interleukin-8 and linsidomine

Macrophages are key inflammatory cells in chronic obstructive pulmonary disease (COPD). The pathophysiology of cigarette smoke-induced lung emphysema is complex but there is a clear role for reactive oxygen species (ROS, such as peroxynitrite), tumor necrosis factor (TNF-alpha) and interleukin (IL)-8. We investigated whether TNF-alpha or cigarette smoke medium (CSM) alone or in combination induces the production of IL-8 by human macrophages or monocyte lymphoma U937. CSM and TNF-alpha induce a dose- and time-dependent increase in IL-8 production. Interestingly, when sub-threshold concentrations of CSM and TNF-alpha were co-incubated, a 1500% increase in IL-8 production was observed compared to either of the compounds alone. Similar results were obtained with TNF-alpha and the peroxynitrite donor SIN-1. Moreover, the overproduction of IL-8 was associated with an enhanced increase in the translocation of NF-kappaB and an enhanced decrease in glutathione levels. Preincubation of the cells with antioxidants, such as N-acetyl-L-cysteine (NAC), prevented the overproduction of IL-8 and activation of NF-kappaB. In conclusion, CSM exposure of macrophages up-regulates the expression and the production of IL-8 via reactive oxygen species and NF-kappaB activation. Moreover, CSM dramatically enhances the production of IL-8 in combination with TNF-alpha. Based upon the strong synergistic action, a combination therapy directed against ROS and TNF-alpha could be a new approach to stop the progression in lung damage during emphysema.

Topics: Antioxidants; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Antagonism; Drug Combinations; Drug Synergism; Emphysema; Glutathione; Humans; Interleukin-8; Macrophages; Molsidomine; NF-kappa B; Nitric Oxide Donors; Reactive Oxygen Species; Smoke; Time Factors; Tumor Necrosis Factor-alpha; Up-Regulation

In vitro stimulation of human intervertebral disc (IVD) cells.. To investigate the oxidative/nitrosative effects of peroxynitrite on human nucleus pulposus (NP) cells.. Peroxynitrite is an important tissue-damaging species generated at sites of inflammation and degeneration. The aim of this study was to examine the effects of oxidative/nitrosative stress caused by peroxynitrite and the peroxynitrite donor SIN-1 in human NP cells.. Degenerated human IVD tissue was analyzed for nitrosylation by immunofluorescence. In addition, human NP cells were isolated from IVDs, expanded and stimulated either with peroxynitrite itself or a stable peroxynitrite donor (SIN-1). Nitrosylation, accumulation of intracellular reactive oxygen species, NF-kappaB nuclear translocation, and cell viability were analyzed by fluorescence. Gene expression of TNF-alpha, IL-1beta, IL-6, IL-8, and IL-10 was quantified by real-time (RT)-PCR.. Degenerated IVD tissue showed strong nitrosylation, especially in the NP. Isolated human NP cells showed a strong signal for nitrosylation and intracellular reactive oxygen species on stimulation with peroxynitrite or SIN-1. NF-kappaB/p65 sustained nuclear translocation of NF-kappaB/p65 and stimulation of IL-1beta, IL-6, and IL-8 expression was noted on treatment of cells with SIN-1.. This study provides evidence that peroxynitrite may play a role in disc degeneration and discogenic back pain development by an increased synthesis of proinflammatory cytokines. Nuclear translocation of NF-kappaB was identified as the potential underlying pathway. Therefore, neutralizing peroxynitrite and its derivatives (e.g., via the use of antioxidants) may be a novel treatment option for discogenic back pain.

Topics: Active Transport, Cell Nucleus; Adolescent; Adult; Cell Nucleus; Cells, Cultured; Female; Gene Expression; Humans; Immunohistochemistry; Interleukin-10; Interleukin-1beta; Interleukin-6; Interleukin-8; Intervertebral Disc; Intervertebral Disc Displacement; Male; Middle Aged; Molsidomine; Nitric Oxide Donors; Peroxynitrous Acid; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; Transcription Factor RelA; Tumor Necrosis Factor-alpha; Tyrosine

Inducible nitric oxide synthase (iNOS) and interleukin-8 (IL-8) mediate gastric inflammation. Nitric oxide (NO) produced by iNOS may activate oxidant-sensitive transcription factors. There are the binding sites for NF-kappaB, AP-1, and C/EBP (CCAAT/enhancer binding protein) in the promoter regions of IL-8 gene. The present study aims to investigate whether NO donors, SIN-1 and NOC-18, activate oxidant-sensitive transcription factors NF-kappaB and AP-1 as well as C/EBP to induce IL-8 expression in gastric epithelial AGS cells. Gastric epithelial AGS cells were treated with NO donors, SIN-1 and NOC-18. mRNA expression and protein level of IL-8 in the medium were determined. Nitrite level in the medium and DNA binding activities of NF-kappaB, AP-1, and C/EBP were assessed. NO donors induced the increase in the levels of IL-8 and nitrite in the medium as well as mRNA expression of IL-8 in AGS cells time-dependently. The induction of IL-8 by NO donors was accompanied with the activation of NF-kappaB and AP-1 but not C/EBP in AGS cells.. Large amount of NO, which may be produced by iNOS, may induce the activation of NF-kappaB and AP-1 and the expression of IL-8 in gastric epithelial cells.

Topics: Anti-Ulcer Agents; CCAAT-Enhancer-Binding Proteins; Cell Line; DNA-Binding Proteins; Electrophoretic Mobility Shift Assay; Epithelial Cells; Gastric Mucosa; Gene Expression Regulation; Genes, Reporter; Humans; Interleukin-8; Molsidomine; NF-kappa B; Nitric Oxide; Nitric Oxide Donors; Nitroso Compounds; RNA, Messenger; Time Factors; Transcription Factor AP-1

Peroxynitrite, formed by the reaction between nitric oxide and superoxide, has been shown to induce protein nitration, which compromises protein function. We hypothesized that peroxynitrite may regulate cytokine function during inflammation. To test this hypothesis, the neutrophil chemotactic activity (NCA) of interleukin-8 (IL-8) incubated with peroxynitrite was evaluated. Peroxynitrite attenuated IL-8 NCA in a dose-dependent manner (p < 0.01) but did not significantly reduce NCA induced by leukotriene B(4) or complement-activated serum. The reducing agents, dithionite, deferoxamine, and dithiothreitol, reversed and exogenous L-tyrosine abrogated the peroxynitrite-induced NCA inhibition. Papa-NONOate [N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium-1, 2-dialase or sodium nitroprusside, NO donors, or a combination of xanthine and xanthine oxidase to generate superoxide did not show an inhibitory effect on NCA induced by IL-8. In contrast, small amounts of SIN-1, a peroxynitrite generator, caused a concentration-dependent inhibition of NCA by IL-8. Consistent with its capacity to reduce NCA, peroxynitrite treatment reduced IL-8 binding to neutrophils. Nitrotyrosine was detected in the IL-8 incubated with peroxynitrite by enzyme-linked immunosorbent assay. These findings are consistent with nitration of tyrosine by peroxynitrite with subsequent inhibition of IL-8 binding to neutrophils and a reduction in NCA and suggest that oxidants may play an important role in regulation of IL-8-induced neutrophil chemotaxis.

Topics: Chemotaxis, Leukocyte; Humans; Interleukin-8; Leukotriene B4; Molsidomine; Neutrophils; Nitrates; Nitric Oxide Donors; Tyrosine

The effects of nitric oxide (NO) on human neutrophil chemotactic responses and release of interleukin (IL)-8 was studied. Neutrophils exposed to chemoattractants (IL-8, FMLP, leukotriene B4, and C5a) failed to show increases in intracellular guanosine 3',5'-cyclic monophosphate (cGMP), an indicator of NO production. Although NO increased cGMP in neutrophils, neither of two NO donors (sodium nitroprusside and 3-morpholino-sydonimine) nor a NO synthase inhibitor (N omega-nitro-L-arginine) altered FMLP- or IL-8-elicited neutrophil chemotaxis (P > .25 for all). However, lipopolysaccharide-induced IL-8 production was increased in a dose-dependent manner by a combination of sodium nitroprusside and N-acetylcysteine (P = .03) or by S-nitrosoglutathione (P = .004). NO-augmented IL-8 release was not reproduced by treating neutrophils with dibutyryl-cGMP. Up-regulation of IL-8 release by NO was associated with increased IL-8 mRNA levels (P = .009). These data suggest that NO does not directly affect neutrophil chemotaxis but may indirectly alter chemotactic responses by increasing IL-8 production via a cGMP-independent pathway.

Topics: Acetylcysteine; Cells, Cultured; Chemotaxis; Complement C5a; Cyclic GMP; Dibutyryl Cyclic GMP; Dose-Response Relationship, Drug; Ferricyanides; Glutathione; Humans; Interleukin-8; Leukotriene B4; Lipopolysaccharides; Molsidomine; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Nitric Oxide; Nitric Oxide Synthase; Nitroprusside; Nitroso Compounds; omega-N-Methylarginine; RNA, Messenger; S-Nitrosoglutathione; Up-Regulation

Inhaled nitric oxide is now widely used in the treatment of hypoxemia and pulmonary hypertension in critically ill patients. Interleukin-8 (IL-8) and neutrophil elastase are important markers of the onset and severity of acute lung injury. We studied the effects of nitric oxide and peroxynitrite on IL-8) and elastase accumulation in lipopolysaccharide-activated whole blood. The nitric oxide donor (GEA-3162) did not affect IL-8 accumulation (P = 0.195) but did cause an increase in elastase accumulation (P = 0.007). The peroxynitrite donor (SIN-1) caused an increase in both IL-8 accumulation (P = 0.0004) and elastase accumulation (P = 0.007). The lack of effect of nitric oxide could be explained by the scavenging of nitric oxide by hemoglobin. These results suggest that modulation of the inflammatory response may occur during inhaled nitric oxide therapy in the critically ill.. Inhaled nitric oxide, used in lung injury, reacts within the lung, forming peroxynitrite. We investigated the effect of nitric oxide and peroxynitrite on interleukin-8 and elastase release by white cells during inflammation. Nitric oxide and peroxynitrite had marked effects on elastase and interleukin-8, which suggests modulation of the inflammatory response.

Topics: Free Radical Scavengers; Humans; In Vitro Techniques; Interleukin-8; Leukocyte Elastase; Lipopolysaccharides; Male; Molsidomine; Nitrates; Nitric Oxide; Triazoles

Patients with acute inflammatory lung injury are commonly treated with inhaled nitric oxide. Nitric oxide has profound immunoregulatory effects. Increased concentrations of the cytokine interleukin-8 (IL-8) in bronchoalveolar lavage fluid has been associated with disease severity. We have investigated the effects of a nitric oxide donor and a combined nitric oxide-superoxide donor on lipopolysaccharide-mediated accumulation of IL-8 from cultured human neutrophils. Interleukin-8 was measured in culture supernatant after 20 h using enzyme immunoassay. The combined nitric oxide-superoxide donor, 3-morpholinosydnonimine (SIN-1), dose-dependently decreased lipopolysaccharide-mediated IL-8 accumulation (P < 0.01). SIN-1 also decreased IL-8 accumulation from unstimulated neutrophils (P < 0.001). In contrast, the pure nitric oxide donor, 1,2,3,4-oxatriazolium 5-amino chloride (GEA-3162), increased stimulated IL-8 accumulation (P < 0.01) and also increased IL-8 accumulation in unstimulated cells (P < 0.002). Nitric oxide and superoxide have profound effects on IL-8. These results have important implications for the treatment of patients with acute lung injury with inhaled nitric oxide.

Topics: Cell Culture Techniques; Dose-Response Relationship, Drug; Humans; Interleukin-8; Lipopolysaccharides; Molsidomine; Neutrophil Activation; Neutrophils; Nitric Oxide; Platelet Aggregation Inhibitors; Superoxides; Triazoles

We investigated the role of nitric oxide (NO) in the expression of interleukin-8 (IL-8) in the human melanoma cell line, G361. Three NO donors, 3-morpholinosydnonimine hydrochloride (SIN-1), S-nitroso-N-acetylpenicillamine (SNAP), and S-nitroso-L-glutathione (SNOG), all caused an increase in both IL-8 protein secretion and promoter activity. Truncation of the promoter showed that 101 bp of the 5' flanking region proximal to the transcription start site are sufficient for the response to NO. Furthermore, mutation of the NF-kappa B and NF-IL-6 binding sites led to a significant decrease in NO-stimulated promoter activity. The nitric oxide synthase inhibitor, NG-amino-L-homoarginine (NAHA), inhibited TNF-alpha-stimulated IL-8 promoter activity by 60%. Addition of excess L- but not D-arginine partially reversed the NAHA-mediated inhibition. These results demonstrate that NO is an endogenous regulator of IL-8 production in G361 melanoma cells.

Topics: Base Sequence; Binding Sites; CCAAT-Enhancer-Binding Proteins; Cell Line; DNA Primers; DNA-Binding Proteins; Gene Expression; Glutathione; Humans; Interleukin-8; Melanoma; Molecular Sequence Data; Molsidomine; Mutagenesis, Site-Directed; NF-kappa B; Nitric Oxide; Nitroso Compounds; Nuclear Proteins; Penicillamine; Platelet Aggregation Inhibitors; Polymerase Chain Reaction; Promoter Regions, Genetic; S-Nitroso-N-Acetylpenicillamine; S-Nitrosoglutathione; Transcription Factors; Transcription, Genetic; Tumor Cells, Cultured; Vasodilator Agents