Regulation of the inducible cyclo-oxygenase pathway in human cultured airway epithelial (A549) cells by nitric oxide (original) (raw)
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Divergent effects of Nitric oxide on airway epithelial cell activation
Biological Research, 2010
Nitric oxide (NO • ) is a gaseous mediator synthesized by nitric oxide sinthases. NO • is involved in the modulation of inflammation, but its role in airway inflammation remains controversial. We investigated the role of NO • in the synthesis of the chemokines Interleukin-8 and Monocyte Chemotactic Protein-1, and of Intercellular Adhesion Molecule-1 by human airway epithelial cells. Normal human bronchial epithelial cells and the bronchial epithelial cell line BEAS-2B were used. Ineterleukin-8 (IL-8) and Monocyte Chemotactic Protein-1 (MCP-1) secretion and Intercellular Adhesion Molecule-1 (ICAM-1) expression were measured by ELISA. mRNA was assessed by semiquantitative RT-PCR. Ineterleukin-8 secretion was significantly reduced after 24h incubation with the NO • donor, sodium nitroprusside. The effect was dose-dependent. Similar results were obtained with S-Nitroso-N-D,L-penicillamine and S-Nitroso-L-glutathione. Inhibition of endogenous NO • with the nitric oxide synthase inhibitor N-Nitro-L-arginine-methyl-esther caused an increase in IL-8 secretion by lypopolisaccharide-and cytokine-stimulated BEAS-2B cells. Sodium nitroprusside also caused a reduction in Monocyte Chemotactic Protein-1 secretion by both cell types. In contrast, Intercellular Adhesion Molecule-1 expression was upregulated by sodium nitroprusside. RT-PCR results indicate that the modulation of protein levels was paralleled by modification in mRNA levels. NO • has divergent effects on the synthesis of different inflammatory mediators in human bronchial epithelial cells.
Nitric oxide amplifies interleukin 1-induced cyclooxygenase-2 expression in rat mesangial cells
Journal of Clinical Investigation, 1996
Interleukin 1 and nitric oxide (NO) from infiltrating macrophages and activated mesangial cells may act in concert to sustain and promote glomerular damage. To evaluate if such synergy occurs, we evaluated the effect if IL-1  and NO on the formation of prostaglandin (PG) E 2 and cyclooxygenase (COX) expression. The NO donors, sodium nitroprusside and S-nitroso-N-acetylpenicillamine, alone did not increase basal PGE 2 formation. However, these compounds amplified IL-1 -induced PGE 2 production. Similarly, sodium nitroprusside and S-nitroso-N-acetylpenicillamine by themselves did not induce mRNA and protein for COX-2, the inducible isoform of COX; however, they both potentiated IL-1 -induced mRNA and protein expression of COX-2. The stimulatory effect of NO is likely to be mediated by cGMP since (a) an inhibitor of the soluble guanylate cyclase, methylene blue, reversed the stimulatory effect of NO donors on COX-2 mRNA expression; (b) the membrane-permeable cGMP analogue, 8-Br-cGMP, mimicked the stimulatory effect of NO donors on COX-2-mRNA expression; and (c) atrial natriuretic peptide, which increases cellular cGMP by activating the membrane-bound guanylate cyclase, also amplified IL-1 -induced COX-2 mRNA expression. These data indicate a novel interaction between NO and COX pathways.
2001
NO produced by the inducible NO synthase (NOS2) and prostanoids generated by the cyclooxygenase (COX) isoforms and terminal prostanoid synthases are major components of the host innate immune and inflammatory response. Evidence exists that pharmacological manipulation of one pathway could result in cross-modulation of the other, but the sense, amplitude, and relevance of these interactions are controversial, especially in vivo. Administration of 6 mg/kg LPS to rats i.p. resulted 6 h later in induction of NOS2 and the membrane-associated PGE synthase (mPGES) expression, and decreased constitutive COX (COX-1) expression. Low level inducible COX (COX-2) mRNA with absent COX-2 protein expression was observed. The NOS2 inhibitor aminoguanidine (50 and 100 mg/kg i.p.) dose dependently decreased both NO and prostanoid production. The LPS-induced increase in PGE 2 concentration was mediated by NOS2-derived NO-dependent activation of COX-1 pathway and by induction of mPGES. Despite absent COX-2 protein, SC-236, a putative COX-2-specific inhibitor, decreased mPGES RNA expression and PGE 2 concentration. Ketoprofen, a nonspecific COX inhibitor, and SC-236 had no effect on the NOS2 pathway. Our results suggest that in a model of systemic inflammation characterized by the absence of COX-2 protein expression, NOS2-derived NO activates COX-1 pathway, and inhibitors of COX isoforms have no effect on NOS2 or NOS3 (endothelial NOS) pathways. These results could explain, at least in part, the deleterious effects of NOS2 inhibitors in some experimental and clinical settings, and could imply that there is a major conceptual limitation to the use of NOS2 inhibitors during systemic inflammation.
Lung sources and cytokine requirements for in vivo expression of inducible nitric oxide synthase
American Journal of Respiratory Cell and Molecular Biology, 1995
Products of inducible nitric oxide synthase (iNOS) are known to be involved in lung injury following intrapulmonary deposition of immunoglobulin G immune complexes (IgG-ICx). In the current studies rat alveolar macrophages stimulated in vitro with murine interferon')' (IFN-')'), tumor necrosis factor a, interleukin Io , (IL-la) , lipopolysaccharide (LPS), or IgG-ICx immunostained for iNOS and produced nitrite/nitrate-(N02-INO)-) in a dose-and time-dependent manner requiring availability of L-arginine. Under the same conditions, IL-4 and IL-I0 reduced N0 2-INO}-generation. Type II alveolar epithelial cells, which were obtained from normal rat lungs and stimulated in vitro with IgG-ICx, LPS, or IFN-')', also immunostained for iNOS and generated N0 2-INO}-. Special techniques of bronchoalveolar lavage (BAL) were used to retrieve alveolar macrophages and type II alveolar epithelial cells. Under these conditions, intrapulmonary deposition of LPS yielded BAL fluids containing increased amounts of N0 2-I NO}-and macrophages that spontaneously released N0 2-INO}-and stained for iNOS. After intrapulmonary deposition of IgG both macrophages as well as type II cells (retrieved by BAL) spontaneously produced N0 2-INO}-and both cell types immunostained for iNOS (approximately 20 % of all type II cells and 35 %of all alveolar macrophages). Using dual fluorescence staining for cell identification, frozen sections of lung tissue after IgG immune complex deposition revealed iNOS in both alveolar macrophages and type II cells. Finally, in the immune complex model of alveolitis, the appearance of iNOS in macrophages as well as macrophage production in vitro of N0 2-INO}-was dependent on the in vivo availability of tumor necrosis factor a, ILl , and IFN-')'. These studies suggest a dual cell source for nitric oxide in inflamed lungs and the requirements for iNOS of several cytokines. Although there is abundant evidence that the pathogenesis of immunoglobulin G immune complex (IgG-ICx)-induced tissue injury requires the role of complement, the participation of phagocytic cells (neutrophils and macrophages) and products from L-arginine oxidation produced by nitric oxide synthase (NOS), precise events that can be linked to the causation of injury, are not clearly defined. It has been postulated that the influx and activation of phagocytic cells result in the local generation of toxic oxygen products (0 2-, H 202 , hydroxyl radical, and hypochlorous acid), toxic products of
The Journal of Immunology, 1999
NO was estimated by its stable end product: nitrite as previously described (9). PGE 2 was estimated by RIA as described previously (8). Fractionation of cell lysate by Dounce method The cells (2 ϫ 10 7) were lysed by using a Dounce homogenizer (Pyrex; Fisher Scientific, Springfield, NJ) to prepare nuclear and cytoplasmic (S-100) fraction as previously reported (10). The lysate was spun down at 3,000 ϫ g to remove unlysed cells in an Eppendorf centrifuge (Fisher Scientific). The resulting supernatant was subjected to 18,000 ϫ g in an Eppendorf centrifuge. This step isolated the intact nuclei from which nuclear extracts were prepared. The supernatant of the nuclear fraction
Modulation of PGE2 and Tnfα by Nitric Oxide in Resting and LPS-Activated Raw 264.7 Cells
Cytokine, 2002
Prostaglandins (PGs), the arachidonic acid (AA) metabolites of the cyclooxygenase (COX) pathway, and the cytokine TNF 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 TNF 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 TNF 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.
The Journal of Immunology, 2001
NO produced by the inducible NO synthase (NOS2) and prostanoids generated by the cyclooxygenase (COX) isoforms and terminal prostanoid synthases are major components of the host innate immune and inflammatory response. Evidence exists that pharmacological manipulation of one pathway could result in cross-modulation of the other, but the sense, amplitude, and relevance of these interactions are controversial, especially in vivo. Administration of 6 mg/kg LPS to rats i.p. resulted 6 h later in induction of NOS2 and the membrane-associated PGE synthase (mPGES) expression, and decreased constitutive COX (COX-1) expression. Low level inducible COX (COX-2) mRNA with absent COX-2 protein expression was observed. The NOS2 inhibitor aminoguanidine (50 and 100 mg/kg i.p.) dose dependently decreased both NO and prostanoid production. The LPS-induced increase in PGE 2 concentration was mediated by NOS2-derived NO-dependent activation of COX-1 pathway and by induction of mPGES. Despite absent COX-2 protein, SC-236, a putative COX-2-specific inhibitor, decreased mPGES RNA expression and PGE 2 concentration. Ketoprofen, a nonspecific COX inhibitor, and SC-236 had no effect on the NOS2 pathway. Our results suggest that in a model of systemic inflammation characterized by the absence of COX-2 protein expression, NOS2-derived NO activates COX-1 pathway, and inhibitors of COX isoforms have no effect on NOS2 or NOS3 (endothelial NOS) pathways. These results could explain, at least in part, the deleterious effects of NOS2 inhibitors in some experimental and clinical settings, and could imply that there is a major conceptual limitation to the use of NOS2 inhibitors during systemic inflammation.
Journal of Clinical Investigation, 1995
We have recently put forward the hypothesis that the dual inhibition of proinflammatory nitric oxide (NO) and prostaglandins (PG) may contribute to the antiinflammatory properties of nitric oxide synthase (NOS) inhibitors. This hypothesis was tested in the present study. A rapid inflammatory response characterized by edema, high levels of nitrites (NOr, a breakdown product of NO), PG, and cellular infiltration into a fluid exudate was induced by the administration of carrageenan into the subcutaneous rat air pouch. The time course of the induction of inducible nitric oxide synthase (iNOS) protein in the pouch tissue was found to coincide with the production of NOR. Dexamethasone inhibited both iNOS protein expression and NO2synthesis in the fluid exudate (IC50 = 0.16 mg/kg). Oral administration of N-iminoethyl-L-lysine (L-NIL) or NG-nitro-L-arginine methyl ester (NO2Arg) not only blocked nitrite accumulation in the pouch fluid in a dose-dependent fashion but also attenuated the elevated release of PG. Finally, carrageenan administration produced a time-dependent increase in cellular infiltration into the pouch exudate that was inhibited by dexamethasone and NOS inhibitors. At early times, i.e., 6 h, the cellular infiltrate is composed primarily of neutrophils (98%). Pretreatment with colchicine reduced both neutrophil infiltration and leukotriene B4 accumulation in the air pouch by 98% but did not affect either NOor PG levels. In conclusion, the major findings of this paper are that (a) selective inhibitors of iNOS are clearly antiinflammatory agents by inhibiting not only NO but also PG and cellular infiltration and (b) that neutrophils are not responsible for high levels of NO and PG produced.
Nitric oxide downregulates lung macrophage inflammatory cytokine production
The Annals of Thoracic Surgery, 1998
Background. Inflammatory cytokine production contributes to lung injury after lung ischemia reperfusion and during lung transplant rejection. Although nitric oxide has been demonstrated to reduce lung injury associated with the adult respiratory distress syndrome, it remains unknown whether the mechanism of nitric oxide's beneficial effects involves reducing lung macrophage inflammatory cytokine production. The purpose of this study was to determine whether nitric oxide downregulates lung macrophage inflammatory cytokine production.