oxide production, and interleukin-1 (IL-1) release via NF-kappaB signaling.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Double-stranded RNA (polyinosinic-polycytidylic acid; poly(I-C)) is a known immune stimulant.
- Macrophages play a critical role in innate and adaptive immunity.
- Interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS) are potent macrophage activators.
Purpose of the Study:
- To investigate the effects of poly(I-C) on macrophage activation.
- To determine the role of NF-kappaB signaling in poly(I-C)-mediated macrophage responses.
- To assess cell-type specificity of poly(I-C) effects.
Main Methods:
- Treatment of RAW 264.7 cells and primary mouse macrophages with poly(I-C), IFN-gamma, and LPS.
- Measurement of nitrite production, iNOS expression, IL-1 release, and IkappaB degradation.
- Inhibition of NF-kappaB activation using pyrrolidinedithiocarbamate.
Main Results:
- Poly(I-C) combined with IFN-gamma additively increased nitrite production and iNOS expression in RAW 264.7 cells.
- Poly(I-C) did not potentiate LPS-induced responses but shared similar NF-kappaB-dependent pathways.
- Poly(I-C) + IFN-gamma stimulated iNOS expression, nitrite production, IkappaB degradation, and IL-1 release in primary macrophages, dependent on NF-kappaB activation.
Conclusions:
- Double-stranded RNA (poly(I-C)), particularly with IFN-gamma, is a potent activator of primary macrophages.
- The mechanism involves NF-kappaB activation, leading to iNOS expression, nitric oxide production, and IL-1 release.
- Poly(I-C) effects on iNOS expression are cell-type specific.