Leishmania donovani-induced macrophages cyclooxygenase-2 and prostaglandin E2 synthesis

Prostaglandin E2 (PGE2) secretion during Leishmania infection has been reported. However, the signalling mechanisms mediating this response are not well understood. Since cyclooxygenase-2 (COX-2) and cytosolic phospholipase A2 (cPLA2) are involved in PGE2 synthesis in response to various stimuli, the implication of these enzymes was evaluated in Leishmania-infected phorbol myristate acetate-differentiated U937 human monocytic cell line. Time-course experiments showed that PGE2 synthesis increased significantly in parallel with COX-2 expression when cells were incubated in the presence of Leishmania donovani promastigotes or lipopolysaccharides (LPS). Increase in cPLA2 mRNA expression was only detected when cells were stimulated with LPS. Indomethacin, genistein, and H7, which are antagonists of COX-2, protein tyrosine kinase (PTK) and protein kinase C (PKC), respectively, inhibited PGE2 production induced by L. donovani and LPS. However, only H7 inhibited COX-2 mRNA synthesis, and there was a significant correlation between PGE2 inhibition and reduced COX-2 expression. Collectively, our results indicate that infection of U937 by L. donovani leads to the generation of PGE2 in part through a PKC-dependent signalling pathway involving COX-2 expression. They further reveal that PTK-dependent events are necessary for Leishmania-induced PGE2 generation, but not for COX-2 expression. A better understanding of the mechanisms by which Leishmania can induce PGE2 production could provide insight into the pathophysiology of leishmaniasis and may help to improve therapeutic approaches.     

The inhibition of spontaneous and tumor necrosis factor-alpha induced neutrophil apoptosis by crystals of calcium pyrophosphate dihydrate and monosodium urate monohydrate

OBJECTIVE: Spontaneous neutrophil apoptosis may be inhibited by various proinflammatory stimuli. which may result in prolonged lifetimes and responses of these phagocytic cells with the potential for extended inflammation. We investigated the effect of short term incubation of opsonized crystals of monosodium urate monohydrate (MSUM) or calcium pyrophosphate dihydrate (CPPD) on both spontaneous and tumor necrosis factor-alpha (TNF-alpha) induced neutrophil apoptosis. METHODS: Peripheral neutrophils were incubated with plasma opsonized crystals of CPPD or MSUM in the presence or absence of TNF-alpha for 4 h at 37 degrees C. Apoptosis was determined using 3 separate assays: (1) an agarose DNA fragmentation assay, (2) a cytoplasmic histone associated DNA fragmentation assay, and (3) a caspase 3 fluorometric assay. RESULTS: All 3 assays showed similar results. Both MSUM and CPPD crystals inhibited spontaneous apoptosis in neutrophils. TNF-alpha induced high levels of apoptosis in neutrophils. However, co-incubation of the cells with TNF-alpha and crystals resulted in the inhibition of apoptosis to levels below those of control cells. Pretreatment of neutrophils with the protein synthesis inhibitor cycloheximide prevented the inhibition of apoptosis by crystals. CONCLUSION: These data support the concept of crystal induced inhibition of neutrophil apoptosis as part of the pathophysiology of the diseases collectively known as crystal induced arthritis.     

Upregulation of synoviocyte COX-2 through interactions with T lymphocytes: role of interleukin 17 and tumor necrosis factor-alpha

OBJECTIVE: T lymphocytes infiltrating rheumatoid synovium may alter the function of resident synoviocytes. We investigated the influence on synoviocyte cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production exerted by soluble factors released by T cells, with particular reference to interleukin 17 (IL-17). METHODS: Human peripheral blood T cells were stimulated with antibodies directed against CD3 and CD28. Harvested T cell supernatants were applied to cultured human fibroblast-like synoviocytes in culture. The effects of IL-17 alone and in combination with tumor necrosis factor-a (TNF-a) were examined using recombinant cytokines and neutralizing antibodies. Synoviocyte COX-2 expression and PGE2 production were examined. RESULTS: Supernatants from stimulated T cells upregulated COX-2 expression and increased PGE2 production by synoviocytes. The T cell supernatants were found to contain IL-17 and TNF-a. Recombinant IL-17 upregulated synoviocyte COX-2 expression and enhanced TNF-a stimulated synoviocyte COX-2 expression. The upregulation of synoviocyte COX-2 expression by supernatants from stimulated T cells was partially inhibited by addition of neutralizing antibodies against IL-17 or TNF-a or by treatment of T cells with cyclosporin A prior to stimulation. CONCLUSION: Activated T cells are capable of paracrine upregulation of synoviocyte COX-2 expression and PGE2 production through release of soluble mediators. T cell derived IL-17, especially in combination with TNF-a, may contribute to ongoing inflammation through its effects on COX-2 expression and PGE2 production. These data provide additional evidence for the contribution of T cells in rheumatoid inflammation and highlight the potential of IL-17 as a therapeutic target.     

Coexpression of microsomal prostaglandin E synthase with cyclooxygenase-2 in human rheumatoid synovial cells

OBJECTIVE: Recently, microsomal prostaglandin (PG) E synthase (mPGES) was cloned as a terminal enzyme catalyzing PGH2 to PGE2. We investigated mPGES as well as cyclooxygenase (COX)-2, catalyzing arachidonic acid to PGH2, in synovial cells from patients with rheumatoid arthritis (RA). The effect of dexamethasone on mPGES expression was also studied. METHODS: Synovial cells were treated with interleukin 1beta (IL-1beta) and dexamethasone under various conditions, and expression of mPGES mRNA and protein was analyzed by Northern blot and Western blot, respectively. Conversions of arachidonic acid or PGH2 to PGE2 were measured by ELISA. Subcellular localization of mPGES and COX-2 was determined by immunofluorescent microscopic analysis. RESULTS: mPGES mRNA and protein expression were significantly upregulated by IL-1beta in synovial cells. COX-2 mRNA and protein were also upregulated by IL-1beta, but with a different time course from that of mPGES. Conversion of PGH2 to PGE2 increased by IL-1beta and was correlated with mPGES expression. Increased conversion of arachidonic acid to PGE2 was maintained when mPGES and COX-2 were coexpressed. Subcellular localization of mPGES and COX-2 overlapped in the perinuclear region in IL-1beta stimulated synovial cells. Dexamethasone inhibited mRNA and protein expression for mPGES and increased conversion of arachidonic acid to PGE2, but inhibition of mPGES was weaker compared with that of COX-2 in IL-1beta stimulated cells. CONCLUSION: The results suggest that abundant PGE2 production at inflammation sites such as rheumatoid synovia is caused by the coordinated upregulation of mPGES and COX-2. Thus mPGES might be a potential new target for therapeutic strategies to control PGE2 synthesis specifically in patients with RA and other inflammatory diseases.     

EP2/EP4 signalling inhibits monocyte chemoattractant protein-1 production induced by interleukin 1beta in synovial fibroblasts

BACKGROUND: Besides its proinflammatory properties, prostaglandin E(2) (PGE(2)) acts as a regulator of the expression of inducible genes. Inhibition of PGE(2) synthesis might thus result in a paradoxical deleterious effect on inflammation. OBJECTIVE: To examine the effect of PGE(2) on monocyte chemoattractant protein-1 (MCP-1) expression in cultured synovial fibroblasts (SF) stimulated with interleukin (IL)1beta. METHODS: MCP-1 expression was assessed in SF stimulated with IL1beta in the presence of PGE(2) or different NSAIDs by RT-PCR or northern blot and immunocytochemistry. Expression of cyclo-oxygenase (COX) isoforms was studied by western blot techniques. The role of PGE(2) receptors (EP) in PGE(2) action was assessed employing EP receptor subtype-specific agonists. RESULTS: PGE(2) significantly inhibited IL1beta induced MCP-1 expression in SF in a dose dependent manner. IL1beta increased COX-2 and did not alter COX-1 synthesis in SF. 11-Deoxy-PGE(1), an EP(2)/EP(4) agonist, reproduced PGE(2) action on MCP-1 expression. Butaprost, a selective EP(2) agonist, was less potent than PGE(2). Sulprostone, an EP(1)/EP(3) agonist, had no effect on IL1beta induced MCP-1 expression. Inhibition of endogenous PGE(2) synthesis by NSAIDs further enhanced MCP-1 mRNA expression in IL1beta stimulated SF, an effect prevented by addition of exogenous PGE(2). CONCLUSION: Activation of EP(2)/EP(4) receptors down regulates the expression of MCP-1 in IL1beta stimulated SF, while PGE(2) pharmacological inhibition cuts off this signalling pathway and results in a superinduction of MCP-1 expression. The data suggest that NSAIDs may intercept a natural regulatory circuit controlling the magnitude of inflammation, which questions their continuous administration in inflammatory joint diseases.     

Cyclooxygenase-2 in myocardium stimulation by angiotensin-II in cultured cardiac fibroblasts and role at acute myocardial infarction.

Myocardial infarction is followed by a complex repair process that includes a significant role for inflammatory cells. Cyclooxygenase-2 (COX-2) plays a key role in mediating inflammation. Contribution of COX-2 to inflammatory response following myocardial infarction is less certain. In an effort to evaluate the function of COX-2 and prostaglandin E(2)(PGE(2)) in myocardial infarction, we examined the role of COX-2 after angiotensin II (Ang II) stimulation in cardiac fibroblasts and in rats with experimental myocardial infarction (MI). We combined Western blot analysis and enzyme immunoassay to demonstrate COX-2 expression and PGE(2)release in cardiac fibroblasts. Isolated cardiac fibroblasts were stimulated with Ang II. Unstimulated fibroblasts showed no COX-2 protein expression. Fibroblasts stimulated with Ang II showed a strong time-dependent expression of COX-2 protein. The p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 but not the p42/44 MAPK-inhibitor PD98059 suppressed Ang II-induced COX-2 protein expression. COX-2 expression correlated with a significantly increased PGE(2)release from cardiac fibroblasts. The COX-2 specific inhibitor NS-398 suppressed the Ang II-stimulated PGE(2)production. We then investigated COX-2 expression and inflammatory cell infiltration in our rat model of myocardial infarction. MI was produced by coronary artery ligation in adult female Wistar rats. The period of coronary artery occlusion was 96 h. The selective COX-2 inhibitor rofecoxib (3 mg/kg/d), administered orally, was given one day before MI and continued for four days. Western blotting showed expression of COX-2 protein in the area of necrosis and the infarct border zone. Immunofluorescence analysis showed macrophage infiltration as well as fibroblast proliferation in the infarct border zone of 4-d infarcted tissue and a significantly reduced cell invasion and fibroblast proliferation in infarcted tissue of rats treated with rofecoxib. MI size at day 4 was comparable in untreated and treated rats. In conclusion, we demonstrate that pharmacological interference with prostaglandin synthesis in myocardial infarction is associated with reduced myocardial invasion of inflammatory cells.     

Prostaglandin E2 inhibits transforming growth factor beta 1-mediated induction of collagen alpha 1(I) in hepatic stellate cells

BACKGROUND/AIMS: Cyclooxygenase-2 (COX-2) has been implicated in a number of hepatic stellate cell (HSC) functions but its relationship to transforming growth factor-beta 1 (TGF-beta 1)-mediated fibrogenesis is unknown. We assessed the impact of COX-2 inhibition and PGE(2) on the regulation of TGF-beta 1-stimulated matrix synthesis in an immortalized human HSC line, LX-1 and corroborated these findings in primary stellate cells. METHODS: Expression of COX-2 was assessed by Western blotting and real time quantitative PCR. The effect of NS398, a selective COX-2 inhibitor, and PGE(2) on TGF-beta 1-mediated fibrogenesis was examined by measuring mRNA levels of collagen alpha1(I). PGE(2) receptor expression was analyzed by RT-PCR. RESULTS: Under basal conditions, NS398 suppressed PGE(2) synthesis and induced collagen alpha 1(I) whereas exogenous PGE(2) suppressed expression of collagen alpha1(I). TGF-beta 1 induced COX-2 mRNA, COX-2 protein and PGE(2) biosynthesis. Importantly, TGF-beta 1-mediated induction of collagen alpha 1(I) was markedly suppressed by the addition of exogenous PGE(2). All four major PGE(2) receptors were expressed in LX-1 cells. CONCLUSIONS: These results suggest that COX-2-derived PGE(2) inhibits both basal and TGF-beta 1-mediated induction of collagen synthesis by HSC. Based on these findings, it will be important to determine whether inhibiting COX-derived PGE(2) synthesis alters the progression of liver fibrosis in vivo.     

Characterization of an activation factor released from human neutrophils after stimulation by triclinic monosodium urate crystals

OBJECTIVE: To determine the presence and characterize the activity of a soluble activation factor rapidly released by human neutrophils after stimulation with monosodium urate (MSU) crystals. METHODS: Supernatants from human neutrophils stimulated by MSU crystals for 5 to 60 min were tested for their ability to stimulate a chemotactic response, induce a mobilization of calcium, and increase the tyrosine phosphorylation levels in naive neutrophils. RESULTS: Supernatant from neutrophils stimulated 相似文献   

Cyclooxygenase (COX) 1 and 2 in normal, inflamed, and ulcerated human gastric mucosa

BACKGROUND AND AIMS: Constitutive cyclooxygenase (COX) 1 is believed to mediate prostaglandin dependent gastric protection. However, gastric mucosa contains cells capable of expressing inducible COX-2. We therefore investigated COX-1 and COX-2 expression, localisation, and activity in normal and abnormal human gastric mucosa. METHODS: COX-1 and COX-2 distribution was investigated by light and electron microscopic immunohistochemistry and by western blot analysis, and their contribution to prostaglandin (PG)E(2) synthesis using selective enzyme inhibitors. RESULTS: There was strong parietal cell COX-1 and COX-2 immunoreactivity in all sections and isolated cells, with macrophage and myofibroblast reactivity in some sections. Immunostaining was specifically abolished by antigen absorption. Western blot analysis confirmed COX-1 and 2 expression. COX-1 and COX-2 immunostaining was increased in Helicobacter pylori gastritis, particularly the mid glandular zone and lamina propria inflammatory cells. This was associated with increased ex vivo PGE(2) synthesis (62.4 (13.5) pg/mg v 36.3 (15.5) pg/mg in uninflamed mucosa; p=0. 017) which was significantly inhibited by COX-1 but not COX-2 inhibition. Increased COX-2 immunostaining in macrophages, endothelial cells, and myofibroblasts (with reduced epithelial expression) was seen at the rim of ulcers. CONCLUSION: COX-2, as well as COX-1, is expressed by normal human gastric mucosa and is increased at the rim of ulcers. Although both are increased with H pylori, COX-1 contributes more than COX-2 to gastric PGE(2) production.     

Monocyte cyclooxygenase-2 overactivity: a new marker of subclinical atherosclerosis in asymptomatic subjects with cardiovascular risk factors?

AIMS: Cyclooxygenase-2 (COX-2)-mediated prostaglandin production by activated macrophages is associated with inflammation and atherosclerosis. We investigated the relationship between COX-2-mediated prostaglandin-E2 (PGE2) release, cardiovascular risk factors, and carotid atherosclerosis in apparently healthy subjects. METHODS AND RESULTS: PGE2 release by lipopolysaccharide-stimulated blood monocytes was measured by ELISA in 291 subjects (76.5% men, mean age 58) who underwent global vascular risk assessment and carotid ultrasonography. COX-2 expression (real-time RT-PCR) was analysed in a subgroup of 100 subjects (76% men, mean age 59). Inducible PGE2 production was associated with smoking and diabetes (P<0.05), but not with arterial hypertension, dyslipidaemia, or obesity. Subjects in the highest tertile of PGE2 (>8.1 ng/mL) had significantly higher mean carotid intima-media thickness (IMT) than those in the lowest tertile (P<0.01). No significant differences among tertiles were observed in the levels of inflammatory markers (C-reactive protein, fibrinogen, and von Willebrand factor). The association between PGE2 and carotid IMT remained statistically significant (P=0.012) after adjustment for a number of cardiovascular and inflammatory risk factors. A correlation between COX-2 expression and PGE2 production was observed (P<0.005). CONCLUSIONS: COX-2-mediated PGE2 overproduction by stimulated monocytes might provide a new marker of subclinical atherosclerosis in asymptomatic subjects exposed to cardiovascular risk factors.     

Vascular endothelial growth factor up-regulates cyclooxygenase-2 expression in human endothelial cells

We investigated the effects of vascular endothelial growth factor (VEGF) on cyclooxygenase-2 (COX-2) expression and prostaglandin E(2) (PGE(2)) synthesis in human microvascular endothelial cells (HMEC-1). Treatment of HMEC-1 with VEGF resulted in a dose- and time-dependent up-regulation of COX-2 mRNA and protein levels. This up-regulation was accompanied by a 1.6-fold increase in PGE(2) synthesis. Pretreatment of HMEC-1 with a selective COX-2 inhibitor, NS-398, abolished VEGF-induced PGE(2) synthesis, suggesting specific up-regulation of COX-2 activity by VEGF in HMEC-1. Transient transfection assays using deletion mutants of the human COX-2 promoter fused to the luciferase reporter gene indicated critical requirement of a regulatory region spanning -828/-123 bp for VEGF induction of COX-2 promoter activity in HMEC-1. Site-directed mutation analysis demonstrated that a GATA cis-acting element at -685/-680 bp was essential for VEGF- induced COX-2 promoter activity in HMEC-1. These observations are of particular importance given the recent demonstrations of critical requirement of COX-2 isoenzyme for tumor growth and angiogenesis.     

Role of prostaglandin E2-dependent angiogenic switch in cyclooxygenase 2-induced breast cancer progression

Overexpression of human cyclooxygenase 2 (COX-2) in the mammary glands of transgenic mice induces tissue-specific tumorigenic transformation. However, the molecular mechanisms involved are not yet defined. Here we show that COX-2 expressed in the epithelial cell compartment regulates angiogenesis in the stromal tissues of the mammary gland. Microvessel density increased before visible tumor growth and exponentially during tumor progression. Inhibition of prostanoid synthesis with indomethacin strongly decreased microvessel density and inhibited tumor progression. Up-regulation of angiogenic regulatory genes in COX-2 transgenic mammary tissue was also potently inhibited by indomethacin treatment, suggesting that prostanoids released from COX-2-expressing mammary epithelial cells induce angiogenesis. G protein-coupled receptors for the major product, prostaglandin E(2) (PGE(2)) EP(1-4), are expressed during mammary gland development, and EP(1,2,4) receptors were up-regulated in tumor tissue. PGE(2) stimulated the expression angiogenic regulatory genes in mammary tumor cells isolated from COX-2 transgenic mice. Such cells are tumorigenic in nude mice; however, treatment with Celecoxib, a COX-2-specific inhibitor, reduced tumor growth and microvessel density. These results define COX-2-derived PGE(2) as a potent inducer of angiogenic switch during mammary cancer progression.     

Calcium pyrophosphate and monosodium urate crystal interactions with neutrophils: effect of crystal size and lipoprotein binding to crystals

"Small" (between 75-98% of crystals less than or equal to 10 microns) and "large" (between 81-93% of crystals greater than 10 microns) size fractions of monosodium urate monohydrate (MSUM) and calcium pyrophosphate dihydrate triclinic (CPPD) crystals were incubated with human neutrophils and crystal induced neutrophil cytolysis monitored by measuring the release of lactate dehydrogenase. "Small" size fractions of MSUM and CPPD gave higher percent lysis values than "large" crystals. The binding of high density lipoproteins (HDL) and low density lipoproteins (LDL) to the crystals was quantitated. HDL and LDL bound in significant amounts to both CPPD and MSUM and strongly inhibited CPPD and MSUM induced neutrophil cytolysis. We propose that HDL and LDL bound to MSUM and CPPD may play important roles in the regulation of gouty inflammation.