Antithrombin III enhances inducible nitric oxide synthase gene expression in vascular smooth muscle cells.

Evidence suggests that antithrombin III (ATIII) exerts anti-inflammatory properties in addition to its anti-coagulative mechanisms. In animal models of sepsis, ATIII affected cytokine plasma concentrations with a decrease of pro-inflammatory cytokines. In addition to cytokines, excessive production of nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) might represent another important mediator of the cytotoxic events during sepsis. Regarding ATIII as a potential anti-inflammatory modulator, one may speculate that ATIII inhibits the synthesis of iNOS-derived NO. However, our data demonstrate that ATIII further stimulates iNOS gene expression when applied together with either interleukin-1 beta or the combination of lipopolysaccharide plus interferon-gamma. The most prominent synergistic effects on NO synthesis were found when ATIII was given at higher concentrations (1, 5, and 10 U/ml). Although the mechanisms of ATIII signal transduction remain to be established, intensification of interleukin-1 beta or interferon-gamma/lipopolysaccharide-induced NO synthesis by ATIII does not attribute to the anti-inflammatory properties of ATIII.

In vitro modulation of inducible nitric oxide synthase gene expression and nitric oxide synthesis by procalcitonin.

OBJECTIVE: Serum procalcitonin (PCT) concentration was recently introduced as valuable diagnostic marker for systemic bacterial infection and sepsis. At present, the cellular sources and biological properties of PCT are unclear. During sepsis and septic shock, inducible nitric oxide synthase (iNOS) gene expression is stimulated followed by the release of large amounts of nitric oxide (NO). We investigated the possible association between PCT and iNOS gene expression in an in vitro cell culture model. DESIGN: Prospective, controlled in vitro cell culture study. SETTING: University research laboratories. INTERVENTIONS: Confluent rat vascular smooth muscle cells (VSMC) were incubated for 24 hrs and 48 hrs with PCT (1 ng/mL, 10 ng/mL, 100 ng/mL, 1,000 ng/mL, 5,000 ng/mL) alone or with the combination of tumor necrosis factor-alpha (TNF-alpha, 500 U/mL) plus interferon-gamma (IFN-gamma, 100 U/mL). iNOS gene expression was measured by qualitative as well as quantitative polymerase chain reaction analysis, NO release was estimated by the modified Griess method. MEASUREMENTS AND MAIN RESULTS: PCT in increasing concentrations had no effect on iNOS gene expression and nitrite/nitrate release for 24 hrs and 48 hrs, respectively. However, PCT ameliorated TNF-alpha/IFN-gamma-induced iNOS gene expression in a dose-dependent manner (maximal inhibition at PCT 100 ng/mL by -66% for 24 hrs and -80% for 48 hrs). This was accompanied by a significantly reduced release of nitrite/nitrate into the cell culture supernatant (maximal reduction at PCT 100 ng/mL by -56% and -45% for 24 hrs and 48 hrs, respectively). CONCLUSIONS: We conclude that recombinant PCT inhibits the iNOS-inducing effects of the proinflammatory cytokines TNF-alpha/ IFN-gamma in a dose-dependent manner. This might be a counter-regulatory mechanism directed against the large production of NO and the concomitant systemic hypotension in severe sepsis and septic shock.

Different patterns of inducible nitric oxide synthase gene expression in ovarian carcinoma cell lines.

BACKGROUND: Stimulation of inducible nitric oxide synthase (iNOS) and release of nitric oxide by tumor cells may play a critical role in cancer development, either by exhibiting tumoricidal effects or by promoting tumor progression. MATERIALS AND METHODS: In the present study, we investigated four ovarian carcinoma cell lines with respect to their iNOS expressing characteristics. RESULTS: Following incubation with interferon-gamma, tumor necrosis factor-alpha and interleukin-1 beta, a marked stimulation of iNOS gene expression was found in SKOV-6 and OVCAR-3 cells, while only minor iNOS synthesis was detectable in HOC-7. Time-dependent accumulation of nitrite/nitrate in the culture supernatant indicated that the three cell lines were stimulated to produce and release nitric oxide. In contrast, no NO generation was observed in the fourth cell line under investigation, 2774. CONCLUSION: Due to the growth regulatory functions attributed to NO, variations in NO production may be of importance e.g. following interferon-gamma therapy in patients with ovarian cancer.

Neopterin-induced expression of intercellular adhesion molecule-1 (ICAM-1) in type II-like alveolar epithelial cells.

Production and release of proinflammatory mediators such as tumour necrosis factor-alpha and neopterin are common events following the activation of the cellular immune system. Concerning inflammatory disorders of the lung, e.g. sepsis or sarcoidosis, high serum neopterin levels have been reported to correlate well with the severity of the disease. These situations are often associated with an increased expression of ICAM-1 reported to be induced in type II alveolar epithelial cells. In our study we investigated the potential effects of neopterin on ICAM-1 synthesis in the type II-like pneumocyte cell line L2. Detection of ICAM-1 gene expression by reverse transcriptase-polymerase chain reaction revealed a dose-dependent effect of neopterin, with maximum impact following 12-h incubations. Comparable results were obtained when ICAM-1 protein synthesis was measured via a cell-based ELISA. In a second set of experiments we were able to show that coincubation of L2 cells with pyrrolidine dithiocarbamate (PDTC) significantly suppressed neopterin-induced ICAM-1 synthesis. Since PDTC is known to be a potent inhibitor of NF-kappaB, the stimulating effects of neopterin on ICAM-1 gene expression and protein generation may be mediated by activation of this transcription factor. From these data we conclude that neopterin stimulates ICAM-1 production in L2 cells. In vivo, these effects may contribute to the prolongation of the inflammatory response, including cytotoxic cell host defence mechanisms that impair the functions of the airway epithelium.

Inhibition of inducible nitric oxide synthase gene expression and nitric oxide synthesis in vascular smooth muscle cells by granulocyte-colony stimulating factor in vitro.

Clinical and experimental evidence suggests that granulocyte-colony stimulating factor (G-CSF) acts as an anti-inflammatory modulator with beneficial effects in severe inflammatory diseases, e.g., sepsis and septic shock. Excessive production of nitric oxide (NO) is regarded as a potent mediator of the vascular changes leading to systemic hypotension that occurs during sepsis. Therefore, the aim of the present study was to investigate the influence of G-CSF on inducible nitric oxide synthase (iNOS) gene expression and NO synthesis in vascular smooth muscle cells (VSMC). Qualitative and quantitative analyses of iNOS cDNA revealed that G-CSF significantly reduced interferon-gamma/lipopolysaccharide (IFN-gamma/LPS) dependent iNOS gene expression (P < 0.05) following 6, 18, 24, and 48 h incubation periods. In addition, the co-application of G-CSF resulted in a decreased IFN-gamma/LPS mediated iNOS protein generation as detected by immunoblotting methods after 24 and 48 h. Measurement of the stable NO metabolites showed a significant reduction of nitrite/nitrate concentrations following co-incubation of VSMC with G-CSF + IFN-gamma/LPS (242.57 +/- 10.73 nmol NO2-/NO3-/mg cell protein, n = 8) as compared to IFN-gamma/LPS treatment (306.20 +/- 19.26 nmol NO2-/NO3-/mg cell protein, n = 8, P < 0.05) following a 24-h incubation protocol. This inhibitory effect of G-CSF was still present after a 48 h incubation period (G-CSF + IFN-gamma/LPS: 319.56 +/- 6.26 nmol NO2-/NO3-/mg cell protein; IFN-gamma/LPS: 489.20 +/- 27.15 nmol NO2-/NO3-/mg cell protein (P < 0.05), n = 8, respectively). The present findings suggest that inhibition of iNOS gene expression and NO generation in VSMC might be one of the protective anti-inflammatory effects of G-CSF during sepsis.

Human dermal microvascular endothelial cells express inducible nitric oxide synthase in vitro.

Stimulation of inducible nitric oxide synthase with subsequent release of nitric oxide in large amounts may play a critical part either in host defense reactions or in the pathophysiology of the inflammatory response syndrome leading to septic shock. The aim of the present study was to investigate whether human dermal microvascular endothelial cells exhibit the typical characteristics of an inducible nitric oxide synthase expressing cell. A strong effect on inducible nitric oxide synthase gene expression could be detected when the cells were coincubated with the proinflammatory cytokines interferon-gamma and tumor necrosis factor-alpha with inducible nitric oxide synthase cDNA concentrations averaging 11.7 +/- 0.6 amol per microg total RNA at 24 h, and 25.0 +/- 1.4 amol per microg total RNA at 48 h, respectively. Intracellular staining with an antibody recognizing inducible nitric oxide synthase protein and subsequent analysis by flow cytometry revealed a 4-fold increase of inducible nitric oxide synthase protein in human dermal microvascular endothelial cells treated with interferon-gamma/tumor necrosis factor-alpha. This was accompanied by a significant elevation in nitrite/nitrate concentrations in the cell-free culture supernatants. Our results indicate that human dermal microvascular endothelial cells are provided with an inducible nitric oxide synthase system and can be regarded as an appropriate cell model for investigating inducible nitric oxide synthase gene expression and nitric oxide properties in microvascular endothelial cells.

Neopterin-induced tumor necrosis factor-alpha synthesis in vascular smooth muscle cells in vitro.

Synthesis and secretion of proinflammatory mediators like tumor necrosis factor-alpha and neopterin are common events in severe systemic inflammatory disorders, e.g. sepsis and septic shock. Recent data suggest that both substances show similarities with respect to their bioactivities. In the present study we investigated the potential interactions of neopterin and tumor necrosis factor-alpha on inducible nitric oxide synthase gene expression and nitric oxide generation in rat vascular smooth muscle cells. In addition, we studied the influence of neopterin on tumor necrosis factor-alpha synthesis in this cell type. Single stimulation of smooth muscle cells with either neopterin or tumor necrosis factor-alpha caused inducible nitric oxide synthase gene expression and nitric oxide production. Coincubation of cells with both compounds resulted in at least additive effects on nitric oxide synthesis. Quantification of tumor necrosis factor-alpha cDNA revealed a dose-dependent effect of neopterin on tumor necrosis factor-alpha gene expression. Similar results were obtained concerning the detection of tumor necrosis factor-alpha protein and the assessment of tumor necrosis factor-alpha bioactivity. These data suggest that neopterin and tumor necrosis factor-alpha are closely associated with regard to synthesis and effects, respectively. The interactions of both inflammatory mediators in vascular smooth muscle cells might contribute to the excessive release of nitric oxide observed during sepsis, thus triggering cellular destruction and multiple organ failure.

Neopterin induces nitric oxide-dependent apoptosis in rat vascular smooth muscle cells.

Numerous studies indicate that proinflammatory substances like tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) as well as macrophage-derived neopterin are increased in atherosclerotic tissue and thus are potentially involved in the process of atherogenesis. Since apoptotic death of vascular smooth muscle cells (VSMC) is reported to occur in atherosclerotic lesions, we investigated the effects of neopterin, TNF-alpha, and IFN-gamma on apoptosis in cultured VSMC. Morphological changes characteristic of apoptosis as well as DNA fragmentation were detected in cells treated with neopterin, TNF-alpha/IFN-gamma, and neopterin + TNF-alpha/IFN-gamma. Simultaneously, neopterin, TNF-alpha/IFN-gamma, and neopterin + TNF-alpha/IFN-gamma led to inducible nitric oxide synthase (iNOS) gene expression and nitric oxide (NO) synthesis. NO generation was significantly reduced when cells were cotreated with the competitive iNOS inhibitor aminoguanidine. This was accompanied by decreased percentual apoptosis as detected by FACS analysis using all kinds of stimuli. We conclude that neopterin as well as TNF-alpha/IFN-gamma are potent mediators of apoptotic death in VSMC which is at least in part triggered by NO synthesis induced by these proinflammatory mediators.