EP4 receptor regulates collagen type-I, MMP-1, and MMP-3 gene expression in human tendon fibroblasts in response to IL-1 beta treatment

Tendinopathy is accompanied by inflammation, tendon matrix degradation, or both. Inflammatory cytokine IL-1beta, which is a potent inflammatory mediator, is likely present within the tendon. The purpose of this study was to determine the biological impact of IL-1beta on tendon fibroblasts by assessing the expression of cPLA(2), COX-2, PGE(2) and its receptors (EPs), collagen type-I, and MMPs. We also studied the role of the p38 MAPK pathway in IL-1beta-induced catabolic effects. We found that IL-1beta increased the expression levels of cPLA(2) and COX-2, and also increased the secretion of PGE(2). Induction of MMPs, such as MMP-1 and MMP-3 at the mRNA level, was also observed after stimulation with IL-1beta. Furthermore, the presence of IL-1beta significantly decreased the level of collagen type-I mRNA in tendon fibroblasts. These effects were found to be mediated by selective upregulation of EP(4) receptor, which is a member of G-protein-coupled receptor that transduces the PGE(2) signal. Blocking EP(4) receptor by a specific chemical inhibitor abolished IL-1beta-induced catabolic effects. These results suggest that IL-1beta-induced catabolic action on tendon fibroblasts occurs via the upregulation of two key inflammatory mediators, cPLA(2) and COX-2, which are responsible for the synthesis of PGE(2). IL-1beta further stimulates the expression of EP(4) receptor, suggesting positive feedback regulation which may lead to accelerated catabolic processes in tendon fibroblasts. Studies using pathway-specific chemical inhibitors suggest that the p38 MAPK pathway is the key signaling cascade transducing IL-1beta-mediated catabolic effects. Collectively, our findings suggest that the EP(4) receptor mediates the IL-1beta-induced catabolic metabolism via the p38 MAPK pathway in human tendon fibroblasts and may play a major role in the tendon's degenerative changes often seen in the later stages of tendinopathy.     

Mechanisms of prostaglandin E2-induced interleukin-6 release in astrocytes: possible involvement of EP4-like receptors, p38 mitogen-activated protein kinase and protein kinase C.

The expression of cyclooxygenase-2 (COX-2) and the synthesis of prostaglandin E2 (PGE2) as well as of cytokines such as interleukin-6 (IL-6) have all been suggested to propagate neuropathology in different brain disorders such as HIV-dementia, prion diseases, stroke and Alzheimer's disease. In this report, we show that PGE2-stimulated IL-6 release in U373 MG human astroglioma cells and primary rat astrocytes. PGE2-induced intracellular cAMP formation was mediated via prostaglandin E receptor 2 (EP2), but inhibition of cAMP formation and protein kinase A or blockade of EP1/EP2 receptors did not affect PGE2-induced IL-6 synthesis. This indicates that the cAMP pathway is not part of PGE2-induced signal transduction cascade leading to IL-6 release. The EP3/EP1-receptor agonist sulprostone failed to induce IL-6 release, suggesting an involvement of EP4-like receptors. PGE2-activated p38 mitogen-activated kinase (p38 MAPK) and protein kinase C (PKC). PGE2-induced IL-6 synthesis was inhibited by specific inhibitors of p38 MAPK (SB202190) and PKC (GF203190X). Although, up to now, EP receptors have only rarely been linked to p38 MAPK or PKC activation, these results suggest that PGE2 induces IL-6 via an EP4-like receptor by the activation of PKC and p38 MAPK via an EP4-like receptor independently of cAMP.     

Prostaglandin E2 Represses Interleukin 1 Beta-Induced Inflammatory Mediator Output from Pregnant Human Myometrial Cells Through the EP2 and EP4 Receptors

Inflammatory mediators, including prostaglandins, cytokines, and chemokines, are strongly implicated in the mechanism of human labor, though their precise roles remain unknown. Here we demonstrate that interleukin 1 beta (IL-1beta) significantly increased the expression and release of interleukin-8 (CXCL8), monocyte chemotactic protein-1 (CCL2), and granulocyte macrophage colony-stimulating factor (CSF2) by primary human myometrial cells. However, this effect was repressed by prostaglandin E(2) (PGE(2)). As PGE(2) can activate four distinct PGE(2) receptors (EP(1), EP(2), EP(3), and EP(4)) to elicit various responses, we sought to define the EP receptor(s) responsible for this repression. Using selective EP receptor agonists and a selective EP(4) antagonist, we show that PGE(2) mediates the repression of IL-1beta-induced release of CXCL8, CCL2, and CSF2 via activation of the EP(2) and EP(4) receptors. The use of siRNA gene-specific knockdown further confirmed a role for both receptors. Real-time RT-PCR demonstrated that EP(2) was the most highly expressed of all four EP receptors at the mRNA level in human myometrial cells, and immunocytochemistry showed that EP(2) protein is abundantly present throughout the cells. Interestingly, PGE(2) does not appear to reduce mRNA expression of CXCL8, CCL2, and CSF2. Our results demonstrate that PGE(2) can elicit anti-inflammatory responses via activation of the EP(2) and EP(4) receptors in lower segment term pregnant human myometrial cells. Further elucidation of the EP receptor-mediated signaling pathways in the pregnant human uterus may be beneficial for optimizing the maintenance of pregnancy, induction of labor or indeed treatment of preterm labor.     

IL-1beta regulation of BDNF expression in rat cultured hypothalamic neurons depends on the presence of glial cells

In the present study, we have shown that IL-1beta increased BDNF mRNA expression in hypothalamic neuron-enriched cultures whereas it reduced this expression in mixed cultures, i.e. containing astrocytes and neurons. Because functional relationships between stress and immunity signals are well documented we investigated the possible interaction between BDNF and IL-1beta in hypothalamic neurons. Notably, we investigated whether IL-1beta affected BDNF expression in vitro either on hypothalamic mixed cultures or on neuron-enriched cultures. We found that the response to IL-1beta was stimulatory when directly examined in neurons but was inhibitory when astrocytes were present in the cultures. Since it has been documented that astrocytes release PGE2 in response to IL-1beta, we examined the effect of indomethacin (a PGE2 synthesis inhibitor) on mixed or neuron-enriched cultures treated with IL-1beta. Indomethacin blocked both stimulatory and inhibitory IL-1beta effects on BDNF mRNA expression whereas picrotoxin (a GABA(A) blocker) or MK-801 (a NMDA receptor blocker) had no effect on BDNF mRNA levels. About 3 and 6h treatments of cells with exogenous PGE2 reproduced the effects of IL-1beta on neuron-enriched or on mixed cultures suggesting that PGE2 was involved in BDNF mRNA regulation. Analysis of PGE2 receptors mRNA expression revealed that the PGE2 receptor pattern was changed when neuron-enriched cultures were treated with conditioned medium produced by astrocytes treated with IL-1beta. Thus, EP3 mRNA levels were increased while EP1 and EP4 messengers were unchanged. This increased expression of the inhibitory prostaglandin receptor under astrocyte influence can explain the inhibition of BDNF mRNA levels observed in mixed cultures following IL-1beta or PGE2 treatment. Finally, we demonstrated by immunocytochemistry that EP3 receptors had a neuronal localization in the hypothalamic cultures. Taken together, these data contribute to underline an emerging physiological concept postulating that a same molecule may have opposite effects as a function of the cellular context.     

Expression of interleukin-1 receptors and their role in interleukin-1 actions in murine microglial cells

Interleukin (IL)-1 is an important mediator of acute brain injury and inflammation, and has been implicated in chronic neurodegeneration. The main source of IL-1 in the CNS is microglial cells, which have also been suggested as targets for its action. However, no data exist demonstrating expression of IL-1 receptors [IL-1 type-I receptor (IL-1RI), IL-1 type-II receptor (IL-1RII) and IL-1 receptor accessory protein (IL-1RAcP)] on microglia. In the present study we investigated whether microglia express IL-1 receptors and whether they present target or modulatory properties for IL-1 actions. RT-PCR analysis demonstrated lower expression of IL-1RI and higher expression of IL-1RII mRNAs in mouse microglial cultures compared with mixed glial or pure astrocyte cultures. Bacterial lipopolysaccharide (LPS) caused increased expression of IL-1RI, IL-1RII and IL-1RAcP mRNAs, induced the release of IL-1beta, IL-6 and prostaglandin-E2 (PGE2), and activated nuclear factor kappaB (NF-kappaB) and the mitogen-activated protein kinases (MAPKs) p38, and extracellular signal-regulated protein kinase (ERK1/2), but not c-Jun N-terminal kinase (JNK) in microglial cultures. In comparison, IL-1beta induced the release of PGE2, IL-6 and activated NF-kappaB, p38, JNK and ERK1/2 in mixed glial cultures, but failed to induce any of these responses in microglial cell cultures. IL-1beta also failed to affect LPS-primed microglial cells. Interestingly, a neutralizing antibody to IL-1RII significantly increased the concentration of IL-1beta in the medium of LPS-treated microglia and exacerbated the IL-1beta-induced IL-6 release in mixed glia, providing the first evidence that microglial IL-1RII regulates IL-1beta actions by binding excess levels of this cytokine during brain inflammation.     

Osteoprotegerin levels increased by interleukin-1beta in human periodontal ligament cells are suppressed through prostaglandin E(2) synthesized de novo

Osteoprotegerin (OPG) is a novel tumor necrosis factor receptor superfamily that inhibits osteoclast differentiation, activity, and survival. Interleukin-1beta (IL-1beta) increases OPG expression. IL-1beta also increases prostaglandin E(2) (PGE(2)) production and stimulates bone resorption. In the present study, we examined the involvement of PGE(2) in IL-1beta-induced increases in OPG levels in human periodontal ligament cells (HPL cells) in an effort to clarify apparently conflicting IL-1beta actions on bone resorption and understand IL-1beta-induced increases in secretion of OPG and PGE(2) in HPL cells. 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole, a mRNA synthesis inhibitor, partly inhibited the increase in OPG mRNA levels induced by IL-1beta. Cycloheximide, a protein synthesis inhibitor, enhanced the stimulatory effect of IL-1beta. Etodolac, a selective cyclooxygenase-2 inhibitor, suppressed the increase in PGE(2) levels. Furthermore, etodolac reinforced the promotion of OPG expression by IL-1beta at the mRNA and protein levels. PGE(2) added to cultures of HPL cells decreased OPG mRNA levels in a dose- and time- dependent manner. These findings suggest that the increase in OPG levels induced by IL-1beta in HPL cells is suppressed through PGE(2) synthesized de novo.     

Cyclooxygenases and prostaglandin E2 receptors in growth plate chondrocytes in vitro and in situ--prostaglandin E2 dependent proliferation of growth plate chondrocytes

Prostaglandin E2 (PGE2) plays an important role in bone development and metabolism. To interfere therapeutically in the PGE2 pathway, however, knowledge about the involved enzymes (cyclooxygenases) and receptors (PGE2 receptors) is essential. We therefore examined the production of PGE2 in cultured growth plate chondrocytes in vitro and the effects of exogenously added PGE2 on cell proliferation. Furthermore, we analysed the expression and spatial distribution of cyclooxygenase (COX)-1 and COX-2 and PGE2 receptor types EP1, EP2, EP3 and EP4 in the growth plate in situ and in vitro. PGE2 synthesis was determined by mass spectrometry, cell proliferation by DNA [3H]-thymidine incorporation, mRNA expression of cyclooxygenases and EP receptors by RT-PCR on cultured cells and in homogenized growth plates. To determine cellular expression, frozen sections of rat tibial growth plate and primary chondrocyte cultures were stained using immunohistochemistry with polyclonal antibodies directed towards COX-1, COX-2, EP1, EP2, EP3, and EP4. Cultured growth plate chondrocytes transiently secreted PGE2 into the culture medium. Although both enzymes were expressed in chondrocytes in vitro and in vivo, it appears that mainly COX-2 contributed to PGE2-dependent proliferation. Exogenously added PGE2 stimulated DNA synthesis in a dose-dependent fashion and gave a bell-shaped curve with a maximum at 10-8 M. The EP1/EP3 specific agonist sulprostone and the EP1-selective agonist ONO-D1-004 increased DNA synthesis. The effect of PGE2 was suppressed by ONO-8711. The expression of EP1, EP2, EP3, and EP4 receptors in situ and in vitro was observed; EP2 was homogenously expressed in all zones of the growth plate in situ, whereas EP1 expression was inhomogenous, with spared cells in the reserve zone. In cultured cells these four receptors were expressed in a subset of cells only. The most intense staining for the EP1 receptor was found in polygonal cells surrounded by matrix. Expression of receptor protein for EP3 and EP4 was observed also in rat growth plates. In cultured chrondrocytes, however, only weak expression of EP3 and EP4 receptor was detected. We suggest that in growth plate chondrocytes, COX-2 is responsible for PGE2 release, which stimulates cell proliferation via the EP1 receptor.     

Microglial expression of prostaglandin EP3 receptor in excitotoxic lesions in the rat striatum

Prostaglandins (PG) are produced by the enzymatic activity of cyclooxygenase (COX). PGs and COX have been implicated in the pathophysiology of excitotoxicity and neurodegeneration in the central nervous system (CNS). The PGE2 receptor EP3 is the most abundantly expressed PGE2 receptor subtype in the brain. So far, in the innate rat brain EP3 receptors have been found exclusively in neurons. The aim of this study was to investigate whether EP3 expression in the brain changes under neurodegenerative circumstances such as an acute excitotoxic lesion. Intrastriatal injection of quinolinic acid (QUIN) resulted in a loss of EP3-positive striatal neurons, while simultaneously small glial-shaped EP3-positive cells appeared. Five days after lesioning, 63% of the glial-shaped EP3-positive cells could be identified as ED-1 expressing microglial cells. This percentage increased to 82% after 10 days, suggesting that most of the EP3-positive ED-1-negative cells on day 5 may be microglia which did not yet express ED-1. ED-1-positive microglia also expressed COX-1. These experiments show for the first time that activated microglial cells in excitotoxic lesions express in vivo the PGE2 receptor EP3 and the PGE2 synthesizing enzyme COX-1. Activation of EP3 receptor downregulates cAMP formation and may counteract the upregulation of cAMP formation via EP2 receptors, which has been linked to the anti-inflammatory effects of PGs. This change in EP3-receptor expression in microglia might participate in acute or chronic microglial activation in a variety of brain diseases such as ischemia or Alzheimer's disease (AD). Investigation of the expression of different PGE2 receptor subtypes might promote a better understanding of the pathophysiology of these diseases as well as leading to a modulation of microglial activation by a more specific interference with selective EP receptors than can be achieved by inhibiting global PG synthesis by selective or non-selective COX inhibitors.     

Interleukin-1beta induces interleukin-6 production through the production of prostaglandin E(2) in human osteoblasts, MG-63 cells.

This study was conducted to investigate the mechanism of interleukin-1beta (IL-1beta)-induced IL-6 production in human osteoblasts (MG-63 cells). Stimulation with IL-1beta resulted in the production of IL-6 and prostaglandin E(2) (PGE(2)). IL-6 production gradually increased and peaked 96 h after stimulation. IL-6 mRNA was detected between 4 and 72 h after IL-1beta stimulation. The patterns of PGE(2) production and the expression of cyclooxygenase-2 (COX-2) mRNA were biphasic after stimulation. Actinomycin D, cycloheximide, indomethacin, and NS-398 (COX-2 inhibitor) suppressed the production of IL-6 and PGE(2). Anti-PGE(2) antibody markedly reduced the production of IL-6. In addition, stimulation with 17-phenyl-PGE(2), a PGE receptor-1 (EP-1 receptor) agonist, led to the expression of IL-6 mRNA after pretreatment with IL-1beta. These findings indicate that IL-1beta-induced IL-6 production in MG-63 cells involves the following sequence of steps: IL-1beta-induced COX-2 activation, PGE(2) production, and EP-1 receptor signaling prior to IL-6 production.     

Interleukin-1beta-induced cyclooxygenase-2 expression is mediated through activation of p42/44 and p38 MAPKS,and NF-kappaB pathways in canine tracheal smooth muscle cells

Interleukin-beta (IL-1beta) was found to induce inflammatory responses in the airways, which exerted a potent stimulus for PG synthesis. This study was to determine the mechanisms of IL-1beta-enhanced cyclooxygenase (COX)-2 expression associated with PGE(2) synthesis in tracheal smooth muscle cells (TSMCs). IL-1beta markedly increased COX-2 expression and PGE(2) formation in a time- and concentration-dependent manner in TSMCs. Both COX-2 expression and PGE(2) formation in response to IL-1beta were attenuated by a tyrosine kinase inhibitor, genistein, a phosphatidylcholine-phospholipase C inhibitor, D609, a phosphatidylinositol-phospholipase C inhibitor, U73122, protein kinase C inhibitors, GF109203X and staurosporine, removal of Ca(2+) by addition of BAPTA/AM plus EGTA, and phosphatidylinositol 3-kinase (PI3-K) inhibitors, LY294002 and wortmannin. IL-1beta-induced activation of NF-kappaB correlated with the degradation of IkappaB-alpha in TSMCs. IL-1beta-induced NF-kappaB activation, COX-2 expression, and PGE(2) synthesis were inhibited by the dominant negative mutants of NIK and IKK-alpha, but not by IKK-beta. IL-1beta-induced COX-2 expression and PGE(2) synthesis were completely inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 (an inhibitor of p38 inhibitor), but these two inhibitors had no effect on IL-1beta-induced NF-kappaB activation, indicating that activation of p42/44 and p38 MAPK and NF-kappaB signalling pathways were independently required for these responses. These findings suggest that the increased expression of COX-2 correlates with the release of PGE(2) from IL-1beta-challenged TSMCs, at least in part, independently mediated through MAPKs and NF-kappaB signalling pathways in canine TSMCs. IL-1beta-mediated responses were modulated by PLC, Ca(2+), PKC, tyrosine kinase, and PI3-K in these cells.     

PGE2 exerts its effect on the LPS-induced release of TNF-alpha, ET-1, IL-1alpha, IL-6 and IL-10 via the EP2 and EP4 receptor in rat liver macrophages

Lipopolysaccharide (LPS) induces a release of tumor necrosis factor (TNF)-alpha, endothelin (ET)-1, interleukin (IL)-1alpha, IL-6 and IL-10 in rat liver macrophages (Kupffer cells). Prostaglandin (PG)E2 inhibits the release of the fibrogenic mediators TNF-alpha, ET-1 and IL-1alpha, and enhances the release of the anti-fibrogenic mediators IL-6 and IL-10. This effect of PGE2 is mimicked by specific agonists for the PGE2 receptors EP2 and EP4; whereas, agonists for the PGE2 receptors EP1 and EP3 are inactive. Rat liver macrophages express mRNA encoding the PGE2 receptors EP2 and EP4 but not the PGE2 receptors EP1 and EP3. These data suggest that PGE2 exerts its anti-fibrogenic effect through the EP2 and EP4 receptor by inhibiting the release of the fibrogenic mediators TNF-alpha, ET-1 and IL-1alpha, and by enhancing the release of the anti-fibrogenic mediators IL-6 and IL-10 in liver macrophages.     

System A neutral amino acid transporter regulation by interleukin-1beta in human osteoarthritic synovial cells: evidence for involvement of prostaglandin E(2) as a second messenger

We studied the long-terms effects of interleukin-1beta (IL-1beta; 3 to 6 h) on alpha-(methylamino) isobutyric acid (MeAIB), a nonmetabolizable amino acid transported by system A. We found that IL-1beta induced a large decrease in MeAIB uptake by human osteoarthritic synovial cells and a concomitant increase in prostaglandin E(2) (PGE(2)) synthesis. Therefore, we investigated whether PGE(2) acts as a mediator for the long-term action of IL-1beta. We found that exogenous PGE(2) inhibited MeAIB uptake, and that AH6809, a PGE(2) receptor antagonist, inhibited IL-1beta-mediated MeAIB uptake. To identify the enzymes involved in the IL-1beta-mediated synthesis of PGE(2) that inhibits MeAIB uptake, we studied the expression of secreted (s) and cytosolic (c) phospholipase A(2) (PLA(2)). Because both were expressed, we selected a broad spectrum of inhibitors to determine which of the two PLA(2)s was involved. We used AACOCF3, a cPLA(2) inhibitor, and dithiothreitol (DTT) and bromophenacyl bromide (BPB), which are sPLA(2) inhibitors. Our results suggest that the PLA(2) involved in the IL-1beta-mediated synthesis of PGE(2) was sPLA(2). We also showed the expression of cyclooxygenase (COX)-2 and its partial involvement using a potent selective COX-2 inhibitor, L-745337. These findings provide insight into the mechanisms underlying the IL-1beta-mediated regulation of transport system A. The Il-1beta-induced inhibition of MeAIB uptake in human osteoarthritic synovial cells thus seems to be essentially mediated by PGE(2) production via the activation of sPLA(2) and the partial activation of COX-2.     

Differential expression of prostaglandin receptor mRNAs during adipose cell differentiation.

To clarify the molecular basis for the prostaglandin (PG) mediated effects in adipose cells at various stages of their development, expression of mRNAs encoding receptors specific for prostaglandin E2, F2alpha and I2 (i.e. EP, FP, and IP receptors) was investigated in differentiating clonal Ob1771 pre-adipocytes, as well as in mouse primary adipose precursor cells and mature adipocytes. We have further characterized the differential expression of mRNAs encoding three subtypes of the EP receptor, i.e. EP1, EP3, and EP4, and examined the expression of mRNAs encoding the three isoforms (alpha, beta, and gamma) of the EP3 receptor. Altogether the results show that the expression of IP, FP, EP1, and EP4 receptor mRNAs was considerably more pronounced in pre-adipose cells than in adipose cells, mRNAs encoding the alpha, beta, and gamma isoforms of the EP3 receptor were all exclusively expressed in freshly isolated mature adipocytes. These data may indicate that PGI2, PGF2alpha, and PGE2 may interact directly with specific receptors in pre-adipose cells, whose transduction mechanisms are known to affect maturation related changes. In mature adipocytes, however, the equipment of mRNAs encoding the EP3 receptor isoforms is in agreement with the well known effect of PGE2 on adenylate cyclase and lipolysis in mature adipocytes.     

Prostaglandin E(2) mediates inhibition of insulin secretion by interleukin-1beta.

Interleukin-1beta (IL-1beta) and prostaglandin E(2) (PGE(2)), frequently co-participants in inflammatory states, are two well recognized inhibitors of glucose-induced insulin secretion. Previous reports have concluded that the inhibitory effects of these two autacoids on pancreatic beta cell function are not related because indomethacin, a potent prostaglandin synthesis inhibitor, does not prevent IL-1beta effects. However, indomethacin is not a specific cyclooxygenase inhibitor, and its other pharmacologic effects are likely to inhibit insulin secretion independently. Since we recently observed that IL-1beta induces cyclooxygenase-2 (COX-2) gene expression and PGE(2) synthesis in islet beta cells, we have reassessed the possibility that PGE(2) mediates IL-1beta effects on beta function. By using two cell lines (HIT-T15 and betaHC13) as well as Wistar rat isolated pancreatic islets, we examined the ability of two COX-2-specific antagonists, NS-398 and SC-236, to prevent IL-1beta inhibition of insulin secretion. Both drugs prevented IL-1beta from inducing PGE(2) synthesis and inhibiting insulin secretion; adding back exogenous PGE(2) re-established inhibition of insulin secretion in the presence of IL-1beta. We also found that EP3, the PGE(2) receptor subtype whose post-receptor effect is to decrease adenylyl cyclase activity and, thereby, insulin secretion, is the dominant mRNA subtype expressed. We conclude that endogenous PGE(2) mediates the inhibitory effects of exogenous IL-1beta on beta cell function.     

Interferon-gamma and interleukin 4 inhibit interleukin 1beta-induced delayed prostaglandin E(2)generation through suppression of cyclooxygenase-2 expression in human fibroblasts

Interleukin (IL-)1 stimulates prostaglandin E(2)(PGE(2)) generation in fibroblasts, and preferential couplings between particular phospholipase A(2)(PLA(2)) and cyclooxygenase (COX) isozymes are implicated with IL-1-induced delayed PGE(2)generation. The regulatory effects of interferon (IFN)-gamma and IL-4 on IL-1beta-induced COX, PLA(2)isoforms expression and terminal delayed PGE(2)generation were examined in three types of human fibroblasts. These human fibroblasts constitutively expressed cytosolic PLA(2)(cPLA(2)) and COX-1 enzymes, and exhibited delayed PGE(2)generation in response to IL-1beta. IL-1beta also stimulated expression of cPLA(2)and COX-2 only, while constitutive and IL-1beta-induced type IIA and type V secretory PLA(2)s (sPLA(2)s) expression could not be detected. A COX-2 inhibitor and cPLA(2)inhibitor markedly suppressed the IL-1beta-induced delayed PGE(2)generation, while a type IIA sPLA(2)inhibitor failed to affect it. IFN-gamma and IL-4 dramatically inhibited the IL-1beta-induced delayed PGE(2)generation; these cytokines apparently suppressed IL-1beta-stimulated COX-2 expression and only weakly suppressed cPLA(2)expression in response to IL-1beta. These results indicate that IL-1beta-induced delayed PGE(2)generation in these human fibroblasts mainly depends on de novo induction of COX-2 and cPLA(2), irrespective of the constitutive presence of COX-1, and that IFN-gamma and IL-4 inhibit IL-1beta-induced delayed PGE(2)generation by suppressing, predominantly, COX-2 expression.     

Lipoxin A4 inhibits IL-1 beta-induced IL-6, IL-8, and matrix metalloproteinase-3 production in human synovial fibroblasts and enhances synthesis of tissue inhibitors of metalloproteinases

Lipoxins are a novel class of endogenous eicosanoid mediators that potently inhibit inflammatory events by signaling via specific receptors expressed on phagocytic cells. Animal models have shown that lipoxin A4 (LXA4) down-regulates inflammation in vivo. Here we demonstrate, for the first time, the expression of LXA4 receptors, and their up-regulation by IL-1 beta, in normal human synovial fibroblasts (SF). We examined whether exogenous LXA4 abrogated IL-1 beta stimulation of SF in vitro. IL-1 beta induced the synthesis of IL-6, IL-8, and matrix metalloproteinases (MMP)-1 and -3. At nanomolar concentrations, LXA4 inhibited these IL-1 beta responses with reduction of IL-6 and IL-8 synthesis, by 45 +/- 7% and 75 +/- 11%, respectively, and prevented IL-1 beta-induced MMP-3 synthesis without significantly affecting MMP-1 levels. Furthermore, LXA4 induced a 2-fold increase of tissue inhibitor of metalloproteinase (TIMP)-1 and a approximately 3-fold increase of TIMP-2 protein levels. LXA4 inhibitory responses were dose dependent and were abrogated by pretreatment with LXA4 receptor antiserum. LXA4-induced changes of IL-6 and TIMP were accompanied by parallel changes in mRNA levels. These results indicate that LXA4 in activated SF inhibits the synthesis of inflammatory cytokines and MMP and stimulates TIMP production in vitro. These findings suggest that LXA4 may be involved in a negative feedback loop opposing inflammatory cytokine-induced activation of SF.