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Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response
Published on: September 15, 2017
Prostacyclin enhances the expression of LPS/INF-gamma-induced nitric oxide synthase in human monocytes
Jörg Plum1, Chunmei Huang, Bernd Grabensee
1Medizinische Klinik und Poliklinik, Klinik für Nephrologie und Rheumatologie, Heinrich-Heine-Universität, Moorenstrasse 5, D-40225 Düsseldorf, Germany. plum@uni-duesseldorf.de
Background:
Nitric oxide (NO) is an important mediator of inflammatory processes, including macrophage-mediated cellular host defense, and is found to be increased in peritonitis. The ability of human mononuclear cells to contribute to the NO production by expression of active inducible NO synthase (iNOS) is still discussed controversely.
Aims:
This study was designed to investigate the influence of prostacyclin receptor (IP receptor) activation on iNOS expression and NO formation in human peripheral blood monocytes.
Method And Results:
Using reverse transcriptase-polymerase chain reaction, we demonstrated that human monocytes express high levels of IP receptor mRNA. Stimulation of monocytes with the IP receptor selective agonist cicaprost (100 nM) significantly induced cellular cyclic adenosine monophosphate formation, indicating functional coupling of the receptor to G(s). Treatment of cells with lipopolysaccharide (LPS)/interferon gamma (IFN-gamma) further enhanced the IP receptor mRNA expression 2.7 +/- 0.1-fold above basal levels (n = 6). Analysis of iNOS expression revealed barely detectable mRNA levels in unstimulated monocytes which were increased 3.75 +/- 0.3-fold (n = 5) after costimulation with 1 microg/ml LPS and 250 U/ml INF-gamma for 16 h. Further increases of iNOS mRNA expression (9.4 +/- 0.9-fold above basal, n = 5) were obtained, if the monocytes were costimulated with 1 microg/ml LPS, 250 U/ml INF-gamma, and 100 nM cicaprost for 16 h. Measurement of the NO generation correlated with the polymerase chain reaction data: treatment of cells with 1 microg/ml LPS plus 250 U/ml INF-gamma increased the NO(2) production to 2.6 microM, being above the basal level of 2.0 microM, as determined in the cell culture medium. Additional treatment with 100 nM cicaprost further significantly increased the NO(2) production to 3.43 microM.
Conclusions:
An IP receptor mediated increase in cyclic adenosine monophosphate formation plays an important role in enhancing LPS/IFN-gamma-induced iNOS expression in human monocytes/macrophages and may, therefore, contribute to the increased production of NO during peritonitis.
Insights
Prostacyclin receptor activation enhances inducible nitric oxide synthase (iNOS) expression in human monocytes. This finding suggests a role for IP receptor signaling in nitric oxide (NO) production during inflammatory conditions like peritonitis.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- Nitric oxide (NO) is a key mediator in inflammatory processes, with elevated levels observed in peritonitis.
- The role of human mononuclear cells in NO production via inducible NO synthase (iNOS) is debated.
Purpose of the Study:
- To investigate how prostacyclin receptor (IP receptor) activation influences iNOS expression and NO formation in human peripheral blood monocytes.
Main Methods:
- Human monocytes were analyzed for IP receptor mRNA expression using reverse transcriptase-polymerase chain reaction.
- Monocytes were stimulated with cicaprost (IP receptor agonist) and/or lipopolysaccharide (LPS)/interferon gamma (IFN-γ).
- iNOS mRNA expression and NO2- production were quantified.
Main Results:
- Human monocytes express significant levels of IP receptor mRNA.
- Cicaprost stimulation induced cyclic adenosine monophosphate (cAMP) formation, confirming functional IP receptor coupling.
- LPS/IFN-γ co-stimulation increased iNOS mRNA expression, which was further significantly enhanced by cicaprost.
- NO2- production correlated with iNOS expression, with cicaprost further increasing LPS/IFN-γ-induced NO2- levels.
Conclusions:
- IP receptor activation, via increased cAMP, enhances LPS/IFN-γ-induced iNOS expression in human monocytes/macrophages.
- This mechanism may contribute to elevated NO production observed in peritonitis.
