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Modulation of PGE(2) and TNFalpha by nitric oxide and LPS-activated RAW 264.7 cells
Cecilia Guastadisegni1, Alessia Nicolini, Maria Balduzzi
1Laboratory of Environmental Hygiene, Institito Superiore di Sanità, Rome, Italy. Cecilia@iss.it
Abstract:
Prostaglandins (PGs), the arachidonic acid (AA) metabolites of the cyclooxygenase (COX) pathway, and the cytokine TNFalpha play major roles in inflammation and they are synthesised mainly by macrophages. Their syntheses have been shown to be regulated by several factors, including nitric oxide, a further important macrophage product. Since both positive and negative regulations of PGs and TNFalpha synthesis by NO have been reported, we sought to understand the mechanisms underlying these opposite NO effects by using a recent class of NO releasing compounds, the NONOates, which have been shown to release NO in a controlled fashion. To this aim, we analysed the effect of NO released from PAPA/NO (t1/2 15 min) and DETA/NO (t1/2 20 h) in RAW 264.7 cells. Both NONOates were used at the same concentrations allowing the cell cultures to be exposed either at high levels of NO for brief time (PAPA/NO) or at low levels of NO for long time (DETA/NO). We found that the two NONOates had opposite effect on basal TNFalpha release, being increased by PAPA/NO and decreased by DETA/NO, while they did not affect the release stimulated by LPS. At variance, both NONOates increased the basal PGE(2) production, while the LPS-stimulated production was slightly increased only by PAPA/NO. The modulation of PGE(2) synthesis was the result of the distinct effects of the two NO-donors on either arachidonic acid (AA) release or cyclooxygense-2 (COX-2) expression, the precursor and synthetic enzyme of PGs, respectively. Indeed, in resting cultures AA release was enhanced only by PAPA/NO whereas COX-2 expression was moderately upregulated by both donors. In LPS activated cells, both NONOates induced AA release, although with different kinetics and potencies, but only DETA/NO significantly increased COX-2 expression. In conclusion, by comparing the activities of these two NONOates, our observations indicate that level and time of exposure to NO are both crucial in determining the molecular target and the final result of the interactions between NO and inflammatory molecules.
Insights
Nitric oxide (NO) has dual effects on inflammation, influencing prostaglandin and TNFalpha synthesis. The duration and concentration of NO exposure, using NONOates, determine its regulatory impact on these inflammatory mediators in macrophages.
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- Prostaglandins (PGs) and TNFalpha are key inflammatory mediators synthesized by macrophages.
- Nitric oxide (NO) from macrophages can either enhance or inhibit PG and TNFalpha synthesis.
- Understanding NO's regulatory mechanisms is crucial for inflammation research.
Purpose of the Study:
- To investigate the differential effects of NO on prostaglandin and TNFalpha synthesis.
- To elucidate the role of NO concentration and exposure time in regulating inflammatory pathways.
- To compare the impact of short-term high NO exposure versus long-term low NO exposure.
Main Methods:
- Utilized RAW 264.7 macrophage cell line.
- Administered two types of nitric oxide-releasing compounds (NONOates): PAPA/NO (short half-life) and DETA/NO (long half-life).
- Analyzed basal and lipopolysaccharide (LPS)-stimulated release of TNFalpha and PGE(2), as well as arachidonic acid (AA) release and cyclooxygenase-2 (COX-2) expression.
Main Results:
- Short-term high NO (PAPA/NO) increased basal TNFalpha release and PGE(2) production, while long-term low NO (DETA/NO) decreased basal TNFalpha and increased PGE(2).
- Both NO donors increased basal AA release, but only PAPA/NO enhanced LPS-stimulated PGE(2).
- DETA/NO significantly upregulated COX-2 expression in LPS-activated cells, whereas PAPA/NO primarily affected AA release.
Conclusions:
- NO's effect on inflammatory mediators like TNFalpha and PGs is highly dependent on concentration and duration of exposure.
- Different NO donors (NONOates) reveal distinct molecular targets and outcomes in macrophage inflammatory responses.
- NO's dual regulatory role in inflammation is mediated by complex interactions with pathways controlling AA release and COX-2 expression.