Cyclooxygenase-1-dependent prostaglandin synthesis modulates tumor necrosis factor-alpha secretion in lipopolysaccharide-challenged murine resident peritoneal macrophages

Comprehensive studies of prostaglandin (PG) synthesis in murine resident peritoneal macrophages (RPM) responding to bacterial lipopolysaccharide (LPS) revealed that the primary PGs produced by RPM were prostacyclin and PGE(2). Detectable increases in net PG formation occurred within the first hour, and maximal PG formation had occurred by 6-10 h after LPS addition. Free arachidonic acid levels rose and peaked at 1-2 h after LPS addition and then returned to baseline. Cyclooxygenase-2 (COX-2) and microsomal PGE synthase levels markedly increased upon exposure of RPM to LPS, with the most rapid increases in protein expression occurring 2-6 h after addition of the stimulus. RPM constitutively expressed high levels of COX-1. Studies using isoform-selective inhibitors and RPM from mice bearing targeted deletions of ptgs-1 and ptgs-2 demonstrated that COX-1 contributes significantly to PG synthesis in RPM, especially during the initial 1-2 h after LPS addition. Selective inhibition of either COX isoform resulted in increased secretion of tumor necrosis factor-alpha (TNF-alpha); however, this effect was much greater with the COX-1 than with the COX-2 inhibitor. These results demonstrate autocrine regulation of TNF-alpha secretion by endogenous PGs synthesized primarily by COX-1 in RPM and suggest that COX-1 may play a significant role in the regulation of the early response to endotoxemia.     

Effects of prostaglandin E2 and lipopolysaccharide on osteoclastogenesis in RAW 264.7 cells

INTRODUCTION: Prostaglandins (PGs) can act on both hematopoietic and osteoblastic lineages to enhance osteoclast formation. METHODS: We examined PGE2 stimulated osteoclastogenesis in RAW 264.7 cells and the role of endogenous PGE2 in lipopolysaccharide (LPS) stimulated osteoclastogenesis. RESULTS: RANKL (1-100 ng/ml) increased formation of osteoclasts, defined as tartrate resistant acid phosphatase multinucleated cells, with peak effects at 30 ng/ml. Addition of PGE2 (0.01-1.0 microM) to RANKL (30 ng/ml) dose dependently increased osteoclast number 30-150%. Use of NS-398 (0.1 microM) or indomethacin (Indo, 1.0 micro M) to block endogenous PG synthesis had little effect on the response to RANKL alone but significantly decreased the response to PGE2. Addition of LPS (100 ng/ml) to RANKL increased osteoclast number 50%, and this response was significantly decreased by NS-398 and Indo. RANKL and PGE2 produced small, additive increases in COX-2 mRNA levels, while LPS produced a larger increase. PG release into the medium was not increased by RANKL and PGE2 but markedly increased by LPS. CONCLUSION: We conclude that RANKL stimulated osteoclastogenesis can be enhanced by PGE2 and LPS though direct effects on the hematopoietic cell lineage and that these effects may be mediated in part by induction of COX-2 and enhanced intracellular PG production.     

RAW264.7 cells lack prostaglandin-dependent autoregulation of tumor necrosis factor-alpha secretion

Studies of the response of RAW264.7 cells (RAW) to lipopolysaccharide (LPS) were carried out to determine why these cells do not demonstrate the prostaglandin (PG)-dependent autocrine regulation of tumor necrosis factor-alpha (TNF-alpha) secretion observed in primary resident peritoneal macrophages (RPMs). The major cyclooxygenase (COX) product of LPS-stimulated RAW was PGD2, with lesser amounts of PGE2. LPS-treated RAW produced PGs more slowly and reached their maximal PG synthetic rate later than did LPS-treated RPMs, as a result of lower constitutive COX-1 expression and a slower rate of COX-2 induction. Cytosolic phospholipase A2 and levels of free arachidonic acid were similar in RAW and RPMs. In contrast to RPMs, LPS-treated RAW produced high quantities of TNF-alpha, which were not altered in the presence of COX inhibitors. This failure of endogenous PGs to suppress TNF-alpha secretion was explained by the absence of the prostaglandin D2 receptor and the low levels of PGE2 produced during the first 2 h of the LPS response. These studies demonstrate that autocrine regulation of TNF-alpha secretion in response to LPS is greatly facilitated by a COX-1-mediated rapid accumulation of PGs as well by a correspondence between the PGs produced and the receptors expressed by the cells.     

Cyclooxygenase-2-mediated prostaglandin E2 production in mesenteric lymph nodes and in cultured macrophages and dendritic cells after infection with Salmonella

Although numerous studies have demonstrated the ability of intestinal epithelial cells to produce PGs after infection with wild-type strains of Salmonella, few studies have focused on Salmonella-induced prostanoids in mucosal lymphoid tissues. This is surprising in view of the profound effects PGs can have on the host response. To begin to address PG production at mucosal sites, mice were orally inoculated with Salmonella, and at varying times postinfection cyclooxygenase-2 (COX-2) mRNA expression and PGE(2) synthesis were investigated. COX-2 mRNA expression was highly inducible in the mesenteric lymph nodes, whereas COX-1 mRNA levels were constitutive. PGE(2) production also increased significantly in the mesenteric lymph nodes following exposure to viable Salmonella, but not after exposure to killed bacteria. This increased PGE(2) response could be blocked by treatment of mice with the selective COX-2 inhibitor, celecoxib. Treatment of mice with celecoxib during salmonellosis resulted in increased viable bacteria in the mesenteric lymph nodes by day 3 postinfection. However, celecoxib treatment prolonged the survival of lethally infected animals. In vitro studies demonstrated Salmonella-induced up-regulation of COX-2 mRNA expression and PGE(2) secretion by both macrophages and dendritic cells, which could also be blocked in the presence of celecoxib. Interestingly, exposure of these cultured APCs to viable Salmonella was a much greater stimulus for induction of PGE(2) synthesis than exposure to Salmonella-derived LPS. The present study demonstrates induction of PGE(2) synthesis in mesenteric lymph nodes, macrophages, and dendritic cells after infection with wild-type salmonella.     

Extracellular matrix-induced cyclooxygenase-2 regulates macrophage proteinase expression

Chronic inflammatory diseases are characterized by the persistent presence of macrophages and other mononuclear cells, tissue destruction, cell proliferation, and the deposition of extracellular matrix (ECM). The tissue degradation is mediated, in part, by enhanced proteinase expression by macrophages. It has been demonstrated recently that macrophage proteinase expression can be stimulated or inhibited by purified ECM components. However, in an intact ECM the biologically active domains of matrix components may be masked either by tertiary conformation or by complex association with other matrix molecules. In an effort to determine whether a complex ECM produced by vascular smooth muscle cells (SMC) regulates macrophage degradative phenotype, we prepared insoluble SMC matrices and examined their ability to regulate proteinase expression by RAW264.7 and thioglycollate-elicited peritoneal macrophages. Here we demonstrate that macrophage engagement of SMC-ECM triggers PKC-dependent activation of MAPK(erk1/2) leading to increased expression of cyclooxygenase (COX)-2 and prostaglandin (PG) E(2) synthesis. The addition of PGE(2) to macrophage cultures stimulates their expression of both urokinase-type plasminogen activator and MMP-9, and the selective COX-2 inhibitor NS-398 blocks ECM-induced proteinase expression. Moreover, ECM-induced PGE(2) and MMP-9 expression by elicited COX-2(-/-) macrophages is markedly reduced when compared with the response of either COX-2(+/-) or COX-2(+/+) macrophages. These data clearly demonstrate that SMC-ECM exerts a regulatory role on the degradative phenotype of macrophages via enhanced urokinase-type plasminogen activator and MMP-9 expression, and identify COX-2 as a targetable component of the signaling pathway leading to increased proteinase expression.     

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.     

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.     

Autoregulation of human monocyte-derived dendritic cell maturation and IL-12 production by cyclooxygenase-2-mediated prostanoid production

PG added to cell culture profoundly affect the in vitro maturation and function of monocyte-derived dendritic cells (MDC). Because unstimulated monocytes express cyclooxygenase (COX)-1, and COX-2 when activated, we examined whether MDC express these enzymes and produce prostanoids that autoregulate maturation and IL-12 production. Immature MDC (I-MDC) and mature MDC express COX-1, but, unlike monocytes, both MDC populations constitutively express COX-2. However, COX-2 regulation in both MDC populations differs from monocytes, as IL-4 does not suppress enzyme expression. COX-2 is functional in MDC as a specific inhibitor, NS-398, significantly reduces PGE(2) production. I-MDC undergoing maturation with soluble CD40 ligand (sCD40L) increase PGE(2) synthesis, but prostanoid synthesis is switched to COX-1. However, with IFN-gamma present, sCD40L-stimulated PG metabolism is redirected to COX-2, and PGE(2) synthesis increases severalfold. Endogenous PG production by MDC does not regulate CD40, CD80, CD86, or HLA DR expression; however, it does promote MDC maturation, as NS-398 significantly reduces CD83 expression in I-MDC matured with sCD40L/IFN-gamma. PG produced through COX-2 also autoregulate IL-12, but the effects are dependent on the MDC maturation state. Blocking COX-2 reduces I-MDC secretion of IL-12p40, whereas it increases IL-12p40 and p70 production by maturing MDC. COX-2-mediated PG production impacts MDC function as maturing these cells in the presence of NS-398 yields MDC that stimulate significantly more IFN-gamma in an allogeneic mixed lymphocyte response than MDC matured without this inhibitor. These studies demonstrate that MDC express both COX isoforms constitutively and produce prostanoids, which autoregulate their maturation and function.     

The effect of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 beta and IL-6 on chorioamnion secretion of prostaglandins (PG)F 2 alpha and E2 in pigs

The aim of the present study was to determine the effect of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) on prostaglandin (PG)F(2 alpha) and PGE(2) secretion as well as cyclooxygenase-2 (COX-2) protein expression in chorioamnion collected on days 25, 30 and 40 of pregnancy in pigs. Fetal membrane slices were incubated for 16 h with TNF-alpha, IL-1 beta, IL-6 (1 or 10 ng/ml of medium) or two combinations of the three cytokines (1 or 10 ng/ml of each cytokine per combination). We demonstrated the stimulatory effect of TNF-alpha, IL-1 beta and/or IL-6 on PGF(2 alpha) and PGE(2) secretion by the porcine fetal membranes. The medium content of these PGs depended on the cytokine type, treatment dose and day of pregnancy. Cytokine stimulation of PGE(2) was more pronounced than that of PGF(2 alpha). In addition, an increase in PGF(2 alpha) and/or PGE(2) secretion was usually associated with an augmentation of COX-2 protein expression. Our results support the notion concerning the possible role of cytokines in modulating production of PGs by fetal membranes during the first trimester of gestation.     

Acetaminophen-sensitive prostaglandin production in rat cerebral endothelial cells

Acetaminophen is a widely used antipyretic and analgesic drug whose mechanism of action has recently been suggested to involve inhibitory effects on prostaglandin synthesis via a newly discovered cyclooxygenase variant (COX-3). Because COX-3 expression is high in cerebral endothelium, we investigated the effect of acetaminophen on the prostaglandin production of cultured rat cerebral endothelial cells (CECs). Acetaminophen dose-dependently inhibited both basal and LPS-induced PGE(2) production in CECs with IC(50) values of 15.5 and 6.9 microM, respectively. Acetaminophen also similarly inhibited the synthesis of 6-keto-PGF(1alpha) and thromboxane B(2). LPS stimulation increased the expression of COX-2 but not COX-1 or COX-3. In addition, the selective COX-2 inhibitor NS398 (1 microM) was equally as effective as acetaminophen in blocking LPS-induced PGE(2) production. Acetaminophen did not influence the expression of the three COX isoforms and the inducible nitric oxide synthase. In LPS-stimulated isolated cerebral microvessels, acetaminophen also significantly inhibited PGE(2) production. Our results show that prostaglandin production in CECs during basal and stimulated conditions is very sensitive to inhibition by acetaminophen and suggest that acetaminophen acts against COX-2 and not COX-1 or COX-3. Furthermore, our findings support a critical role for cerebral endothelium in the therapeutic actions of acetaminophen in the central nervous system.     

Upregulation of iNOS by COX-2 in muscularis resident macrophage of rat intestine stimulated with LPS

We investigated the effect of lipopolysaccharide (LPS) on the induction of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in muscularis resident macrophages of rat intestine in situ. When the tissue was incubated with LPS for 4 h, mRNA levels of iNOS and COX-2 were increased. The majority of iNOS and COX-2 proteins appeared to be localized to the dense network of muscularis resident macrophages immunoreactive to ED2. LPS treatment also increased the production of nitric oxide (NO), PGE(2), and PGI(2). The increased expression of iNOS mRNA by LPS was suppressed by indomethacin but not by N(G)-monomethyl-L-arginine (L-NMMA). The increased expression of COX-2 mRNA by LPS was affected neither by indomethacin nor by L-NMMA. Muscle contractility stimulated by 3 microM carbachol was significantly inhibited in the LPS-treated muscle, which was restored by treatment of the tissue with L-NMMA, aminoguanidine, indomethacin, or NS-398. Together, these findings show that LPS increases iNOS expression and stimulates NO production in muscularis resident macrophages to inhibit smooth muscle contraction. LPS-induced iNOS gene expression may be mediated by autocrine regulation of PGs through the induction of COX-2 gene expression.     

Glycerylprostaglandin synthesis by resident peritoneal macrophages in response to a zymosan stimulus

Cyclooxygenase (COX)-2 oxygenates arachidonic acid (AA) and 2-arachidonylglycerol (2-AG) to endoperoxides, which are subsequently transformed to prostaglandins (PGs) and glycerylprostaglandins (PG-Gs). PG-G formation has not been demonstrated in intact cells treated with a physiological agonist. Resident peritoneal macrophages, which express COX-1, were pretreated with lipopolysaccharide to induce COX-2. Addition of zymosan caused release of 2-AG and production of the glyceryl esters of PGE2 and PGI2 over 60 min. The total quantity of PG-Gs (16 +/- 6 pmol/10(7) cells) was much lower than that of the corresponding PGs produced from AA (21,000 +/- 7,000 pmol/10(7) cells). The differences in PG-G and PG production were partially explained by differences in the amounts of 2-AG and AA released in response to zymosan. The selective COX-2 inhibitor, SC236, reduced PG-G and PG production by 49 and 17%, respectively, indicating a significant role for COX-1 in PG-G and especially PG synthesis. Time course studies indicated that COX-2-dependent oxygenation rapidly declined 20 min after zymosan addition. When exogenous 2-AG was added to macrophages, a substantial portion was hydrolyzed to AA and converted to PGs; 1 microm 2-AG yielded 820 +/- 200 pmol of PGs/10(7) cells and 78 +/- 41 pmol of PG-Gs/10(7) cells. SC236 reduced PG-G and PG production from exogenous 2-AG by 88 and 76%, respectively, indicating a more significant role for COX-2 in the utilization of exogenous substrate. In conclusion, lipopolysaccharide-pretreated macrophages produce PG-Gs from endogenous 2-AG during zymosan phagocytosis, but PG-G formation is limited by substrate hydrolysis and inactivation of COX-2.     

EP2 and EP4 receptors on muscularis resident macrophages mediate LPS-induced intestinal dysmotility via iNOS upregulation through cAMP/ERK signals

Intestinal resident macrophages play an important role in gastrointestinal dysmotility by producing prostaglandins (PGs) and nitric oxide (NO) in inflammatory conditions. The causal correlation between PGs and NO in gastrointestinal inflammation has not been elucidated. In this study, we examined the possible role of PGE(2) in the LPS-inducible inducible NO synthase (iNOS) gene expression in murine distal ileal tissue and macrophages. Treatment of ileal tissue with LPS increased the iNOS and cyclooxygenase (COX)-2 gene expression, which lead to intestinal dysmotility. However, LPS did not induce the expression of iNOS and COX-2 in tissue from macrophage colony-stimulating factor-deficient op/op mice, indicating that these genes are expressed in intestinal resident macrophages. iNOS and COX-2 protein were also expressed in dextran-phagocytized macrophages in the muscle layer. CAY10404, a COX-2 inhibitor, diminished LPS-dependent iNOS gene upregulation in wild-type mouse ileal tissue and also in RAW264.7 macrophages, indicating that PGs upregulate iNOS gene expression. EP(2) and EP(4) agonists upregulated iNOS gene expression in ileal tissue and isolated resident macrophages. iNOS mRNA induction mediated by LPS was decreased in the ileum isolated from EP(2) or EP(4) knockout mice. In addition, LPS failed to decrease the motility of EP(2) and EP(4) knockout mice ileum. EP(2)- or EP(4)-mediated iNOS expression was attenuated by KT-5720, a PKA inhibitor and PD-98059, an ERK inhibitor. Forskolin or dibutyryl-cAMP mimics upregulation of iNOS gene expression in macrophages. In conclusion, COX-2-derived PGE(2) induces iNOS expression through cAMP/ERK pathways by activating EP(2) and EP(4) receptors in muscularis macrophages. NO produced in muscularis macrophages induces dysmotility during gastrointestinal inflammation.     

Cross-regulation of iNOS and COX-2 by its products in murine macrophages under stress conditions.

Exposure of macrophages to heat shock induces rapid synthesis of heat shock proteins (HSPs) which are important for cell homeostasis. Prostaglandins (PGs) and nitric oxide (NO) are important cell regulatory molecules. We have therefore investigated the interactions between these molecules in the LPS-induced expression of iNOS and COX-2 and in the mitochondrial activity of macrophages. Cultures of the murine macrophage cell line, J774, were exposed to heat shock (43 degrees C, 30 min) and stimulated with LPS (1 microg/ml), concomitantly or after 8h of cell recovery. NO production was measured by Griess reaction; PGE(2) by ELISA; HSP70, iNOS and COX-2 by immunobloting; mitochondrial activity by MTT assay. Heat shock induced HSP70, but not iNOS or COX-2 whereas LPS induced iNOS and COX-2 but not HSP70. When heat shock and LPS were given concomitantly, iNOS but not COX-2 expression was reduced. When a period of 8h was given between heat shock and LPS stimulation, iNOS, COX-2, PGE(2) and NO levels were significantly increased. Under these conditions, the expression of COX-2 was reduced by L-NAME (NO-synthesis inhibitor) and of iNOS by nimesulide (PGs-synthesis inhibitor). Such cross-regulation was not observed in cells at 37 degrees C. These treatments significantly reduced MTT levels in cells at 37 degrees C but not in cells submitted to heat shock. These results suggest that HSPs and cross-regulation of iNOS and COX-2 by their products might be of relevance in the control of cell homeostasis during stress conditions.     

Il-10 is a central regulator of cyclooxygenase-2 expression and prostaglandin production

IL-10 is a potent anti-inflammatory and immune regulatory cytokine. IL-10(-/-) mice produce exaggerated amounts of inflammatory cytokines when stimulated with LPS, indicating that endogenous IL-10 is a central regulator of inflammatory cytokine production in vivo. PGs are lipid mediators that are also produced in large amounts during the inflammatory response. To study the role of IL-10 in the regulation of PG production during the acute inflammatory response, we evaluated LPS-induced cyclooxygenase (COX) expression and PG production in wild-type (wt) and IL-10(-/-) mice. LPS-induced PGE(2) production from IL-10(-/-) spleen cells was 5.6-fold greater than that from wt spleen cells. LPS stimulation resulted in the induction of COX-2 mRNA and protein in both wt and IL-10(-/-) spleen cells; however, the magnitude of increase in COX-2 mRNA was 5.5-fold greater in IL-10(-/-) mice as compared with wt mice. COX-1 protein levels were not affected by LPS stimulation in either wt or IL-10(-/-) mice. Neutralization of IFN-gamma, TNF-alpha, or IL-12 markedly decreased the induction of COX-2 in IL-10(-/-) spleen cells, suggesting that increased inflammatory cytokine production mediates much of the COX-2 induction in IL-10(-/-) mice. Treatment of IL-10(-/-) mice with low doses of LPS resulted in a marked induction of COX-2 mRNA in the spleen, whereas wt mice had minimal expression of COX-2 mRNA. These findings indicate that, in addition to IL-10's central role in the regulation of inflammatory cytokines, endogenous IL-10 is an important regulator of PG production in the response to LPS.     

Targeting prostaglandin E2 receptors as an alternative strategy to block cyclooxygenase-2-dependent extracellular matrix-induced matrix metalloproteinase-9 expression by macrophages

COX-2-dependent prostaglandin (PG) E2 synthesis regulates macrophage MMP expression, which is thought to destabilize atherosclerotic plaques. However, the administration of selective COX-2 inhibitors paradoxically increases the frequency of adverse cardiovascular events potentially through the loss of anti-inflammatory prostanoids and/or disturbance in the balance of pro- and anti-thrombotic prostanoids. To avoid these collateral effects of COX-2 inhibition, a strategy to identify and block specific prostanoid-receptor interactions may be required. We previously reported that macrophage engagement of vascular extracellular matrix (ECM) triggers proteinase expression through a MAPKerk1/2-dependent increase in COX-2 expression and PGE2 synthesis. Here we demonstrate that elicited macrophages express the PGE2 receptors EP1-4. When plated on ECM, their expression of EP2 and EP4, receptors linked to PGE2-induced activation of adenylyl cyclase, is strongly stimulated. Forskolin and dibutryl cyclic-AMP stimulate macrophage matrix metalloproteinase (MMP)-9 expression in a dose-dependent manner. However, an EP2 agonist (butaprost) has no effect on MMP-9 expression, and macrophages from EP2 null mice exhibited enhanced COX-2 and MMP-9 expression when plated on ECM. In contrast, the EP4 agonist (PGE1-OH) stimulated macrophage MMP-9 expression, which was inhibited by the EP4 antagonist ONO-AE3-208. When compared with COX-2 silencing by small interfering RNA or inhibition by celecoxib, the EP4 antagonist was as effective in inhibiting ECM-induced proteinase expression. In addition, ECM-induced MMP-9 expression was blocked in macrophages in which EP4 was silenced by small interfering RNA. Thus, COX-2-dependent ECM-induced proteinase expression is effectively blocked by selective inhibition of EP4, a member of the PGE2 family of receptors.     

Type IIA secretory phospholipase A2 up-regulates cyclooxygenase-2 and amplifies cytokine-mediated prostaglandin production in human rheumatoid synoviocytes

Human type IIA secretory phospholipase A2 (sPLA2-IIA) is induced in association with several immune-mediated inflammatory conditions. We have evaluated the effect of sPLA2-IIA on PG production in primary synovial fibroblasts from patients with rheumatoid arthritis (RA). At concentrations found in the synovial fluid of RA patients, exogenously added sPLA2-IIA dose-dependently amplified TNF-alpha-stimulated PGE2 production by cultured synovial fibroblasts. Enhancement of TNF-alpha-stimulated PGE2 production in synovial cells was accompanied by increased expression of cyclooxygenase (COX)-2 and cytosolic phospholipase A2 (cPLA2)-alpha. Blockade of COX-2 enzyme activity with the selective inhibitor NS-398 prevented both TNF-alpha-stimulated and sPLA2-IIA-amplified PGE2 production without affecting COX-2 protein induction. However, both sPLA2-IIA-amplified PGE2 production and enhanced COX-2 expression were blocked by the sPLA2 inhibitor LY311727. Colocalization studies using triple-labeling immunofluorescence microscopy showed that sPLA2-IIA and cPLA2-alpha are coexpressed with COX-2 in discrete populations of CD14-positive synovial macrophages and synovial tissue fibroblasts from RA patients. Based on these findings, we propose a model whereby the enhanced expression of sPLA2-IIA by RA synovial cells up-regulates TNF-alpha-mediated PG production via superinduction of COX-2. Therefore, sPLA2-IIA may be a critical modulator of cytokine-mediated synovial inflammation in RA.     

Endogenous prostaglandins E2 and F 2alpha serve as an anti-apoptotic factor against apoptosis induced by tumor necrosis factor-alpha in mouse 3T3-L1 preadipocytes

Adipocytes can function as endocrine cells secreting a variety of adipocytokines including tumor necrosis factor (TNF)-alpha. Treatment of cultured mouse 3T3-L1 preadipocytes with TNF-alpha induced apoptosis, as was evident from increases in nuclear condensation and caspase-3 activity, but differentiated adipocytes during the maturation phase showed resistance to apoptosis by TNF-alpha. Antioxidants effectively reduced TNF-alpha-induced apoptosis in preadipocytes, indicating the involvement of reactive oxygen species. Exposure of preadipocytes to calcium ionophore A23187 reduced TNF-alpha-induced apoptosis, which was accompanied by increased production of prostaglandins (PGs) E2 and PGF 2alpha. TNF-alpha preferentially promoted gene expression of cyclooxygenase (COX)-2 without affecting that of COX-1. Consistently, NS-398, a COX-2 inhibitor, stimulated TNF-alpha-induced apoptosis, which was reversed by exogenous PGE2 and PGF 2alpha. These results indicate that endogenous PGE2 and PGF 2alpha synthesized by preadipocytes through the induction of COX-2 can serve as anti-apoptotic factors against apoptosis by TNF-alpha.     

Expression of enzymes of cyclooxygenase pathway and secretion of prostaglandin E2 and F2alpha by porcine myometrium during luteolysis and early pregnancy

Past studies of uterine prostaglandin (PGs) and pig reproduction have focused on endometrial rather than myometrial PGs. This study documents the synthesis and secretion of myometrial prostaglandins (PGs) in pigs and the involvement of oxytocin (OT) in these processes. Cyclooxygenase-2 (COX-2) expression was similar in myometrial explants from cyclic and pregnant pigs (days 14-16) and OT (10(-7) M) in vitro significantly increased COX-2 protein regardless of reproductive state. Basal expression of prostaglandin E2 synthase (PGES) was higher during pregnancy than during luteolysis. Conversely, prostaglandin F synthase (PGFS) was highest during luteolysis and lower in myometrium from gravid animals. OT had no influence on the expression of PGES and PGFS. In another tissue culture experiment, myometrial slices produced more PGE2 than PGF2alpha regardless of reproductive state of the female. OT stimulated PGE2 production in myometrium harvested during luteolysis and increased PGF2alpha production in all tissues examined. Progesterone (P4; 10(-5) M) blocked stimulatory effect of OT on myometrial PG release. Myometrial OTr mRNA was higher (P=0.03) during luteolysis than during pregnancy. In conclusion: (1) oxytocin increases myometrial COX-2 expression, but does not influence the expression of terminal enzymes of PGs synthesis (PGES and PGFS); (2) porcine myometrium preferentially produces PGs during early pregnancy and secretes more PGE2 than PGF2alpha; (3) myometrial OT and OTr support secretion of PGs from myometrium during luteolysis.