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CELLULAR AND MOLECULAR

cDNA Microarray Analysis Reveals a Nuclear Factor-B-Independent Regulation of Macrophage Function by Adenosine

Departments of Surgery (Z.H.N., E.A.D., C.S., G.H.) and Cell Biology and Molecular Medicine (S.J.L.), University of Medicine and Dentistry-New Jersey Medical School, Newark, New Jersey; Department of Pharmacology (E.S.V., G.H.), Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary; and Institute of Human Physiology and Clinical Experimental Research (C.S.), Semmelweis University of Medicine, Budapest, Hungary

Received for publication April 10, 2003
Accepted May 15, 2003.

	Abstract

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Adenosine is released into the extracellular space from nerve terminals and cells subjected to ischemic stress. This nucleoside modulates a plethora of cellular functions via occupancy of specific receptors. Adenosine is also an important endogenous regulator of macrophage function, because it suppresses the production of a number of proinflammatory cytokines by these cells. However, the mechanisms of this anti-inflammatory effect have not been well characterized. We hypothesized that adenosine may exert some of its anti-inflammatory effects by decreasing activation of the transcription factor nuclear factor-B (NF-B), because gene expression of most of the proinflammatory cytokines inhibited by adenosine is dependent on NF-B activation. Using bacterial lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages, we found that adenosine as well as adenosine receptor agonists decreased the production of tumor necrosis factor (TNF)-, a typical NF-B-regulated cytokine. This effect of adenosine was not due to an action on the process of TNF- release, because adenosine suppressed also the intracellular levels of TNF-. However, cDNA microarray analysis revealed that mRNA levels of neither TNF- nor other cytokines were altered by adenosine in either LPS-activated or quiescent macrophages. In addition, although LPS induced expression of a number of other, noncytokine genes, including the adenosine A2b receptor, adenosine did not affect the expression of these genes. Furthermore, adenosine as well as adenosine receptor agonists failed to decrease LPS-induced NF-B DNA binding, NF-B promoter activity, p65 nuclear translocation, and inhibitory B degradation. Together, our results suggest that the anti-inflammatory effects of adenosine are independent of NF-B.

Monocytes/macrophages have recently emerged as prime targets of the immunomodulatory effects of adenosine (Haskó and Szabó, 1998; Haskó et al., 2002b). Adenosine exerts its biological effects by engaging cell surface receptors. Adenosine receptors have been subdivided according to molecular, biochemical, and pharmacological evidence into four sub-types, which are the A1, A2a, A2b, and A3 receptors (Ralevic and Burnstock, 1998). Cells of the monocyte/macrophage lineage have been documented to express all four adenosine receptors (Haskó and Szabó, 1998; Haskó et al., 2002b). In most in vitro and in vivo studies using macrophages, stimulation of adenosine receptors has been shown to result in an anti-inflammatory, deactivated macrophage phenotype (Haskó and Szabó, 1998; Haskó et al., 2002b). In bacterial lipopolysaccharide (LPS)-stimulated monocytes/macrophages, adenosine receptor stimulation reduces the production of a variety of proinflammatory cytokines, including tumor necrosis factor (TNF)- (Bouma et al., 1994; Haskó et al., 1996; McWhinney et al., 1996; Sajjadi et al., 1996; Mayne et al., 1999; Elenkov et al., 2000; Mayne et al., 2001; Leibovich et al., 2002), interleukin-12 (Haskó et al., 2000; Link et al., 2000; Khoa et al., 2001; Bshesh et al., 2002), and macrophage inflammatory protein (MIP)-1 (Szabó et al., 1998). Furthermore, adenosine receptor stimulation prevents the induction of inducible nitric-oxide synthase and the formation of nitric oxide (Haskó et al., 1996; Xaus et al., 1999). In addition to the suppressive effect of adenosine on the production of these various soluble macrophage products, adenosine decreases expression of the membrane protein major histocompatibility complex II (Edwards et al., 1994; Xaus et al., 1999).

Based on the above-mentioned evidence that adenosine suppresses the expression of proinflammatory chemokines, inducible nitric-oxide synthase, and major histocompatibility complex II, which are all dependent on the transcription factor nuclear factor-B (NF-B) (Baeuerle and Henkel, 1994; Karin and Ben-Neriah, 2000; Haddad, 2002), we hypothesized that adenosine exerts its anti-inflammatory effects by diminishing the activation of this transcription factor system.

	Materials and Methods

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Cell Culture. The mouse macrophage cell line RAW 264.7 was grown in Dulbeccos's modified Eagle's medium supplemented with 10% fetal bovine serum, 50 U/ml penicillin, 50 µg/ml streptomycin, and 1.5 mg/ml sodium bicarbonate in a humidified atmosphere of 95% air and 5% CO₂.

Drugs and Reagents. The nonselective adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine; the selective A₁ receptor agonists N⁶-cyclopentyladenosine, 2-chloro-N⁶-cyclopentyladenosine, N⁶-cyclohexyladenosine, and R-(-)-N⁶-phenylisopropyladenosine; the A_2a receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethyl-carboxamidoadenosine (CGS-21680); and the A₃ receptor agonist N⁶-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) were obtained from Sigma/RBI (Natick, MA). Adenosine, MTT, and LPS (Escherichia coli 055:B5) were purchased from Sigma-Aldrich (St. Louis, MO).

TNF- Determination from Cell Supernatants and Cell Extracts. Cells in 96-well plates were treated with adenosine or various adenosine receptor agonists 30 min before the addition of 10 µg/ml LPS. Supernatants for TNF- determination were obtained 4 h after stimulation with LPS. For the determination of intracellular TNF-, macrophages in 24-well plates were pretreated with adenosine followed by LPS (10 µg/ml) stimulation 30 min later. After an additional 6-h incubation, the supernatants were removed and the cells were lysed as described previously (Haskó et al., 2002a). TNF- levels in cell supernatants or cell lysates were determined by ELISA, as we have described previously (Haskó et al., 2002a).

NF-B Electromobility Shift Assay (EMSA). RAW 264.7 cells were stimulated with LPS (10 µg/ml) for 45 min and nuclear protein extracts were prepared as described previously (Németh et al., 2002). To determine the effect of adenosine receptor agonists, cells were pretreated with these agents or their vehicle 30 min before stimulation. All nuclear extraction procedures were performed on ice with ice-cold reagents. Cells were washed with PBS and harvested by scraping into 1.5 ml of PBS and pelleted at 1,500g for 10 min. The pellet was resuspended in 60 µl of cytosolic lysis buffer [20% (v/v) glycerol, 10 mM HEPES pH 8.0, 10 mM KCl, 0.5 mM EDTA pH 8.0, 1.5 mM MgCl₂, 0.5% (v/v) Nonidet P-40, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin A] and incubated for 15 min on ice with occasional vortexing. After centrifugation at 4,500g for 10 min, supernatants (cytosolic extracts) were discarded. Two cell pellet volume of nuclear extraction buffer [20% (v/v) glycerol, 20 mM HEPES pH 8.0, 420 mM NaCl, 0.5 mM EDTA pH 8.0, 1.5 mM MgCl₂, 50 mM glycerol-phosphate, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin A] was added to the nuclear pellet and incubated on ice for 30 min with occasional vortexing. Nuclear proteins were isolated by centrifugation at 14,000g for 15 min. Protein concentrations were determined using the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA). Nuclear extracts were aliquoted and stored at -80°C until used for EMSA. The NF-B consensus oligonucleotide probe used for the EMSA was purchased from Promega (Madison, WI). Oligonucleotide probes were labeled with [-³²P]ATP using T4 polynucleotide kinase (Invitrogen, Carlsbad, CA) and purified in MicroSpin G-50 columns (Amersham Biosciences, Inc., Piscataway, NJ). For the EMSA analysis, 8 to 12 µg of nuclear proteins was preincubated with EMSA binding buffer [8% glycerol (v/v), 10 mM Tris-HCl pH 8.0, 2 mM MgCl₂, 0.5 mM EDTA pH 8.0, and 0.5 mM dithiothreitol) as well as 15 ng/µl poly(dI)-poly(dC), 0.4 ng/µl of single-stranded DNA, and 0.2 mg/ml of bovine serum albumin at room temperature for 10 min before addition of the radiolabeled oligonucleotide for an additional 25 min. Protein-nucleic acid complexes were resolved using a non-denaturing polyacrylamide gel consisting of 4% acrylamide (29:1 ratio of acrylamide/bisacrylamide) and run in 0.5x Tris borate-EDTA buffer (44.5 mM Tris-base, 44.5 mM boric aid, and 1 mM EDTA) for approximately 2.5 h at constant current (35 mA). Gels were transferred to 3M paper (Whatman, Maidstone, UK), dried under vacuum at 80°C for 40 min, and exposed to photographic film at -80°C with an intensifying screen. For supershift studies, before addition of the radiolabeled probe, samples were incubated for 30 min with 4 µg of isotype control (rabbit polyclonal IgG Mad 1 antibody, sc-222X), p65 (sc-109X), or p50 (sc-114X) antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Transient Transfection and Luciferase Activity. For transient transfections, 3 x 10⁵ RAW 264.7 cells were seeded per well of a 24-well tissue culture dish 1 day before transient transfection. Cells were transfected with 15 µl/ml FuGENE 6 transfection reagent (Roche Diagnostics, Indianapolis, IN) in 160 µl of medium per well. The medium contained 5 µg/ml DNA containing a NF-B luciferase promoter construct (BD Biosciences Clontech, San Diego, CA). After an overnight transfection, the cells were pretreated with adenosine (100 µM) or its vehicle (medium) for 30 min, which was followed by stimulation with LPS (10 µg/ml) for 6 h. Luciferase activity was measured by the luciferase reporter assay system (Promega) and normalized relative to micrograms of protein, as we have described previously (Németh et al., 2002).

Western Blot Analysis of Inhibitory B (IB) and p65. p65 levels were analyzed using the nuclear extracts prepared for the EMSAs. For IB Western blotting, RAW cells in six-well plates were pretreated with adenosine (100 µM) or vehicle and 30 min later the cells were stimulated with LPS (10 µg/ml) for 30 min. After washing with PBS, the cells were lysed by the addition of modified radioimmunoprecipitation (radioimmunoprecipitation assay) buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 0.25% Na-deoxycholate, 1% Nonidet P-40, 1 µg/ml pepstatin, 1 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 1 mM Na₃VO₄). The lysates were transferred to Eppendorf tubes, centrifuged at 15,000g, and the supernatant was recovered. Protein concentrations were determined using the Bio-Rad protein assay. Sample (10-20 µg) was separated on an 8 to 16% Tris-glycine gel (Invitrogen) and transferred to a nitrocellulose membrane. The membranes were probed with anti-p65 (Santa Cruz Biotechnology, Inc.) or anti-IB (Cell Signaling Technology, Inc., Beverly, MA) and subsequently incubated with a secondary horseradish peroxidase-conjugated donkey anti-rabbit antibody (Roche Diagnostics). Bands were detected using enhanced chemiluminescence Western blotting detection reagent (Amersham Biosciences, Inc.).

Affymetrix Gene Chip Analysis. RAW cells were plated in six-well plates 1 day before the experiment. The cells were activated with LPS for 3 h or exposed to the vehicle for LPS (medium). Adenosine (100 µM) or its vehicle was added to the cells 30 min before the LPS challenge. Thus, the following four experimental groups were designed: vehicle for adenosine + vehicle for LPS, vehicle for adenosine + LPS, adenosine + LPS, and adenosine + vehicle for LPS. With the exception of the adenosine + vehicle group, where n was 3, all groups contained an n of 4. These numbers were the result of two independent experiments performed on two different experimental days (two samples from both experiments for all the groups with the exception of the adenosine + vehicle group, where two samples were obtained from the first experiment and one from the other). Total RNA was prepared using TRIzol (Invitrogen) and further purified using RNeasy mini kit (QIAGEN, Valencia, CA). Thereafter, total RNA was reverse transcribed in 20 µl using superscript II (Invitrogen). Double-stranded DNA was created using a replacement reaction involving RNase H, ligase, and DNA polymerase I. The in vitro transcription was done using the Enzo high yield transcription kit, which incorporates the biotinylated ribonucleotides UTP and CTP. Equal amounts of fragmented cRNA were then hybridized to MGU74Av2 gene chips according to Affymetrix protocols (Santa Clara, CA) at the Biopolymers Facility (Harvard Medical School, Boston, MA). Chips were scanned and analyzed using GeneChip Analysis Suite software. Data sets of intensities of 12,488 probe sets per array were compared using Microsoft Excel (Microsoft, Redmond, WA) software. To identify differentially expressed genes, we excluded all genes from the analysis that were scored absent in any of the samples. Furthermore, the extended sequence tags were excluded from the analysis.

RT-PCR. These experiments were performed using RNA isolated for the microarray experiment. RNA (5 µg) was transcribed in a 20-µl reaction containing 10.7 µl of RNA (5 µg), 2 µl of 10x PCR buffer, 2 µl of 10 mM dNTP mix, 2 µl of 25 mM MgCl₂, 2 µl of 100 mM dithiothreitol, 0.5 µl of RNase inhibitor (20 U/µl; PerkinElmer Life Sciences, Boston, MA), 0.5 µl of 50 mM oligo d(T), and 0.3 µl of reverse transcriptase (Roche Diagnostics). The reaction mix was incubated at 42°C for 15 min for reverse transcription. Thereafter, the reverse transcriptase was inactivated at 99°C for 5 min. Reverse transcriptase-generated DNA was amplified using Expand high fidelity PCR system (Roche Diagnostics). The reaction buffer (25 µl) contained 2 µl of cDNA, water, 2.5 µl of PCR buffer, 1.5 µl of 25 mM MgCl₂, 1 µl of 10 mM dNTP mix, 0.5 µl of 10 µM oligonucleotide primer (each), and 0.2 µl of Taq polymerase. cDNA was amplified using the following primers and conditions: TNF- (Murray et al., 1990), 5'-GGCAGGTCTACTTTGGAGTCATTGC-3' (sense) and 5'-ACATTCGAGGCTCCAGTGAATTCGG-3' (antisense); A2b receptor (Zhao et al., 2002), 5'-TGGCGCTGGAGCTGGTTA-3' (sense) and 5'-GCAAAGGGGATGGCGAAG-3' (antisense); and A2a receptor (Hoskin et al., 2002) 5'-CACGCAGAGTTCCATCTTCA-3' (sense) and 5'-AGCAGTTGATGATGTGCAGG-3' (antisense); an initial denaturation at 94°C x 5 min, 22, 30, and 30 cycles of 94°C x 30 s for TNF-, A2b receptor, and A2a receptor, respectively, 58°C x 45 s, 72°C x 45 s; a final dwell at 72°C x 7 min. PCR products were resolved on a 1.5% agarose gel and stained with ethidium bromide (Németh et al., 2002).

Measurement of Mitochondrial Respiration. Mitochondrial respiration, an indicator of cell viability, was assessed by the mitochondria-dependent reduction of MTT to formazan (Németh et al., 2002). After the various treatments for cytokine measurements (see above), supernatants were removed and cells incubated with MTT (0.5 mg/ml) for 60 min at 37°C. Culture medium was removed by aspiration and the cells were solubilized in Me₂SO (100 µl/well). The extent of reduction of MTT to formazan within cells was quantitated by measurement of optical density at 550 nm using a Spectramax 250 microplate reader.

Statistical Evaluation. Values in the figures, tables, and text are expressed as mean ± S.E.M. of n observations. Statistical analysis of the data were performed by Student's t test or one-way analysis of variance followed by Dunnett's test, as appropriate.

	Results

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Adenosine Receptor Agonists Decrease TNF- Production and Intracellular TNF- Levels in LPS-Stimulated RAW 264.7 Macrophages. First, we examined whether adenosine receptor stimulation decreased the production of the NF-B-regulated cytokine TNF- by macrophages. Stimulation of cells with LPS for 4 h induced the release of TNF- into the medium. Adenosine (10-100 µM) pretreatment of cells 30 min before the LPS challenge reduced the release of TNF-, which occurred in a concentration-dependent manner (Fig. 1A). The adenosine receptor agonists N⁶-cyclopentyladenosine, 2-chloro-N⁶-cyclopentyladenosine, CGS-21680, 5'-N-ethylcarboxamidoadenosine, and IB-MECA all mimicked the effect of adenosine in suppressing the production of TNF- by LPS-stimulated RAW 264.7 cells (Fig. 1B). None of these purinergic agents had any effect on cell viability, as determined using the MTT assay (data not shown). These data obtained using LPS-stimulated RAW 264.7 cells confirm the previous observations of studies using other macrophage systems (Bouma et al., 1994; Haskó et al., 1996; McWhinney et al., 1996; Sajjadi et al., 1996; Mayne et al., 1999, 2001; Leibovich et al., 2002) that adenosine receptor stimulation attenuates the production of TNF-.

View larger version (20K): [in this window] [in a new window]   Fig. 1. A, adenosine inhibits TNF- production by RAW 264.7 macrophages stimulated with LPS (10 µg/ml) for 4 h. B, effect of various adenosine receptor agonists (10 µM) on LPS-induced TNF- production by RAW cells. Adenosine or the adenosine agonists were added to the cells 30 min before LPS administration. TNF- was measured from the supernatants using ELISA. Data are expressed as the mean ± S.E.M. of six wells. *, p < 0.05; **, p < 0.01.

Next, we asked the question, whether adenosine acted by decreasing the accumulation of intracellular TNF- or whether it affected the release of this cytokine. The results of this experiment showed that treatment of the cells with LPS induced the appearance of intracellular TNF-, which was suppressed by adenosine pretreatment (Fig. 2). These results indicate that adenosine does not interfere with the release process of TNF-.

View larger version (14K): [in this window] [in a new window]   Fig. 2. Adenosine decreases intracellular TNF- levels in RAW 264.7 macrophages. Cells were pretreated with adenosine (100 µM) and 30 min later the cells were exposed to LPS (10 µg/ml) for another 6 h. At the end of the incubation period, the adherent cells were lysed for the determination of intracellular TNF-. TNF- levels were determined by ELISA. Data are expressed as the mean ± S.E.M. of eight wells. **, p < 0.01.

Lack of Effect of Adenosine on NF-B Activation in RAW 264.7 Macrophages. Because NF-B is an important regulator of TNF- production by macrophages (Baeuerle and Henkel, 1994), we next tested the possibility that adenosine decreased activation of the NF-B transcription factor system. As shown in Fig. 3, A and B, using nuclear extracts from LPS-treated RAW 264.7 cells, we observed an increase in NF-B binding, compared with LPS-untreated cells. Supershift studies confirmed the observation by previous reports (Baeuerle and Henkel, 1994) that the DNA binding complex induced by LPS contained both p65 and p50 (Fig. 3A, right). However, neither adenosine (Fig. 3A) nor adenosine receptor agonists (Fig. 3B) affected this induction of NF-B DNA binding. Furthermore, adenosine did not prevent either the LPS-induced accumulation of p65 in the nucleus or LPS-elicited IB degradation (Fig. 4).

View larger version (50K): [in this window] [in a new window]   Fig. 3. Adenosine (ADO) pretreatment (100 µM) fails to decrease LPS (10 µg/ml)-induced NF-B DNA binding in RAW 264.7 cells (A). Pretreatment with a variety of adenosine receptor agonists (10 µM) does not affect LPS (10 µg/ml)-induced NF-B DNA binding in these cells (B). a, no LPS; b, LPS; c, agonist + LPS. Adenosine or the adenosine agonists were added to the cells 30 min before LPS administration. NF-B DNA binding was assessed from nuclear extracts obtained 45 min after LPS (10 µg/ml) stimulation using EMSA. This figure is representative of three separate experiments. CHA, cyclohexyladenosine; R-PIA, phenylisopropyladenosine; CCPA, chlorocyclopentyladenosine.

View larger version (25K): [in this window] [in a new window]   Fig. 4. Lack of effect of adenosine (100 µM) on LPS (10 µg/ml)-induced degradation of IB and the nuclear translocation of p65. RAW 264.7 cells were pretreated with vehicle (c) or adenosine (ADO) for 30 min followed by an LPS challenge for 30 min. The degradation of IB and p65 nuclear translocation activation were determined using Western blotting. This figure is representative of three separate experiments.

The possibility still existed that adenosine could prevent NF-B transcriptional activity without interfering with NF-B DNA binding. To test this hypothesis, we transiently transfected cells with a NF-B-luciferase reporter construct. Then, the transfectants were pretreated with adenosine or its vehicle for 30 min, which was followed by stimulation with LPS for 6 h. The effect of adenosine on NF-B-dependent gene transcription was assessed using the luciferase assay. Similar to results of the DNA binding experiments, adenosine failed to suppress LPS-stimulated NF-B-dependent gene transcription (Fig. 5). Finally, adenosine alone failed to affect NF-B-dependent gene transcription (data not shown).

View larger version (13K): [in this window] [in a new window]   Fig. 5. Adenosine pretreatment (100 µM) has no effect on LPS-induced NF-B-dependent transcriptional activity in RAW 264.7 cells transiently transfected with a NF-B-luciferase promoter construct. Adenosine was added to the cells 30 min before LPS administration. Luciferase activity was measured from cells lysed 6 h after LPS treatment and normalized to protein content. Data are mean ± S.E.M. of n = 14 to 16 wells from two separate experiments. **, p < 0.01 versus control.

Microarray Analysis of Gene Expression in RAW 264.7 Cells Treated with Adenosine and/or LPS. Stimulation with LPS induced a 2-fold induction of 98 genes after 3 h (Table 1), whereas 32 genes were repressed 2-fold by LPS at this time point (Table 2). However none of the LPS-induced genes, including the NF-B-regulated ones, such as TNF-, IB, and interleukin-1 receptor antagonist were altered by at least 1.5-fold by adenosine. In addition, none of the LPS-repressed genes was changed (at least 1.5-fold) by adenosine treatment. Adenosine (no LPS) treatment did not affect gene expression compared with treatment with vehicle (no LPS). Interestingly, although the A2a receptor mRNA was not expressed in either LPS-untreated or LPS-treated cells, the mRNA for A2b receptor was not present in LPS nonstimulated cells, but became detectable in LPS-stimulated cells (data not shown).

View this table: [in this window] [in a new window]   TABLE 2 Genes down-regulated after 3 h in LPS-stimulated RAW 264.7 macrophages Data are the average of four independent experiments and were analyzed as described under Materials and Methods. Genes shown were decreased 2-fold.

RT-PCR Analysis of TNF- and A2b Receptor Gene Expression. As shown in Fig. 6, RT-PCR analysis confirmed that TNF- mRNA was induced by LPS but was not affected by adenosine pretreatment. Furthermore, the A2b receptor was up-regulated in response to LPS, but was unchanged in adenosine-pretreated cells. Finally, it was confirmed using RT-PCR that the A2a receptor was not expressed in RAW cells (data not shown).

View larger version (21K): [in this window] [in a new window]   Fig. 6. LPS treatment for 3 h induces mRNA accumulation of both TNF- and the A2b adenosine receptor in RAW 264.7 cells. Adenosine pretreatment 30 min before LPS fails to affect mRNA levels of either TNF- or the A2b adenosine receptor. Furthermore, adenosine alone has no effect on mRNA levels of either TNF- or the A2b adenosine receptor compared with LPS-unstimulated (c) cells. mRNA levels of both TNF- and the A2b adenosine receptor were quantitated using RT-PCR.

	Discussion

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In this article, we examined the possibility that some of the anti-inflammatory effects of extracellular adenosine and adenosine receptor agonists observed in macrophages are mediated by a suppressive effect on the NF-B transcription factor system. The major finding of our study is that despite the fact that adenosine receptor stimulation decreased both extracellular and intracellular concentrations of TNF-, a prototype NF-B-regulated proinflammatory cytokine, adenosine did not interfere with NF-B activation. There are three lines of evidence to support this proposition. First, adenosine as well as a series of adenosine receptor agonists failed to decrease DNA binding of NF-B. Second, adenosine was unable to decrease NF-B-driven promoter activity of a luciferase construct. Finally, global analysis of gene expression using cDNA microarray demonstrated that although LPS induced expression of a number of NF-B-regulated genes, adenosine failed to alter this response.

Although these results argue against a role of NF-B and even a transcriptional effect of adenosine in macrophages, there are several caveats that need to be discussed. First, gene expression was assessed only at the 3-h time point, whereas it is possible that adenosine may affect gene expression at other time points. Second, although adenosine itself had no effect on the expression of cytokine mRNAs in the current study using RAW 264.7 macrophages, we found that the selective A3 adenosine receptor agonist IB-MECA decreased MIP-1 mRNA levels in the same cell type in a previous study (Szabó et al., 1998). Because adenosine itself is a relatively week agonist at A3 receptors (Linden, 2001), it is possible that selective A3 receptor stimulation can decrease the levels of cytokine mRNAs.

The mechanism of action for the macrophage-deactivating effect of adenosine is incompletely understood. A recent study by Sajjadi et al. (1996) demonstrated that adenosine decreased TNF- mRNA steady-state levels in an LPS-stimulated human monocytic cell line, which results are contradictory to our findings in LPS-stimulated mouse macrophages showing a failure of adenosine to inhibit TNF- mRNA accumulation. Nevertheless, this reduction in TNF- mRNA steady-state levels after adenosine receptor stimulation in human macrophages was not associated with a decrease in NF-B activation. On the other hand, it seems that under certain conditions, adenosine can decrease NF-B activation. For example, adenosine suppressed NF-B activation in both myeloid and lymphocytic, as well as epithelial cells, when TNF- but not when LPS was used to stimulate the cells (Majumdar and Aggarwal, 2003). Clearly, further studies will be necessary to dissect the signaling pathways whereby adenosine exerts its anti-inflammatory effects.

It is also important to point out that at this point it is unclear which receptors mediated the suppressive effect of adenosine on TNF- production in the current study. Although the general view is that the A2a receptor may be the most important one in regulating cytokine production and macrophage activation (Cronstein, 1998), it is clear that this was not the case here. That is because the microarray analysis found no A2a receptor agonist mRNA expression in the RAW cells. In addition, in our previous study (Szabó et al., 1998), the selective A2a receptor CGS-21680 was much less potent (EC₅₀ value in the low micromolar range) in suppressing MIP-1 production by RAW cells than would have been expected. On the other hand, in a study using primary peritoneal macrophages (Haskó et al., 2000), we found that the potency of CGS-21680 in decreasing cytokine production was much more consistent with an effect on A2a receptors (EC₅₀ value in the nanomolar range). A further support for the role of A2a receptors in peritoneal cells came from the observation that CGS-21680 lost its efficacy in cells taken from A2a receptor knockout mice (Haskó et al., 2000). Nevertheless, adenosine itself, although to a lesser extent, was still capable of decreasing cytokine production by peritoneal cells from A2a knockout mice, suggesting that both A2a and other receptors are involved in the anti-inflammatory effects of adenosine. Because RAW 264.7 cell do not seem to express A2a receptors, this cell line may be a powerful tool to study the A2a receptor-independent effects of adenosine on macrophage function.

Interestingly, the results presented in the current study found evidence, for the first time, for a profound up-regulation of A2b receptors after LPS stimulation. Thus, the A2b receptor may have been a possible mediator of the anti-inflammatory effects of adenosine in RAW cells. Of note, it has been reported that interferon- up-regulates A2b receptor expression in murine bone marrow-derived macrophages, and through this receptor, adenosine suppresses the induction of inducible nitric-oxide synthase expression (Xaus et al., 1999).

Together, it seems that the anti-inflammatory effects of adenosine may be mediated by different receptors depending on the experimental conditions. Consequently, it can be proposed that the intracellular pathways mediating the anti-inflammatory effects of adenosine in macrophages may vary with the adenosine receptors expressed and/or coupling mechanisms.

	Footnotes

 
This work was supported by the National Institutes of Health Grants 1R01 GM66189-01 (to G.H.) and R01-GM57982 (to S.J.L.).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

DOI: 10.1124/jpet.103.052944.

ABBREVIATIONS: LPS, lipopolysaccharide; TNF, tumor necrosis factor; MIP, macrophage inflammatory protein; NF-B, nuclear factor-B; CGS-21680, 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethyl-carboxamidoadenosine; IB-MECA, N⁶-(3-iodobenzyl)-adenosine-5'-N-methyluronamide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; ELISA, enzyme-linked immunosorbent assay; EMSA, electromobility shift assay; PBS, phosphate-buffered saline; IB, inhibitory B; RT-PCR, reverse transcription-polymerase chain reaction.

Address correspondence to: Dr. György Haskó, Department of Surgery, University of Medicine and Dentistry-New Jersey Medical School, 185 South Orange Ave., University Heights, Newark, NJ 07103. E-mail: haskoge{at}umdnj.edu

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