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The Journal of Immunology, 2002, 169: 5279-5286.
Copyright © 2002 by The American Association of Immunologists

Adenosine Up-Regulates Cyclooxygenase-2 in Human Granulocytes: Impact on the Balance of Eicosanoid Generation1

Marc Pouliot2, Marie-Élaine Fiset, Mireille Massé, Paul H. Naccache and Pierre Borgeat

Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du Centre Hospitalier Universitaire de Québec, and Faculté de Médecine, Université Laval, Québec, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Polymorphonuclear neutrophils (granulocytes; PMNs) are often the first blood cells to migrate toward inflammatory lesions to perform host defense functions. PMNs respond to specific stimuli by releasing several factors and generate lipid mediators of inflammation from the 5-lipoxygenase and the inducible cyclooxygenase (COX)-2 pathways. In view of adenosine’s anti-inflammatory properties and suppressive impact on the 5-lipoxygenase pathway, we addressed in this study the impact of this autacoid on the COX-2 pathway. We observed that adenosine up-regulates the expression of the COX-2 enzyme and mRNA. Production of PGE2 in response to exogenous arachidonic acid was also increased by adenosine and correlated with COX-2 protein levels. The potentiating effect of adenosine on COX-2 could be mimicked by pharmacological increases of intracellular cAMP levels, involving the latter as a putative second messenger for the up-regulation of COX-2 by adenosine. Specific COX-2 inhibitors were used to confirm the predominant role of the COX-2 isoform in the formation of prostanoids by stimulated PMNs. Withdrawal of extracellular adenosine strikingly emphasized the inhibitory potential of PGE2 on leukotriene B4 formation and involved the EP2 receptor subtype in this process. Thus, adenosine may promote a self-limiting regulatory process through the increase of PGE2 generation, which may result in the inhibition of PMN functions. This study identifies a new aspect of the anti-inflammatory properties of adenosine in leukocytes, introducing the concept that this autacoid may exert its immunomodulatory activities in part by modifying the balance of lipid mediators generated by PMNs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Polymorphonuclear neutrophils (PMNs)3 are often the first blood cells to migrate toward inflammatory lesions where they accumulate and perform host defense functions, including phagocytosis of invading microorganisms and cell debris, release of proteolytic enzymes, and generation of oxygen-derived reactive agents. PMNs respond to specific stimuli by releasing cytokines and chemokines, such as IL-1{beta}, IL-6, IL-8, G- and M-CSF, macrophage inflammatory peptides 1{alpha} and 1{beta}, and IFN-{alpha} (reviewed in Ref. 1), each of which distinctly contributes to the regulation of inflammatory responses. In addition, PMNs are an important source of arachidonic acid (AA)-derived lipid mediators of inflammation, collectively termed eicosanoids, in particular leukotriene (LT)B4, PGE2, and thromboxane (TX)A2 (2, 3, 4).

It is likely that the overall profile of eicosanoids generated by leukocytes under distinct physiological or pathophysiological conditions will determine in part the way these cells impact on inflammatory responses. LTB4, a metabolite of the 5-lipoxygenase (LO) pathway, is a potent leukocyte chemoattractant and activator (5). In contrast, TXA2 and PGE2 are generated by the cyclooxygenase (COX) pathway. The former is recognized for stimulating platelet aggregation and activation, while the latter participates in regulating blood flow and vascular permeability, bronchial airway contraction, nociceptor activation, and hyperresponsiveness (6). In PMNs, the expression of enzymes that are pivotal for the generation of these eicosanoids is tightly regulated. Differential induction of the 5-LO, the 5-LO-activating protein (FLAP), and the COX-2 by selected cytokines, inflammatory agents, and growth factors has been observed in PMNs (7, 8, 9, 10, 11, 12). Therefore, it is clear that these cells have the potential to actively influence the onset as well as the resolution of an inflammatory response.

COX catalyzes two reactions by which AA is converted to PGH2, the common precursor of all prostanoids. Two COX isoforms, COX-1 and COX-2, are known; COX-1 appears to support prostanoid biosynthesis required for maintaining organ and tissue homeostasis (13, 14), whereas COX-2 expression is restricted in basal conditions and is up-regulated during inflammation (15, 16). Both isoforms contribute to the inflammatory process, but COX-2 was considered of particular therapeutic interest, as it is specifically induced during acute as well as chronic inflammation. Incubation of PMNs with a range of agonists, including phorbol esters, whole bacteria, LPS, fMLP, TNF-{alpha}, and opsonized particles, leads to rapid up-regulation of COX-2 (9, 10, 11, 12). In addition to its association with inflammatory conditions, COX-2 plays a role in regulating cellular proliferation, differentiation, and tumorigenesis (17, 18), and is emerging as a therapeutic target in the treatment and prevention of many human cancers, including colon, prostate, esophageal, and breast cancers (19, 20, 21, 22). In contrast, there is increasing evidence that COX-2 also supports a number of physiological functions. Investigations with COX-2-deficient mice suggest that COX-2 is important for kidney functions (23), postnatal development, and female reproductive processes (23, 24, 25). Biochemical evidence that COX-2 inhibitors may worsen a tendency for thrombosis is supported by clinical trials and case reports (reviewed in Ref. 26). In addition, COX-2 inhibition was found to exacerbate inflammation in vivo, in a model of carrageenan-induced pleurisy (27). In leukocytes, there is considerable evidence in support of anti-inflammatory activities of PGE2. Indeed, PGE2 has been shown to inhibit chemotaxis, aggregation, superoxide production, lysosomal enzyme release, and LTB4 generation from activated PMNs (28, 29, 30, 31, 32), suggesting that up-regulation of COX-2 in these cells may contribute to the down-regulation of their inflammatory functions. Thus, COX-2 appears to be a multifaceted enzyme associated both with physiology and pathophysiology.

Adenosine is a ubiquitous autacoid with a large spectrum of biological activities, including the attenuation of selected leukocyte functions. In vivo studies have demonstrated a protective role of adenosine in several models of acute inflammation (reviewed in Ref. 33), and activation of A2A adenosine receptor has been reported to accelerate wound healing (34). Studies on A2AR knockout mice revealed that A2AR are a critical part of the physiological negative feedback mechanism for limitation and termination of both tissue-specific and systemic inflammatory responses (35). In turn, adenosine is increasingly being considered as a potent anti-inflammatory agent, and its receptors, as potential therapeutic targets (36). Through engagement of A2AR, adenosine inhibits selected PMN functions: adhesion to endothelial cells, generation of superoxide anions, and phagocytosis (37). Our laboratory has recently shown that endogenous adenosine as well as agonists of A2AR potently inhibit the generation of LTB4 (33) and platelet-activating factor (P. Borgeat, unpublished data), both in isolated PMN preparations and in whole blood.

In view of adenosine’s anti-inflammatory properties and suppressive impact on the 5-LO pathway, it appeared of interest to assess its putative involvement in the generation of COX-derived lipid mediators. In the present study, we investigated the impact of A2AR activation on COX-2 and the consequent generation of prostanoids. We observed that adenosine potentiates the expression of COX-2 and increases the capacity of PMNs to generate PGE2, identifying a new immunomodulatory facet of adenosine toward leukocytes. These results support the concept that adenosine may exert its anti-inflammatory activities in part by modulating the profile of eicosanoids produced by PMNs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

5,5-Dimethyl-3-(3-fluorophenyl)-4-(4-methylsulfonyl)phenyl-2(5H)-furanone (DFU) was a generous gift of Merck-Frosst Labs (Pointe-Claire, Quebec, Canada). NS-398, PGE2, and TXB2 were purchased from Cayman Chemicals (Ann Arbor, MI). 2-p-(2-Carboxyethyl)phenethylamino-5'-N-ethylcarboxamindoadenosine hydrochloride (CGS 21680) and 9-chloro-2-(2-furyl)[1,2,4]triazolo[1,5-c]quinazolin-5-amine (CGS 15934) were purchased from Research Biochemicals International (Natick, MA). Cytochalasin B, fMLP, forskolin, RO-20-1724, AA, 8-(3-chlorostyryl)caffeine (CSC) were all obtained from Sigma-Aldrich (Oakville, Ontario, Canada). Butaprost, AH 6809, I-BOP, and U-46619 were purchased from Cedarlane Laboratories (Hornsby, Ontario, Canada). HPLC solvents (acetonitrile and methanol) were purchased from Fisher (Ville St.-Laurent, Quebec, Canada) and from VWR Canlab (Ville Mont-Royal, Quebec, Canada), respectively.

PMN preparation

PMN suspensions were prepared as previously described (38) with modifications. Briefly, venous blood was collected from healthy volunteers on EDTA anticoagulant solution and centrifuged (250 x g, 15 min); the platelet-rich plasma was collected and centrifuged (1000 x g, 10 min) to reduce the number of platelets in the preparation. The platelet-depleted plasma was used to resuspend the RBC/leukocyte pellet, which was centrifuged once more (250 x g, 15 min). The resulting plasma was discarded, and the RBC/leukocyte pellet was then subjected to dextran sedimentation of erythrocytes, and PMN were isolated by centrifugation over a 10-ml Ficoll-Paque cushion. Contaminating erythrocytes were removed by a 25-s hypotonic lysis. Resulting PMN preparations (>95% neutrophils, <5% eosinophils) contained fewer than 0.2% monocytes, as determined by esterase staining. Viability was >98%, as determined by trypan blue dye exclusion. The whole cell isolation procedure was conducted under sterile conditions at RT (room temperature).

Cell incubations

PMNs were resuspended at a concentration of 10 x 106 cells/ml in HBSS containing 10 mM HEPES (pH 7.4), 1.6 mM Ca2+, and no Mg2+. When mentioned in this paper, adenosine deaminase (ADA; 0.1 U/ml) was added to cell suspensions 20 min before cell stimulation. Pharmacological agents dissolved in DMSO were added to PMN suspensions 15 min before incubation with agonist(s); the final organic solvent concentration never exceeded 0.1%. Cytochalasin B was added 5 min before incubations with fMLP. Unless stated otherwise, AA was added to a final concentration of 3 µM for 5-min incubations (39).

Immunoblots

Cell incubations were stopped in an ice-water bath, and samples were briefly centrifuged. Cell pellets were resuspended in 100 µl of cold (4°C) HEPES-buffered HBSS without Ca2+ (containing the following antiprotease mixture: 10 µg/ml phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin); then 150 µl of warm (65°C) 2x sample buffer (125 mM Tris-HCl (pH 6.8), 8% SDS, 10% 2-ME, 17% glycerol, with antiprotease mixture) were added, and the mixtures were boiled for 7 min. Aliquots (50 µl) were then subjected to 9% SDS-PAGE and transferred to Immobilon membranes (Millipore, Bedford, MA). Equal protein loading and transfer efficiency were visualized by Ponceau Red staining. The membranes were soaked for 30 min at RT in TBS (25 mM Tris-HCl (pH 7.6), 0.2 M NaCl, and 0.15% Tween 20) containing 5% (w/v) dried milk, and exposed for 30 min either to an anti-COX-2 mouse mAb or an anti-TX-synthase polyclonal Ab (Cayman Chemicals). Membranes were then washed twice in TBS and incubated for 30 min with a 1/5000 dilution either of a HRP-linked donkey anti-mouse Ab, for COX-2, or a sheep anti-rabbit Ab, for TX synthase (Santa Cruz Biotechnology, Santa Cruz, CA). Enzyme expression was revealed with ECL-Plus (NEN-Mandel, Mississauga, Ontario, Canada).

Northern blots

Total RNA was isolated using TRIzol (Life Technologies, Burlington, VT) according to the manufacturer’s protocol, with modifications. Briefly, 25 x 106 PMNs were homogenized in 1 ml TRIzol, and 200 µl of chloroform were added. After a brief mixing by vortex, samples were centrifuged at 10,000 x g for 15 min at 4°C. Following centrifugation, the upper aqueous phase was transferred to a tube containing an equal volume of isopropanol. The mixtures were thoroughly vortexed and centrifuged at 12,000 x g for 10 min at 4°C. Supernatants were carefully discarded, and the precipitated RNA pellets were washed using 1 ml of 70% ethanol. RNA pellets were centrifuged at 12,000 x g for 5 min at RT. The supernatants were discarded, and the pellets were allowed to air-dry for 2–3 min. RNA pellets were resuspended in diethyl pyrocarbonate-treated water, and RNA was quantitated by measuring the OD260. Total RNA (5–10 µg/well) was migrated on an agarose/formaldehyde gel and transferred onto a nylon filter (Hybond-XL), using a Vacugene XL transfer apparatus (Amersham Pharmacia Biotech, Baie d’Urfé, Quebec, Canada). RNA was fixed using a UV crosslinker according to the manufacturer’s specifications (Amersham Pharmacia Biotech). The filters were hybridized with a human COX-2 cDNA probe, labeled with [{alpha}-32P]dCTP using a Neblot kit (New England Biolabs, Beverly, MA). The COX-2 cDNA probe was generated by RT-PCR as described previously (12). Integrity of the RNA and equal loading were assessed by hybridization of the filters with GAPDH. Bands were revealed and analyzed with a BAS-1800 bio-imaging analyzer (Fuji Medical Systems, Stamford, CT).

Measurement of 5-LO metabolites by reversed phase (RP)-HPLC

Following stimulation, reactions were stopped and the samples were processed for RP-HPLC analysis, as previously described (40). RP-HPLC analyses were performed using an on-line extraction procedure with detection limits of 0.2 ng at 280 nm and 1 ng at 229 nm (41).

Measurement of prostanoids by ELISA

Production of PGE2 and TXA2 was measured using commercial ELISA kits (Cayman Chemicals). Cross-reactivities in the PGE2 ELISA were <0.04% for 6-keto PGF1{alpha} and <0.01% for LTB4, TXB2, and AA.

Densitometry

Immunoblot autoradiograms were digitalized using a Snapscan 1236 scanner (Agfa, Woburn, MA). Densitometric analyses of autoradiograms were performed with NIH Image (http://rsb.info.nih.gov).

Statistical analysis

Where applicable, statistical analysis was performed by Student’s nonpaired t test (two-tailed), and significance (*) was considered to be attained when p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Adenosine increases the expression of COX-2 in stimulated PMNs

To evaluate the impact of adenosine on COX-2 expression, PMNs were incubated with a surrogate of microbial-derived formylated proteins, fMLP, alone or with ADA, an enzyme which prevents accumulation of extracellular adenosine and thus activation of the adenosine receptors. Alternatively, PMNs were incubated with ADA and CGS 21680, a stable agonist of the A2AR resistant to the action of ADA (42). As earlier observed (12), fMLP induced a significant COX-2 protein expression in PMNs after 60 min of incubation (Fig. 1GoA). Enzymatic elimination of extracellular adenosine with ADA decreased the fMLP-stimulated expression of COX-2 by ~25%, as determined by densitometric analysis of the COX-2 doublets. In contrast, the presence of CGS 21680 in the incubation medium caused a superinduction of COX-2 expression. In nonstimulated cells, ADA and CGS 21680 had no detectable effect on COX-2 levels. Densitometric analyses revealed a 3-fold increase of protein expression in fMLP plus CGS 21680-treated cells when compared with fMLP-treated PMNs. Comparable results were observed after 4 h of incubation (Fig. 1GoB), pointing to a sustained impact of adenosine on COX-2 expression. In each of the above-mentioned conditions, protein expression of the TX-synthase enzyme remained essentially unchanged. Superinduction of COX-2 by adenosine was not unique to fMLP and was obtained in cells stimulated with other agonists, including LPS, a phorbol ester, or with a mixture of GM-CSF and TNF-{alpha}, where withdrawal of adenosine consistently resulted in decreased levels of COX-2 (data not shown).



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  		FIGURE 1. Adenosine potentiates the stimulation of COX-2 expression by fMLP. A, PMNs were incubated for 1 h with fMLP, alone or in combination with 0.1 U/ml ADA alone or ADA plus 1 µM CGS 21680, a selective agonist of the A2AR. Reactions were stopped and samples were processed for determination of COX-2 and TX-synthase protein expression by Western immunoblotting as described in Materials and Methods. The bar graph depicts the expression of COX-2 protein levels relative to that from fMLP-treated PMNs, as determined by densitometric analysis of the bands in three independent experiments (mean ± SEM; n = 3). B, PMNs were treated as indicated in A but stimulated with fMLP for 4 h. Samples were processed for determination of COX-2 protein expression by Western immunoblotting. In each panel, the immunoblots shown are from one experiment representative of three independent experiments performed with PMNs from different donors.

 
To confirm the involvement of the A2AR on COX-2 protein expression, cells were incubated with CSC, a highly selective antagonist of the A2AR (43), or with CGS 15943, an antagonist of both A1 and A2A receptor subtypes, before incubation with fMLP. In these experiments, COX-2 protein levels were significantly reduced by either of the antagonists (Fig. 2Go), supporting the concept of an A2AR-mediated event.



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  		FIGURE 2. Potentiation of COX-2 expression by adenosine implicates activation of the A2AR. PMNs were incubated with fMLP for the indicated times, alone or in combination with 1 µM CSC, a selective antagonist of the A2AR, or with 1 µM CGS 15943, an antagonist of both A1 and A2A receptors. Reactions were stopped and samples were processed for determination of COX-2 protein expression. Immunoblots are from one experiment representative of three independent experiments performed with PMNs from different donors.

 
Potentiation of COX-2 expression by adenosine could also be observed at the mRNA level. In PMN preparations treated with fMLP, withdrawal of extracellular adenosine efficiently prevented the up-regulation of COX-2 mRNA levels, while activation of the A2AR with CGS 21680 maintained the up-regulation of the COX-2 mRNA levels (Fig. 3Go). Maximal increase was observed after 15 min of incubation with fMLP. In fMLP-treated cells, potentiation of COX-2 mRNA levels by CGS 21680 was comparable to that caused by endogenous adenosine (observed in the absence of ADA). Of interest, removal of adenosine completely prevented the fMLP-induced up-regulation of COX-2 mRNA, while it decreased that of the protein only in part, suggesting that activation of the A2AR may have an impact on COX-2 mRNA steady-state levels as well as on translational processes. Together, these results indicate that adenosine can increase COX-2 levels in inflammatory PMNs through activation of the A2AR, and raise the possibility that adenosine has an impact on the capacity of inflammatory PMNs to generate prostanoids.



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  		FIGURE 3. Adenosine up-regulates COX-2 mRNA steady-state levels. PMNs were incubated for the indicated times with fMLP, alone or in combination with ADA or ADA and CGS 21680. Reactions were stopped and samples were processed for the determination of COX-2 mRNA levels by Northern blotting, as described in Materials and Methods. In each panel, results shown are from one experiment representative of three independent experiments performed with PMNs from different donors.

 
Intracellular cAMP levels may mediate the impact of adenosine on COX-2

The immediate intracellular events following activation of the A2AR are not fully characterized. However, positive coupling of the A2AR with the adenylyl cyclase pathway in PMNs has been reported (44). Also, a recently published study showed that activation of the A2AR can significantly delay degradation of cAMP in stimulated PMNs (45), supporting the concept that intracellular cAMP may mediate some of adenosine’s activities. Therefore, we examined the impact that intracellular cAMP levels have on the expression of COX-2 in PMNs. COX-2 was induced by incubation of the cells with fMLP, alone or in combination with a cell-permeable stable analog of cAMP, S-P-cAMP-acetoxymethyl, or a mixture containing a direct activator of adenylyl cyclase (forskolin) and an inhibitor of phosphodiesterase IV (RO-20-1724). Pharmacological elevation of intracellular cAMP levels by either of these two distinct approaches caused an induction of COX-2 in otherwise nonstimulated PMNs, and mimicked activation of the A2AR in causing a superinduction of COX-2 in fMLP-stimulated PMNs (Fig. 4Go). In contrast, TX-synthase levels remained relatively stable. These results support the hypothesis that an elevation in intracellular cAMP may mediate the adenosine-driven potentiation of COX-2 expression.



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  		FIGURE 4. Agents that elevate intracellular cAMP levels potentiate the fMLP-stimulated expression of COX-2. PMNs were incubated for 1 h with fMLP, alone or in combination with an activator of adenylyl cyclase and an inhibitor of phosphodiesterase IV, forskolin (50 µM) and RO-20-1724 (10 µM), respectively; or in combination with a cAMP analog, S-P-cAMP-AM (50 µM). Reactions were stopped, and samples were processed for determination of COX-2 and TX synthase protein expression by Western immunoblotting. The autoradiogram shown is from one experiment that is representative of three independent experiments, each performed with PMNs from different donors.

 
Adenosine increases the generation of PGE2 from AA-stimulated PMNs

In view of the impact of adenosine on COX-2 expression, we investigated the effect of adenosine on the capacity of COX-2-expressing PMNs to generate PGE2 from AA. For these experiments, PMNs were incubated for 60 min in the absence or presence of fMLP, before stimulating them with 3 µM AA for 5 min. Induction of COX-2 by fMLP significantly enhanced the capacity of PMNs to generate PGE2 in response to exogenous AA, and CGS 21680 augmented this response by 2- to 3-fold (Fig. 5Go). Removal of extracellular adenosine caused a 25% decrease in PGE2 production, in correlation with the COX-2 protein levels observed in this condition. In contrast to the decrease in PGE2, generation of TXA2 remained stable; neither ADA nor CGS 21680 significantly affected its generation as determined by levels of its stable metabolite TXB2. This latter result suggests that TX-synthase may constitute a limiting step, a hypothesis supported by the fact that in PMNs, PGE2 generation correlates with increasing concentrations of exogenous AA, up to 50 µM, while that of TXA2 reaches a plateau at a concentration of 3 µM (M. Pouliot, unpublished observations). Together, these results show that, in addition to potentiating the expression of COX-2, the activation of the PMN A2AR can have a positive regulatory effect on the generation of COX-2-derived PGE2.



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  		FIGURE 5. Activation of the A2AR increases the capacity of PMNs to generate COX-2-derived PGE2 from AA. PMNs were incubated for 1 h with fMLP, alone or in combination with ADA and CGS 21680, then stimulated with 3 µM AA for 5 min. Reactions were stopped, and samples processed for PGE2 and TXA2 determination, as described in the Materials and Methods section. Bars illustrate the generation of PGE2 (light bars) and TXB2 (dark bars), the stable metabolite of TXA2, from AA-stimulated PMNs. Data are expressed as percentage of control (mean ± SEM; n = 3), i.e., the generation of PGE2 obtained from stimulated PMNs incubated with fMLP alone. PGE2, 100% = 4914 ± 355 pg/107 PMNs (mean ± SEM, n = 6). TXB2, 100% = 23.7 ± 2.1 ng/107 PMNs (mean ± SEM, n = 3). *, Significantly different from the fMLP-treated group.

 
Extracts from stimulated PMNs were analyzed by liquid chromatography-tandem mass spectrometry to confirm identity of the metabolites. Analysis of the parent ion/daughter ion transitions (M/Z- 351/175 for PGE2 and M/Z- 369/169 for TXB2) (46) confirmed the presence of these eicosanoids in extracts from stimulated PMNs, as reported earlier (12). In contrast, the M/Z- 319/179 transition, which is specific to 12-HETE, was not detected in any of the analyzed samples, showing that PMN preparations were relatively free of platelets that contain high levels of 12-LO activity (data not shown).

A preeminent role of COX-2 over COX-1 for the generation of PGE2 and TXA2 is now well established for PMNs incubated with a number of agonists (9, 10, 12, 47). We addressed the relative involvement of these two COX isoforms from PMNs incubated with fMLP in PGE2 and TXA2 biosynthesis from endogenous and exogenous AA. To this end, the following selection of inhibitors of COX activity were used: NS-398 and DFU, two inhibitors showing high specificity for the COX-2 isoform, and indomethacin, a less-specific inhibitor that preferentially inhibits COX-1. These compounds were used in experiments in which PMNs were incubated with fMLP for 60 min and then stimulated with either exogenous AA or the Ca2+-ionophore A23187 for the release of endogenous AA. All inhibitors prevented PGE2 and TXA2 production from AA with comparable potency in a concentration-dependent fashion (Fig. 6Go, A and B). These concentration-response experiments allowed us to determine the minimal concentration required to obtain optimal inhibition of prostanoid generation by PMNs. At that concentration (10 µM), the magnitude of inhibition of PGE2 formation ranged between 60 and 70% for either NS-398 and DFU (Fig. 6GoC). In AA-stimulated PMNs, the less-specific COX inhibitor, indomethacin, was more potent, with an inhibition of 92%. All inhibitors were equally efficient at blocking PGE2 and TXA2 production from endogenous AA (Fig. 6GoD). These results, together with the facts that the COX-1 isoform is weakly detected in PMNs (9, 10, 11, 12) and its expression remains relatively stable regardless of the treatment (9, 11, 12), support an important role for the PMN inducible COX isoform (COX-2) in the generation of PGE2 and TXA2 from exogenous and endogenous AA.



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  		FIGURE 6. COX-2 is the preeminent isoform accounting for the generation of prostanoids from stimulated PMNs. A and B, PMNs were incubated with fMLP for 1 h and stimulated with AA for 5 min, alone or in the presence of the indicated concentrations of the following COX inhibitors: indomethacin (Indo), NS 398, or DFU. Reactions were stopped and samples processed for PGE2 (A) and TXB2 (B) determination by ELISA. Results presented are from one experiment that is representative of three independent experiments performed in identical conditions with PMNs from different donors. C and D, fMLP-treated PMNs were stimulated in the presence of inhibitors at the concentration of 10 µM; prostanoid generation from PMNs stimulated with 3 µM AA (C) or 1 µM A23187 (D) was determined. Results are expressed as the percentage of inhibition of prostanoid synthesis (mean ± SEM, n = 3).

 
PGE2 inhibits LTB4 generation from exogenous AA via activation of the EP2 receptor

Because PGE2 and TXA2 were found to be the major metabolites resulting from COX-2 activity in stimulated PMNs (9, 12), we sought to determine the impact of PGE2 and TXA2 on the generation of LTB4, a potent chemoattractant for PMNs. PGE2 was identified earlier as an inhibitory agent for LTB4 generation; however, the inhibition was partial and observed at very high concentrations (32). In view of the studies of Krump and colleagues (48, 49, 50), which thoroughly documented the inhibitory effect of extracellular adenosine on LTB4 generation, it seemed likely that adenosine accumulating in cell suspensions could mask the inhibitory effect of PGE2 on LTB4. Therefore, we determined the effect of PGE2 and TXA2 on LTB4 generation in the absence of extracellular adenosine, a condition allowing optimal formation of LTB4 (39). Fresh PMNs were preincubated in presence of ADA and incubated with increasing concentrations of PGE2 before stimulation with AA. As shown in Fig. 7GoA, PGE2 almost completely inhibited LTB4 formation in a concentration-dependent fashion; a significant inhibition was observed with a concentration as low as 100 pM PGE2, and inhibition was nearly total at 100 nM (apparent IC50 = 2 nM). I-BOP and U 46619, two stable mimetics of TXA2, had no significant effect on LTB4 generation, up to the highest concentration tested. Available pharmacological evidence strongly supports an important role for the EP2 subtype in mediating the activities of PGE2 in PMNs. Accordingly, butaprost, a stable PGE2 analog showing specificity toward the EP2 receptor subtype, had an effect comparable to that of PGE2 in preventing LTB4 formation in adenosine-free conditions (Fig. 7GoB). Preincubation of the cells with AH 6809, an antagonist of the EP1 and EP2 receptor subtypes, reversed the inhibitory effect of an intermediate effective concentration of PGE2 (3 nM) on LTB4 formation, also in a concentration-dependent manner (Fig. 7GoC). Together, these results emphasize the potent inhibitory impact of PGE2 on LTB4 generation and support an EP2 receptor-mediated event. Moreover, these results suggest that adenosine and PGE2 can act in concert in limiting LTB4 generation by PMNs.



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  		FIGURE 7. PGE2 decreases LTB4 formation from AA-stimulated PMNs via activation of the EP2 receptor. A, Freshly isolated PMNs were preincubated with ADA and the indicated concentrations of PGE2 for 15 min and then stimulated with 3 µM AA for 5 min. B, PMNs were treated with 3 nM PGE2 in combination with the indicated concentrations of butaprost, a specific agonist of the PG EP2 receptor subtype, before stimulation with AA. C, PMNs were treated with 3 nM PGE2 in combination with the indicated concentrations of AH 6809, a specific antagonist of the EP1-EP2 receptor subtypes, before stimulation with AA. Reactions were stopped, and samples processed for LTB4 determination by RP-HPLC, as described in Materials and Methods. For each panel, results are presented as the mean ± SEM from three independent experiments and are expressed as the percentage of control, i.e., the generation of LTB4 obtained from PMNs incubated with ADA and stimulated with AA alone. 100% = 55.2 ± 15.1 ng/107 PMNs (mean ± SEM; n = 14). In the absence of ADA, the generation of LTB4 was 4.1 ± 1.3 ng/107 PMNs (mean ± SEM; n = 4).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The receptors mediating adenosine’s actions are of one or more of the following four subtypes: A1, A2A, A2B, and A3 (51). PMNs express both A1 and A2 (now known as A2A) receptors: A1 are stimulatory, while the A2A subtype mediates adenosine-induced inhibition of a number of PMN functions (36). Studies demonstrating the presence of A2AR in human circulating blood cells strongly suggest that adenosine could play an important role in modulating aggregatory, immune, and inflammatory processes and that activation of A2AR may have therapeutic potential (49, 52). In particular, studies with PMNs have indicated that adenosine inhibits adhesion, generation of superoxide anions, phagocytosis, as well as generation of LTB4 (37, 50) and platelet-activating factor (P. Borgeat, unpublished data). Results from the present study indicate that, in inflammatory PMNs, the potentiation of the COX-2 pathway by adenosine is also obtained via activation of the A2AR subtype.

Intracellular signaling events that mediate the actions of adenosine on PMNs are not thoroughly characterized. Activation of the A2AR often results in an elevation of intracellular cAMP, which in turn results in an inhibition of functions, such as degranulation, locomotion, and H2O2 synthesis (53). However, it is not clear whether all of the inhibitory activities of adenosine on PMN functions depend on an elevation of intracellular cAMP levels (54). In fact, some studies have shown that the inhibition of superoxide anion synthesis was not blocked by a protein kinase A inhibitor (55), nor could the inhibition be mimicked by cAMP, underlining the probable involvement of other kinases or signaling pathways. It was also recently proposed that adenosine suppresses superoxide anion formation in PMNs through activation of a serine/threonine protein phosphatase (56). This does not exclude the possibility that other inhibitory effects of adenosine are mediated by cAMP, and our results further support a mediating role for intracellular cAMP in the superinduction of COX-2 in PMNs, a situation previously observed in other cell types (57).

In addition to the expression of COX-2, adenosine also augmented the capacity of PMNs to generate PGE2 from endogenous and exogenous AA sources, as expected. Therefore, PMNs exposed to high concentrations of adenosine are likely to produce more PGE2 and less LTB4 in response to free AA available at inflammatory sites. This shift in lipid mediator generation caused by adenosine may well represent a new facet of the anti-inflammatory spectrum of activities attributed to this autacoid.

PGE2 has a well-documented inhibitory impact on leukocyte inflammatory functions, both in vitro and in vivo. In PMNs, PGE2 has been shown to inhibit chemotaxis, aggregation, superoxide production, lysosomal release, and LTB4 generation, by raising intracellular cAMP above basal levels (28, 29, 30). However, the original observation for inhibition of LTB4 by PGE2 required relatively high concentrations of the latter (32). In our experiments, withdrawal of extracellular adenosine allowed us to further define the action of PGE2, which potently inhibited LTB4 formation from stimulated PMNs in a concentration-dependent fashion. In contrast, TXA2 formation was not significantly affected in any of the experimental conditions tested, and TXA2 mimetics did not interfere with LTB4 generation. Taken together, these data suggest that adenosine and PGE2 both constitute potent anti-inflammatory signals for PMNs and share some common mechanisms of action, possibly the elevation of intracellular cAMP. It is reasonable to submit that an increase of PGE2 generation from PMNs by adenosine may contribute to limiting further leukocyte activation and that the up-regulation of COX-2 in inflammatory PMNs is actually part of a "stop signal," notably against further leukocyte recruitment. Accordingly, a recent study (58) demonstrated that PMNs exposed to PGE2 switched eicosanoid biosynthesis, via the second messenger cAMP, from predominantly LTB4- and 5-LO-initiated pathways to lipoxin A4, a 15-LO product that terminated PMN infiltration. Therefore, PGE2 may cause a shift to anti-inflammatory lipid generation by leukocytes, thus promoting resolution.

Accumulation of extracellular adenosine in inflamed and damaged tissues and the immunosuppressive properties of cAMP-elevating adenosine receptors indicate that signaling via A2AR possibly constitutes an endogenous mechanism of inhibition of inflammation (35). Adenosine concentration measured in different biological media, including the synovial fluids of rheumatoid arthritis patients (59), ranged from low to high micromolar concentration, suggesting that the physiological modulation of PMN functions through A2AR is possible and even likely. In the generic murine air-pouch model, the anti-inflammatory effects of aspirin and sodium salicylate have been shown to be largely mediated by adenosine, rather than through COX inhibition (60). In turn, a pharmacological control of extracellular concentrations of adenosine or the use of adenosine analogs selectively activating the A2AR may lead to a novel class of potent anti-inflammatory agents capable of controlling a wide range of inflammatory processes (61).

Significant progress has been achieved over the past two decades in the therapy of inflammation. The introduction of methotrexate, selective COX-2 inhibitors, and anti-cytokine drugs represented major steps and improved our abilities to treat inflammatory diseases such as rheumatoid arthritis. However, there is still an urgent need for more efficient and more targeted treatments that present fewer side effects. Phosphodiesterase inhibitors, which inhibit the degradation of cAMP, are currently under development, and adenosine A2AR agonists are also currently being investigated for use in inflammatory diseases (35). Interestingly, PG mimetics represent a third class of compound with similar anti-inflammatory potential. These three classes of compounds activate a common anti-inflammatory signaling pathway involving rises of cAMP in inflammatory cells.

In conclusion, data from this study demonstrate that adenosine potentiates the COX-2 pathway in human inflammatory PMNs, a situation that may have anti-inflammatory consequences. Adenosine may promote a self-limiting regulatory process through the increase of PGE2 generation, which may result in a reduction of LTB4 generation as well as inhibition of PMN-dependent pathophysiological events. This study identifies a new aspect of the anti-inflammatory properties of adenosine in leukocytes and supports the concept that this autacoid may exert some of its immunomodulatory activities in part by modifying the balance of lipid mediators generated by PMNs.


   Acknowledgments
 
We thank Serge Picard and Line Bouchard for excellent technical assistance and Nicolas Flamand for helpful discussions.


   Footnotes
 
1 This work was supported by grants from the Canadian Institutes of Health Research and the Canadian Arthritis Network (to P.B. and M.P.) and from the Banting Research Foundation (to M.P.). Back

2 Address correspondence and reprint requests to Dr. Marc Pouliot, Centre de Recherche en Rhumatologie et Immunologie, Centre Hospitalier de l’Université Laval, 2705 Laurier Boulevard, Office T1-49, Sainte-Foy, Québec, Canada, G1V 4G2. E-mail address: Marc.Pouliot{at}crchul.ulaval.ca Back

3 Abbreviations used in this paper: PMN, polymorphonuclear neutrophil (granulocyte); LO, lipoxygenase; AA, arachidonic acid; ADA, adenosine deaminase; COX, cyclooxygenase; CSC, 8-(3-chlorostyryl)caffeine; DFU, 5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulfonyl)phenyl-2(5H)-furanone; LT, leukotriene; RP-HPLC, reversed phase-HPLC; TX, thromboxane; RT, room temperature; GCS 21680, 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride; CGS 15943, 9-chloro-2-(2-furyl)[1,2,4]triazolo[1,5-c]quinazolin-5-amine. Back

Received for publication May 8, 2002. Accepted for publication September 4, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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