Inhibitory effect of the coffee diterpene kahweol on carrageenan-induced inflammation in rats

Previous studies reported that kahweol, a coffee-specific diterpene, inhibits cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression in cultured lipopolysaccharide-activated macrophages. The aim of this study was to confirm the anti-inflammatory effects of kahweol by examining its effect on the inflammatory response induced by carrageenan in a rat using an acute air pouch inflammation model. Kahweol significantly reduced the levels of the inflammatory process markers in the air pouch, such as the volume of exudates, the amount of protein and the number of leukocytes and neutrophils. The levels of nitrite, TNF-alpha and prostaglandin E2 (PGE2) were also markedly lower in the air pouch of the kahweol-treated animals than in the controls. Immunoblot analysis showed that kahweol reduced the COX-2 and iNOS expression level in the exudate cells. The histological examination showed that there was a lower inflammatory response in the pouch tissues from the kahweol-treated animals. In addition, kahweol significantly reduced the paw edema induced by carrageenan and also markedly reduced the level of PGE2 production in the inflamed paw. These results suggest that kahweol has significant anti-inflammatory effects in vivo, which might be due to the inhibition of iNOS and COX-2 expression in the inflammatory sites.     

Lipopolysaccharide-induced up-regulation of triggering receptor expressed on myeloid cells-1 expression on macrophages is regulated by endogenous prostaglandin E2

Triggering receptor expressed on myeloid cells-1 (TREM-1) is a recently identified cell surface molecule that is expressed by neutrophils and monocytes. TREM-1 expression is modulated by various ligands for TLRs in vitro and in vivo. However, the influence of PGE(2), a potential mediator of inflammation, on TREM-1 expression has not been elucidated. In this study, we examined the effects of PGE(2) on LPS-induced TREM-1 expression by resident murine peritoneal macrophages (RPM) and human PBMC. PGE(2) significantly induced murine TREM-1 (mTREM-1) expression by RPM. Up-regulation of TREM-1 expression was specific to PGE(2) among arachidonic acid metabolites, while ligands for chemoattractant receptor-homologous molecule expressed on Th2 cells and the thomboxane-like prostanoid receptor failed to induce mTREM-1 expression. PGE(2) also increased expression of the soluble form of TREM-1 by PBMC. LPS-induced TREM-1 expression was regulated by endogenous PGE(2) especially in late phase (>2 h after stimulation), because cyclooxygenase-1 and -2 inhibitors abolished this effect at that points. A synthetic EP4 agonist and 8-Br-cAMP also enhanced mTREM-1 expression by RPM. Furthermore, protein kinase A, PI3K, and p38 MAPK inhibitors prevented PGE(2)-induced mTREM-1 expression by RPM. Activation of TREM-1 expressed on PGE(2)-pretreated PBMC by an agonistic TREM-1 mAb significantly enhanced the production of IL-8 and TNF-alpha. These findings indicate that LPS-induced TREM-1 expression on macrophages is mediated, at least partly, by endogenous PGE(2) followed by EP4 and cAMP, protein kinase A, p38 MAPK, and PI3K-mediated signaling. Regulation of TREM-1 and the soluble form of TREM-1 expression by PGE(2) may modulate the inflammatory response to microbial pathogens.     

Inhibition of nitric oxide synthase inhibitors and lipopolysaccharide induced inducible NOS and cyclooxygenase-2 gene expressions by rutin, quercetin, and quercetin pentaacetate in RAW 264.7 macrophages

Several natural flavonoids have been demonstrated to perform some beneficial biological activities, however, higher-effective concentrations and poor-absorptive efficacy in body of flavonoids blocked their practical applications. In the present study, we provided evidences to demonstrate that flavonoids rutin, quercetin, and its acetylated product quercetin pentaacetate were able to be used with nitric oxide synthase (NOS) inhibitors (N-nitro-L-arginine (NLA) or N-nitro-L-arginine methyl ester (L-NAME)) in treatment of lipopolysaccharide (LPS) induced nitric oxide (NO) and prostaglandin E2 (PGE2) productions, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) gene expressions in a mouse macrophage cell line (RAW 264.7). The results showed that rutin, quercetin, and quercetin pentaacetate-inhibited LPS-induced NO production in a concentration-dependent manner without obvious cytotoxic effect on cells by MTT assay using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide as an indicator. Decrease of NO production by flavonoids was consistent with the inhibition on LPS-induced iNOS gene expression by western blotting. However, these compounds were unable to block iNOS enzyme activity by direct and indirect measurement on iNOS enzyme activity. Quercetin pentaacetate showed the obvious inhibition on LPS-induced PGE2 production and COX-2 gene expression and the inhibition was not result of suppression on COX-2 enzyme activity. Previous study demonstrated that decrease of NO production by L-arginine analogs effectively stimulated LPS-induced iNOS gene expression, and proposed that stimulatory effects on iNOS protein by NOS inhibitors might be harmful in treating sepsis. In this study, NLA or L-NAME treatment stimulated significantly on LPS-induced iNOS (but not COX-2) protein in RAW 264.7 cells which was inhibited by these three compounds. Quercetin pentaacetate, but not quercetin and rutin, showed the strong inhibitory activity on PGE2 production and COX-2 protein expression in NLA/LPS or L-NAME/LPS co-treated RAW 264.7 cells. These results indicated that combinatorial treatment of L-arginine analogs and flavonoid derivates, such as quercetin pentaacetate, effectively inhibited LPS-induced NO and PGE2 productions, at the same time, inhibited enhanced expressions of iNOS and COX-2 genes.     

Inflammation and pain sensitivity: effects of leukotrienes D4, B4 and prostaglandin E1 in the rat paw

Leukotrienes (LT's) and prostaglandins (PG's) have been proposed as mediators of vascular permeability changes in inflammatory reactions. Also, prostaglandins, especially of the E-type, have been shown to enhance pain responses. In the present studies in rats, the effects of LTB4 and LTD4 on edema and pain thresholds were examined in combination with PGE1 and/or brewer's yeast. Subplantar injections of LTD4 or LTB4 induced small increases in paw thickness which were potentiated by the co-administration of PGE1. LTD4 alone had no significant effect on the development of the yeast paw edema. LTB4 was found to reduce significantly the yeast edema and this reduction could be reversed by administration PGE1. A small but significant decrease in pain threshold was caused by PGE1 and this was significantly enhanced in the presence of LTD4. LTB4, like PGE1, was found to cause slight hyperalgesia but no synergy between the two agents was observed. LTD4 was found to have no effect on the initial hypoalgesia or subsequent development of hyperalgesia caused by brewer's yeast. Both LTB4 and PGE1, however, prevented the initial hypoalgesia and significantly reduced the latency for development of yeast induced hyperalgesia. These effects of LTB4 are discussed in terms of possible release of cyclooxygenase products.     

Downregulation of COX-2 and iNOS by amentoflavone and quercetin in A549 human lung adenocarcinoma cell line

Flavonoids are natural polyphenolic compounds ubiquitously present in the plant kingdom. They are reported to exhibit numerous beneficial health effects. In the present study, we demonstrate the potential effects of different flavonoids on cytokines mediated cyclooxygenase-2 and inducible nitric oxide synthase expression and activities in A549 cell line using quercetin, amentoflavone and flavanone. Our data revealed that quercetin, at 50 micro M concentration inhibited PGE(2) biosynthesis by A549 very strongly with little effect on COX-2 mRNA and protein expression. Unlike quercetin, amentoflavone inhibited both PGE(2) biosynthesis and COX-2 mRNA and protein expression strongly. In another set of experiment, quercetin inhibited iNOS protein expression completely without affecting iNOS mRNA expression. In contrast, amentoflavone although exerted no inhibitory effect on iNOS mRNA expression, did inhibit weakly iNOS protein expression. Flavanone had no inhibitory effect on either enzyme at the same concentration. Taken together, our data indicated that amentoflavone and quercetin differentially exerted supression of PGE(2) biosynthesis via downregulation of COX-2/iNOS expression.     

PAF is involved in the Mycoplasma arthritidis superantigen-triggering pathway for iNOS and COX-2 expression in murine peritoneal cells

We investigated the capacity of Mycoplasma arthritidis mitogen (MAM) to induce (a) expression of the inducible enzymes cyclo-oxygenase (COX-2) and nitric oxide synthase (iNOS), (b) production of prostaglandin E2 (PGE2) and nitric oxide (NO), and (c) involvement of platelet-activating factor (PAF) in the MAM-induced activation pathway. Resident peritoneal cells from C3H/HePas mice were incubated with MAM in the presence or absence of a PAF-antagonist (WEB2170) or COX-2 inhibitors (nimesulide or NS398). Enzyme expression was evaluated by immunoblotting, PGE2 by EIA, and NO by Griess reaction. Following MAM-stimulation of peritoneal cells, expression of COX-2 was detected at 3 h (peak levels at 12 h) and of iNOS at 6 h (peak levels at 20 h). PGE2 increased till 20 h, decreasing thereafter, whereas NO increased with time. WEB2170 (5 x 10(-5) M) treatment caused 44% inhibition of NO output and reduced iNOS expression (48% at the peak of expression). Concomitant treatment with WEB2170 and nimesulide (10(-5) M) reversed these inhibitory effects. WEB2170 reduced COX-2 expression (43% at the peak of expression) and prevented the decline in PGE2 levels after 20 h. These results suggest the involvement of PAF in the signaling pathway triggered by MAM that leads to expression of iNOS and COX-2, and show that PAF regulates the production of NO, possibly by controlling levels of PGE2.     

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.     

Acetylbritannilatone suppresses NO and PGE2 synthesis in RAW 264.7 macrophages through the inhibition of iNOS and COX-2 gene expression

In order to elucidate the mechanism of anti-inflammatory effect of 1-o-acetylbritannilatone (ABL) isolated from Inula Britannica-F, we investigated ABL for its ability to inhibit the inflammatory factor production in RAW 264.7 macrophages. The studies showed that ABL not only inhibited LPS/IFN-gamma-mediated nitric oxide (NO) production and inducible nitric synthase (iNOS) expression, but also decreased LPS/IFN-gamma-induced prostaglandin E2 (PGE2) production and cyclo-oxygenase-2 (COX-2) expression in a concentration-dependent manner. EMSA demonstrated that ABL inhibited effectively the association of NF-kappaB, which is necessary for the expression of iNOS and COX-2, with its binding motif in the promoter of target genes. These data suggest that ABL suppress NO and PGE2 synthesis in RAW 264.7 macrophages through the inhibition of iNOS and COX-2 gene expression, respectively. The anti-inflammatory effect of ABL involves blocking the binding of NF-kappaB to the promoter in the target genes and inhibiting the expression of iNOS and COX-2.     

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.     

Role of nitric oxide in D-galactosamine-induced cell death and its protection by PGE1 in cultured hepatocytes.

Prostaglandin E(1) (PGE(1)) reduces cell death in experimental and clinical manifestations of liver dysfunction. Nitric oxide (NO) has been shown to exert a protective or noxious effect in different experimental models of liver injury. The aim of the present study was to investigate the role of NO during PGE(1) protection against D-galactosamine (D-GalN) citotoxicity in cultured hepatocytes. PGE(1) was preadministered to D-GalN-treated hepatocytes. The role of NO in our system was assessed by iNOS inhibition and a NO donor. Different parameters related to apoptosis and necrosis, NO production such as nitrite+nitrate (NO(x)) release, iNOS expression, and NF-kappaB activation in hepatocytes were evaluated. The inhibition of iNOS reduced apoptosis induced by D-GalN in hepatocytes. PGE(1) protection against D-GalN injury was associated with its capacity to reduce iNOS expression and NO production induced by D-GalN. Nevertheless, iNOS inhibition showed that protection by PGE(1) was also mediated by NO. Low concentrations of a NO donor reduced D-GalN injury with a decrease in the extracellular NO(x) concentration. High concentrations of the NO donor enhanced NO(x) concentration and increased cell death by D-GalN. The present study suggests that low NO production induced by PGE(1) preadministration reduces D-GalN-induced cell death through its capacity to reduce iNOS expression and NO production caused by the hepatotoxin.     

Dynamic compression influences interleukin-1beta-induced nitric oxide and prostaglandin E2 release by articular chondrocytes via alterations in iNOS and COX-2 expression

Interleukin-1beta (IL-1beta) induces the release of nitric oxide (.NO) and prostaglandin E2 (PGE2) by chondrocytes and this effect can be reversed with the application of dynamic compression. Previous studies have indicated that integrins may play a role. In addition, IL-1beta upregulates the expression of iNOS and COX-2 mRNA via upstream activation of p38 MAPK. The current study examines the involvement of these pathways in mediating .NO and PGE2 release in IL-1beta stimulated bovine chondrocytes subjected to dynamic compression. Bovine chondrocytes were seeded in agarose constructs and cultured with 0 or 10 ng.ml(-1) IL-1beta with or without the application of 15% dynamic compressive strain at 1 Hz. Selected inhibitors were used to interrogate the role of alpha5beta1 integrin signalling and p38 MAPK activation in mediating the release of .NO and PGE2 in response to both IL-1beta and dynamic compression. The relative expression levels of iNOS and COX-2 were assessed using real-time quantitative PCR. Nitrite, a stable end product of .NO, was measured using the Griess assay and PGE2 release was measured using an enzyme immunoassay. IL-1beta enhanced .NO and PGE2 release and this effect was reversed by the application of dynamic compression. Co-incubation with an integrin binding peptide (GRGDSP) abolished the compression-induced effect. Real-time quantitative PCR analysis revealed that IL-1beta enhanced iNOS and COX-2 mRNA levels, with the maximum expression at 6 or 12 hours. Dynamic compression reduced this effect via a p38 MAPK sensitive pathway. These results suggest that dynamic compression acts to abrogate of .NO and PGE2 release by directly influencing the expression levels of iNOS and COX-2.     

Inhibition of inducible nitric oxide synthase and cyclooxygenase-2 expression by flavonoids in macrophage J774A.1

The present study focuses on the effect of various naturally occurring flavonoids (apigenin, galangin, morin, naringenin, quercetin, and silymarin) on nitric oxide (NO) and prostaglandin E2 (PGE2) production induced by lipopolysaccharide (LPS) in the macrophage cell line J774A.1. Moreover, we evaluated flavonoid modulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) enzyme expression by western blot analysis. Apigenin and quercetin (0.5-50 microM) were the most potent inhibitors of NO production and this effect was concentration-dependent and significant at 5 and 50 microM. These data were consistent with the modulation of iNOS enzyme expression. A similar pattern was observed considering the inhibitory effect of flavonoids on LPS-induced PGE2 release and COX-2 expression. Quercetin, galangin, apigenin, and naringenin markedly decreased PGE2 release and COX-2 expression in a concentration-dependent manner. This study suggests that inhibition of iNOS and COX-2 expression by flavonoids may be one of the mechanisms responsible for their anti-inflammatory effects.     

Green tea polyphenol epigallocatechin-3-gallate inhibits the IL-1 beta-induced activity and expression of cyclooxygenase-2 and nitric oxide synthase-2 in human chondrocytes

We have previously shown that green tea polyphenols inhibit the onset and severity of collagen II-induced arthritis in mice. In the present study, we report the pharmacological effects of green tea polyphenol epigallocatechin-3-gallate (EGCG), on interleukin-1 beta (IL-1 beta)-induced expression and activity of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in human chondrocytes derived from osteoarthritis (OA) cartilage. Stimulation of human chondrocytes with IL-1 beta (5 ng/ml) for 24 h resulted in significantly enhanced production of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) when compared to untreated controls (p <.001). Pretreament of human chondrocytes with EGCG showed a dose-dependent inhibition in the production of NO and PGE(2) by 48% and 24%, respectively, and correlated with the inhibition of iNOS and COX-2 activities (p <.005). In addition, IL-1 beta-induced expression of iNOS and COX-2 was also markedly inhibited in human chondrocytes pretreated with EGCG (p <.001). Parallel to these findings, EGCG also inhibited the IL-1 beta-induced LDH release in chondrocytes cultures. Overall, the study suggests that EGCG affords protection against IL-1 beta-induced production of catabolic mediators NO and PGE(2) in human chondrocytes by regulating the expression and catalytic activity of their respective enzymes. Furthermore, our results also indicate that ECGC may be of potential therapeutic value for inhibiting cartilage resorption in arthritic joints.     

Carrageenan-induced paw edema in rat elicits a predominant prostaglandin E2 (PGE2) response in the central nervous system associated with the induction of microsomal PGE2 synthase-1

Peripheral inflammation involves an increase in cyclooxygenase-2 (COX-2)-mediated prostaglandin (PG) synthesis in the central nervous system (CNS), which contributes to allodynia and hyperalgesia. In the present study we have determined the changes in prostanoid tissue levels and in expression of terminal prostanoid synthases in both the CNS and inflamed peripheral tissue during carrageenan-induced paw inflammation in the rat. Prostanoid levels were measured by liquid chromatography-mass spectrometry and enzyme expression at the RNA level by quantitative PCR analysis during both the early (1-6 h) and late (12 and 24 h) phases of the inflammatory response. In the paw, the early phase was associated with increases in PGE(2) and thromboxane (TX)B(2) levels and with a peak of COX-2 expression that preceded that of microsomal prostaglandin-E(2) synthase-1 (mPGES-1). COX-2 and mPGES-1 remained elevated during the late phase, and PGE(2) continued to further increase through 24 h. The cytosolic PGE(2) synthase (cPGES) showed a small transient increase during the early phase, whereas mPGES-2 expression was not affected by inflammation. In the cerebrospinal fluid, elevated levels of PGE(2), 6-keto-PGF(1alpha), PGD(2), and TXB(2) were detected during the early phase. PGE(2) levels also increased in the spinal cord and, to a lesser extent, in the brain and remained elevated in both the cerebrospinal fluid and the spinal cord during the late phase. The expression of mPGES-1 was strongly up-regulated in the brain and spinal cord during inflammation, whereas no change was detected for the expression of cPGES, mPGES-2, COX-1, and terminal PGD, TX, or PGI synthases. The results show that the carrageenan-induced edema in the paw elicits an early phase of COX-2 induction in the CNS leading to an increase synthesis in PGD(2), 6-keto-PGF(1alpha), and TXB(2) in addition to the major PGE(2) response. The data also indicate that the up-regulation of mPGES-1 contributes to COX-2-mediated PGE(2) production in the CNS during peripheral inflammation.     

Regulatory interconnections of cyclooxigenase and inducible no-synthase in urinary bladder epithelial cells of the frog Rana temporaria under action of bacterial stimuli

Earlier we have shown that in epithelial cells of the frog urinary bladder under action of bacterial lipopolysaccharides (LPS) there is activated expression of inducible NO-synthase (iNOS) and there is increased the NO production, which can play an important role in providing protective cell reactions from pathogens. The goal of the present work consisted in study of cyclooxigenase (cOG) products and mechanisms of their regulatory effect on expression of iNOS under action of LPS. In experiments on urinary bladder epithelial cells on the frog Rana temporaria it has been shown that incubation of the cells for 21 h with LPS leads to a rise in production of PGE2 and nitrites, stable NO metabolites. Inhibitor of iNOS 1400W decreased sharply production of nitrites, but did not affect the PGE2 level. Both the basal and the LPS-stimulated level of PGE2 and nitrites were inhibited in the presence of selective cOG inhibitors--SC-560 (cOG-1) and NS-398 (cOG-2). The IC50 value amounted to 90, 220, and 470 microM for NS-398, SC-560, and diclofenac (unspecific inhibitor of both isoforms), respectively. PGE2 and butaprost, the EP2-receptor agonist, but not agonists of EP1/EP3 or EP1 receptors, partially eliminated the inhibitory action of diclofenac on production of nitrites. Action of PGE2 was accompanied by an increase in the intracellular cAMP. Analysis of expression of iNOS mRNA in the epithelial cells incubated with LPS or LPS + inhibitor of cOG has shown the LPS-stimulated rise in expression of iNOS mRNA to decrease sharply in the presence of SC-560 or NS-398. Thus, the epithelial cells of the frog urinary bladder have the effectively functioning system of the congenital immune protection against bacterial pathogens, the most important component of this system being PGE2 and NO. Analysis of mechanisms of regulatory interactions of cOG and iNOS indicates that in this cell type the main regulators of iNOS expression and of the nitrogen oxide level are products of the cOG catalytic activity.     

TLR3-dependent induction of nitric oxide synthase in RAW 264.7 macrophage-like cells via a cytosolic phospholipase A2/cyclooxygenase-2 pathway

dsRNA is a by-product of viral replication capable of inducing an inflammatory response when recognized by phagocyte cells. In this study, we identify group IVA cytosolic phospholipase A2 (cPLA2alpha) as an effector of the antiviral response. Treatment of RAW 264.7 murine macrophage-like cells with the dsRNA analog polyinosinic:polycytidylic acid (poly-IC) promotes the release of free arachidonic acid that is subsequently converted into PGE2 by the de novo-synthesized cyclooxygenase-2 (COX-2) enzyme. These processes are blocked by the selective cPLA2alpha inhibitor pyrrophenone, pointing out to cPLA2alpha as the effector involved. In keeping with this observation, the cPLA2alpha phosphorylation state increases after cellular treatment with poly-IC. Inhibition of cPLA2alpha expression and activity by either small interfering RNA (siRNA) or pyrrophenone leads to inhibition of the expression of the inducible NO synthase (iNOS) gene. Moreover, COX-2-derived PGE2 production appears to participate in iNOS expression, because siRNA inhibition of COX-2 also leads to inhibition of iNOS, the latter of which is restored by exogenous addition of PGE2. Finally, cellular depletion of TLR3 by siRNA inhibits COX-2 expression, PGE2 generation, and iNOS induction by poly-IC. Collectively, these findings suggest a model for macrophage activation in response to dsRNA, whereby engagement of TLR3 leads to cPLA2alpha-mediated arachidonic acid mobilization and COX-2-mediated PGE2 production, which cooperate to induce the expression of iNOS.     

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.     

Inhibition of p38 mitogen-activated protein kinase attenuates interleukin-1beta-induced thermal hyperalgesia and inducible nitric oxide synthase expression in the spinal cord

We have reported recently that intrathecal (i.t.) injection of interleukin-1beta (IL-1beta), at a dose of 100 ng, induces inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in the spinal cord and results in thermal hyperalgesia in rats. This study further examines the role of mitogen-activated protein kinase (MAPK) in i.t. IL-1beta-mediated iNOS-NO cascade in spinal nociceptive signal transduction. All rats were implanted with an i.t. catheter either with or without an additional microdialysis probe. Paw withdrawal latency to radiant heat is used to assess thermal hyperalgesia. The iNOS and MAPK protein expression in the spinal cord dorsal horn were examined by western blot. The [NO] in CSF dialysates were also measured. Intrathecal IL-1beta leads to a time-dependent up-regulation of phosphorylated p38 (p-p38) MAPK protein expression in the spinal cord 30-240 min following IL-1beta injection (i.t.). However, neither the phosphorylated extracellular signal-regulated kinase (p-ERK) nor phosphorylated c-Jun NH2-terminal kinase (p-JNK) was affected. The total amount of p38, ERK, and JNK MAPK proteins were not affected following IL-1beta injection. Intrathecal administration of either selective p38 MAPK, or JNK, or ERK inhibitor alone did not affect the thermal nociceptive threshold or iNOS protein expression in the spinal cord. However, pretreatment with a p38 MAPK inhibitor significantly reduced the IL-1beta-induced p-p38 MAPK expression by 38-49%, and nearly completely blocked the subsequent iNOS expression (reduction by 86.6%), NO production, and thermal hyperalgesia. In contrast, both ERK and JNK inhibitor pretreatments only partially (approximately 50%) inhibited the IL-1beta-induced iNOS expression in the spinal cord. Our results suggest that p38 MAPK plays a pivotal role in i.t. IL-1beta-induced spinal sensitization and nociceptive signal transduction.