Anti-inflammatory effect of two isoforms of COX in H. pylori-induced gastritis in mice: possible involvement of PGE2

Neutrophil infiltration mediated by TNF-alpha is associated with various types of gastric injury, whereas PGs play a crucial role in gastric defense. We examined roles of two isoforms of cyclooxygenase (COX) and PGE2 in Helicobacter pylori-induced gastritis in mice. Mice infected with H. pylori were given selective COX-1 inhibitor SC-560 (10 mg/kg), selective COX-2 inhibitor NS-398 (10 mg/kg), or nonselective COX inhibitor indomethacin (2 mg/kg) with or without 16,16-dimethyl PGE2 for 1 wk. H. pylori infection increased levels of mRNA for COX-1 and -2 in gastric tissue by 1.2-fold and 3.3-fold, respectively, accompanied by a significant increase in PGE2 production by gastric tissue. H. pylori infection significantly elevated MPO activity, a marker of neutrophil infiltration, and epithelial cell apoptosis in the stomach. SC-560 augmented MPO activity and epithelial cell apoptosis with associated reduction in PGE2 production, whereas NS-398 had the same effects without affecting PGE2 production. Inhibition of both COX-1 and -2 by indomethacin or concurrent treatment with SC-560 and NS-398 resulted in a stronger increase in MPO activity and apoptosis than inhibition of either COX-1 or -2 alone. H. pylori infection elevated TNF-alpha mRNA expression in the stomach, which was further increased by indomethacin. Effects of COX inhibitors on neutrophil infiltration, apoptosis, and TNF-alpha expression in H. pylori-infected mice were abolished by exogenous 16,16-dimethyl PGE2. In conclusion, PGE2 derived from either COX-1 or -2 is involved in regulation of gastric mucosal inflammation and contributes to maintenance of mucosal integrity during H. pylori infection via inhibition of TNF-alpha expression.     

Effect of H. pylori infection on the expression of cyclooxygenase-2 in human gastric mucosa

Cyclooxygenase-1 is the primary isoform responsible for the production of cytoprotective prostaglandins (PGE(2) and PGI(2)) in the stomach. In contrast COX-2 is induced at the sites of inflammation. Using Helicobacter pylori infection as a model of inflammation, this study was designed to evaluate the effects of H. pylori infection on prostanoid synthesis and expression of COX-2 in human gastric mucosa.Prostaglandin (PGE(2)) and prostacyclin (PGI(2)) synthesis in gastric biopsies obtained from 21 patients undergoing diagnostic endoscopy, were determined. H. pylori was detected by CLO test, histology and culture. Biopsy samples were incubated either with NS-398, selective COX-2 inhibitor or aspirin. Samples were also treated with endotoxin (LPS) in order to induce COX-2 expression. Tissue was also analysed for COX-2 expression in vivo by immunohistochemistry.In 15 out of 21 patients, H. pylori was detected by at least two of the three methods. Higher levels of PGE(2) and PGI(2) were seen in patients infected with H. pylori (191+/-30 and 245+/-88ng/mg protein, respectively) compared with non-infected patients (77+/-17 and 120+/-36ng/mg protein, respectively). There was significant inhibition of PGE(2) and PGI(2) with aspirin in both H. pylori infected (28+/-6.6 and 53+/-43ng/mg, respectively) and in non-infected patients (16+/-7 and 12.5+/-3.5ng/mg protein, respectively). However, NS-398 and LPS did not alter prostaglandin function significantly. Immunohistochemistry in all patients irrespective of Hp status demonstrated expression of COX-2.Lower concentration of constitutive expression of COX-2 was detected in human gastric mucosa by immunohistochemistry, however, H. pylori infection failed to induce COX-2 protein. In addition, increased prostaglandin synthesis in Hp-infected patients appears to be COX-1 mediated rather than COX-2. Furthermore, failure of endotoxaemia-treated sample to produce more PGE(2) in the face of enhanced COX-2 expression in gastric mucosa further suggests that increased prostanoids in human gastric stomach are COX-1 mediated.     

COX-2 induction during murine gammaherpesvirus 68 infection leads to enhancement of viral gene expression

The murine gammaherpesvirus 68 (MHV-68 or gammaHV-68) model provides many advantages for studying virus-host interactions involved in gammaherpesvirus replication, including the role of cellular responses to infection. We examined the effects of cellular cyclooxygenase-2 (COX-2) and its by-product prostaglandin E(2) (PGE(2)) on MHV-68 gene expression and protein production following de novo infection of cultured cells. Western blot analyses revealed an induction of COX-2 protein in MHV-68-infected cells but not in cells infected with UV-irradiated MHV-68. Luciferase reporter assays demonstrated activation of the COX-2 promoter during MHV-68 replication. Two nonsteroidal anti-inflammatory drugs, a COX-2-specific inhibitor (NS-398) and a COX-1-COX-2 inhibitor (indomethacin), substantially reduced MHV-68 protein production in infected cells. Inhibition of viral protein expression and virion production by NS-398 was reversed in the presence of exogenous PGE(2). Global gene expression analysis using an MHV-68 DNA array showed that PGE(2) increased production of multiple viral gene products, and NS-398 inhibited production of many of the same genes. These studies suggest that COX-2 activity and PGE(2) production may play significant roles during MHV-68 de novo infection.     

Expression of cyclooxygenase (COX)-1 and COX-2 in adaptive cytoprotection induced by mild stress.

Prostaglandins (PG) derived from COX-1 play an important role in the maintenance of mucosal integrity but the role of COX-2-derived products in mucosal defence mechanism has not been fully explained. Mild stress is known to prevent gastric mucosal lesions induced by severe stress via the phenomenon of adaptive cytoprotection but it remains unknown which COX is involved in this adaptation. In this study, the mucosal expression of COX-1 and COX-2 was examined and the inhibitors of these enzymes were used to determine the contribution of these enzymes in adaptive cytoprotection induced by mild stress. Male Wistar rats were exposed to mild water immersion and restraint stress (WRS) at various time intervals ranging from 5 min up to 2 h followed 1 h later by exposure to severe 3.5 h WRS with or without pretreatment with: 1) NS-398 (10 mg x kg(-1) i.g.), a selective COX-2 inhibitor; 2) resveratrol (5 mg x kg(-1) i.g.), a selective COX-1 inhibitor; 3) meloxicam (2 mg x kg(-1) i.g.), preferential COX-2 inhibitor; and 4) indomethacin (5 mg x kg(-1) i.p), non-selective inhibitor of COX. The number of WRS lesions was counted, gastric blood flow (GBF) was measured by H2-gas clearance technique, mucosal biopsy samples were taken for the assessment of PGE2 by radioimmunoassay, and the expression of COX-1 and COX-2 mRNA by RT-PCR. WRS for 3.5 h produced numerous gastric lesions, decreased GBF by 48% and inhibited formation of PGE2 by 68% as compared to intact mucosa. Exposure to mild WRS during 5-30 min by itself failed to affect mucosal integrity but significantly attenuated gastric lesions induced by exposure to severe 3.5 h stress; the maximal protective effect being achieved with mild WRS during 15 min. This protective effect was accompanied by the rise in GBF and the generation of PGE2 in the gastric mucosa. After extension of mild WRS from 15 min up to 1 or 2 h before more severe 3.5 h WRS, the loss of cytoprotective effect of mild WRS against severe stress accompanied by significant fall in the GBF were observed. Pretreatment with NS-398 (10 mg x kg(-1) i.g.) that failed to affect mucosal PGE2 generation, reduced significantly the protection and accompanying rise in GBF produced by mild WRS whereas resveratrol partly reduced the protection and the rise in GBF induced by mild WRS. Meloxicam or indomethacin significantly inhibited PGE2 generation and completely abolished the hyperemia and protection induced by mild WRS against more severe stress. The protective and hyperemic effects of mild WRS were completely restored by the addition of 16,16 dm PGE2 (5 microg x kg(-1) i.g.) to NS-398 or resveratrol, while the deleterious effects of meloxicam and indomethacin were significantly attenuated by the concomitant treatment with this PGE2 analogue. We conclude that PG derived from both, COX-1 and COX-2 appear to be involved in adaptive cytoprotection developed in response to mild stressors.     

Cyclooxygenase-2 selective and nitric oxide-releasing nonsteroidal anti-inflammatory drugs and gastric mucosal responses.

Occurrence of gastrointestinal damage and delayed healing of pre-existing ulcer are commonly observed in association with clinical use of nonsteroidal antiinflammatory drugs (NSAIDs). We examined the effects of NS-398, the cyclooxygenase (COX)-2 selective inhibitor, and nitric oxide (NO)- releasing aspirin (NCX-4016) on gastric mucosal ulcerogenic and healing responses in experimental animals, in comparison with those of nonselective COX inhibitors such as indomethacin and aspirin. Indomethacin and aspirin given orally were ulcerogenic by themselves in rat stomachs, while either NS-398 or NCX-4016 was not ulcerogenic at the doses which exert the equipotent antiinflammatory action with indomethacin or aspirin. Among these NSAIDs, only NCX-4016 showed a dose-dependent protection against gastric lesions induced by HCl/ethanol in rats. On the other hand, the healing of gastric ulcers induced in mice by thermal-cauterization was significantly delayed by repeated administration of these NSAIDs for more than 7 days, except NCX-4016. Gastric mucosal prostaglandin contents were reduced by indomethacin, aspirin and NCX-4016 in both normal and ulcerated mucosa, while NS-398 significantly decreased prostaglandin generation only in the ulcerated mucosa. Oral administration of NCX-4016 in pylorus-ligated rats and mice increased the levels of NO metabolites in the gastric contents. In addition, both NS-398 and NCX-4016 showed an equipotent anti-inflammatory effect against carrageenan-induced paw edema in rats as compared with indomethacin and aspirin. These results suggest that both indomethacin and aspirin are ulcerogenic by themselves and impair the healing of pre-existing gastric ulcers as well. The former action is due to inhibition of COX-1, while the latter effect may be accounted for by inhibition of COX-2 and mimicked by NS-398, the COX-2 selective NSAID. NCX-4016, despite inhibiting both COX-1 and COX-2, protects the stomach against damage and preserves the healing response of gastric ulcers, probably because of the beneficial action of NO.     

Cyclooxygenase-2-regulated vascular endothelial growth factor release in gastric fibroblasts

VEGF is a highly specific stimulator of endothelial cells and may play an important role in angiogenesis in the process of tissue regeneration. We previously showed that cyclooxygenase-2 (COX-2) expressed in mesenchymal cells of the ulcer bed is involved in the ulcer repair process. To clarify the role of COX-2 in angiogenesis during gastric ulcer healing, we investigated the relation between COX-2 expression and VEGF production in human gastric fibroblasts in vivo and in vitro. Gastric fibroblasts were cultured in RPMI 1640 with and without IL-1alpha or IL-1beta in the presence or absence of NS-398, a selective COX-2 inhibitor. Supernatant VEGF and PGE(2) concentrations were measured by enzyme-linked immunosorbent assay. COX-2 expression in fibroblasts was determined by Western blot analysis. VEGF and COX-2 expression in surgical resections of human gastric ulcer tissue was examined immunohistochemically. IL-1 dose dependently enhanced VEGF release in cultured gastric fibroblasts after a 24-h stimulation. IL-1 also stimulated PGE(2) production in gastric fibroblasts via COX-2 induction. NS-398 significantly suppressed VEGF and PGE(2) release from IL-1-stimulated gastric fibroblasts; concurrent addition of PGE(2) restored NS-398-inhibited VEGF release. COX-2 and VEGF immunoreactivity were colocalized in fibroblast-like cells in the ulcer bed of gastric tissues. These results suggest that COX-2 plays a key role in VEGF production in gastric fibroblasts stimulated by IL-1 in vitro and that angiogenesis induced by the COX-2-VEGF pathway might be involved in gastric ulcer healing.     

Dendritic cells issued in vitro from bone marrow produce PGE(2) that contributes to the immunomodulation induced by antigen-presenting cells.

Given that preliminary work has indicated that prostaglandins can play a role in modulating dendritic cell (DC) functions, we addressed the prostaglandin E(2) (PGE(2)) biosynthetic capacity of mouse DC produced in vitro from bone marrow cells. We observed production of significant amounts of PGE(2), which was reduced by at least 80% when cells were incubated in the presence of indomethacin, a COX-1 preferential inhibitor. Indeed, when tested by Western blot analysis with specific COX-1 and COX-2 antibodies, only COX-1 expression could be detected in the bone marrow (BM)-DC. For lipopolysaccharide (LPS)-treated BM-DC, inhibition of PGE(2) production by indomethacin or by NS-398 (a COX-2-selective inhibitor) used alone was less potent. After LPS treatment of BM-DC, COX-1 and COX-2 expression was potent, and inhibition of PGE(2) synthesis needed the presence of both indomethacin and NS-398. We also observed that exogenous PGE(2) diminished the expression of MHC class II molecules by BM-DC and that prostaglandin and indomethacin had antagonistic effects on cell proliferation during the mixed lymphocyte reaction using BM-DC as stimulatory cells. This assessment of PGE(2) suggests that endogenous PGE(2) produced by DC might play a role as an immunomodulating factor during the immune response. This hypothesis is sustained by the fact that IL-12 production by BM-DC is modulated by exogenous PGE(2) as well as endogenous prostaglandin, since either the addition of exogenous PGE(2) or the presence of LPS (which increases endogenous PGE(2) synthesis) decreases IL-12 production, while NS-398 (which decreases LPS-induced PGE(2) synthesis) increases IL-12 synthesis.     

The effect of ascorbic acid on Helicobacter pylori induced cyclooxygenase 2 expression and prostaglandin E2 production by gastric epithelial cells in vitro

BACKGROUND: Cyclooxygenase 2 (COX-2) is induced by the presence of Helicobacter pylori (H. pylori) on the gastric mucosa as part of the inflammatory response; this results in the synthesis of prostaglandins that amplify the local inflammatory response. The presence of H. pylori inhibits the secretion of ascorbate into the gastric lumen. Interestingly, ascorbate inhibits the growth of H. pylori and low dietary levels are associated with an increased risk of gastric adenocarcinoma. We therefore investigated the effect of ascorbate on H. pylori mediated COX-2 induction and prostaglandin production in vitro. METHODS: H. pylori was cocultured with gastric epithelial cells in the presence of ascorbate at physiological concentrations. The expression of COX-2 was assessed by Western blotting and prostaglandin E(2) (PGE(2)) was assessed by ELISA. RESULTS: Ascorbate inhibited gastric cell PGE(2) synthesis but not in COX-2 expression in response to H. pylori. In the absence of the organism, ascorbate also reduced PGE(2) expression in cells that constitutively express COX-2, again with no reduction of COX-2 protein expression. CONCLUSIONS: Physiological concentrations of ascorbate inhibit PGE(2) but not COX-2 expression in response to H. pylori in gastric epithelial cells.     

Increased production of matrix metalloproteinases in Helicobacter pylori-associated human gastritis

BACKGROUND AND AIMS: Helicobacter pylori infection results in an active, chronic inflammation of the gastric mucosa. Previous studies have highlighted the importance of matrix metalloproteinases (MMPs) in diseases involving mucosal inflammation, prompting us to investigate MMP activity in H. pylori-induced gastritis. METHODS: Gastric biopsies were obtained from H. pylori-infected and uninfected volunteers, and MMP activity was assessed using substrate gel electrophoresis. MMP production was also evaluated by immunohistochemistry and real time-polymerase chain reaction. In parallel, tissue inhibitors of MMPs (TIMP) levels and TIMP-MMP complexes were examined in corresponding tissues using enzyme-linked immunosorbent assays and Western blotting. Finally, MMP production by gastric macrophages was determined after stimulation with H. pylori. RESULTS: Antral mucosa of H. pylori-infected subjects demonstrated a 19-fold higher MMP-9 activity than that of uninfected individuals. MMP-2 was present at lower levels, but was also increased in H. pylori-infected individuals, while there was no difference in the total levels of TIMP-1 and TIMP-2 between the groups of volunteers. Significant numbers of MMP-9-containing cells were only found in the H. pylori-infected antral mucosa. Tissue-resident macrophages were significantly increased in H. pylori-infected individuals, and double-staining showed MMP-9 colocalized to macrophages. Furthermore, gastric macrophages secreted MMP-9 in response to H. pylori bacteria. A corresponding 10-fold increase of gene expression of MMP-9 was seen in patients infected with H. pylori compared to uninfected individuals. CONCLUSIONS: Helicobacter pylori infection results in a substantial increase in MMP-9 and MMP-2 activity in the gastric mucosa, probably contributed to in large part by tissue-resident macrophages, while no changes were seen in the TIMP levels. The net increase in gastric MMP activity is likely to contribute to tissue damage during H. pylori-associated gastritis.     

Lipoteichoic acid-induced nitric oxide synthase expression in RAW 264.7 macrophages is mediated by cyclooxygenase-2, prostaglandin E2, protein kinase A, p38 MAPK, and nuclear factor-kappaB pathways

We recently reported that lipoteichoic acid (LTA), a cell wall component of the gram-positive bacterium Staphylococcus aureus, stimulated inducible nitric oxide synthase (iNOS) expression, nitric oxide (NO) release, and cyclooxygenase-2 (COX-2) expression in RAW 264.7 macrophages. This study was carried out to further investigate the roles of COX-2 and prostaglandin E2 (PGE2) in LTA-induced iNOS expression and NO release in RAW 264.7 macrophages. Treatment of RAW 264.7 macrophages with LTA caused a time-dependent increase in PGE2 release. LTA-induced iNOS expression and NO release were inhibited by a non-selective COX inhibitor (indomethacin), a selective COX-2 inhibitor (NS-398), an adenylyl cyclase (AC) inhibitor (dideoxyadenosine, DDA), and a protein kinase A (PKA) inhibitor (KT-5720). Furthermore, both PGE2 and the direct PKA activator, dibutyryl-cAMP, also induced iNOS expression in a concentration-dependent manner. Stimulation of RAW 264.7 macrophages with LTA, PGE2, and dibutyryl-cAMP all caused p38 MAPK activation in a time-dependent manner. LTA-mediated p38 MAPK activation was inhibited by indomethacin, NS-398, and SB 203580, but not by PD 98059. The PGE2-mediated p38 MAPK activation was inhibited by DDA, KT-5720, and SB 203580, but not by PD 98059. LTA caused time-dependent activation of the nuclear factor-kappaB (NF-kappaB)-specific DNA-protein complex formation. The LTA-induced increase in kappaB-luciferase activity was inhibited by indomethacin, NS-398, KT-5720, and a dominant negative mutant of p38 alphaMAPK (p38 alphaMAPK DN). These results suggest that LTA-induced iNOS expression and NO release involve COX-2-generated PGE2 production, and AC, PKA, p38 MAPK, and NF-kappaB activation in RAW 264.7 macrophages.     

COX-2 inhibition affects growth rate of Chlamydia muridarum within epithelial cells

Chlamydiae alter apoptosis of host target cells, which regulates their growth. Cyclooxygenase-2 (COX-2), the rate-limiting enzyme for prostaglandin E2 (PGE2) production, modulates epithelial cell survival. We addressed whether endogenous PGE2 alters chlamydial growth or apoptosis of epithelial cells infected with Chlamydia muridarum. PGE2 is secreted by infected host cells in the genital tract (GT). Using immunohistochemical techniques, we found that COX-2 enzyme was localized to epithelial cells in the GT in vivo. Pellets of the COX-2 enzyme inhibitor, NS-398, and placebo were implanted in mice subcutaneously and released a constant amount of these chemicals throughout the infection. NS-398-treated mice were found to exhibit 10-fold lower bacterial load than the placebo group on day 3 post infection, suggesting disruption of the chlamydial developmental cycle. To prove this, the human lung adenocarcinoma cell line A549 was then infected with different MOIs of C. muridarum in the presence of multiple concentrations of NS-398 in vitro. There was no difference in inclusion forming units (IFUs) between NS-389-treated and untreated cells. We also found no alterations in C. muridarum IFUs in A549 cells transfected with a 2.0 kb cDNA fragment of human COX-2 cloned in the sense (S) or anti-sense (AS) orientation. However, the inclusion size was reduced and the number of EB was significantly diminished during reinfection in AS-transfected cells. In addition, the absence of COX-2 did not significantly modify apoptosis in infected cells. In total, COX-2 deficiency reduces the infectious burden in vivo and may modulate transmission of the organism.     

Respiratory syncytial virus (RSV) infection induces cyclooxygenase 2: a potential target for RSV therapy

Cyclooxygenases (COXs) are rate-limiting enzymes that initiate the conversion of arachidonic acid to prostanoids. COX-2 is the inducible isoform that is up-regulated by proinflammatory agents, initiating many prostanoid-mediated pathological aspects of inflammation. The roles of cyclooxygenases and their products, PGs, have not been evaluated during respiratory syncytial virus (RSV) infection. In this study we demonstrate that COX-2 is induced by RSV infection of human lung alveolar epithelial cells with the concomitant production of PGs. COX-2 induction was dependent on the dose of virus and the time postinfection. PG production was inhibited preferentially by NS-398, a COX-2-specific inhibitor, and indomethacin, a pan-COX inhibitor, but not by SC-560, a COX-1-specific inhibitor. In vivo, COX-2 mRNA expression and protein production were strongly induced in the lungs and cells derived from bronchioalveolar lavage of cotton rats infected with RSV. The pattern of COX-2 expression in vivo in lungs is cyclical, with a final peak on day 5 that correlates with maximal histopathology. Treatment of cotton rats with indomethacin significantly mitigated lung histopathology produced by RSV. The studies described in this study provide the first evidence that COX-2 is a potential therapeutic target in RSV-induced disease.     

Effects of dexamethasone and FK506 on Helicobacter pylori-induced gastritis and bacterial viability in Mongolian gerbils.

FK506 and dexamethasone were used to investigate whether or not immunosuppression affects H. pylori colonization and gastric mucosal damage induced by Helicobacter pylori in Mongolian gerbils. Two weeks after H. pylori infection, FK506 and dexamethasone or vehicle alone were subcutaneously administered once daily for the following 2 weeks. FK506 or vehicle alone was administered subcutaneously once daily for 5 weeks (1 week before and 4 weeks after infection). In H. pylori-infected animals for 4 weeks, hemorrhagic erosions and inflammatory responses (neutrophil infiltration and lymphoid follicle formation) were induced in gastric mucosa at an incidence of 100%. Both FK506 and dexamethasone administered for 2 weeks markedly reduced such mucosal changes. In these animals, H. pylori viability in the stomach was significantly elevated. FK506 administered for 5 weeks also significantly inhibited the hemorrhagic erosions, edema and neutrophil infiltration in the stomach. H. pylori viability was slightly elevated as compared with the control. It was concluded that the host immune responses might play dual roles both by deteriorating gastritis induced by H. pylori and by protecting against H. pylori infection in its early stage.     

Gastrin enhances gastric mucosal integrity through cyclooxygenase-2 upregulation in rats

Gastrin, PGs, and growth factors have important roles in maintaining gastrointestinal mucosal integrity. Cyclooxygenases (COX-1 and COX-2) are the key enzymes involved in PG synthesis. This study aimed to clarify the mechanisms of gastric mucosal protection by gastrin. Fasted rats were administered subcutaneous gastrin 17 with or without gastrin receptor antagonist YM022 pretreatment. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) and COX-2 expression were examined using Western blot analysis. Another series of experiments investigated 1) PGE(2) levels in gastric mucosa, 2) the protective action of gastrin against gastric damage by acidified ethanol, 3) the effects of a specific HB-EGF-neutralizing antibody on gastrin-induced COX-2 expression, and 4) the effects of a specific COX-2 inhibitor NS-398 on PGE(2) synthesis and the mucosal protection afforded by gastrin. Gastrin dose-dependently increased HB-EGF, COX-2 expression, and PGE(2) levels and reduced gastric damage. However, pretreatment with YM022 dose-dependently abolished such effects of gastrin. A specific HB-EGF- neutralizing antibody and an EGF receptor inhibitor decreased gastrin-induced COX-2 expression. NS-398 blocked gastrin-induced PGE(2) synthesis and mucosal protection. In conclusion, this study demonstrates that gastrin enhances gastric mucosal integrity through COX-2, which is partially mediated by HB-EGF, and PGE(2) upregulation in rats.     

Induced microsomal PGE synthase-1 is involved in cyclooxygenase-2-dependent PGE2 production in gastric fibroblasts

We have previously shown that the cyclooxygenase (COX)-2/PGE2 pathway plays a key role in VEGF production in gastric fibroblasts. Recent studies have identified three PGE synthase (PGES) isozymes: cytosolic PGES (cPGES) and microsomal PGES (mPGES)-1 and -2, but little is known regarding the expression and roles of these enzymes in gastric fibroblasts. Thus we examined IL-1beta-stimulated mPGES-1 and cPGES mRNA and protein expression in gastric fibroblasts by quantitative PCR and Western blot analysis, respectively, and studied both their relationship to COX-1 and -2 and their roles in PGE2 and VEGF production in vitro. IL-1beta stimulated increases in both COX-2 and mPGES-1 mRNA and protein expression levels. However, COX-2 mRNA and protein expression were more rapidly induced than mPGES-1 mRNA and protein expression. Furthermore, MK-886, a nonselective mPGES-1 inhibitor, failed to inhibit IL-1beta-induced PGE2 release at the 8-h time point, while totally inhibiting PGE2 at the later stage. However, MK-886 did inhibit IL-1beta-stimulated PGES activity in vitro by 86.8%. N-(2-cyclohexyloxy-4-nitrophenyl)-methanesulfonamide (NS-398), a selective COX-2 inhibitor, totally inhibited PGE2 production at both the 8-h and 24-h time points, suggesting that COX-2-dependent PGE2 generation does not depend on mPGES-1 activity at the early stage. In contrast, NS-398 did not inhibit VEGF production at 8 h, and only partially at 24 h, whereas MK-886 totally inhibited VEGF production at each time point. These results suggest that IL-1beta-induced mPGES-1 protein expression preferentially coupled with COX-2 protein at late stages of PGE2 production and that IL-1beta-stimulated VEGF production was totally dependent on membrane-associated proteins involved in eicosanoid and glutathione metabolism (MAPEG) superfamily proteins, which includes mPGES-1, but was partially dependent on the COX-2/PGE2 pathway.     

Inducible types of cyclooxygenase and nitric oxide synthase in adaptive cytoprotection in rat stomachs.

Roles of cyclooxygenases (COX-1 and COX-2) and nitric oxide (NO) synthases (nNOS and iNOS) in adaptive cytoprotection induced by 20 mM taurocholate dissolved in 50 mM HCl (TC) were investigated in rat stomachs. Intragastric administration of 0.6 N HCl caused haemorrhagic damage in the stomach. These lesions were prevented by pretreatment of the animals with TC p.o. 0.5 h before 0.6 N HCl, and a significant protection persisted for more than 5 h. The protection afforded by TC given 0.5 h before HCl was almost totally reversed by indomethacin and slightly mitigated by N(G)-nitro-L-arginine methyl ester (L-NAME) but not affected by NS-398 or aminoguanidine. By contrast, the mucosal protective action of TC given 5 h before HCl was significantly reversed by NS-398, L-NAME and aminoguanidine as well as indomethacin. Mucosal prostaglandin E2 (PGE2) contents were significantly increased for over 5 h after TC, while luminal NOx output tended to elevate at 0.5 h and be significantly increased at 5 h after TC. The increased PGE2 generation observed 0.5 h after TC was attenuated only by indomethacin, while that observed 5 h after TC was inhibited by NS-398 as well as indomethacin. On the other hand, the NOx output determined at 5 h after TC was significantly reduced by both L-NAME and aminoguanidine. The expression of mRNA for both COX-2 and iNOS was apparently detected in the stomach from 3 h after TC treatment. These results suggest that TC induced adaptive cytoprotection in the rat stomach against 0.6 N HCl, the effect lasting for over 5 h, and the underlying mechanism differs depending on the period after the irritation. The early phase is mediated mainly by COX-1/PGs, while the later phase is mediated by iNOS/NO, in addition to prostaglandins (PGs) produced by both COX-1 and COX-2.     

Sensitivity difference to the suppressive effect of prostaglandin E2 among mouse strains: a possible mechanism to polarize Th2 type response in BALB/c mice

PGE2 has been shown to play a prominent role in regulating Th1 and Th2 type responses. We studied the role of PGE2 in IFN-gamma production by Staphylococcus aureus Cowan I-stimulated spleen cells from several mouse strains such as BALB/c, C3H/HeN, and C57BL/6. When spleen cells were pretreated with indomethacin (cyclooxygenase (COX)-1 and COX-2 inhibitor) or NS-398 (COX-2-specific inhibitor), S. aureus Cowan I -induced IFN-gamma production was increased more markedly in spleen cells from BALB/c mice than from C3H/HeN and C57BL/6 mouse. However, PGE2 production was not significantly different among spleen cells from three mouse strains. When various concentrations of PGE2 were exogeneously added to spleen cells, PGE2 showed a stronger suppressive effect on IFN-gamma production in spleen cells from BALB/c mice than from other strains of mice. This suppressive effect of PGE2 in BALB/c mice mainly depended on IL-12p70 production by APCs. More PGE2 binding sites were found in BALB/c spleen cells than in C3H/HeN spleen cells, indicating that the sensitivity difference to the suppressive effect of PGE2 was due to the difference of the number of PGE2 receptors. The administration of NS-398 into BALB/c mice enhanced Ag-specific IFN-gamma production, but not IL-4 production. This effect is the same as IL-12 administration in vivo. From these results, we propose that the modulation of PGE2 is important for Th1 activation via IFN-gamma and IL-12p70 production in vitro and in vivo and that PGE2 is one of the pivotal factors in the Th2-dominant immune response in BALB/c mice.     

Effects of selective cyclooxygenase-2 inhibitor and non-selective NSAIDs on Helicobacter pylori-induced gastritis in Mongolian gerbils

Background. It is still a point of controversy whether Helicobacter pylori‐infected patients are more likely to develop mucosal damage while taking NSADIs. Selective cyclooxygenase (COX‐2) inhibitors may be associated with less severe gastric mucosal damage than conventional NSAIDs, but this association is undefined in H. pylori‐induced gastritis. The aim of this study was to evaluate the effects of selective COX‐2 and nonselective NSAIDs on H. pylori‐induced gastritis. Methods. After intragastric administration of indomethacin, NS‐398 or vehicle alone, once daily for 5 days in H. pylori‐infected and uninfected Mongolian gerbils, we evaluated gastric mucosal damage, inflammatory cell infiltration and prostaglandin E₂ (PGE₂) concentration. We investigated whether H. pylori infection induced the COX‐2 expression. Results. In H. pylori‐uninfected groups, the indomethacin‐treated group showed the highest mucosal damage score and the lowest PGE₂ concentration. There was no difference in mucosal damage scores and PGE₂ concentration between NS‐398 and vehicle‐alone treated group. In H. pylori‐infected groups, there was no difference in mucosal damage scores, irrespective of the type of drugs administered. The indomethacin‐treated group showed the lowest PGE₂ concentration, similar to that of the NS‐398 and vehicle‐alone treated groups, both without H. pylori infection. Gastric neutrophil and monocyte infiltration scores were higher in H. pylori‐infected groups than in uninfected groups. However, there was no difference in these scores according to the type of drugs administered, within H. pylori‐infected or uninfected groups. COX‐2 protein expression was observed in H. pylori‐infected Mongolian gerbils but not in uninfected ones. Conclusions. Our animal study showed that H. pylori infection induced COX‐2 expression and increased prostaglandin concentration. Administration of NSAIDs decreased the prostaglandin concentration, but did not increase mucosal damage in H. pylori‐induced gastritis. Selective COX‐2 inhibitors, instead of conventional NSIADs, had no beneficial effect on preventing mucosal damage in H. pylori‐induced gastritis.