Nitric oxide modulates pro- and anti-inflammatory cytokines in lipopolysaccharide-activated macrophages

Chih-Hsiung Wu, Ta-Liang Chen, Tyng-Guey Chen, Wei-Pin Ho, Wen-Ta Chiu, Ruei-Ming Chen

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Abstract

Background: Sepsis is a serious and life-threatening syndrome that occurs in intensive care unit patients. Lipopolysaccharide (LPS) has been implicated as one of major causes of sepsis. Nitric oxide (NO) and cytokines are involved in sepsis-induced inflammatory responses. This study is aimed at evaluating the effects of NO on the modulation of pro- and antiinflammatory cytokines in LPS-activated macrophages and its possible mechanism. Methods: N-Monomethyl arginine (NMMA), an inhibitor of NO synthase, was used in this study to suppress NO production. Mouse macrophage-like Raw 264.7 cells were exposed to LPS, NMMA, or a combination of NMMA and LPS. Cell viability was determined by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The amounts of nitrite, an oxidative product of NO, in the culture medium were quantified according to the Griess reaction method. Enzyme-linked immunosorbent assay and reverse-transcriptase polymerase chain reaction were carried out to determine the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-10 in macrophages. Results: Exposure of macrophages to LPS, NMMA, and a combination of NMMA and LPS for 24 hours did not affect cell viability. LPS significantly increased the amounts of nitrite in macrophages (p <0.01). Treatment with NMMA decreased LPS-enhanced nitrite (p <0.01) in a concentration-dependent manner. Analyses of enzyme-linked immunosorbent assays and reverse-transcriptase polymerase chain reaction revealed that LPS significantly induced TNF-α, IL-1β, and IL-10 proteins and mRNA (p <0.01). A combined treatment with NMMA and LPS significantly blocked LPS-induced TNF-α and IL-1β (p <0.01), but synergistically enhanced LPS-induced IL-10 (p <0.05) protein and RNA. Conclusion: This study has shown that NO suppression can inhibit LPS-induced TNF-α and IL-1β but enhance IL-10, and the modulation occurs at a pretranslational level.

Original languageEnglish
Pages (from-to)540-545
Number of pages6
JournalJournal of Trauma - Injury, Infection and Critical Care
Volume55
Issue number3
DOIs
Publication statusPublished - Sep 1 2003

Fingerprint

Lipopolysaccharides
Nitric Oxide
Anti-Inflammatory Agents
Macrophages
Cytokines
Arginine
Interleukin-1
Interleukin-10
Nitrites
Tumor Necrosis Factor-alpha
Sepsis
Reverse Transcriptase Polymerase Chain Reaction
Cell Survival
Enzyme-Linked Immunosorbent Assay
Nitric Oxide Synthase
Intensive Care Units
Culture Media
Proteins
RNA
Messenger RNA

Keywords

  • Cytokines
  • Lipopolysaccharide
  • N-Monomethyl arginine
  • Nitric oxide
  • Sepsis

ASJC Scopus subject areas

  • Surgery

Cite this

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title = "Nitric oxide modulates pro- and anti-inflammatory cytokines in lipopolysaccharide-activated macrophages",
abstract = "Background: Sepsis is a serious and life-threatening syndrome that occurs in intensive care unit patients. Lipopolysaccharide (LPS) has been implicated as one of major causes of sepsis. Nitric oxide (NO) and cytokines are involved in sepsis-induced inflammatory responses. This study is aimed at evaluating the effects of NO on the modulation of pro- and antiinflammatory cytokines in LPS-activated macrophages and its possible mechanism. Methods: N-Monomethyl arginine (NMMA), an inhibitor of NO synthase, was used in this study to suppress NO production. Mouse macrophage-like Raw 264.7 cells were exposed to LPS, NMMA, or a combination of NMMA and LPS. Cell viability was determined by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The amounts of nitrite, an oxidative product of NO, in the culture medium were quantified according to the Griess reaction method. Enzyme-linked immunosorbent assay and reverse-transcriptase polymerase chain reaction were carried out to determine the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-10 in macrophages. Results: Exposure of macrophages to LPS, NMMA, and a combination of NMMA and LPS for 24 hours did not affect cell viability. LPS significantly increased the amounts of nitrite in macrophages (p <0.01). Treatment with NMMA decreased LPS-enhanced nitrite (p <0.01) in a concentration-dependent manner. Analyses of enzyme-linked immunosorbent assays and reverse-transcriptase polymerase chain reaction revealed that LPS significantly induced TNF-α, IL-1β, and IL-10 proteins and mRNA (p <0.01). A combined treatment with NMMA and LPS significantly blocked LPS-induced TNF-α and IL-1β (p <0.01), but synergistically enhanced LPS-induced IL-10 (p <0.05) protein and RNA. Conclusion: This study has shown that NO suppression can inhibit LPS-induced TNF-α and IL-1β but enhance IL-10, and the modulation occurs at a pretranslational level.",
keywords = "Cytokines, Lipopolysaccharide, N-Monomethyl arginine, Nitric oxide, Sepsis",
author = "Chih-Hsiung Wu and Ta-Liang Chen and Tyng-Guey Chen and Wei-Pin Ho and Wen-Ta Chiu and Ruei-Ming Chen",
year = "2003",
month = "9",
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doi = "10.1097/01.TA.0000033496.62796.3B",
language = "English",
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TY - JOUR

T1 - Nitric oxide modulates pro- and anti-inflammatory cytokines in lipopolysaccharide-activated macrophages

AU - Wu, Chih-Hsiung

AU - Chen, Ta-Liang

AU - Chen, Tyng-Guey

AU - Ho, Wei-Pin

AU - Chiu, Wen-Ta

AU - Chen, Ruei-Ming

PY - 2003/9/1

Y1 - 2003/9/1

N2 - Background: Sepsis is a serious and life-threatening syndrome that occurs in intensive care unit patients. Lipopolysaccharide (LPS) has been implicated as one of major causes of sepsis. Nitric oxide (NO) and cytokines are involved in sepsis-induced inflammatory responses. This study is aimed at evaluating the effects of NO on the modulation of pro- and antiinflammatory cytokines in LPS-activated macrophages and its possible mechanism. Methods: N-Monomethyl arginine (NMMA), an inhibitor of NO synthase, was used in this study to suppress NO production. Mouse macrophage-like Raw 264.7 cells were exposed to LPS, NMMA, or a combination of NMMA and LPS. Cell viability was determined by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The amounts of nitrite, an oxidative product of NO, in the culture medium were quantified according to the Griess reaction method. Enzyme-linked immunosorbent assay and reverse-transcriptase polymerase chain reaction were carried out to determine the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-10 in macrophages. Results: Exposure of macrophages to LPS, NMMA, and a combination of NMMA and LPS for 24 hours did not affect cell viability. LPS significantly increased the amounts of nitrite in macrophages (p <0.01). Treatment with NMMA decreased LPS-enhanced nitrite (p <0.01) in a concentration-dependent manner. Analyses of enzyme-linked immunosorbent assays and reverse-transcriptase polymerase chain reaction revealed that LPS significantly induced TNF-α, IL-1β, and IL-10 proteins and mRNA (p <0.01). A combined treatment with NMMA and LPS significantly blocked LPS-induced TNF-α and IL-1β (p <0.01), but synergistically enhanced LPS-induced IL-10 (p <0.05) protein and RNA. Conclusion: This study has shown that NO suppression can inhibit LPS-induced TNF-α and IL-1β but enhance IL-10, and the modulation occurs at a pretranslational level.

AB - Background: Sepsis is a serious and life-threatening syndrome that occurs in intensive care unit patients. Lipopolysaccharide (LPS) has been implicated as one of major causes of sepsis. Nitric oxide (NO) and cytokines are involved in sepsis-induced inflammatory responses. This study is aimed at evaluating the effects of NO on the modulation of pro- and antiinflammatory cytokines in LPS-activated macrophages and its possible mechanism. Methods: N-Monomethyl arginine (NMMA), an inhibitor of NO synthase, was used in this study to suppress NO production. Mouse macrophage-like Raw 264.7 cells were exposed to LPS, NMMA, or a combination of NMMA and LPS. Cell viability was determined by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The amounts of nitrite, an oxidative product of NO, in the culture medium were quantified according to the Griess reaction method. Enzyme-linked immunosorbent assay and reverse-transcriptase polymerase chain reaction were carried out to determine the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-10 in macrophages. Results: Exposure of macrophages to LPS, NMMA, and a combination of NMMA and LPS for 24 hours did not affect cell viability. LPS significantly increased the amounts of nitrite in macrophages (p <0.01). Treatment with NMMA decreased LPS-enhanced nitrite (p <0.01) in a concentration-dependent manner. Analyses of enzyme-linked immunosorbent assays and reverse-transcriptase polymerase chain reaction revealed that LPS significantly induced TNF-α, IL-1β, and IL-10 proteins and mRNA (p <0.01). A combined treatment with NMMA and LPS significantly blocked LPS-induced TNF-α and IL-1β (p <0.01), but synergistically enhanced LPS-induced IL-10 (p <0.05) protein and RNA. Conclusion: This study has shown that NO suppression can inhibit LPS-induced TNF-α and IL-1β but enhance IL-10, and the modulation occurs at a pretranslational level.

KW - Cytokines

KW - Lipopolysaccharide

KW - N-Monomethyl arginine

KW - Nitric oxide

KW - Sepsis

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M3 - Article

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AN - SCOPUS:0141633832

VL - 55

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JO - Journal of Trauma and Acute Care Surgery

JF - Journal of Trauma and Acute Care Surgery

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