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      Prostacyclin Enhances the Expression of LPS/INF-γ-Induced Nitric Oxide Synthase in Human Monocytes

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          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 n M) significantly induced cellular cyclic adenosine monophosphate formation, indicating functional coupling of the receptor to G<sub>s</sub>. Treatment of cells with lipopolysaccharide (LPS)/interferon gamma (IFN-γ) 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 µg/ml LPS and 250 U/ml INF-γ 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 µg/ml LPS, 250 U/ml INF-γ, and 100 n M cicaprost for 16 h. Measurement of the NO generation correlated with the polymerase chain reaction data: treatment of cells with 1 µg/ml LPS plus 250 U/ml INF-γ increased the NO<sub>2</sub> production to 2.6 µ M, being above the basal level of 2.0 µ M, as determined in the cell culture medium. Additional treatment with 100 n M cicaprost further significantly increased the NO<sub>2</sub> production to 3.43 µ M. Conclusions: An IP receptor mediated increase in cyclic adenosine monophosphate formation plays an important role in enhancing LPS/IFN-γ-induced iNOS expression in human monocytes/macrophages and may, therefore, contribute to the increased production of NO during peritonitis.

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          Most cited references 7

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          Altered immune responses in mice lacking inducible nitric oxide synthase.

          Nitric oxide (NO) is important in many biological functions. It is generated from L-arginine by the enzyme NO synthase (NOS). The cytokine-inducible NOS (iNOS) is activated by several immunological stimuli, leading to the production of large quantities of NO which can be cytotoxic. To define the biological role of iNOS further, we generated iNOS mutant mice. These are viable, fertile and without evident histopathological abnormalities. However, in contrast to wild-type and heterozygous mice, which are highly resistant to the protozoa parasite Leishmania major infection, mutant mice are uniformly susceptible. The infected mutant mice developed a significantly stronger Th1 type of immune response than the wild-type or heterozygous mice. The mutant mice showed reduced nonspecific inflammatory response to carrageenin, and were resistant to lipopolysaccharide-induced mortality.
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            Inducible nitric oxide synthase in pulmonary alveolar macrophages from patients with tuberculosis

            The high-output pathway of nitric oxide production helps protect mice from infection by several pathogens, including Mycobacterium tuberculosis. However, based on studies of cells cultured from blood, it is controversial whether human mononuclear phagocytes can express the corresponding inducible nitric oxide synthase (iNOS;NOS2). The present study examined alveolar macrophages fixed directly after bronchopulmonary lavage. An average of 65% of the macrophages from 11 of 11 patients with untreated, culture-positive pulmonary tuberculosis reacted with an antibody documented herein to be monospecific for human NOS2. In contrast, a mean of 10% of bronchoalveolar lavage cells were positive from each of five clinically normal subjects. Tuberculosis patients' macrophages displayed diaphorase activity in the same proportion that they stained for NOS2, under assay conditions wherein the diaphorase reaction was strictly dependent on NOS2 expression. Bronchoalveolar lavage specimens also contained NOS2 mRNA. Thus, macrophages in the lungs of people with clinically active Mycobacterium tuberculosis infection often express catalytically competent NOS2.
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              Interferon (IFN)-α Activation of Human Blood Mononuclear Cells In Vitro and In Vivo for Nitric Oxide Synthase (NOS) Type 2 mRNA and Protein Expression: Possible Relationship of Induced NOS2 to the Anti–Hepatitis C Effects of IFN-α In Vivo

              Although researchers have noted high level activation of rodent mononuclear phagocytes for nitric oxide (NO) synthase type 2 (S2) expression and NO production with a variety of agents such as interferon (IFN) γ and endotoxin, it has been difficult to demonstrate activation of human mononuclear phagocytes. The purpose of this study was to determine if IFN-α serves as an activator in vitro and in vivo in humans. Treatment of normal monocytes or mononuclear cells in vitro with IFN-α caused a dose-dependent increase in monocyte NOS2 activity and NO production, and increased expression of NOS2 protein and mRNA expression. To determine if in vivo administration of IFN-α also modulated NOS2, we studied blood cells from patients with hepatitis C before and after IFN-α therapy. Untreated patients with chronic hepatitis C virus infection had levels of NOS activity and NOS2 antigen in freshly isolated mononuclear cells similar to those of healthy subjects, and they expressed minimal or no NOS2 mRNA. However, IFN-α treatment of patients with hepatitis C infection was associated with a significant elevation in mononuclear cell NOS activity, NOS2 antigen content, and NOS2 mRNA content. IFN-α–treated patients had significant decreases in levels of serum alanine aminotransferase and plasma hepatitis C mRNA. The degree of IFN-α–enhanced mononuclear cell NOS2 antigen content correlated significantly with the degree of reduction in serum alanine aminotransferase levels. Thus, IFN-α treatment of cells in vitro or administration of IFN-α to hepatitis C patients in vivo increases expression of mononuclear cell NOS2 mRNA expression, NOS activity, NOS2 antigen expression, and NO production. Since NO has been reported to have antiviral activity for a variety of viruses, we speculate that induced NO production may be related to the antiviral action(s) of IFN-α in hepatitis C infection.

                Author and article information

                S. Karger AG
                July 2002
                01 July 2002
                : 91
                : 3
                : 391-398
                aMedizinische Klinik und Poliklinik, Klinik für Nephrologie und Rheumatologie, und bInstitut für Pharmakologie und Klinische Pharmakologie, Heinrich-Heine-Universität, Düsseldorf, Deutschland
                64278 Nephron 2002;91:391–398
                © 2002 S. Karger AG, Basel

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                Figures: 4, References: 40, Pages: 8
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