Suppressive effect of selective cyclooxygenase-2 inhibitor on cytokine release in human neutrophils

To clarify whether a selective cyclooxygenase-2 (COX-2) inhibitor can affect various functions in human peripheral blood neutrophils. For this purpose, the effects of selective COX-2 inhibitors, NS-398 and nimesulide, on the expression of COX-2, PGE2 release and respiratory burst, degranulation and cytokine release in activated neutrophils were examined. Peripheral blood neutrophils were stimulated with formyl-methionyl-leucyl-phenylalanine (FMLP; 100 nM) or opsonized zymosan (OZ; 200 microg/ml). Then, the expression of COX-2 at protein and mRNA levels was detected by Western blot analysis and RT-PCR. The concentration of prostaglandin E2 (PGE2) and cytokines in the culture supernatant of neutrophils was determined using ELISA. Superoxide generation was measured by the cytochrome c reduction method. Elastase activity was measured using a chromogenic substrate assay specific for human neutrophil elastase. FMLP and OZ enhanced PGE2 release through induction of COX-2 protein and mRNA expression. FMLP- or OZ-induced PGE2 release was abolished by the addition of NS-398 or nimesulide; nevertheless, even a high concentration of COX-2 inhibitor did not change FMLP- or OZ-induced expression of COX-2 at message and protein levels. Although FMLP- or OZ-induced superoxide generation and elastase release were not affected by the addition of COX-2 inhibitor, cytokine release such as interleukin (IL)-1beta, IL-6 and IL-8 was significantly inhibited by high concentration of COX-2 inhibitor, but tumor necrosis factor-alpha (TNF-alpha) was partially attenuated. These studies showed that selective COX-2 inhibitors, NS-398 and nimesulide, suppressed PGE2 and proinflammatory cytokine release in activated neutrophils. These results suggest that selective COX-2 inhibitors may contribute to resolution of acute inflammation through the reduction of inflammatory cytokine release in activated neutrophils.     

Inhibition by licochalcone A, a novel flavonoid isolated from liquorice root, of IL-1beta-induced PGE2 production in human skin fibroblasts

Licochalcone A, a novel flavonoid isolated from the root of Glycyrrhiza inflata, has been reported to exhibit anti-inflammatory activity in animal models. In this study, we examined the effect of licochalcone A on the production of chemical mediators such as prostaglandin (PG)E2 and cytokines by interleukin (IL)-1beta in human skin fibroblasts. Licochalcone A (IC50 15.0 nM) inhibited PGE2 production, but not IL-6 and IL-8 production, in response to IL-1beta. NS-398 (IC50 1.6 nM), a COX-2 selective inhibitor, also suppressed the PGE2 production. Furthermore, licochalcone A and NS-398 suppressed PGF(2alpha) production by IL-1beta. However, licochalcone A (1 microM) had no effect on increased levels of cyclooxygenase (COX)-2 mRNA and protein in cells. Dexamethasone (100 nM) not only inhibited PGE2, PGF(2alpha), IL-6 and IL-8 production but also strongly suppressed the expression of COX-2 mRNA and protein. Licochalcone A had no effect on COX-1-dependent PGE2 production, whereas indometacin (100 nM), a dual inhibitor of COX-1 and COX-2, was very effective. These results suggest that licochalcone A induces an anti-inflammatory effect through the inhibition of COX-2-dependent PGE2 production. Furthermore, it appears that the inhibitory effect of licochalcone A on PGE2 production in response to IL-1beta is quite different from that of the steroid.     

Oxidative stress and cyclooxygenase-2 induction mediate cyanide-induced apoptosis of cortical cells

Cyanide (KCN)-induced generation of reactive oxygen species (ROS) involves cyclooxygenase-2 (COX-2)-mediated reactions in some neurons. The present study examines the extent to which COX isoforms are involved in KCN-induced apoptotic cell death processes of cultured cortical cells. After treatment with KCN (10-300 microM), COX-2 was expressed in a time- and concentration-dependent manner increasing markedly over a 4-h period. However, no significant changes were observed in COX-1 levels at any cyanide concentration. Correlated with COX-2 up-regulation, KCN induced a time-dependent apoptotic death. TUNEL staining showed that the COX-2 inhibitor NS-398 (30 microM) blocked KCN-induced apoptosis, whereas the selective COX-1 inhibitor valeryl salicylate did not affect the level of apoptotic cell death. Exposure of cells to KCN (300 microM) for 24 h resulted in DNA fragmentation, which was also reduced by NS-398. Prostaglandin E(2) (PGE(2)) accumulation in cell culture supernatants was increased by KCN and NS-398 blocked PGE(2) generation. PCR studies further confirmed that COX-2 expression was increased by KCN. Antioxidants phenyl-N-test-butylnitrone, superoxide dismutase, and catalase significantly inhibited KCN-induced COX-2 up-regulation and subsequent apoptosis. N(G)-nitro-L-arginine methylester an inhibitor of nitric oxide synthase, blocked KCN-induced PGE(2) production and apoptosis, but not COX-2 expression. Increased nitric oxide levels caused by cyanide may directly activate the COX-2 enzyme. These data show that cyanide treatment of cortical cells involves increased COX-2 expression, PGE(2) accumulation, and ROS generation, resulting in apoptotic cell death.     

Regulation by PGE2 of the production of interleukin-6, macrophage colony stimulating factor,and vascular endothelial growth factor in human synovial fibroblasts

1. We examined the effects of endogenous prostaglandin E(2) (PGE(2)) on the production of interleukin-6 (IL-6), macrophage colony stimulating factor (M-CSF), and vascular endothelial growth factor (VEGF) by interleukin-1beta (IL-1beta)-stimulated human synovial fibroblasts. 2. NS-398 (1 microM), a cyclo-oxygenase-2 (COX-2) inhibitor, inhibited IL-6 and VEGF production (35+/-4% and 26+/-2%, respectively) but enhanced M-CSF production (38+/-4%) by IL-1beta (1 ng ml(-1)) in synovial fibroblasts isolated from patients with osteoarthritis (OA) and rheumatoid arthritis (RA). Exogenous PGE(2) completely abolished the effects of NS-398 on the production of each mediator by OA fibroblasts stimulated with IL-1beta. 3. 8-Bromo cyclic AMP and dibutyryl cyclic AMP, cyclic AMP analogues, mimicked the effects of PGE(2) on IL-6, M-CSF, and VEGF production by OA fibroblasts. 4. The EP(2) selective receptor agonist ONO-AE1-259 (2 nM) and the EP(4) selective receptor agonist ONO-AE1-329 (2 or 20 nM), but not the EP(1) selective receptor agonist ONO-DI-004 (1 microM) and the EP(3) selective receptor agonist ONO-AE-248 (1 microM), replaced the effects of PGE(2) on IL-6, M-CSF, and VEGF production by OA and RA fibroblasts stimulated with IL-1beta in the presence of NS-398. 5. Both OA and RA fibroblasts expressed mRNA encoding EP(2) and EP(4) but not EP(1) receptors. In addition, up-regulation of EP(2) and EP(4) receptor mRNAs was observed at 3 h after IL-1beta treatment. 6. These results suggest that endogenous PGE(2) regulates the production of IL-6, M-CSF, and VEGF by IL-1beta-stimulated human synovial fibroblasts through the activation of EP(2) and EP(4) receptors with increase in cyclic AMP.     

Byakangelicol,isolated from Angelica dahurica,inhibits both the activity and induction of cyclooxygenase-2 in human pulmonary epithelial cells

We examined the inhibitory mechanism of byakangelicol, isolated from Angelica dahurica, on interleukin-1beta (IL-1beta)-induced cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) release in human pulmonary epithelial cell line (A549). Byakangelicol (10-50 microM) concentration-dependently attenuated IL-1beta-induced COX-2 expression and PGE2 release. The selective COX-2 inhibitor, NS-398 (0.01-1 microM), and byakangelicol (10-50 microM) both concentration-dependently inhibited the activity of the COX-2 enzyme. Byakangelicol, at a concentration up to 200 microM, did not affect the activity and expression of COX-1 enzyme. IL-1beta-induced p44/42 mitogen-activated protein kinase (MAPK) activation was inhibited by the MAPK/extracellular signal-regulated protein kinase (MEK) inhibitor, PD 98059 (30 microM), while byakangelicol (50 microM) had no effect. Treatment of cells with byakangelicol (50 microM) or pyrrolidine dithiocarbamate (PDTC; 50 microM) partially inhibited IL-1beta-induced degradation of IkappaB-alpha in the cytosol, translocation of p65 NF-kappaB from the cytosol to the nucleus and the NF-kappaB-specific DNA-protein complex formation. Taken together, we have demonstrated that byakangelicol inhibits IL-1beta-induced PGE2 release in A549 cells; this inhibition may be mediated by suppression of COX-2 expression and the activity of COX-2 enzyme. The inhibitory mechanism of byakangelicol on IL-1beta-induced COX-2 expression may be, at least in part, through suppression of NF-kappaB activity. Therefore, byakangelicol may have therapeutic potential as an anti-inflammatory drug on airway inflammation.     

Interleukin-1beta induces MUC2 and MUC5AC synthesis through cyclooxygenase-2 in NCI-H292 cells

Interleukin-1beta (IL-1beta) has been implicated in the pathogenesis of inflammatory diseases of the airway. In this study, we investigated the regulation of MUC2 and MUC5AC expression and of their regulatory mechanisms through cyclooxygenase-2 (COX-2) and prostaglandin E(2) (PGE(2)). Cells activated by IL-1beta showed increased COX-2, MUC2, and MUC5AC expressions at both the mRNA and protein levels. Mucin production was blocked by the selective COX-2 inhibitor NS398, and PGE(2) directly induced MUC2 and MUC5AC expression at both the mRNA and protein levels in a dose-dependent manner. These results suggest a role for PGE(2) in IL-1beta-induced mucin synthesis in NCI-H292 cells. To investigate the roles of molecules upstream of COX-2 in mucin regulation, we examined the role of mitogen-activated protein kinases (MAPKs). Cells activated by IL-1beta showed increased extracellular signal-regulated kinase (ERK)1/2 and p38 phosphorylation, and IL-1beta-induced MUC2 and MUC5AC production was blocked by the ERK pathway inhibitor PD98059 or the p38 inhibitor SB203580. The inhibition of both MAPKs reduced IL-1beta-induced COX-2 expression and PGE(2) synthesis. Furthermore, the addition of PGE(2) to cells overcame the inhibitory effects of both MAPK inhibitors in IL-1beta-induced mucin production. These results indicate that in human pulmonary epithelial cells, IL-1beta activates ERK or p38 to induce COX-2 production, which in turn induces MUC2 and MUC5AC production.     

Meloxicam inhibits prostaglandin E(2) generation via cyclooxygenase 2 in the inflammatory site but not that via cyclooxygenase 1 in the stomach

We studied the effects of meloxicam on prostanoid levels, both in the inflammatory site in rat carrageenin-induced pleurisy and in the rat stomach injected with 1 mol/l NaCl solution, to clarify the relationship between its low gastric toxicity and its relative cyclooxygenase (COX) 2 selectivity. NS-398 (3 mg/kg), a highly selective COX-2 inhibitor, and meloxicam (3 mg/kg) exhibited anti-inflammatory effects in the pleurisy model. Prostaglandin (PG) E(2) thromboxane (TX) B(2) and 6-keto-PGF(1alpha) were detectable in the inflammatory site. Anti-inflammatory doses of NS-398 and meloxicam each suppressed the intrapleural PGE(2) level at 5 h as potently as piroxicam (3 mg/kg) as aspirin (100 mg/kg), both of which are nonselective COX inhibitors. NS-398 was much less potent than the other three in suppressing the levels of TXB(2) and 6-keto-PGF(1alpha). These results suggest that PGE(2) may be produced mainly via COX-2 in this model and that meloxicam may inhibit COX-2 in the inflammatory site. Piroxicam completely inhibited the increase in gastric PGE(2) induced by administering 1 mol/l NaCl solution into the rat stomach. Nimesulide (3 mg/kg), another selective COX-2 inhibitor, however, never affected this increase, suggesting that the gastric PGE(2) may be produced via COX-1. The anti-inflammatory dose of meloxicam caused statistically nonsignificant suppression of the PGE(2) level, by approximately 50%. These results suggest that the potent anti-inflammatory effect of meloxicam, accompanied with low gastric toxicity, may be related to its relative selectivity for COX-2 over COX-1.     

选择性COX-2抑制剂NS-398对人胃腺癌细胞增殖和凋亡的影响

目的 研究环氧化酶2(COX-2)抑制剂NS-398对胃癌培养细胞系BGC-823增殖及凋亡的影响.方法 应用MTT法检测NS-398对胃癌细胞BGC-823细胞增殖的影响;应用流式细胞术、逆转录聚合酶链反应(RT-PCR)和Western blot法检测NS-398对胃癌细胞BGC-823细胞凋亡的影响.结果 NS-398随剂量的增加及作用时间延长对胃癌细胞呈抑制作用;体外NS-398能减少BGC-823细胞株COX-2的表达,对BGC-823有细胞毒作用,可增加细胞凋亡率.结论 NS-398可抑制胃癌BGC-823细胞的增殖,促进胃癌BGC-823细胞的凋亡.其可能机制是与抑制COX-2表达有关.     

Induction of COX-2 and PGE(2) biosynthesis by IL-1beta is mediated by PKC and mitogen-activated protein kinases in murine astrocytes

Interleukin-1 (IL-1) is an important mediator of immunoinflammatory responses in the brain. In the present study, we examined whether prostaglandin E(2) (PGE(2)) production after IL-1beta stimulation is dependent upon activation of protein kinases in astroglial cells. Astrocyte cultures stimulated with IL-1beta or the phorbol ester, PMA significantly increased PGE(2) secretion. The stimulatory action of IL-1beta on PGE(2) production was totally abolished by NS-398, a specific inhibitor of cyclo-oxygenase-2 activity, as well as by the protein synthesis inhibitor cycloheximide, and the glucocorticoid dexamethasone. Furthermore, IL-1beta induced the expression of COX-2 mRNA. This occurred early at 2 h, with a maximum at 4 h and declined at 12 h. IL-1 beta treatment also induced the expression of COX-2 protein as determined by immunoblot analysis. In that case the expression of the protein remained high at least up to 12 h. Treatment of cells with protein kinase C inhibitors (H-7, bisindolylmaleimide and calphostin C) inhibited IL-1beta stimulation of PGE(2). In addition, PKC-depleted astrocyte cultures by overnight treatment with PMA no longer responded to PMA or IL-1. The ablation of the effects of PMA and IL-1beta on PGE(2) production, likely results from down-regulation of phorbol ester sensitive-PKC isoenzymes. Immunoblot analysis demonstrated the translocation of the conventional isoform cPKC-alpha from cytosol to membrane following treatment with IL-1beta. In addition, IL-1beta treatment led to activation of extracellular signal-regulated kinase (ERK1/2) and p38 subgroups of MAP kinases in astroglial cells. Interestingly, the inhibition of ERK kinase with PD 98059, as well as the inhibition of p38 MAPK with SB 203580, prevented IL-1beta-induced PGE(2) release. ERK1/2 activation by IL-1beta was sensitive to inhibition by the PKC inhibitor bisindolylmaleimide suggesting that ERK phosphorylation is a downstream signal of PKC activation. These results suggest key roles for PKC as well as for ERK1/2 and p38 MAP kinase cascades in the biosynthesis of PGE(2), likely by regulating the induction of cyclo-oxygenase-2, in IL-1beta-stimulated astroglial cells.     

Cyclo-oxygenase-2 enhances basic fibroblast growth factor-induced angiogenesis through induction of vascular endothelial growth factor in rat sponge implants

Angiogenesis is reportedly enhanced by prostaglandins (PGs). In the present study, we investigated whether or not cyclo-oxygenase (COX)-2 mediated angiogenesis in chronic and proliferate granuloma. In rat sponge implants, angiogenesis was gradually developed over a 14-day experimental period as granuloma formed. This angiogenesis was enhanced by topical injections of human recombinant basic fibroblast growth factor (bFGF). In sponge granuloma, mRNA of COX-1 was constitutively expressed, whereas that of COX-2 was increased with neovascularization in parallel with that of vascular endothelial growth factor (VEGF). Topical injections of bFGF increased the expression of COX-2 mRNA. bFGF-stimulated angiogenesis was inhibited by indomethacin or selective COX-2 inhibitors, NS-398, nimesulide, and JTE-522. The levels of PGE(2) and 6-keto-PGF(1alpha) in the sponge granuloma were increased with bFGF 13 fold and 9 fold, respectively, and these levels were markedly reduced by NS-398. The expression of VEGF mRNA in the granuloma was also enhanced by bFGF, and was reduced by NS-398. Topical injections of PGE(2) and beraprost sodium, a PGI(2) analogue, increased the expression of VEGF mRNA, with angiogenesis enhancement. The enhanced angiogenesis by bFGF was significantly inhibited by topical injections of VEGF anti-sense oligonucleotide. These results suggested that COX-2 may enhance bFGF-induced neovascularization in sponge granuloma by PG-mediated expression of VEGF, and that a COX-2 inhibitor would facilitate the management of conditions involving angiogenesis.     

Caspase-1 inhibitors abolish deleterious enhancement of COX-2 expression induced by HIV-1 gp120 in human neuroblastoma cells

The human CHP100 neuroblastoma cell line has been shown to provide an useful in vitro model to elucidate the mechanisms underlying HIV-1 gp120 neurotoxicity. Here we report western blotting evidence demonstrating that exposure to a cytotoxic concentration of the viral coat protein up-regulates expression of the inducible isoform of cyclooxygenase (COX-2) in neuroblastoma cells and this seems to be due to the previously observed increase in secreted IL-1beta. In fact, here we show that acetyl-Tyr-Val-Ala-Asp-chloromethylketone (Ac-YVAD-CMK) and t-butoxycarbonyl-L-aspartic acid benzyl ester-chloromethylketone (Boc-Asp-(OBzl)-CMK), two inhibitors of Interleukin-1 Converting Enzyme (ICE; also referred to as caspase-1), abolish COX-2 expression enhanced by gp120 and consequent cell death. In addition, NS-398, a selective inhibitor of COX-2 activity, affords neuroprotection strengthening the role of COX-2 in the mechanisms of death. In conclusion, the present data support the notion that IL-1beta is the signal through which gp120 elevates COX-2 expression and the latter is strongly implicated in the mechanisms underlying cytotoxicity.     

Acute pulmonary inflammation induced by exposure of the airways to staphylococcal enterotoxin type B in rats

Staphylococcus aureus is a gram-positive bacterium that produces several enterotoxins, which are responsible for most part of pathological conditions associated to staphylococcal infections, including lung inflammation. This study aimed to investigate the underlying inflammatory mechanisms involved in leukocyte recruitment in rats exposed to staphylococcal enterotoxin B (SEB). Rats were anesthetized with pentobarbital sodium and intratracheally injected with either SEB or sterile phosphate-buffered saline (PBS, 0.4 ml). Airways exposition to SEB (7.5-250 ng/trachea) caused a dose- and time-dependent neutrophil accumulation in BAL fluid, the maximal effects of which were observed at 4 h post-SEB exposure (250 ng/trachea). Eosinophils were virtually absent in BAL fluid, whereas mononuclear cell counts increased only at 24 h post-SEB. Significant elevations of granulocytes in bone marrow (mature and immature forms) and peripheral blood have also been detected. In BAL fluid, marked elevations in the levels of lipid mediators (LTB(4) and PGE(2)) and cytokines (TNF-alpha, IL-6 and IL-10) were observed after SEB instillation. The SEB-induced neutrophil accumulation in BAL fluid was reduced by pretreatment with dexamethasone (0.5 mg/kg), the COX-2 inhibitor celecoxib (3 mg/kg), the selective iNOS inhibitor compound 1400 W (5 mg/kg) and the lipoxygenase inhibitor AA-861 (200 microg/kg). In separate experiments carried out with rat isolated peripheral neutrophils, SEB failed to induce neutrophil adhesion to serum-coated plates and chemotaxis. In conclusion, rat airways exposition to SEB causes a neutrophil-dependent lung inflammation at 4 h as result of the release of proinflammatory (NO, PGE(2), LTB(4), TNF-alpha, IL-6) and anti-inflammatory mediators (IL-10).     

An Anacardiaceae preparation reduces the expression of inflammation-related genes in murine macrophages

This study investigated the effects of an aqueous extract of the stem bark of Mangifera indica L. (Anacardiaceae; Vimang), which contains a defined mixture of components including polyphenols (principally mangiferin, MA), triterpenes, phytosteroids, fatty acids and microelements, on expression of inflammation mediators in inflammatory murine macrophages after stimulation in vitro with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). In vitro treatment with Vimang at 4 microg/ml reduced levels of NOS-2 mRNA and NOS-2, while treatment at 40 microg/ml also reduced levels of COX-2 mRNA, COX-2, and prostaglandin E2 (PGE2). Results suggested that MA is involved in these effects. In vitro treatment with Vimang at 40 microg/ml also inhibited mRNA levels of the proinflammatory cytokines interleukin 1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha) and colony-stimulating factor (GM-CSF), but did not affect mRNA levels of IL-6 or tumor growth factor-beta (TGF-beta). Extracellular release of TNF-alpha by inflammatory macrophages was inhibited by in vitro treatment with Vimang at the same concentrations that showed inhibition of TNF-alpha mRNA levels. The inhibition of TNF-alpha production appears to be at least partially attributable to MA. Vimang at 4 microg/ml decreased mRNA levels of nuclear factor-kappaB (NF-kappaB) but did not affect expression of the NF-kappaB inhibitor (IkappaB). These data indicate that the potent anti-inflammatory effects of Vimang are due to selective modulation of the expression of inflammation-related genes, leading to attenuation of macrophage activation.     

The effects of meloxicam, indomethacin or NS-398 on eicosanoid synthesis by fresh human gastric mucosa

BACKGROUND: In the stomach, constitutive cyclooxygenase (COX-1) synthesizes prostaglandins that maintain the integrity of the gastric mucosa, while their inhibition contributes to gastric mucosal damage. In contrast COX-2, an inducible enzyme, forms prostanoids involved in pain and inflammation. AIM: To compare prostaglandin synthesis inhibition by meloxicam, a selective COX-2 NSAID reported to have better gastric tolerability, with indomethacin and NS-398 in human gastric mucosa and in whole blood assays. METHODS: Meloxicam, indomethacin or NS-398 were incubated with fresh human gastric mucosa pieces (100 mg in 1 mL phosphate buffered saline, pH 7.4, 37 degrees C, 30 min), clotting human blood (1 mL, 37 degrees C, 60 min) or with lipopolysaccharide-stimulated heparinized blood (1 mL, 37 degrees C, 24 h). Prostanoids were analysed by radioimmunoassay. RESULTS: Meloxicam was a less potent inhibitor of gastric mucosal eicosanoid compared to indomethacin, showing a sixfold difference in IC50 with gastric mucosal prostaglandin E (PGE) (11.8 and 1.8 microM, respectively). In the whole blood assays, the COX-2/COX-1 ratio for meloxicam was 0.2 compared to 0.9 for indomethacin confirming meloxicam's COX-2 selectivity. CONCLUSION: The results with human mucosa pieces would suggest that the better gastric tolerability of meloxicam compared to indomethacin is related to its relatively lower inhibition of gastric mucosal PGE synthesis by COX-1.     

Interleukin-1alpha and tumour necrosis factor-alpha modulate airway smooth muscle DNA synthesis by induction of cyclo-oxygenase-2: inhibition by dexamethasone and fluticasone propionate

1. Previous studies have established that glucocorticoids inhibit airway smooth muscle DNA synthesis. The effects of a combination of the pro-inflammatory cytokines, interleukin-1alpha (IL-1alpha) and tumour necrosis factor-alpha (TNF-alpha) on the inhibition of DNA synthesis by glucocorticoids in human cultured airway smooth muscle have now been investigated, since these cytokines are chronically expressed in asthmatic airways. 2. Thrombin (0.3 u ml(-1)) and basic fibroblast growth factor (bFGF, 300 pM) stimulated increases in DNA synthesis which were concentration-dependently inhibited by dexamethasone (1-1000 nM). 3. The cytokine mixture, comprising IL-1alpha (0.01 and 0.1 pM) and TNF-alpha (3 and 30 pM), directly evoked increases in DNA synthesis which were attenuated by dexamethasone. However, the cytokine mixture prevented responses to bFGF or thrombin. 4. Paradoxically, in the presence of the cytokine mixture and bFGF, dexamethasone (1-1000 nM) concentration-dependently increased DNA synthesis. Furthermore, neither dexamethasone (100 nM) nor fluticasone propionate (1 nM) inhibited DNA synthesized in response to bFGF/cytokine mixture combination and dexamethasone was similarly inactive against the thrombin/cytokine mixture. 5. The levels of prostaglandin E2 (PGE2), an established inhibitor of airway smooth muscle DNA synthesis, remained below the limits of assay detection (0.05 nM) under basal conditions or following stimulation with either thrombin or bFGF. In contrast, the cytokine mixture alone, and in the presence of thrombin or bFGF, induced biologically active levels of PGE2. Dexamethasone (100 nM), the non-selective cyclo-oxygenase (COX) inhibitor indomethacin (3 microM) or the selective COX-2 inhibitor L-745,337 (0.3 microM) completely inhibited synthesis of PGE2. 6. Neither indomethacin (3 microM) nor L-745,337 (0.3 microM) influenced thrombin- or bFGF-induced DNA synthesis. However, each COX inhibitor enhanced DNA synthesis in cytokine-treated cells. 7. In unstimulated airway smooth muscle cells, COX-1, but not COX-2 protein was detectable by Western blotting. The induction of COX-2 protein by the cytokine mixture was attenuated by dexamethasone (100 nM), whereas the level of COX-1 protein was unaffected by either the cytokines or by dexamethasone. 8. Cytokine-induced, COX-2-dependent eicosanoid production inhibits DNA synthesis. The paradoxical increase in DNA synthesis observed in glucocorticoid treated airway smooth muscle stimulated by cytokine/bFGF combinations may be explained by the ability of glucocorticoids to repress COX-2 induction and prevent cytokine-induction of the DNA synthesis inhibitor, PGE2.     

Cyclo-oxygenase-2 mediates P2Y receptor-induced reactive astrogliosis

Excessive cyclo-oxygenase-2 (COX-2) induction may play a role in chronic neurological diseases characterized by inflammation and astrogliosis. We have previously identified an astroglial receptor for extracellular nucleotides, a P2Y receptor, whose stimulation leads to arachidonic acid (AA) release, followed, 3 days later, by morphological changes resembling reactive astrogliosis. Since COX-2 may be upregulated by AA metabolites, we assessed a possible role for COX-2 in P2Y receptor-mediated astrogliosis. A brief challenge of rat astrocytes with the ATP analogue alpha,beta-methylene ATP (alpha,beta(me)ATP) resulted, 24 h later, in significantly increased COX-2 expression. The selective COX-2 inhibitor NS-398 completely abolished alpha,beta(me)ATP-induced astrocytic activation. Constitutive astroglial COX-1 or COX-2 did not play any role in purine-induced reactive astrogliosis. PGE2, a main metabolite of COX-2, also induced astrocytic activation. These data suggest that a P2Y receptor mediates reactive astrogliosis via induction of COX-2. Antagonists selective for this receptor may counteract excessive COX-2 activation in both acute and chronic neurological diseases.     

Involvement of cyclooxygenase-2 and EP3 prostaglandin receptor in acute herpetic but not postherpetic pain in mice

The precise mechanisms of zoster-associated pain and postherpetic neuralgia remain unknown. Inoculation of mice with herpes simplex virus type-1 elicits acute herpetic pain- and delayed postherpetic pain-related responses. We investigated the role of prostaglandins (PGs) and their synthases in both types of pain. Deficiency in EP3 but not EP1, IP or TP prostanoid receptor markedly diminished the acute herpetic pain and resulted in the decrease of the incidence of the delayed postherpetic pain. Preventive but not therapeutic administration of the EP3 antagonist ONO-AE3-240 inhibited the acute herpetic pain. The non-selective cyclooxygenase (COX) inhibitor diclofenac and the selective COX-2 inhibitors NS-398 and JTE-522 dose dependently reduced the acute herpetic pain, and NS-398 was without effect on delayed postherpetic pain. COX-2 was induced and PGE2 content was increased in the affected dorsal root ganglia at the stage of acute herpetic pain. COX-2-like immunoreactivities were found around the nuclear membrane of many dorsal root ganglion neurons that were negative for herpesvirus antigen. COX-2 mRNA expression and PGE2 content in the affected dorsal root ganglia at the stage of delayed postherpetic pain were similar to those of naive mice. The propagation of herpes virus in dorsal root ganglion may induce COX-2 and produce PGE2 in uninfected neurons. The results suggest the important roles of COX-2 induction and the PGE2-EP3 receptor system in the dorsal root ganglia in the development but not maintenance of acute herpetic pain. It was further confirmed that the PG systems do not play a key role in delayed postherpetic pain.     

Up-regulation of cyclooxygenase-2 expression is involved in R(+)-methanandamide-induced apoptotic death of human neuroglioma cells

Cannabinoids have been implicated in the reduction of glioma growth. The present study investigated a possible relationship between the recently shown induction of cyclooxygenase (COX)-2 expression by the endocannabinoid analog R(+)methanandamide [R(+)-MA] and its effect on the viability of H4 human neuroglioma cells. Incubation with R(+)-MA for up to 72 h decreased the cellular viability and enhanced accumulation of cytoplasmic DNA fragments in a time-dependent manner. Suppression of R(+)-MA-induced prostaglandin (PG) E2 synthesis with the selective COX-2 inhibitor celecoxib (0.01-1 microM) or inhibition of COX-2 expression by COX-2-silencing small-interfering RNA was accompanied by inhibition of R(+)-MA-mediated DNA fragmentation and cell death. In contrast, the selective COX-1 inhibitor SC-560 was inactive in this respect. Cells were also protected from apoptotic cell death by other COX-2 inhibitors (NS-398 [[N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide]] and diclofenac) and by the ceramide synthase inhibitor fumonisin B1, which interferes with COX-2 expression by R(+)-MA. Moreover, the proapoptotic action of R(+)-MA was mimicked by the major COX-2 product PGE2. Apoptosis and cell death by R(+)-MA were not affected by antagonists of cannabinoid receptors (CB1, CB2) and vanilloid receptor 1. In further experiments, celecoxib was demonstrated to suppress apoptotic cell death elicited by anandamide, which is structurally similar to R(+)-MA. As a whole, this study defines COX-2 as a hitherto unknown target by which a cannabinoid induces apoptotic death of glioma cells. Furthermore, our data show that pharmacological concentrations of celecoxib may interfere with the proapoptotic action of R(+)-MA and anandamide, suggesting that cotreatment with COX-2 inhibitors could diminish glioma regression induced by these compounds.     

Stimulation of cyclooxygenase-2-activity by nitric oxide-derived species in rat chondrocyte: lack of contribution to loss of cartilage anabolism

Cross-talk between inducible nitric oxide synthase (NOS II) and cyclooxygenase-2 (COX-2) was investigated in rat chondrocytes. In monolayers, interleukin-1beta (IL-1beta) induced COX-2 and NOS II expression in a dose- and time-dependent manner, to produce high prostaglandin E(2) (PGE(2)) and nitrite (NO(2)(-)) levels in an apparently coordinated fashion. COX-2 mRNA was induced earlier (30 min. versus 4 hr) and less markedly (4-fold versus 12-fold at 24 hr) than NOS II, and was poorly affected by the translational inhibitor cycloheximide (CHX). IL-1beta did not stabilize COX-2 mRNA in contrast to CHX. Indomethacin and NS-398 lacked any effect on NO(2)(-) levels whereas L-NMMA and SMT reduced PGE(2) levels at concentration inhibiting NO(2)(-) production from 50 to 90%, even when added at a time allowing a complete expression of both enzymes (8 hr). Basal COX activity was unaffected by NO donors. The SOD mimetic, CuDips inhibited COX-2 activity by more than 75% whereas catalase did not. Inhibition of COX-2 by CuDips was not sensitive to catalase, consistent with a superoxide-mediated effect. In tridimensional culture, IL-1beta inhibited radiolabelled sodium sulphate incorporation while stimulating COX-2 and NOS II activities. Cartilage injury was corrected by L-NMMA or CuDips but not by NSAIDs, consistent with a peroxynitrite-mediated effect. These results show that in chondrocytes: (i) COX2 and NOS II genes are induced sequentially and distinctly by IL-1beta; (ii) COX-1 and COX-2 activity are affected differently by NO-derived species; (iii) peroxynitrite accounts likely for stimulation of COX-2 activity and inhibition of proteoglycan synthesis induced by IL-1beta.