Low Molecular Weight Hyaluronan Activates Cytosolic Phospholipase A2α and Eicosanoid Production in Monocytes and Macrophages

Hyaluronan (HA) is the major glycosaminoglycan in the extracellular matrix. During inflammation, there is an increased breakdown of HA, resulting in the accumulation of low molecular weight (LMW) HA and activation of monocytes and macrophages. Eicosanoids, derived from the cytosolic phospholipase A₂ group IVA (cPLA₂α) activation, are potent lipid mediators also attributed to acute and chronic inflammation. The aim of this study was to determine the effect of LMW HA on cPLA₂α activation, arachidonic acid (AA) release, and subsequent eicosanoid production and to examine the receptors and downstream mechanisms involved in these processes in monocytes and differently polarized macrophages. LMW HA was a potent stimulant of AA release in a time- and dose-dependent manner, induced cPLA₂α, ERK1/2, p38, and JNK phosphorylation, as well as activated COX2 expression and prostaglandin (PG) E₂ production in primary human monocytes, murine RAW 264.7, and wild-type bone marrow-derived macrophages. Specific cPLA₂α inhibitor blocked HA-induced AA release and PGE₂ production in all of these cells. Using CD44, TLR4, TLR2, MYD88, RHAMM or STAB2 siRNA-transfected macrophages and monocytes, we found that AA release, cPLA₂α, ERK1/2, p38, and JNK phosphorylation, COX2 expression, and PGE₂ production were activated by LMW HA through a TLR4/MYD88 pathway. Likewise, PGE₂ production and COX2 expression were blocked in Tlr4^−/− and Myd88^−/− mice, but not in Cd44^−/− mice, after LMW HA stimulation. Moreover, we demonstrated that LMW HA activated the M1 macrophage phenotype with the unique cPLA₂α/COX2^high and COX1/ALOX15/ALOX5/LTA4H^low gene and PGE₂/PGD₂/15-HETE^high and LXA₄^low eicosanoid profile. These findings reveal a novel link between HA-mediated inflammation and lipid metabolism.     

Characterisation of (R)-2-(2-Fluorobiphenyl-4-yl)-N-(3-Methylpyridin-2-yl)Propanamide as a Dual Fatty Acid Amide Hydrolase: Cyclooxygenase Inhibitor

Background

Increased endocannabinoid tonus by dual-action fatty acid amide hydrolase (FAAH) and substrate selective cyclooxygenase (COX-2) inhibitors is a promising approach for pain-relief. One such compound with this profile is 2-(2-fluorobiphenyl-4-yl)-N-(3-methylpyridin-2-yl)propanamide (Flu-AM1). These activities are shown by Flu-AM1 racemate, but it is not known whether its two single enantiomers behave differently, as is the case towards COX-2 for the parent flurbiprofen enantiomers. Further, the effects of the compound upon COX-2-derived lipids in intact cells are not known.

Methodology/Principal Findings

COX inhibition was determined using an oxygraphic method with arachidonic acid and 2-arachidonoylglycerol (2-AG) as substrates. FAAH was assayed in mouse brain homogenates using anandamide (AEA) as substrate. Lipidomic analysis was conducted in unstimulated and lipopolysaccharide + interferon γ- stimulated RAW 264.7 macrophage cells. Both enantiomers inhibited COX-2 in a substrate-selective and time-dependent manner, with IC₅₀ values in the absence of a preincubation phase of: (R)-Flu-AM1, COX-1 (arachidonic acid) 6 μM; COX-2 (arachidonic acid) 20 μM; COX-2 (2-AG) 1 μM; (S)-Flu-AM1, COX-1 (arachidonic acid) 3 μM; COX-2 (arachidonic acid) 10 μM; COX-2 (2-AG) 0.7 μM. The compounds showed no enantiomeric selectivity in their FAAH inhibitory properties. (R)-Flu-AM1 (10 μM) greatly inhibited the production of prostaglandin D₂ and E₂ in both unstimulated and lipopolysaccharide + interferon γ- stimulated RAW 264.7 macrophage cells. Levels of 2-AG were not affected either by (R)-Flu-AM1 or by 10 μM flurbiprofen, either alone or in combination with the FAAH inhibitor URB597 (1 μM).

Conclusions/Significance

Both enantiomers of Flu-AM1 are more potent inhibitors of 2-AG compared to arachidonic acid oxygenation by COX-2. Inhibition of COX in lipopolysaccharide + interferon γ- stimulated RAW 264.7 cells is insufficient to affect 2-AG levels despite the large induction of COX-2 produced by this treatment.     

Identification of prostaglandin F-producing cells in the liver

Prostaglandin (PG) F synthase forms PGF_2α and 9α, 11β-PGF₂ from PGH₂ and PGD₂, respectively. PGH₂ is synthesized from arachidonic acid by cyclooxygenase (COX) and then metabolized to various PGs and thromboxane by specific enzymes. PGD₂ is synthesized from PGH₂ by PGD synthase. To identify PGF₂-producing cells in the rat liver, the occurrence and localization of PGF synthase and COX were studied with immunochemical and immunohistochemical techniques using anti-liver-type PGF synthase and anti-COX antibodies. In Western blot analyses, positive bands of liver-type PGF synthase and constitutive COX-1 were observed at positions approximately 37 kDa and 70–72 kDa, respectively. However, inducible COX-2 was not detected. In the immunohistochemical study, PGF synthase was present in the cytoplasm of the sinusoidal endothelial cells. COX-1 was present on the membranes of the nucleus and endoplasmic reticulum of the endothelial cells and Kupffer cells. Double immunostaining for PGF synthase and COX-1 showed that both enzymes were present in the same endothelial cells. These results suggest that the main site of PGF₂ synthesis in the liver is the sinusoidal endothelial cell. Accepted: 12 October 1999     

Increased cyclooxygenase-2 and 5-lipoxygenase activating protein expression in peritoneal macrophages during ovalbumin immunization of mice and cytosolic phospholipase A2 activation after antigen challenge

The present study investigates phenotypic and functional differentiation of peritoneal macrophages during ovalbumin-induced subcutaneous immunization of mice. For the first time we show that, in mouse peritoneal macrophages, ovalbumin immunization induces an increase in cyclooxygenase-2 (COX-2) and 5-lipoxygenase activating protein (FLAP) expression whereas it inhibits cytosolic phospholipase A₂ (cPLA2) expression. The study of arachidonic acid (AA) metabolism in peritoneal macrophages from control (cPM) and ovalbumin-immunized (iPM) mice shows that the reduced cPLA2 expression is correlated to a reduced basal AA metabolism, but is not a limiting factor for the opsonized zymosan-, PMA-, or A23187-triggered AA metabolism. We also show that in vitro ovalbumin challenge induces, only in iPM, cPLA2 activation through phosphorylation of serine residues, via a mechanism involving MAP kinases, and through increased intracellular calcium concentrations, leading to eicosanoid production. In parallel, we report that, in peritoneal macrophages, ovalbumin immunization induces the expression of CD23, the low affinity receptor for IgEs known for its involvement in allergic diseases. Thus, the modified expression of the enzymes involved in AA metabolism and the difference of response of cPM and iPM toward the antigen are important elements to understand the underlying mechanisms of ovalbumin-induced allergic responses.     

Corn Silk Induced Cyclooxygenase-2 in Murine Macrophages

Stimulation of murine macrophages with corn silk induced cyclooxygenase (COX)-2 with secretion of PGE₂. Expression of COX-2 was inhibited by pyrolidine dithiocarbamate (PDTC), and increased DNA binding by nuclear factor kappa B (NF-κB), indicating that COX-2 induction proceeds also via the NF-κB signaling pathway. A specific inhibitor of COX-2 decreased the expression level of inducible nitric oxide synthase (iNOS) stimulated by corn silk. PGE₂ elevated the expression level of iNOS, probably via EP2 and EP4 receptors on the surface of the macrophages.     

Coupling between cyclooxygenases and terminal prostanoid synthases

Biosynthesis of prostanoids is regulated by three sequential enzymatic steps, namely phospholipase A2, cyclooxygenase (COX), and terminal prostanoid synthase. Recent evidence suggests that lineage-specific terminal prostanoid synthases, including prostaglandin (PG) E2, PGD2, PGF2alpha, PGI2, and thromboxane synthases, show distinct functional coupling with upstream COX isozymes, COX-1 and COX-2. This can account, at least in part, for segregated utilization of the two COX isozymes in distinct phases of PG-biosynthetic responses. In terms of their localization and COX preference, terminal prostanoid synthases are classified into three categories: (i) the perinuclear enzymes that prefer COX-2, (ii) the cytosolic enzyme that prefers COX-1, and (iii) the translocating enzyme that utilizes both COXs depending on the stimulus. Additionally, altered supply of arachidonic acid by phospholipase A2s significantly affects the efficiency of COX-terminal prostanoid synthase coupling. In this review, we summarize our recent understanding of the coupling profiles between the two COXs and various terminal prostanoid synthases.     

Coupling between cyclooxygenases and prostaglandin F2α synthase: Detection of an inducible,glutathione-activated,membrane-bound prostaglandin F2α-synthetic activity

Distinct functional coupling between cyclooxygenases (COXs) and specific terminal prostanoid synthases leads to phase-specific production of particular prostaglandins (PGs). In this study, we examined the coupling between COX isozymes and PGF synthase (PGFS). Co-transfection of COXs with PGFS-I belonging to the aldo-keto reductase family into HEK293 cells resulted in increased production of PGF_2α only when a high concentration of exogenous arachidonic acid (AA) was supplied. However, this enzyme failed to produce PGF_2α from endogenous AA, even though significant increase in PGF_2α production occurred in cells transfected with COX-2 alone. This poor COX/PGFS-I coupling was likely to arise from their distinct subcellular localization. Measurement of PGF_2α-synthetic enzyme activity in homogenates of several cells revealed another type of PGFS activity that was membrane-bound, glutathione (GSH)-activated, and stimulus-inducible. In vivo, membrane-bound PGFS activity was elevated in the lung of lipopolysaccharide-treated mice. Taken together, our results suggest the presence of a novel, membrane-associated form of PGFS that is stimulus-inducible and is likely to be preferentially coupled with COX-2.     

Effects of non-steroidal anti-inflammatory drugs on prostaglandin E2 production by cyclooxygenase-2 from endogenous and exogenous arachidonic acid in rat peritoneal macrophages stimulated with lipopolysaccharide

Lipopolysaccharide (LPS) stimulated prostaglandin E₂ (PGE₂) formation and induction of cyclooxygenase-2 (COX-2) expression without changing the levels of COX-1 protein in rat peritoneal macrophages. Non-steroidal anti-inflammatory drugs (NSAIDs) (nimesulide, indomethacin and ibuprofen) strongly inhibited LPS-stimulated PGE₂ production without any effect on COX-2 protein expression, suggesting that NSAIDs are active in inhibiting the ability of COX-2 to convert arachidonic acid (AA) endogenously released in response to LPS stimulation. Exogenous AA can be converted to PGE₂ by both COX isoforms even in LPS-stimulated macrophages. NSAIDs inhibited PGE₂ production from exogenous AA mediated by both COX-1 and COX-2. However, the two isoforms interacted differentially with different NSAIDs. Furthermore, NSAIDs were distinctly more active in inhibiting PGE₂ production from endogenous AA than that from exogenous AA. These data suggest that PGE₂ production through COX-2 from exogenous AA may not be subject to the same regulatory processes as that from endogenous AA and the two metabolic processes may be differentially sensitive to different NSAIDs.     

Protein kinase B in the diabetic heart

Cytosolic phospholipases A₂ (cPLA₂) and cyclooxygenases-1 and -2 (COX-1 and -2) play a pivotal role in the metabolism of arachidonic acid (AA) and in eicosanoid production. The coordinate regulation and expression of these enzymes is not well defined. In this study, the effect of phorbol 12-myristate 13-acetate (PMA), tumor necrosis factor (TNF), lipopolysaccharide (LPS) and macrophage-colony stimulating factor (M-CSF) on AA release and prostaglandin E₂ (PGE₂) production and the expression of cPLA₂ and COX-1 and -2 were investigated in U937 human pre-monocytic cells and fully differentiated macrophages. Treatment of U937 cells with PMA or macrophages with LPS increased AA release and PGE₂ production. Incubation of U937 cells or macrophages for 8 h with all stimuli elevated cPLA₂ expression. In contrast, cPLA₂ expression was reduced upon further incubation of U937 cells or macrophages for 24 h with all stimuli indicating a bi-phasic expression pattern of this enzyme. PMA induced COX-1 expression in U937 cells whereas LPS induced COX-2 expression in macrophages. Although TNF and M-CSF induced a significant amount of AA release in both cell models, they failed to induce a comparable production of PGE₂ since they were unable to induce the coordinate expression of the downstream key enzymes, COX-1 or COX-2. The results suggest that the enhancement of AA release in both U937 cells and macrophages may be caused by both increased cPLA₂ activity and elevated cPLA₂ protein expression. In addition, PMA stimulates PGE₂ production via up-regulation of COX-1, and likely COX-2, expression in U937 cells whereas LPS stimulates PGE₂ production via induction of COX-2 expression in macrophages.     

Palmitoleate Reverses High Fat-induced Proinflammatory Macrophage Polarization via AMP-activated Protein Kinase (AMPK)

A rise in tissue-embedded macrophages displaying “M1-like” proinflammatory polarization is a hallmark of metabolic inflammation during a high fat diet or obesity. Here we show that bone marrow-derived macrophages (BMDM) from high fat-fed mice retain a memory of their dietary environment in vivo (displaying the elevated proinflammatory genes Cxcl1, Il6, Tnf, Nos2) despite 7-day differentiation and proliferation ex vivo. Notably, 6-h incubation with palmitoleate (PO) reversed the proinflammatory gene expression and cytokine secretion seen in BMDM from high fat-fed mice. BMDM from low fat-fed mice exposed to palmitate (PA) for 18 h ex vivo also showed elevated expression of proinflammatory genes (Cxcl1, Il6, Tnf, Nos2, and Il12b) associated with M1 polarization. Conversely, PO treatment increased anti-inflammatory genes (Mrc1, Tgfb1, Il10, Mgl2) and oxidative metabolism, characteristic of M2 macrophages. Therefore, saturated and unsaturated fatty acids bring about opposite macrophage polarization states. Coincubation of BMDM with both fatty acids counteracted the PA-induced Nos2 expression in a PO dose-dependent fashion. PO also prevented PA-induced IκBα degradation, RelA nuclear translocation, NO production, and cytokine secretion. Mechanistically, PO exerted its anti-inflammatory function through AMP-activated protein kinase as AMP kinase knockout or inhibition by Compound C offset the PO-dependent prevention of PA-induced inflammation. These results demonstrate a nutritional memory of BMDM ex vivo, highlight the plasticity of BMDM polarization in response to saturated and unsaturated fatty acids, and identify the potential to reverse diet- and saturated fat-induced M1-like polarization by administering palmitoleate. These findings could have applicability to reverse obesity-linked inflammation in metabolically relevant tissues.     

Vitamin D Inhibits COX-2 Expression and Inflammatory Response by Targeting Thioesterase Superfamily Member 4

Inadequate vitamin D status has been linked to increased risk of type 2 diabetes and cardiovascular disease. Inducible cyclooxygenase (COX) isoform COX-2 has been involved in the pathogenesis of such chronic inflammatory diseases. We found that the active form of vitamin D, 1,25(OH)₂D produces dose-dependent inhibition of COX-2 expression in murine macrophages under both basal and LPS-stimulated conditions and suppresses proinflammatory mediators induced by LPS. Administration of 1,25(OH)₂D significantly alleviated local inflammation in a carrageenan-induced paw edema mouse model. Strikingly, the phosphorylation of both Akt and its downstream target IκBα in macrophages were markedly suppressed by 1,25(OH)₂D in the presence and absence of LPS stimulation through up-regulation of THEM4 (thioesterase superfamily member 4), an Akt modulator protein. Knockdown of both vitamin D receptor and THEM4 attenuated the inhibitory effect of 1,25(OH)₂D on COX-2 expression in macrophages. A functional vitamin D-responsive element in the THEM4 promoter was identified by chromatin immunoprecipitation and luciferase reporter assay. Our results indicate that vitamin D restrains macrophage-mediated inflammatory processes by suppressing the Akt/NF-κB/COX-2 pathway, suggesting that vitamin D supplementation might be utilized for adjunctive therapy for inflammatory disease.     

Oxygenation of the endocannabinoid, 2-arachidonylglycerol, to glyceryl prostaglandins by cyclooxygenase-2

Cyclooxygenases (COX) play an important role in lipid signaling by oxygenating arachidonic acid to endoperoxide precursors of prostaglandins and thromboxane. Two cyclooxygenases exist which differ in tissue distribution and regulation but otherwise carry out identical chemical functions. The neutral arachidonate derivative, 2-arachidonylglycerol (2-AG), is one of two described endocannabinoids and appears to be a ligand for both the central (CB1) and peripheral (CB2) cannabinoid receptors. Here we report that 2-AG is a substrate for COX-2 and that it is metabolized as effectively as arachidonic acid. COX-2-mediated 2-AG oxygenation provides the novel lipid, prostaglandin H(2) glycerol ester (PGH(2)-G), in vitro and in cultured macrophages. PGH(2)-G produced by macrophages is a substrate for cellular PGD synthase, affording PGD(2)-G. Pharmacological studies reveal that macrophage production of PGD(2)-G from endogenous sources of 2-AG is calcium-dependent and mediated by diacylglycerol lipase and COX-2. These results identify a distinct function for COX-2 in endocannabinoid metabolism and in the generation of a new family of prostaglandins derived from diacylglycerol and 2-AG.     

Prostaglandin E(2) regulates wound closure in airway epithelium

Repair of the airway epithelium after injury is critical for the maintenance of barrier function and the limitation of airway hyperreactivity. Airway epithelial cells (AECs) metabolize arachidonic acid to biologically active eicosanoids via the enzyme cyclooxygenase (COX). We investigated whether stimulating or inhibiting COX metabolites would affect wound closure in monolayers of cultured AECs. Inhibiting COX with indomethacin resulted in a dose-dependent inhibition of wound closure in human and feline AECs. Specific inhibitors for both COX-1 and COX-2 isoforms impaired wound healing. Inhibitors of 5-lipoxygenase did not affect wound closure in these cells. The addition of prostaglandin E(2) (PGE(2)) eliminated the inhibition due to indomethacin treatment, and the exogenous application of PGE(2) stimulated wound closure in a dose-dependent manner. Inhibition of COX with indomethacin only at initial time points resulted in a sustained inhibition of wound closure, indicating that prostanoids are involved in early wound repair processes such as spreading and migration. These differences in wound closure may be important if arachidonic acid metabolism and eicosanoid concentrations are altered in disease states such as asthma.     

Up-regulation of microsomal prostaglandin E synthase-1 in COX-1 and COX-2 knock-out mouse fibroblast cell lines

In this paper we investigated the possible involvement of prostaglandin E synthases (PGESs) in compensatory mechanism. Our findings showed that microsomal (m)PGES-1 expression was significantly up-regulated in COX knock-out (K/O) cells whereas the expression of cytosolic PGES was not changed indicating that the induction of mPGES-1 may, at least in part, contribute to the substantial increase of PGE₂ production in COX K/O cell lines. The selective up-regulation of mPGES-1 in COX-2 K/O cells suggests that mPGES-1 may be metabolically coupled with COX-1 for PGE₂ formation. Addition of arachidonic acid caused significant induction of mPGES-1 and COX-2 in WT cells, whereas COX-1 and cPGES were not affected. Our earlier and the current studies demonstrate the coregulation of cPLA₂, COX, and mPGES-1, in PGE₂ synthesis pathway, and that these enzymes contribute to the elevation of PGE₂ level when one COX isoform is absent.     

Effect of cyclooxygenase genotype and dietary fish oil on colonic eicosanoids in mice

Dietary ω3 fatty acids can modulate substrate availability for cyclooxygenases (COXs) and lipoxygenases, thus modulating downstream eicosanoid formation. This could be an alternative approach to using nonsteroidal anti-inflammatory drugs and other COX inhibitors for limiting Prostaglandin E(2) (PGE(2)) synthesis in colon cancer prevention. The aims of this study were to evaluate to what extent COX- and lipoxygenase-derived products could be modulated by dietary fish oil in normal colonic mucosa and to evaluate the role of COX-1 and COX-2 in the formation of these products. Mice (wild-type, COX-1 null or COX-2 null) were fed a diet supplying a broad mixture of fatty acids present in European/American diets, supplemented with either olive oil (oleate control diet) or menhaden (fish) oil ad libitum for 9-11 weeks. Colonic eicosanoid levels were measured by liquid chromatography tandem mass spectroscopy (LC-MS/MS), and proliferation was assessed by Ki67 immunohistochemistry. For the dietary alteration of colonic arachidonic acid: eicosapentaenoic ratios resulted in large shifts in formation of COX and lipoxygenase metabolites. COX-1 knockout virtually abolished PGE(2) formation, but interestingly, 12-hydroxyeicosatetraenoic (12-HETE) acid and 15-HETE formation was increased. The large changes in eicosanoid profiles were accompanied by relatively small changes in colonic crypt proliferation, but such changes in eicosanoid formation might have greater biological impact upon carcinogen challenge. These results indicate that in normal colon, inhibition of COX-2 would have little effect on reducing PGE(2) levels.