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The Journal of Immunology, 1999, 163: 4140-4149.
Copyright © 1999 by The American Association of Immunologists

Adenosine Inhibits Macrophage Colony-Stimulating Factor-Dependent Proliferation of Macrophages Through the Induction of p27kip-1 Expression1

Jordi Xaus2,*, Annabel F. Valledor2,*, Marina Cardó*, Laura Marquès*, Jorge Beleta{dagger}, José M. Palacios{dagger} and Antonio Celada3,*

* Departamento de Fisiologia (Biologia del Macrófag), Facultat de Biologia and Fundació August Pi i Sunyer, Campus de Bellvitge, Universitat de Barcelona, Barcelona, Spain; and {dagger} Laboratorios Almirall Prodesfarma SA, Research Center, Barcelona, Spain


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Adenosine is produced during inflammation and modulates different functional activities in macrophages. In murine bone marrow-derived macrophages, adenosine inhibits M-CSF-dependent proliferation with an IC50 of 45 µM. Only specific agonists that can activate A2B adenosine receptors such as 5'-N-ethylcarboxamidoadenosine, but not those active on A1 (N6-(R)-phenylisopropyladenosine), A2A ([p-(2-carbonylethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine), or A3 (N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide) receptors, induce the generation of cAMP and modulate macrophage proliferation. This suggests that adenosine regulates macrophage proliferation by interacting with the A2B receptor and subsequently inducing the production of cAMP. In fact, both 8-Br-cAMP (IC50 85 µM) and forskolin (IC50 7 µM) inhibit macrophage proliferation. Moreover, the inhibition of adenylyl cyclase and protein kinase A blocks the inhibitory effect of adenosine and its analogues on macrophage proliferation. Adenosine causes an arrest of macrophages at the G1 phase of the cell cycle without altering the activation of the extracellular-regulated protein kinase pathway. The treatment of macrophages with adenosine induces the expression of p27kip-1, a G1 cyclin-dependent kinase inhibitor, in a protein kinase A-dependent way. Moreover, the involvement of p27kip-1 in the adenosine inhibition of macrophage proliferation was confirmed using macrophages from mice with a disrupted p27kip-1 gene. These results demonstrate that adenosine inhibits macrophage proliferation through a mechanism that involves binding to A2B adenosine receptor, the generation of cAMP, and the induction of p27kip-1 expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Macrophages are generated through a process of differentiation known as myelopoiesis. In the bone marrow, pluripotent stem cells differentiate into monocytes in the presence of M-CSF and other cytokines. Monocytes leave the bone marrow and, by circulating through blood vessels, reach different tissues where they terminally differentiate into macrophages and perform their specialized functions (1, 2, 3). Tissue macrophages are able to proliferate thanks to the autocrine production of M-CSF (4). Macrophages require M-CSF for proliferation, differentiation, and survival (5). M-CSF is the main growth factor for macrophages and also the only one specific for these cells. After interacting with the tyrosine kinase receptor c-fms, M-CSF triggers the activation of several signal transducing molecules in macrophages (5, 6, 7, 8), such as some protein kinases of the Src family (9), the transcription factors Stat-1, Stat-3, and Stat-5 (10, 11), protein kinase C (12), and phosphatidylinositol 3-kinase (13, 14). M-CSF also activates the Raf/mitogen-activated protein/extracellular signal-related kinase (ERK)4 kinase pathway in macrophages (15). The activation of ERK-1/2 is required for macrophage proliferation in response to M-CSF.5 Active ERKs phosphorylate and regulate several cellular proteins (16), including other protein kinases, cytoskeletal components, phospholipase A2, and nuclear transcription factors, such as Elk1/TCF and c-Jun, which regulate the expression of immediate early genes (17, 18).

Mitogen-activated protein kinase phosphatase-1 (MKP-1) is a member of a family of dual-specificity phosphatases (19, 20) that dephosphorylate both phosphotyrosine and phosphothreonine residues on target proteins. MKP-1 dephosphorylates and inactivates ERK-1 and -2 both in vitro and in vivo, suggesting that this phosphatase has a critical effect on maintaining the balance between ERK phosphorylation and dephosphorylation (21). The overexpression of MKP-1 inhibits ERK-regulated reporter gene expression, the synthesis of DNA induced by Ras and G1-specific gene transcription, and entry of fibroblasts to the S phase in response to mitogenic stimuli (22, 23). The induction of the expression of MKP-1 is a mechanism used by the cell to control and attenuate proliferative signaling pathways.

All these signaling pathways allow macrophages to enter the cell cycle in response to M-CSF. Passage through the cell cycle is regulated through the action of a family of protein kinase complexes. Each complex is composed of at least a catalytic subunit, a cyclin-dependent kinase (cdk), and its essential activating partner, which is a cyclin (reviewed in Refs. 24 and 25). These complexes are activated at various checkpoints after specific intervals during the cell cycle, but they can also be modulated by exogenous factors. The cdks are inhibited when associated with a group of proteins known as cdk inhibitors (cki) (26). The ckis best described are p16Ink-4, which belongs to the INK 4 family, p21waf-1, and p27Kip-1, the latter belonging to the CIP/KIP family of cdk inhibitors (27, 28, 29). The presence of growth factors is only required during the G1 phase. Once the cell reaches the restriction point that appears late during the G1 phase and at the beginning of the S phase, the growth factors are no longer necessary and the cell is committed to complete the cell cycle (30, 31). This may explain why several regulatory steps of the cell cycle, such as the p53 checkpoint, pRb phosphorylation, and the activity of the cdk inhibitors, take place at this point (32, 33, 34, 35).

Adenosine is a purine nucleoside produced and secreted to the extracellular media by cells during normal intracellular ATP metabolization and degradation. Nevertheless, in stress situations like ischemia or hypoxia, massive ATP degradation increases local adenosine concentration to micromolar values (36, 37). In these situations, adenosine modulates several physiological functions, acting mainly as an endogenous antiinflammatory agent (reviewed in Ref. 38).

Most functional activities of extracellular adenosine are mediated through binding to specific surface receptors. However, it has been reported that adenosine needs to be internalized to induce some functions such as NO production and inhibition of LPS-induced TNF-{alpha} expression in some models (39). So far, four different adenosine receptors have been described and called A1, A2A, A2B, and A3 depending on their structural, functional, and pharmacological characteristics (40). Recently, all four adenosine receptor subtypes have been cloned from several species (41, 42, 43). All belong to the G-protein-coupled receptor superfamily. The type and density of adenosine receptors present on the cell surface are characteristic of each cell type.

The antiinflammatory role of adenosine has been associated to its effects on neutrophil activity. Additionally, adenosine may also play an important role in the attenuation of macrophage activity, as it modulates several functions of macrophages, such as the regulation of nitrite production (39, 44), the inhibition of LPS-induced TNF-{alpha} expression (39, 45), and the induction of IL-6 (46) and IL-10 production (39, 47).

We have found that adenosine inhibits M-CSF-dependent proliferation of murine bone marrow-derived macrophages (BMDM). To do this, adenosine interacts with the A2B receptor at the cell surface and induces a subsequent increase of cAMP levels. Treatment of macrophages with adenosine does not inhibit the activation of the ERK pathway. Instead, adenosine induces the expression of p27kip-1 in a protein kinase A (PKA)-dependent pathway, thus causing the growth arrest at the G1 phase of the cell cycle without inducing apoptosis. These results reveal the molecular mechanism involved in the adenosine-mediated inhibition of the M-CSF-dependent proliferation of macrophages and remark the relevance of this nucleoside as an immunosuppressor of macrophage activity and proliferation.


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

Adenosine, 2-chloroadenosine (CADO), 5'-N-ethylcarboxamidoadenosine (NECA), N6-(R)-phenylisopropyladenosine (R-PIA), and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) were obtained from Sigma (St. Louis, MO). 2-[p-(2-Carbonylethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680) and N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA) were purchased from Research Biochemicals (Natick, MA). 8-Br-cAMP and forskolin were obtained from Fluka Biochemika (Buchs, Switzerland). [3H]Thymidine was obtained from Amersham (Buckinghamshire, U.K.). 4,6-Diamidino-2-phenylindole (DAPI), SQ 22536, and KT 5720 were purchased from Calbiochem (La Jolla, CA). All the other products were of the best grade available and were purchased from Sigma. Deionized water further purified with a Millipore Milli-Q system (Bedford, MA) was used.

Cell culture

BMDM were isolated from 6-wk-old BALB/c mice (Charles River Laboratories, Wilmington, MA) as previously described (48). The cells were cultured in plastic tissue culture dishes (150 mm) in 40 ml DMEM containing 20% FBS and 30% L cell-conditioned media as a source of M-CSF. The cells were incubated at 37°C in a humidified 5% CO2 atmosphere. After 7 days of culture, an homogeneous population of adherent macrophages was obtained. To render the cells quiescent, when the macrophages were 80% confluent they were deprived of L cell-conditioned medium for 14–16 h before the experiment. BMDM from p27kip-1 knockout mice were isolated in the same conditions. These mice were kindly donated by Dr. J. Roberts from Howard Hughes Medical Institute (Seattle, WA) (49).

Antibodies

Surface expression of the M-CSF receptor (c-fms) was analyzed by using affinity-purified rabbit Abs anti-mouse c-fms (Upstate Biotechnology, Lake Placid, NY). Fluorescein-conjugated rat anti-rabbit IgG Ab from Sigma was used as a secondary Ab. To block Fc receptors, we used an anti-CD16/CD32 Ab (PharMingen, San Diego, CA). For the analysis of p27kip-1 expression by Western blotting, we used a monoclonal anti-mouse p27kip-1 Ab (PharMingen). The rabbit anti-mouse MKP-1 Ab was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). For ERK mobility shift assays, we used an anti-ERK-1/2 Ab, which was a kind gift of Dr. M. J. Weber (University of Virginia School of Medicine, Charlottesville, VA). Peroxidase-conjugated anti-mouse and anti-rabbit IgGs (Cappel, Turnhout, Belgium) were used as secondary Abs. Primary Abs against mouse ß-actin were purchased from Sigma.

Plasmids and constructs

The pMH117 plasmid corresponds to the mouse p21waf-1 full-length cDNA cloned in pEx-lox and was kindly provided by Dr. Massague (Sloan Kettering Institute, Howard Hughes Medical Institute, New York, NY). The pET-3d plasmid corresponds to the D1 cyclin cDNA cloned in pET-12 as described (50). The pCMJ3/cdk-4 plasmid contains the mouse cdk-4 full-length cDNA cloned in pBluescript KS as described (51). The probe for the 18S rRNA was obtained as described (52).

Proliferation assay

Cell proliferation was measured as previously described (53, 54) with minor modifications. The cells were deprived of M-CSF for 18 h and then 105 BMDM were incubated for 24 h in 24-well plates (3424 MARK II; Costar, Cambridge, MA) in 1 ml of complete medium in the presence or absence of the indicated adenosine analogues or derivatives. After this period of time, the medium was removed and replaced with 0.5 ml of media containing [3H]thymidine (1 µCi/ml). After two additional h of incubation at 37°C, the medium was removed and the cells were fixed in ice-cold 70% methanol. After three washes in ice-cold 10% TCA, the cells were solubilized in 1% SDS, 0.3 N NaOH. Radioactivity was counted by liquid scintillation using a 1500 Packard Tri-Carb scintillation counter (Meriden, CT). Each experiment was performed three times, and the results were expressed as the mean ± SD.

Determination of cAMP

The production of cAMP was measured using a standard procedure. Briefly, 106 macrophages were cultured in 24-well plates in complete media. The cells were stimulated with the indicated adenosine agonists for 15 min. Extraction of cAMP from the cells was conducted using a liquid phase extraction method. Ice-cold ethanol was added to the cell suspension to a final concentration of 65% (v/v) ethanol. After being allowed to settle, the supernatants were transferred to test tubes and centrifuged at 2000 x g for 15 min at 4°C. The supernatants were transferred to new tubes and dried using a speed-vac system (Bio-Rad, Hercules, CA). The dried extracts were dissolved in assay buffer, and the amount of cAMP was analyzed using a nonacetylation cAMP enzyme immunoassay system (Amersham). Each sample was analyzed in triplicate, and the results were represented as the mean ± SD.

Determination of c-fms cell-surface expression

Cell-surface staining was conducted using specific Abs and cytofluorometric analysis. After treatment with adenosine or its analogues for 24 h, 106 cells were harvested and washed in cold PBS. After fixing with 2% paraformaldehyde during 30 min at 4°C, the cells were resuspended in 50 ml PBS containing 5% FBS and then incubated at 4°C for 15 min with 1 µg/106 cells of anti-CD16/CD32 mAb to block Fc receptors. Then, the cells were incubated for 1 h at room temperature with murine c-fms-specific Ab (1 µg/106 cells). The cells were then washed by centrifugation through a FBS cushion. Finally, cells were incubated with FITC-conjugated anti-mouse IgG Ab for 1 h at 4°C. Stained cell suspensions were analyzed using an Epics XL flow cytometer (Coulter, Hialeah, FL). FITC excitation was obtained using a 488-nm Argon laser lamp, and its fluorescence was collected with a 525-nm band-pass filter. The parameters used to select cell populations for analysis were forward and side light scatter. As a control for specificity, we used a nonrelated Ab.

Analysis of DNA content with DAPI

A total of 106 cells previously subjected to a specific treatment were resuspended and fixed in ice-cold 70% ethanol. The cells were then washed in PBS, resuspended in 0.2 ml of a solution containing 150 mM NaCl, 80 mM HCl, and 0.1% Triton X-100, and incubated at 4°C for 10 min. Afterward, 1 ml of a solution containing 180 mM Na2HPO4, 90 mM citric acid, and 2 µg/ml DAPI, pH 7.4, was added to each sample. After incubating the cells at 4°C for 24 h, their fluorescence was measured with an Epics Elite flow cytometer (Coulter). For this analysis, we used an UV laser with an excitation beam of 25 mW at 333–364 nm, and fluorescence was collected with a 525-nm band-pass filter. Cell doublets were gated out by comparing the pulse area vs the pulse width. A total of 12,000 cells were counted for each histogram, and cell cycle distributions were analyzed with the Multicycle program (Phoenix Flow Systems, San Diego, CA).

Chromatin fragmentation assay

Fragmentation of DNA due to internucleosomal cleavage was determined as described previously (55). Briefly, 3 x 106 cells were harvested and washed in ice-cold PBS. The cells were lysed in 0.5 ml of lysis buffer (50 mM Tris-HCl, 10 mM EDTA, 1% SDS, pH 8.0) for 16 h at 4°C, and the lysates were centrifuged (15,000 x g) to separate high m.w. DNA (pellet) from cleaved low m.w. DNA (supernatant). The DNA supernatants were phenol-extracted twice and precipitated. The pellets were resuspended in Tris-EDTA buffer containing 250 µg/ml RNase (Boehringer Mannheim, Mannheim, Germany). The samples were heated at 65°C for 10 min and subjected to electrophoresis in a 2% agarose gel containing ethidium bromide.

Protein extraction and Western blot analysis

Cells were washed twice in cold PBS and lysed on ice with lysis solution (1% Triton X-100, 10% glycerol, 50 mM HEPES, pH 7.5, 150 mM NaCl, protease inhibitors). Then, 100 µM sodium orthovanadate was added to inhibit the activity of tyrosine phosphatases when necessary. The protein concentration of the samples was determined by the Bio-Rad protein assay. The proteins from the cell lysates (100–150 µg) were boiled at 95°C in Laemmli SDS-loading buffer, separated in 12% SDS-PAGE, and electro-transferred to nitrocellulose membranes (Hybond-ECL; Amersham). The membranes were blocked for at least 1 h at room temperature in TBS-T containing 5% nonfat dry milk and then incubated with TBS-T containing the primary Ab. For p27kip-1 and ß-actin immunoblotting, incubation was performed for 1 h at room temperature. After three washes of 15 min each in TBS-T, the membranes were incubated with peroxidase-conjugated anti-mouse or anti-rabbit IgG Abs (Cappel) for 1 h. After three washes of 15 min with TBS-T, enhanced chemiluminescence detection was performed (Amersham) and the membranes were exposed to x-ray films (Amersham). Quantification of the blot was conducted by densitometric analysis.

Determination of the ERK phosphorylation state by mobility shift assay

This assay was performed as described for the Western blot analysis with slight modifications (56). Proteins from cell lysates (50–100 µg) were subjected to 7.5% SDS-PAGE to allow efficient separation of the phosphorylated and dephosphorylated forms of ERK. The incubation of the membranes with anti-ERK-1/2 primary Ab or peroxidase-conjugated anti-mouse IgG Ab were done sequentially in TBS-T for 1 h at room temperature.

Determination of ERK activity by in-gel-kinase assay

First, 50 µg of total protein were separated by 12.5% SDS-PAGE in the presence of 0.1 mg/ml of myelin basic protein (Sigma) copolymerized in the gel. After electrophoresis, SDS was removed by washing the gel with two changes of 20% 2-propanol in 50 mM Tris-HCl (pH 8.0) for 1 h at room temperature. The gel was then incubated with 50 mM Tris-HCl (pH 8.0) containing 5 mM 2-ME (buffer A) for 1 h at room temperature. The proteins were denatured by incubating the gel with two changes of 6 M guanidine-HCl for 1 h at room temperature and then renatured by incubating with five changes of buffer A containing 0.04% Tween 20 for 16 h at 4°C. To perform the phosphorylation assay, the gel was first equilibrated in 40 mM HEPES-NaOH (pH 7.4) containing 2 µM DTT, 0.1 mM EGTA, 15 mM MgCl2, 300 µM sodium orthovanadate for 30 min at 25°C and then incubated for 1 h in the same solution containing 50 µM ATP and 100 µCi [{gamma}-32P]ATP (ICN Pharmaceuticals, Costa Mesa, CA). The reaction was stopped by washing the gel with 5% TCA containing 10 mM sodium pyrophosphate to inhibit phosphatase activity. The gel was dried, exposed to x-ray films (Kodak, Rochester, NY), and quantitated with a Bio-Rad molecular analyst system.

Northern blot analysis

Total cellular RNA (20 µg), extracted with the acid guanidinium thiocyanate-phenol-chloroform method (57), were separated in 1% agarose with 5 mM 3-[N-morpholino]propanesulfonic acid (pH 7.0)/1 M formaldehyde buffer. The RNA was transferred overnight to a GeneScreen nitrocellulose membrane (Life Science Products, Boston, MA) and fixed by UV irradiation (150 mJ). For p21waf-1 mRNA detection, we obtained the full-length cDNA of p21waf-1 by digesting pMH117 with EcoRI/HindIII and used it as a probe. The D1 cyclin probe was prepared by digesting the pET-3d construct with Bgl2/EcoRV. PCMJ3/cdk-4 digestion with EcoRI allowed us to obtain a cdk-4 probe. To check for differences in RNA loading, we analyzed the expression of the 18S rRNA transcript. All probes were labeled with [{alpha}-32P]dCTP (ICN Pharmaceuticals) with the oligolabeling kit method (Pharmacia Biotech, Uppsala, Sweden). After incubating the membranes for 18 h at 65°C in hybridization solution (20% formamide, 5x Denhart’s, 5x SSC, 10 mM EDTA, 1% SDS, 25 mM Na2HPO4, 25 mM NaH2PO4, 0.2 mg/ml salmon sperm DNA, and 106 cpm/ml of 32P-labeled probe), they were exposed to Kodak X-AR films. The bands of interest were quantified with a molecular analyst system (Bio-Rad).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Adenosine induces a serie of functional alterations in macrophages. We used macrophages obtained from bone marrow cultures, because they represent an homogeneous population of macrophages that require M-CSF to proliferate and survive. Under the effect of M-CSF, macrophages proliferate in a dose-dependent manner (Fig. 1GoA). The addition of 100 µM adenosine inhibited macrophage proliferation almost completely. The adenosine-induced inhibition of M-CSF-dependent proliferation was a dose-dependent process with an IC50 of 45 µM (Fig. 1GoB).



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  		FIGURE 1. Adenosine inhibits M-CSF-dependent proliferation of BMDM. A, BMDM were obtained after 7 days of culture in the presence of M-CSF. A total of 105 macrophages were incubated in 24-well plates in the presence of the indicated amounts of M-CSF either alone (Control) or with 10-4 M adenosine. Proliferation was determined as indicated in Materials and Methods. B, Adenosine inhibits macrophage proliferation in a dose-dependent manner. A total of 105 macrophages were incubated in 24-well plates in the presence of 1000 U/ml of M-CSF and the indicated amounts of adenosine. Control cells were incubated with M-CSF alone. Each determination was made in triplicate, and the values represented correspond to the mean ± SD of one representative of four independent experiments.

 
Some responses to adenosine are mediated through its direct interaction with target molecules inside the macrophages, whereas others are caused by its recognition by specific cell-surface receptors. Adenosine internalization is mediated through mechanisms of nucleoside transport. Recently, four types of adenosine receptors have been characterized and they have been classified according to their biochemical properties. These receptors are seven transmembrane-domain proteins coupled to G proteins (58). Recently, using specific Abs and radiolabeled ligands we have identified the presence of A2B and A3, but not A1 or A2A, adenosine receptors in bone marrow macrophages (59). To determine the type of adenosine receptor that mediates the inhibition of macrophage proliferation, we measured the activity of adenosine analogues that at small concentrations are recognized specifically by each type of receptors. However, at higher concentrations these analogues could also affect other adenosine receptors. NECA (60) and CADO (61) are synthetic adenosine analogues recognized by all types of adenosine receptors. NECA and CADO inhibited M-CSF-dependent proliferation with an IC50 <10 µM (Fig. 2GoA). In contrast, confirming our previous results, other analogues such as R-PIA, specific for A1 receptor (62), IB-MECA, specific for A3 (63) and CGS21680, recognized by A2A receptors (64), showed little or no effect as inhibitors of macrophage proliferation at those concentrations at which these compounds are specific for their respective receptors, which are at nanomolar range (Fig. 2GoA); however, at higher concentrations (>10 µM) they could also bind to A2B receptors and thus inhibit macrophage proliferation. The order of potency of adenosine analogues as inhibitors was: NECA > CADO >= R-PIA > adenosine > IB-MECA > CGS21680. Therefore, these results suggested that adenosine inhibits M-CSF-dependent proliferation by interacting with the A2B receptor. These data were confirmed by the fact that the adenosine antagonist DPCPX, which inhibits A2B receptors (65), blocked the inhibitory effect of NECA on the proliferation of macrophages in a dose-dependent manner (Fig. 2GoB).



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  		FIGURE 2. Adenosine inhibits M-CSF-dependent proliferation through activating the A2B adenosine receptors. A, The effects of synthetic adenosine analogues on macrophage proliferation were analyzed by [3H]thymidine incorporation. A total of 105 macrophages grown in the presence of 1000 U/ml of M-CSF were treated with NECA, CADO, R-PIA, CGS 21680, and IB-MECA at the indicated concentrations. *, At this concentration, R-PIA binds to A2B receptors. B, The A2B adenosine receptor antagonist DPCPX blocks the inhibitory effect of NECA on macrophage proliferation. A total of 105 cells cultured in 24-well plates were treated with NECA at the indicated concentrations in the presence or absence of 10-6 or 10-8 M DPCPX. Control cells from A and B were incubated in the presence of M-CSF alone. Each determination was made in triplicate, and the values represented correspond to the mean ± SD of one representative of three independent experiments.

 
The mechanism mediated by adenosine to inhibit macrophage proliferation was further investigated. As adenosine receptors are coupled to adenylyl cyclase (66, 67), we stimulated macrophages with different agonists and measured the intracellular production of cAMP. The treatment of BMDM with 10-5 M NECA induced a marked increase in the intracellular levels of cAMP (Fig. 3GoA). Similar results were obtained when macrophages were treated with 5 x 10-5 M adenosine or 10-5 M CADO, although in these cases the increase was lower than that mediated by NECA, which was in accordance with their relative potency as inhibitors of macrophage proliferation. Treatment of macrophages with 10-5 M CGS 21680, 10-6 M R-PIA, or 10-5 M IB-MECA did not induce a detectable increase of intracellular cAMP levels. To date, there are no specific agonists for A2B receptors. The results, obtained with adenosine, CADO, or NECA, three nonspecific agonists, indicate that the effect of adenosine is mediated through their interaction with the A2B adenosine receptor, because specific agonists for the other adenosine receptor subtypes did not have any effect on cAMP production at those concentrations specific for their receptors, while at higher concentrations they could bind to A2B receptors and induce cAMP production, thus inhibiting macrophage proliferation. This is the case of R-PIA, an inhibitor of A1 receptors (data not shown). Furthermore, 10-6 M DPCPX inhibited completely the cAMP increase induced by 10-5 M NECA (data not shown). Although we cannot exclude the presence of other adenosine receptors, all our results suggest that the A2B adenosine receptor is the main responsible for the cAMP increases found in response to adenosine in BMDM.



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  		FIGURE 3. Induction of cAMP production by adenosine agonists. A, A total of 106 BMDM were stimulated for 15 min with 5 x 10-5 M adenosine (Ado), 10-5 M NECA, 10-5 M CADO, 10-5 M IB-MECA, 10-5 M CGS 21680, or 10-6 M R-PIA. The cultures were liquid phase extracted (see Materials and Methods), and the cAMP content was measured with an enzyme immunoassay system (Amersham). B, The production of cAMP in BMDM stimulated with NECA is dose-dependent. The cells were treated for 15 min with the indicated concentrations of NECA. C, Time-course of NECA-induced cAMP production in macrophages. BMDM were treated with 10-5 M NECA for the indicated periods of times. Each sample was analyzed in triplicate, and the data are represented as the mean ± SD of triplicate determinations of two independent experiments.

 
The natural ligand adenosine induced a lower macrophage response than NECA, probably due to the surface expression of ecto-adenosine deaminase, a molecule that degrades adenosine before it can be metabolized (68). Therefore, we used NECA in the following experiments. The NECA-induced production of intracellular cAMP in BMDM was both dose- and time-dependent (Fig. 3Go, B and C), with an EC50 of 5 µM similar to that observed in previous reports (69).

So far we have shown that the adenosine-induced inhibition of M-CSF-dependent proliferation is mediated through the A2B receptors, and that the interaction of adenosine with these receptors induces the production of cAMP. We were also interested in studying the role of cAMP in the inhibition of M-CSF-dependent proliferation of macrophages. 8-Br-cAMP, a cell membrane-permeable and nonmetabolizable cAMP analogue, inhibited macrophage proliferation in response to 1200 U/ml of M-CSF. The IC50 for this inhibition was 85 µM (Fig. 4GoA). Treatment of macrophages with forskolin, a drug that directly activates adenylyl cyclase and induces the generation of cAMP, also inhibited M-CSF-dependent macrophage proliferation in a dose-dependent manner (Fig. 4GoB). This suggested that the production of cAMP was sufficient to inhibit macrophage proliferation. To confirm that the activation of adenylyl cyclase and the production of cAMP was responsible for the inhibitory effect of NECA on macrophage proliferation, we analyzed the effect of the inhibitor of adenylyl cyclase SQ 22536. This drug blocked the inhibitory effect on macrophage proliferation induced by NECA (Fig. 4Go, C and D), thus indicating that NECA inhibits M-CSF-dependent proliferation through activating the adenylyl cyclase and the subsequent production of cAMP. Moreover, treatment of macrophages with KT 5720, a PKA inhibitor, also blocked the anti-proliferative effect of NECA (Fig. 4Go, C and D). This confirms that NECA inhibits macrophage proliferation through a PKA-dependent pathway.



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  		FIGURE 4. cAMP inhibits M-CSF-dependent proliferation of macrophages. Macrophages were treated with 8-Br-cAMP (A) or forskolin (B) at the indicated concentrations, and thymidine incorporation was measured as described in Materials and Methods. Control cells were grown in the presence of 1000 U/ml of M-CSF with no other treatment. C, NECA inhibits macrophage proliferation through an adenylyl cyclase- and PKA-dependent pathway. A total of 105 macrophages grown in the presence of the indicated concentrations of M-CSF (Control) were treated with 10-5 M NECA alone or combined with either 10-4 M SQ 22536 or 10-7 M KT 5720. D, Dose-dependent effect of PKA and adenylyl cyclase inhibitors on NECA-induced inhibition of macrophage proliferation. Macrophages were treated with 1000 U/ml of M-CSF alone or combined with 10-5 M NECA and the indicated concentrations of SQ 22536 or KT 5720. Each determination was made in triplicate, and the values represented correspond to the mean ± SD of one representative of three independent experiments.

 
We next determined the molecular mechanism used by adenosine and its analogues to inhibit macrophage proliferation. First, we studied the effect of adenosine on the modulation of M-CSF receptors. An 18-h starvation of M-CSF induced a 4- to 5-fold increase in the level of expression of M-CSF receptors compared with that observed in cells growing in the presence of M-CSF (control) (Fig. 5Go). In contrast, no modification in the number of M-CSF receptors was detected in macrophages incubated for 24 h with adenosine, NECA, forskolin, or 8-Br-cAMP (Fig. 5Go). Therefore, the adenosine-induced inhibition of M-CSF-dependent proliferation was not due to an altered expression of M-CSF receptors.



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  		FIGURE 5. Adenosine analogues do not modify c-fms surface expression in macrophages. A, A total of 106 macrophages were deprived of M-CSF for 24 h or treated with 5 x 10-5 M adenosine in the presence of M-CSF. The control cells were grown in the presence of M-CSF with no additional treatment. The c-fms surface expression was analyzed by flow cytometry using mAbs. B, Quantification of c-fms surface expression after M-CSF starvation or treatment with 5 x 10-5 M adenosine (Ado), 10-5 M NECA, 10-5 M forskolin (Forsk), or 10-4 M 8-Br-cAMP for 24 h. Data are represented as the mean ± SD of two experiments.

 
Bone marrow macrophage cultures grown in the presence of M-CSF are not cell cycle-synchronized and showed a random distribution, with 51% of cells in G0/G1, 30% in S, and 17% in G2/M (Fig. 6Go). In response to adenosine, macrophages appeared to be distributed homogeneously (88% of total cells) in a peak corresponding to the G1 phase of the cell cycle (Fig. 6GoA). We did not find any subdiploid peak corresponding to apoptotic cells. Besides, cells treated with adenosine, NECA, or forskolin did not show DNA fragmentation in comparison to cells in which apoptosis had been induced by treatment with actinomycin D (70) (Fig. 6GoB). These results indicated that adenosine-mediated inhibition of proliferation was not due to a massive induction of apoptosis. Instead, the cell cycle stop induced by adenosine explains the inhibition of proliferation. Therefore, we were interested in studying the mechanisms used by adenosine to stop the cell cycle at the G1 phase.



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  		FIGURE 6. Adenosine causes a growth arrest of macrophages at the G0/G1 phase of the cell cycle and does not induce apoptosis. A, A total of 106 macrophages grown in the presence of M-CSF were treated or not with 5 x 10-5 M adenosine for 24 h and their DNA content was measured by flow cytometry. Cell cycle distribution was analyzed using Immuno-4 software (Coulter). The apoptotic cells (Apop) appear as a subdiploid fraction. B, A total of 3 x 106 BMDM were treated with 5 x 10-5 M adenosine, 10-5 M NECA, or 10-5 M forskolin for 24 h, and chromatin fragmentation was analyzed by electrophoresis in an agarose gel. The positive control cells for apoptosis correspond to macrophages treated with 5 µg/ml actinomycin D. Negative control cells correspond to macrophages grown in the presence of M-CSF with no additional treatment. Both analyses were performed twice with identical results.

 
In an initial approach, we first analyzed the effects of adenosine on the activation of the ERK pathway. The activation of ERK-1/2 is required for M-CSF-dependent proliferation of macrophages (5). Phosphorylation of ERK-2 was analyzed by a mobility shift assay. ERK-2 phosphorylation can be used as an indicator of ERK activation in macrophages, because there is a close correlation between the phosphorylation state of ERK-1/2 and their activity in an in-gel-kinase assay (data not shown). M-CSF induced ERK-2 phosphorylation in bone marrow macrophages (Fig. 7GoA). No differences in ERK-2 phosphorylation were observed when M-CSF-treated macrophages were incubated in the presence of adenosine, NECA, or 8-Br-cAMP (Fig. 7GoA). We also analyzed the effect of NECA on the kinetics of ERK activation by in-gel-kinase assays using myelin basic protein as a substrate for ERK-dependent phosphorilation (Fig. 7GoB). The addition of M-CSF to quiescent macrophages induced the rapid activation of ERK proteins. ERK activation peaked after 5 min and then the activity of these kinases decreased progressively to basal levels. Treatment of macrophages with 10-5 M NECA did not modify the kinetics of the M-CSF-induced ERK activation. We also analyzed the expression of MKP-1, a dual specificity phosphatase responsible for the inactivation of ERK-1/2. According to the results on ERK phosphorylation and activity, treatment of macrophages with NECA, forskolin, or 8-Br-cAMP did not modify the mRNA nor protein expression of MKP-1 (data not shown). These results indicated that adenosine and related drugs did not affect the activation/deactivation of the ERK pathway in macrophages.



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  		FIGURE 7. An increase in the intracellular levels of cAMP does not inhibit the M-CSF-induced activation of the ERK pathway. A, Cell extracts were obtained after stimulation of quiescent macrophages with or without M-CSF for 10 min in the presence or absence of 5 x 10-5 M adenosine, 10-5 M NECA, 10-5 M forskolin, or 10-4 M 8-Br-cAMP. The induction of ERK-2 phosphorylation by M-CSF was assessed with a mobility shift assay. Then, 80 µg of total protein were loaded per lane. The positions of phosphorylated and dephosphorylated forms of ERK-2 are indicated with arrows. B, cAMP does not modify the kinetics of the M-CSF-induced ERK activation. Quiescent macrophages were either left untreated or preincubated with 10-5 M NECA for 15 min and then stimulated with M-CSF (1200 U/ml) for the indicated times. ERK activity was analyzed with an in-gel-kinase assay.

 
We next determined the effects of adenosine on different elements involved in cell cycle control. Because adenosine and its analogues cause an arrest of macrophages at the G1 phase of the cell cycle, we studied the expression of G1 cdks. The expression of the mRNA for the components of the cyclin D/cdk-4 (Fig. 8Go) or cyclin E/cdk-2 (data not shown) complex were not modified by treatment with adenosine and its analogues. Moreover, the analysis of the immunoprecipitates and blotting of the cyclin D/cdk-4 complex formation showed no differences between control and NECA-treated cells (data not shown).We also analyzed the expression of cdk inhibitors. The expression of p21waf-1, a dual inhibitor of cdk required for the passage through the cell cycle (28), was not modified by adenosine or analogues (Fig. 8Go).



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  		FIGURE 8. Effect of adenosine agonists on the cell cycle machinery. A, The effect of adenosine and related agents on the cell cycle machinery was analyzed by Northern blotting. Macrophages were treated for 24 h with 10-5 M of each of the indicated adenosine analogues and related agents in the presence of M-CSF. Then, 20 µg of total RNA from each sample was blotted for the expression of p21waf-1, D1 cyclin, and cdk-4 kinase. In this figure, we show the expression and quantification of one representative of three independent experiments.

 
In contrast, adenosine, NECA, forskolin, and 8-Br-cAMP induced a 3- to 8-fold increase of p27kip-1 protein levels (Fig. 9Go). p27kip-1 is another G1 cki of the CIP/KIP family that binds to cyclin/cdk complexes and inhibits their activity (29). NECA induced the expression of p27kip-1 in a time- and dose-dependent manner (Fig. 9Go, B and C). The induction of p27kip-1 by NECA was observed after 6 h of treatment and it was maintained during the whole time-course of 24 h. Besides, the expression of p27kip-1 induced by NECA was inhibited by the treatment with either QS 22536 or KT 5720 (Fig. 9GoD); therefore, the expression of p27kip-1 depended on the production of cAMP by activated adenylyl cyclase and on the activation of the PKA pathway. Thus, the induction of p27kip-1 expression mediated by adenosine and its analogues may be responsible for the adenosine-induced arrest of macrophages at the G1 phase of the cell cycle.



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  		FIGURE 9. Adenosine induces the expression of p27kip-1. A, The induction of p27kip-1 by adenosine and related agents was measured by Western blotting after treating the macrophages for 24 h with 10-5 M of each of the indicated adenosine analogues and related agents in the presence of M-CSF. The blots were quantified by densitometry, and the results are indicated as fold-induction of p27kip-1 expression compared with the level of expression in control cells grown in the presence of M-CSF with no additional treatment. B, The induction of p27kip-1 by NECA is dose-dependent. BMDM were treated for 24 h with the indicated concentrations of NECA. C, The induction of p27kip-1 by NECA is time-dependent. BMDM were treated with 10-5 M NECA at the indicated times. D, NECA-induced p27kip-1 expression is adenylyl cyclase- and PKA-dependent. Macrophages were treated or not with 10-5 M NECA alone or in combination with 10-4 M SQ 22536 or 10-7 M KT 5720. Then, 100 µg of total protein were loaded per lane and p27 expression was analyzed as indicated in Materials and Methods. The expression of ß-actin was used as a control of sample loading and transfer efficiency. Each experiment was performed twice.

 
To confirm this hypothesis, we analyzed the inhibitory effect of NECA on macrophage proliferation using macrophages from mice with the p27kip-1 gene disrupted. In these macrophages, in contrast with that observed in macrophages from normal mice, NECA, but not IFN-{gamma}, did not inhibit cell proliferation (Fig. 10Go), demonstrating that the expression of p27kip-1 is necessary for the inhibition by NECA of the M-CSF-dependent proliferation of macrophages.



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  		FIGURE 10. NECA did not inhibit M-CSF-dependent proliferation of BMDM from p27kip-1 knockout mice. BMDM were obtained after 7 days of culture in the presence of M-CSF. A total of 105 macrophages from control (A) and p27kip-1 knockout (B) mice were incubated in 24-well plates in the presence of the indicated amounts of M-CSF alone or with 10-5 M NECA or 300 U/ml IFN-{gamma}. Proliferation was determined as indicated in Materials and Methods. Each determination was made in triplicate, and the values represented correspond to the mean ± SD of one representative of two independent experiments.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Most experiments on proliferation and cell cycling have been conducted using transformed cell lines. In this report, we have used primary cultures of BMDM, which is an homogeneous population that responds to physiological proliferative or activating stimuli (54).

We have found that adenosine blocks M-CSF-dependent proliferation of macrophages. The effect of adenosine seems to be mediated through the engagement of the A2B adenosine receptor. This is supported by the use of specific agonists and antagonists for the different types of adenosine receptors. NECA, an agonist for the four types of adenosine receptors so far described, induces a stronger inhibition of M-CSF-dependent proliferation than the agonists specific for A1, A3, and A2A adenosine receptors. The NECA-induced increase of the intracellular levels of cAMP is likely mediated by A2B receptors, because DPCPX, an inhibitor of this type of receptors, inhibits the production of cAMP in response to NECA. This confirms our previous observations about the expression of adenosine receptors in macrophages (59). By using binding assays with radiolabeled NECA in competition with different agonists and immunoblotting with specific Abs, we have identified the presence of A2B and A3 adenosine receptors on the cell surface of macrophages, whereas A1 and A2A receptors are poorly represented in these cells.

The inhibition of M-CSF-dependent proliferation by adenosine may be caused by an increase in the production of cAMP. Both cAMP and PKA activators inhibit the proliferation of macrophages. Treatment of BMDM with adenosine arrests them in the G1 phase of the cell cycle, as it has been already demonstrated with cAMP analogues (71, 72, 73). Moreover, the inhibition of adenylyl cyclase and PKA blocks the antiproliferative effect of NECA in macrophages.

The first step in the induction of macrophage proliferation in response to M-CSF is the interaction of this growth factor with its specific cell-surface receptor, c-fms. IFN-{gamma}, the major activator of macrophages, inhibits M-CSF-induced proliferation and also down-modulates the expression of c-fms (74). However, neither adenosine nor cAMP-increasing agents modulated the expression of c-fms, thus indicating that growth arrest was not due to a reduction in M-CSF recognition at the cell surface.

One of the earliest events in the signal transduction of M-CSF is the activation of ERK-1/2 (15, 75). The inhibition of ERK activation by using PD98059, a specific inhibitor of the mitogen-activated protein/ERK kinase, blocked macrophage proliferation in response to M-CSF.5 Recently, several reports have shown that the capability of Ras to activate Raf-1 was impaired in cells treated with cAMP-elevating agents, leading to a loss in the capability to activate ERKs (76). Therefore, the activation of PKA may prevent growth factor-mediated cell division by interfering with the activation of ERK-1/2. However, cAMP enhances the M-CSF-induced ERK activity in macrophages (77). In our experiments, neither adenosine nor cAMP altered the phosphorylation state or the activation kinetics of ERK-2 in response to M-CSF. Besides, the expression of the phosphatase MKP-1 was not modified by cAMP-increasing agents. Therefore, cAMP does not seem to inhibit macrophage proliferation by reducing the capability of M-CSF to activate the ERK pathway.

Macrophages incubated with cAMP are blocked at the G1 phase despite synthesizing normal amounts of cyclin D1 and cdk-4. The phosphorylation of cdk4 at threonine 172 is necessary for cdk-4 activation. The cdk-activating kinase precipitated from cAMP-treated cells was as active as that obtained from nontreated proliferating cells. This suggested the presence of an inhibitory activity present in cell lysates of cAMP-treated cells. Recently p27kip-1, an inhibitor of cdk-4, has been identified (72, 78). We have studied the effects of adenosine analogues on the expression of two cdk-4 inhibitors, p21waf-1 and p27kip-1. We detected p27kip-1 expression only in those cells treated with cAMP-increasing agents, which may account for the cAMP-mediated arrest of the cell cycle at the G1 phase. Moreover, cAMP-increasing agents induce the expression of p27kip-1 in an adenylyl cyclase- and PKA-dependent pathway. The involvement of p27 in the adenosine-induced inhibition of the M-CSF-dependent proliferation was confirmed using macrophages from mice with the p27kip-1 gene disrupted (49), which are unresponsive to the inhibitory effect of NECA on macrophage proliferation.

The data presented in this report allow us to suggest a model for adenosine-mediated inhibition of macrophage proliferation. Adenosine binds to A2B receptors on the surface of macrophages and subsequently induces the production of cAMP by activating adenylyl cyclase. As a consequence of the cAMP increase, PKA is activated and the expression of p27kip-1 is induced, thus blocking the activity of G1 cyclin/cdk complexes. As a result, macrophages cannot progress through the G1 phase of the cell cycle and their proliferation is blocked even in the presence of M-CSF.

These results could have clinical relevance. In fact, the inhibition of macrophage proliferation could be part of the immunosuppressive effect of adenosine. The inhibition of macrophage proliferation and activation could result in the modulation and the resolution of the inflammatory process.


   Acknowledgments
 
We acknowledge the help received from Jaume Comas and Rosario González in charge of the flow cytometry facility of the Serveis Científico Técnics de la Universitat de Barcelona (Barcelona, Spain). We thank Dr. Michael Weber (University of Virginia, Charlottesville, VA) for the anti-ERK1/2 Abs. We also thank Dr. J. Massagué (Sloan Kettering Institute, Howard Hughes Medical Institute, New York, NY) for the pMH117 plasmid and Dr. J. Roberts (Howard Hughes Medical Institute, Seattle, WA) for the the p27kip-1 knockout mice. We especially thank Dr. Gabriel Gil (Institut Municipal d’Investigaciones Biomédiques, Barcelona, Spain) for his help with the p27kip-1 knockout mice. We also thank Martín Cullell-Young for the revision of the manuscript.


   Footnotes
 
1 This work was supported by grants from Comisión Interministerial de Ciencia y Tecnologia (SAF98-102 and PM 98/0200 to A.C.). J.X. and A.F.V. are recipients of fellowships from the Comissió Interdepartamental de Recerca i Innovació Tecnológica. Back

2 J.X. and A.F.V. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Antonio Celada, Departamento de Fisiologia, Facultat de Biologia, Av. Diagonal 645, 08028 Barcelona, Spain. E-mail address: Back

4 Abbreviations used in this paper: ERK, extracellular signal-related kinase; NECA, 5'-N-ethylcarboxamidoadenosine; R-PIA, N6-(R)-phenylisopropyladenosine; DPCPX, 1,3-dipropyl-8-cyclopentylxanthine; CGS 21680, 2-[p-(2-carbonyl-ethyl)phenyl-ethylamino]-5'-N-ethylcarboxamidoadenosine; IB-MECA, N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide; BMDM, bone marrow-derived macrophages; PKA, protein kinase A; MKP, mitogen-activated protein kinase phosphatase; cdk, cyclin-dependent kinse; cki, cdk inhibitor; CADO, 2-choloradenosine; DAPI, 4,6-diamidino-2-phenylindole. Back

5 A. F. Valledor, J. Xaus, L. Marquès, and A. Celada. M-CSF induces the expression of MKP-1 through a PKC-dependent mechanism. Submitted for publication. Back

Received for publication February 24, 1999. Accepted for publication July 28, 1999.


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