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. 2010 Aug;24(8):2631–2640. doi: 10.1096/fj.10-155192

Adenosine A2A receptor activation protects CD4+ T lymphocytes against activation-induced cell death

Leonóra Himer *, Balázs Csóka †, Zsolt Selmeczy *, Balázs Koscsó *, Tímea Pócza *, Pál Pacher ‡, Zoltán H Németh †,§, Edwin A Deitch †, E Sylvester Vizi *, Bruce N Cronstein ‖, György Haskó †,¶,1
PMCID: PMC2909295  PMID: 20371613

Abstract

Activation-induced cell death (AICD) is initiated by T-cell receptor (TCR) restimulation of already activated and expanded peripheral T cells and is mediated through Fas/Fas ligand (FasL) interactions. Adenosine is a purine nucleoside signaling molecule, and its immunomodulatory effects are mediated by 4 G-protein-coupled receptors: A1, A2A, A2B, and A3. In this study, we investigated the role of A2A receptors in regulating CD4+ T lymphocyte AICD. Our results showed that the selective A2A receptor agonist CGS21680 (EC50=15.2–32.6 nM) rescued mouse CD4+ hybridomas and human Jurkat cells from AICD and that this effect was reversed by the selective A2A receptor antagonist ZM241385 (EC50=2.3 nM). CGS21680 decreased phosphatidylserine exposure on the membrane, as well as the cleavage of caspase-3, caspase-8 and poly(ADP-ribose) polymerase indicating that A2A receptor stimulation blocks the extrinsic apoptotic pathway. In addition, CGS21680 attenuated both Fas and FasL mRNA expression. This decrease in FasL expression was associated with decreased activation of the transcription factor systems NF-κB, NF-ATp, early growth response (Egr)-1, and Egr-3. The antiapoptotic effect of A2A receptor stimulation was mediated by protein kinase A. Together, these results demonstrate that A2A receptor activation suppresses the AICD of peripheral T cells.—Himer, L., Csóka, B., Selmeczy, Z., Koscsó, B., Pócza, T., Pacher, P., Németh, Z. H., Deitch, E. A., Vizi, E. S., Cronstein, B. N., Haskó, G. Adenosine A2A receptor activation protects CD4+ T lymphocytes against activation-induced cell death.

Keywords: inflammation, apoptosis, anergy

T-cell receptor(TCR) restimulation of previously activated and expanded mature T cells or hybridoma cells by an antigen or mitogen causes activation-induced cell death (AICD), which is mediated by the induction of the expression of Fas ligand (FasL) and its interaction with Fas (1,2,3). FasL-dependent lymphocyte death plays an essential role in maintaining peripheral tolerance and in limiting an ongoing immune response. The significance of the Fas-FasL pathway is highlighted by the fact that both Fas- and FasL-deficient mice develop massive lymphoproliferation and autoimmunity (4, 5).

Adenosine is a purine nucleoside signaling molecule that is released from metabolically active cells into the extracellular space. Extracellular adenosine exerts a variety of immunomodulatory effects, which are mediated by 4 G-protein-coupled receptor subtypes: A1, A2A, A2B, and A3(6,7,8,9,10,11). Although resting mouse and human T lymphocytes express all four adenosine receptors, A2A receptors (A2ARs) are the dominant adenosine receptors in governing lymphocyte responses (7, 12,13,14,15,16,17,18). A2AR stimulation inhibits TCR-mediated proliferation, cytokine production, and up-regulation of positive costimulatory molecules (reviewed in ref. 7). In addition, A2AR stimulation on T cells increases the expression of negative costimulatory molecules (19) and regulatory markers such as forkhead box P3 (Foxp3) and lymphocyte-activation gene 3 (LAG3) (20). A2AR-mediated inhibition of lymphocyte function is ensured by up-regulation of A2AR levels in CD4+ T lymphocytes after TCR stimulation (14, 21).

Previous studies have suggested that adenosine and its analogs induce apoptosis through A2A or A3 receptors in a variety of cell types including lymphoid cells (22,23,24). Mouse thymocytes were shown to exhibit internucleosomal DNA cleavage and lactate dehydrogenase (LDH) release in the presence of adenosine, 2-chloroadenosine, or the selective A2AR agonist CGS21680 (25,26,27,28,29). Adenosine and CGS21680 induced apoptosis preferentially in immature CD4+CD8+ thymocytes and did not substantially affect the viability of CD4−CD8−, CD4+CD8−, or CD4−CD8+ thymocytes (27, 28).

It is unclear what role A2ARs have in regulating the TCR ligation-induced, FasL-dependent cell death of mature CD4+ T cells. In this report we demonstrate that A2AR activation prevents TCR cross-linking-induced CD4+ T-cell apoptosis by down-regulating FasL gene expression.

MATERIALS AND METHODS

Cell lines and drugs

The murine T hybridoma cell line 7.5 (16, 30) and the human T-cell line Jurkat (gift from A. Erdei, Department of Immunology, Eötvös Loránd University, Budapest, Hungary) were grown in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-inactivated FBS (Invitrogen) and 100 U/ml penicillin/100 μg/ml streptomycin (Invitrogen).

The selective A2AR agonist 2-p-(2-carboxyethyl) phenethyl-amino-5′-N-ethylcarboxamidoadenosine (CGS21680) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The selective A2AR antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl) phenol (ZM241385) was purchased from Tocris Bioscience (Ellisville, MO, USA). Stock solutions of the various agents were prepared using DMSO (Sigma-Aldrich). PKA inhibitors H-89 and KT5720 were purchased from Sigma-Aldrich, and Rp-isomer 8-bromoadenosine-3′,5′-cyclic monophosphorothioate (Rp-8-Br-cAMPS) was obtained from Alexis (Lausen, Switzerland) The selective exchange protein activated by cAMP (EPAC) activator 8-(4-chlorophenylthio-2′-O-methyladenosine-3′,5′-cyclic monophosphate was purchased from Tocris. FLIM58 anti-mouse FasL neutralizing antibody was purchased from MBL International (Woburn, MA, USA), and hamster IgG isotype control was from BD Pharmingen (San Diego, CA, USA).

Induction of AICD

Murine 7.5 hybridoma or Jurkat cells (5×105 cells/ml) were cultured in 6- or 96-well plates (Corning Incorporated, Corning, NY, USA) and treated for various time periods with increasing concentrations (1 nM–10 μM) of CGS21680 followed by stimulation with 5 μg/ml concanavalin A (ConA) (Sigma-Aldrich) 30 min later.

Assessment of cell viability and apoptosis

The viability of 7.5 hybridoma or Jurkat cells was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay and lactate dehydrogenase (LDH) release assay (31). In the MTT assay, the yellow tetrazolium salt (MTT) is reduced in metabolically active cells to form insoluble purple formazan crystals, which are solubilized by the addition of a detergent. The color can then be quantified by spectrophotometric means. In the MTT experiments, 100 μl of cell suspension was placed in one well of a 96-well tissue culture plate, and 10 μl of MTT solution (2.5 mg/ml; Sigma-Aldrich) was added. After incubation for 4 h at 37°C, 100 μl of acid-isopropanol (0.04 N HCl in isopropanol) was added and mixed by gentle pipetting to solubilize the cells. The optical density of the solution was read at 550 nm using a microplate reader (Victor3 V 1420 Multilabel Counter; PerkinElmer Wallac Victor, Turku, Finland).

LDH is a soluble cytosolic enzyme that is released into the culture medium after loss of membrane integrity. The LDH assay is based on the reduction of the tetrazolium salt 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium in a NADH-coupled enzymatic reaction to formazan. LDH activity was measured using the In Vitro Toxicology Assay Kit (Sigma-Aldrich) according to the manufacturer’s protocol. In brief, 50 μl/well of cell-free supernatant from each sample was transferred into one well of a 96-well plate, and 100 μl of LDH assay reaction mixture (Sigma-Aldrich) was added to each well. After 20–30 min of incubation at room temperature in the dark, the reaction was terminated by the addition of 0.1 vol of 1 M HCl to each well. The OD490 was read using a microplate reader (PerkinElmer Wallac Victor).

Determination of apoptosis and necrosis by flow cytometry

Murine 7.5 hybridoma cells were treated in the same way as described above, and the percentages of apoptotic and necrotic cells were determined by flow cytometry using an FITC annexin V Apoptosis Detection Kit (BD Pharmingen, San Diego, CA, USA) according to the manufacturer’s protocol. In brief, 105 cells/sample were washed with PBS and then resuspended in 100 μl of binding buffer containing 5 μl of FITC-annexin V and 10 μl of propidium iodide and incubated at room temperature for 15 min. Then 400 μl of binding buffer was added, and the cells were analyzed in a FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA, USA). Cells were gated for lymphocytes using forward and side scatter. Ten thousand events were recorded from each treatment group. The samples were analyzed using Becton-Dickinson CellQuest and WinMDI 2.8 (Windows Multiple Document Interface for Flow Cytometry) software.

Cell fractionation and protein extraction

AICD in murine 7.5 hybridoma cells was induced in the same way as described above. For the whole cell lysate preparation, after the end of the incubation period, the cells were washed with PBS and pelleted at 1200 g for 5 min. The pellet was resuspended in modified RIPA lysis buffer [0.05 M Tris-HCl, pH 6.8; 0.25% sodium deoxycholate; 0.15 M NaCl; 1 mM EDTA, pH 7.4; 1 mM Na3VO4; 1 mM NaF; 1% Nonidet P-40; 1 mM PMSF; and protease inhibitor cocktail mix (Sigma-Aldrich)] (32) and incubated on ice for 15 min. The lysates were centrifuged at 15,000 g for 15 min, and the supernatants were recovered.

For nuclear extract preparation, after the end of the incubation period, the cells were washed with PBS and pelleted at 1200 g for 5 min. The pellet was resuspended in cytosolic lysis buffer (20% glycerol; 10 mM HEPES, pH 8.0; 10 mM KCl; 0.5 mM EDTA, pH 8.0; 1.5 mM MgCl2; 0.5% Nonidet P-40; 0.5 mM DTT; 0.2 mM PMSF; and proteinase inhibitor cocktail mix) and incubated for 30 min. After centrifugation at 5000 g for 10 min, supernatants (cytosolic extracts) were saved, and the nuclear pellets were processed further. Then 20–30 μl of nuclear extraction buffer (20% 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 DTT, 0.2 mM PMSF, and 100× diluted proteinase inhibitor cocktail mix) was added to the nuclear pellet and incubated on ice for 30 min. Nuclear proteins were isolated by centrifugation at 15,000 g for 15 min. All extraction procedures were performed on ice with ice-cold reagents. Protein concentrations were determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA).

Western blot analysis

Whole-cell lysates, cytoplasmic fractions, or nuclear extracts, all containing 30–50 μg of protein, were subjected to reducing SDS-PAGE (10–12%). After electrophoresis, the gels were electroblotted in Tris-glycine buffer containing 10% methanol onto a Hybond nitrocellulose membrane (GE Healthcare, Amersham Place, Little Chalfont, UK). The membranes were probed with polyclonal rabbit anti-mouse primary Abs against NF-κB p65, cleaved caspase-3, and cleaved poly(ADP-ribose) polymerase (PARP; Cell Signaling Technology, Danvers, MA, USA) or Abs against cleaved caspase-8, Fas, NF-ATp, and early growth response (Egr) family members Egr-1, Egr-2, and Egr-3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Thereafter, the membranes were incubated with a secondary horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (Santa Cruz Biotechnology). HRP-conjugated polyclonal goat anti-β-actin antibody from Santa Cruz Biotechnology was used to assess equal loading. Bands were detected using ECL Western Blotting Detection Reagent (GE Healthcare).

Real-time PCR for the detection of Fas and FasL mRNA

Murine 7.5 hybridoma and Jurkat cells were activated in the same way as described above. Six hours after activation, RNA was extracted using TRIzol (Invitrogen) according to the manufacturer’s protocol. For each sample, 5 μg of appropriately diluted RNA was reverse-transcribed into cDNA using 1 μl of oligo(dT)18 primer (0.5 μg/μl), 2 μl of 10× RT-PCR buffer, 2 μl of 25 mM MgCl2, 1.5 μl of 10 mM dNTP, and 1 μl of Omniscript reverse transcriptase (Qiagen, Valencia, CA, USA), supplemented with diethyl pyrocarbonate water to a 20-μl final volume. This reaction mixture was incubated for 1 h at 42°C and then heated to 99°C for 5 min using a Mastercycler (Eppendorf North America, Westbury, NY, USA). cDNA was stored at −20°C. Real-time PCR was performed according to standard protocols using the SensiMix HRM Kit (Quantace, London, UK). cDNA samples were used as a template, and data were normalized for 18S (endogenous housekeeping gene) levels. The following primers were used for Fas and FasL mRNA detection: mouse Fas, 5′-GAGGACTGCAAAATGAATGGGG-3′ (forward) and 5′-ACAACCATAGGCGATTTCTGGG-3′ (reverse); mouse FasL, 5′-CAGCAGTGCCACTTCATCTTGG-3′ (forward) and 5′-TTCACTCCAGAGATCAGAGCGG-3′ (reverse) (33); human Fas, 5′-ACTGTGACCCTTGCACCAAAT-3′(forward) and 5′-GCCACCCCAAGTTAGATCTGG-3′ (reverse); human FasL, 5′-AAAGTGGCCCATTTAACAGGC-3′ (forward) and 5′-AAAGCAGGACAATTCCATAGGTG-3′ (reverse); and 18S, 5′-GTAACCCGTTGAACCCCATT-3′ (forward) and 5′-CCATCCAATCGGTAGTAGCG-3′ (reverse) (34, 35). PCR conditions were optimized for primers, templates, and MgCl2.

Transfection and luciferase assay

Human Jurkat cells (4×105) were transfected with the firefly luciferase reporter plasmid NF-κB-Luc (Clontech, San Diego, CA) or its control vector (empty pTAL) using Lipofectamine 2000 transfection reagent (Invitrogen) dissolved in antibiotic-free Opti-MEM medium (Invitrogen) in 24-well plates according to the manufacturer’s protocol. Cells were incubated at 37°C for 20–24 h with the transfection reagent, after which procedure the transfection reagent-containing medium was replaced with fresh RPMI 1640 growth medium. The cells were then treated with 100 nM CGS21680 or its vehicle for 30 min and then activated with ConA (5 μg/ml) for another 8 h at 37°C. After the incubation, the cells were lysed in passive lysis buffer (80 μl) (Promega, Madison, WI, USA), and a Luciferase 1000 assay system (Promega) was added to the lysates. The luciferase activity of the lysates was determined using a luminometer (PerkinElmer Wallac Victor). Protein concentrations were determined using the Bio-Rad protein assay kit, and luciferase activity was normalized for protein content.

Statistical analysis

Values in the figures are expressed as means ± se of n observations. Statistical analysis of the data was performed by Student’s t test or 1-way analysis of variance followed by the Newman-Keuls test, as appropriate. Statistical significance was assigned to P < 0.05. EC50 values were calculated using Prism software (GraphPad, San Diego, CA, USA).

RESULTS

A2AR activation decreases AICD of murine and human T cells

We first established that exposure of both murine 7.5 hybridoma and human Jurkat cells to ConA induced cell death, as assessed using the MTT and LDH assays (Fig. 1). Pretreatment of the cells with the A2AR agonist CGS21680 for 30 min before ConA administration markedly reduced the death of both 7.5 hybridoma (Fig. 1A, B) and Jurkat cells (Fig. 1C, D). The protective effect of CGS21680 against cell death in both cellular systems was concentration dependent. The inhibitory effect of CGS21680 on T-cell death was completely prevented by the selective A2AR antagonist ZM241385 (Fig. 1E).

Figure 1.

Figure 1

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Selective A2AR agonist CGS21680 promotes the survival of murine and human T cells during AICD. A, B) Murine 7.5 T-hybridoma cells were treated with various doses of CGS21680 or vehicle 30 min before activation by ConA for 18–20 h. Cell viability was determined by the MTT (A) and LDH (B) assays. EC50 for CGS21680 was 21.65 and 15.19 nM in the MTT and LDH assays, respectively. C, D) CGS21680 also decreases AICD of human Jurkat T cells. Jurkat cells were treated with various doses of CGS21680 or vehicle, 30 min before activation by ConA for 24 h. Cell viability was determined using the MTT (C) and LDH (D) assays. EC50 for CGS21680 was 32.58 and 23.78 nM in the MTT and LDH assays, respectively. E) Pretreatment of 7.5 T cells with the A2AR antagonist ZM241385 (ZM) 30 min before addition of CGS21680 (100 nM) reversed the protective effect of CGS21680 against AICD. EC50 for ZM was 2.26 nM. Results are means ± se of a representative experiment from 3 separate experiments; n = 5 wells/group. **P < 0.01, ***P < 0.001 vs. 0 μM CGS21680; ###P < 0.001 vs. 0 nM ZM + CGS21680.

A2AR activation suppresses T-cell apoptosis

The primary mechanism of cell death in TCR-activated lymphocytes is apoptosis (36,37,38). We next set out to explore the effect of A2AR activation on the apoptotic process of TCR-stimulated T cells. We first evaluated the effect of A2AR activation on translocation of phosphatidylserine from the inner to the outer leaflet of the cell membrane, a hallmark of apoptosis. Using FITC-annexin V staining of the cells, we found that CGS21680 significantly down-regulated exposure of phosphatidylserine on the outer cell membrane, which occurred in a concentration-dependent manner (Fig. 2A, B).

Figure 2.

Figure 2

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Selective A2AR agonist CGS21680 (CGS) decreases AICD in T cells. Murine 7.5 T-hybridoma cells were incubated with various doses of CGS or vehicle, 30 min before activation by ConA; 18–20 h later, T-cell death was measured using FITC-annexin V and propidium iodide (PI) staining and flow cytometry. Selection of gates was based on use of controls with calibration staining. A) Dot plots show percentages of gated live, dead, and apoptotic cells. B) Average percentage of annexin V-positive cells (both PI-positive and -negative). Results are means ± se of one representative experiment from 3 separate experiments; n = 3 samples/group. *P < 0.05; ***P < 0.001. C) Effect of CGS on activation-induced caspase-3 and PARP cleavage. Murine 7.5 T-hybridoma cells were treated with 100 nM CGS or vehicle 30 min before activation by ConA; 18–20 h later, whole-cell proteins were extracted, and Western blot analysis was performed for cleaved caspase-3 and cleaved PARP. Western blots in one figure were done on the same membrane. One representative experiment of 3 is shown. V, vehicle; M, medium.

The cleavage/activation of caspase-3, one of the key executioners of apoptosis, is an important indicator of apoptosis. PARP is a major downstream target of activated caspase-3 and is cleaved by this enzyme during apoptosis. To further establish the antiapoptotic effect of A2AR activation, we next investigated the effect of CGS21680 on ConA-induced cleavage of caspase-3 and PARP. We found that 7.5 hybridoma cells exhibited substantial cleavage of both caspase-3 and PARP when measured 18 h after ConA stimulation. The cleavage of both caspase-3 and PARP was markedly suppressed by pretreatment with CGS21680 (Fig. 2C).

Antiapoptotic effect of A2AR activation is mediated by inhibition of the FasL/Fas/caspase-8 system

Previous studies have documented that AICD in T cells, including Jurkat cells and hybridomas, proceeds via up-regulation of FasL and subsequent FasL/Fas interaction (39, 40). We first sought to confirm the role of the FasL/Fas system in activation-induced apoptosis in our hybridoma cells. For this purpose, 7.5 hybridoma cells were stimulated with ConA in the presence of 10 μg/ml FLIM58 anti-murine FasL inhibitory mAb (41) or its isotype control Ab. We found that neutralizing FasL significantly attenuated AICD, as detected using the MTT and LDH tests (Fig. 3A, B). This observation confirms that FasL is primarily responsible for the apoptotic effect of TCR cross-linking in the 7.5 hybridoma cells. We then studied the effect of A2AR activation on the gene expression of FasL. In agreement with previous data (37, 42), the expression of FasL mRNA was up-regulated after ConA activation of both 7.5 hybridoma and Jurkat cells (Fig. 3C, D). In addition, pretreatment with CGS21680 markedly reduced FasL mRNA expression in both murine 7.5 T cells (Fig. 3C) and human Jurkat T cells (Fig. 3D). We then analyzed the effect of A2AR activation on Fas expression. ConA up-regulated both Fas mRNA (Fig. 3E) and protein (Fig. 3F) in 7.5 hybridoma cells, and pretreatment of the cells with CGS21680 prevented these increases in Fas mRNA and protein expression (Fig. 3E, F). However, neither ConA nor CGS21680 affected Fas levels in Jurkat cells (data not shown).

Figure 3.

Figure 3

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A, B) AICD of T cells is mediated by Fas-FasL interactions. Murine 7.5 T-hybridoma cells were stimulated with ConA in the presence of 10 μg/ml FLIM58 anti-mouse FasL (a-FasL) neutralizing antibody or its isotype control. After 18–20 h, cell viability was assessed using the MTT (A) and LDH (B) assays. Results are means ± se of a representative experiment from 3 separate experiments; n = 5 wells/group. ** P < 0.01. C–E) Selective A2AR agonist CGS21680 (CGS) decreases FasL mRNA levels in ConA-stimulated murine 7.5 hybridoma (C) and human Jurkat (D) T-cell lines, and also decreases Fas mRNA levels in ConA-stimulated 7.5 murine T hybridoma cells (E). Cells were stimulated with ConA for 6 h, and CGS (100 nM) or its vehicle (veh) was added to the cells 30 min before stimulation. Fas and FasL mRNA levels were determined using real-time PCR. Results are means ± se of one representative experiment from 3 separate experiments; n = 4 samples/group. **P < 0.01; ***P < 0.001. F) CGS decreases the activation-induced expression of Fas protein levels in T cells. Murine 7.5 T-hybridoma cells were treated with 100 nM CGS or vehicle (V) 30 min before activation by ConA. Whole cell proteins were extracted at various time points after stimulation and analyzed by Western blotting. β-Actin, used as an internal control, is shown in Fig. 4. Western blots in one figure were done on the same membrane. One representative experiment of 3 is shown. unstim, unstimulated; M, medium.

Trimerization of the death receptor Fas by FasL leads to the recruitment of procaspase-8 by the adaptor protein Fas-associated death domain and the formation of the death-inducing signaling complex through homologous domain interactions, resulting in the activation of caspase-8. Active caspase-8 then cleaves and activates caspase-3 and other executioner caspases, which target vital cellular substrates and induce cell death (1). ConA activated caspase-8 in 7.5 hybridoma cells as determined by the appearance of cleaved caspase-8, and CGS21680 decreased the abundance of cleaved, active caspase-8 protein (Fig. 4).

Figure 4.

Figure 4

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Selective A2AR agonist CGS21680 (CGS) decreases the activation-induced up-regulation of cleaved caspase-8 levels in T cells. Murine 7.5 T-hybridoma cells were treated with 100 nM CGS or vehicle (V) 30 min before activation by ConA. At different time points after ConA administration, whole-cell proteins were extracted and analyzed by Western blotting. β-Actin was used as an internal control. Western blots in one figure were done on the same membrane. One representative experiment of 3 is shown. M, medium.

These results support the concept that A2AR activation limits T-cell AICD, at least in part, by down-regulating the FasL/Fas/caspase-8 system.

A2AReceptor activation inhibits NF-κB activation in T cells

It has been previously demonstrated that the NF-κB transcription factor system plays a critical role in triggering the gene expression of FasL and subsequent apoptosis in T cells (43, 44). To delineate the effect of A2AR activation on NF-κB activation, we first transiently transfected Jurkat cells with a NF-κB-luciferase reporter construct. Then, the transfectants were pretreated with CGS21680 or its vehicle for 30 min, which was followed by stimulation with ConA for 20–24 h. The effect of CGS21680 on NF-κB-dependent gene transcription was assessed using the luciferase assay. Our results showed that CGS21680 suppressed ConA-stimulated NF-κB-dependent gene transcription (Fig. 5A).

Figure 5.

Figure 5

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A) Selective A2AR agonist CGS21680 (CGS) decreases NF-κB-dependent transcriptional activity in Jurkat cells. To determine NF-κB-dependent transcriptional activity, cells were transfected with pNF-κB-Luc reporter construct or its control vector (pTAL) for 20–24 h, which was followed by treatment of the cells with ConA in the presence or absence of 100 nM CGS21680. Luciferase activity was determined from cell extracts that were obtained 8 h after ConA treatment. Results are means ± se of one representative experiment from 3 separate experiments; n = 3 wells/group. ***P < 0.001. B) CGS suppresses the activation-induced nuclear translocation of NF-κB p65 subunit. Murine 7.5 T-hybridoma cells were treated with 100 nM CGS or vehicle (V) 30 min before activation by ConA, and cytosolic and nuclear proteins were extracted 4 h after ConA activation. NF-κB p65 levels in the cytoplasmic and nuclear extracts were detected using Western blot analysis. β-Actin, used as an internal control, was extracted from the cytosolic fraction; the nuclear fraction was not contaminated with β-actin. Western blots in one figure were done on the same membrane. One representative experiment of 3 is shown. M, medium.

Because NF-κB activation involves its nuclear translocation, in another approach we measured the levels of p65 NF-κB subunit in both the cytoplasm and nucleus before and after ConA activation. ConA triggered the disappearance of p65 from the cytoplasm (Fig. 5B), which was paralleled by the translocation of p65 into the nucleus (Fig. 5B). Pretreatment of the cells with CGS21680 inhibited this NF-κB p65 nuclear translocation (Fig. 5B), confirming the results of the luciferase assay.

A2AR activation inhibits NF-ΑΤp and Egr activation in T cells

NF-ATp is activated by the engagement of the TCR and is a key regulator of FasL expression after activation (45, 46). NF-ATp has been directly implicated in FasL transcription by its binding to a transactivating site in the FasL promoter (47). NF-ATp (also called NF-AT1) is located in the cytoplasm in unstimulated T cells, and TCR signaling leads to its nuclear translocation. We found that ConA stimulation of 7.5 hybridoma cells resulted in nuclear translocation of NF-ATp as indicated by its reduced levels in the cytoplasm and increased abundance in the nucleus (Fig. 6A). CGS21680 pretreatment of the cells before ConA activation inhibited this nuclear translocation of NF-ATp (Fig. 6A).

Figure 6.

Figure 6

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Selective A2AR agonist CGS21680 (CGS) decreases the activation-induced nuclear translocation of NF-ATp (A) and the activation-induced expression of Egr-1 and Egr-3 transcription factors (B). ConA treatment significantly decreased expression of cytosolic NF-ATp and significantly increased expression of NF-ATp. In contrast, in the presence of CGS, ConA was unable to significantly decrease cytosolic NF-ATp and increase NF-ATp expression. ConA treatment significantly increased Egr-1 and Egr-3 expression of the cells. In contrast, in the presence of CGS, ConA was unable to significantly increase expression of Egr1 and Egr3. Murine 7.5 T-hybridoma cells were treated with 100 nM CGS or vehicle (V) 30 min before activation by ConA. Cytosolic and nuclear proteins were extracted, and Western blot analysis was performed for Egr proteins (from extracts taken 3–4 h after stimulation) in whole-cell lysates, and for NF-ATp (from extracts taken 1–2 h after stimulation) in cytoplasmic and nuclear extracts. β-Actin, used as an internal control, was extracted from cytosolic fractions; nuclear fractions were not contaminated with β-actin. Western blots in one figure were done on the same membrane. One representative experiment of 3 is shown. Densitometric results are means ± se of ≥3 separate experiments. *P < 0.05, **P < 0.01 vs. V + M. M, medium.

The Egr family of transcription factors is also important for FasL expression in T cells, because Egr proteins and NF-AT form DNA-binding complexes and can cooperatively regulate FasL transcription (48, 49). We therefore investigated the effect of CGS21680 on Egr-1, Egr-2, and Egr-3 expression, because inducible Egr expression correlates with its transcriptional activity. The expression of Egr-1 and Egr-3 protein was increased 3–4 h after ConA stimulation, and CGS21680 reduced the abundance of these Egr family members (Fig. 6B). However, neither ConA nor CGS21680 affected Egr-2 levels (data not shown).

PKA mediates the protective effect of A2AR activation against T-cell AICD

A2AR signaling traditionally proceeds through elevation of cAMP, which leads to PKA activation and downstream signaling (50). PKA is a tetrameric structure consisting of two regulatory and two catalytic subunits. The binding of cAMP to the regulatory subunits results in the dissociation of the holoenzyme complex. Pharmacological agents can inhibit PKA either by blocking directly the activity of the catalytic units or by blocking the dissociation and function of the regulatory subunits (51). We first determined the role of the catalytic subunits in directly mediating the antiapoptotic effect of A2AR activation. The selective catalytic subunit inhibitors H89 (0.001–10 μM) and KT5720 (0.001–10 μM) failed to block the ability of CGS21680 to inhibit AICD (data not shown). We next tested Rp-8-Br-cAMPS, which not only prevents the activation of catalytic subunits but also blocks the release of the regulatory subunits of the PKA holoenzyme. We found that Rp-8-Br-cAMPS (500 μM) reversed the inhibitory effect of CGS21680 on AICD as assessed using the MTT and LDH assays (Fig. 7).

Figure 7.

Figure 7

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Protective effect of the selective A2AR agonist CGS21680 (CGS) against T-cell AICD is mediated by the cAMP-PKA pathway. Murine 7.5 T-hybridoma cells were preincubated for 30 min with the PKA inhibitor Rp-8-Br-cAMPs (PKA i; 500 μM) or its vehicle (veh) and then were treated with 100 nM CGS or its vehicle (no CGS21680); 30 min later, the cells were activated with ConA. After an additional 18–20 h, cell viability was assessed using the MTT (A) and LDH (B) assays. Results are means ± se of one representative experiment from 3 separate experiments; n = 5 wells/group. ***P < 0.001 vs. veh-no CGS21680; ###P < 0.001 vs. veh-CGS21680.

More recent studies have identified an alternative cAMP target, which is independent of PKA: EPAC (52). We excluded the role of EPAC in mediating the antiapoptotic effect of A2AR activation, because AICD was not reversed by the potent EPAC activator 8-CPT-2-Me-cAMP (1–500 μM) (data not shown).

In summary, PKA regulatory subunit mediates the antiapoptotic effect of A2AR stimulation in T cells.

DISCUSSION

The present study demonstrates that A2AR stimulation rescues TCR-stimulated CD4+ T cells from AICD by down-regulating the expression of both Fas and FasL. AICD, particularly in mature CD4+ T cells, is mediated through Fas/FasL interactions. By using the neutralizing anti-FasL antibody FLIM58, we showed that blockade of the Fas-FasL system prevented the death of T cells, indicating a role for the Fas/FasL system in mediating AICD also in our system.

The mechanism behind the antiapoptotic actions of A2ARs involves pretranslational effects, because A2AR activation reduced mRNA levels of both Fas and FasL in activated murine T-hybridoma cells and mRNA levels of FasL in activated human Jurkat cells. The FasL promoter is fairly well characterized, with binding sites for interaction with the transcription factors NF-κB, NF-AT, activator protein-1, early growth response family proteins Egr-1, Egr-2, Egr-3, c-Myc, secretory protein-1, and interferon regulatory factors, all of which have been shown to be functionally important for FasL expression (1,2,3, 43,44,45, 47, 53). Our results indicate that inhibition of NF-κB activation by A2AR activation may be an important mechanism eliciting down-regulation of FasL expression and preventing apoptosis. This proposition is supported by results from previous studies, in which cAMP-elevating neuropeptides (54, 55), antioxidants (34), or immunosuppressive sesquiterpenes (35) inhibited both NF-κB activation and AICD in T cells. Because A2AR activation has been shown to prevent antigen- and LPS-driven NF-κB activation in B lymphocytes (56), it appears that NF-κB is a major target of A2AR signaling in lymphoid cells. In contrast, the role of A2ARs in regulating the NF-κB pathways is controversial in monocytes/macrophages, as we reported a lack of effect of A2A agonists in RAW264.7 cells (57, 58) and peritoneal macrophages (59), whereas others found that NF-κB activation in LPS-stimulated monocytic THP-1 cells was strongly attenuated in the presence of CGS21680 (60).

We also documented that A2AR signaling decreased the nuclear translocation of NF-ATp in T cells, providing a further explanation for the suppressive effect of A2AR activation on FasL gene expression. We speculate that because the A2AR is up-regulated after TCR stimulation in an NF-ATp-dependent manner (20), the decrease in NF-ATp activation after A2AR activation may blunt the further up-regulation of the A2AR, resulting in preservation of at least some T-cell function in the face of high extracellular levels of adenosine. Finally, we noted that A2AR activation decreased Egr-1 and Egr-3 up-regulation, which probably contributes to the decrease in FasL gene expression after A2AR activation.

Signaling through A2AR stimulates adenylyl cyclase with a consequent increase of cAMP levels and PKA activity (50). The A2AR-mediated inhibition of T-cell AICD was reversed by the PKA RI inhibitor Rp-8-Br-cAMPS. These data confirm that regulatory subunits of PKA have functional activity and that PKA has biological significance beyond its catalytic activity (51).

The effect of adenosine on lymphocyte apoptosis appears to depend on several factors including the maturity of T cells, the apoptotic stimulus, and the inflammatory environment. In this regard, numerous studies have demonstrated that immature double-positive thymocytes display apoptosis after A2AR engagement in the absence of TCR stimulation (26,27,28,29). In contrast, extracellular adenosine protected the same double-positive thymocytes from TCR-induced apoptosis (61, 62). This protection was more evident when ADA was inhibited by either pharmacological means (61) or knockout (62), but the receptor subtype mediating the protective effect was not identified. Our results demonstrate that A2ARs inhibit TCR-induced apoptosis of mature CD4+ cells. In addition, A2AR activation in the absence of TCR ligation fails to affect the apoptosis of mature CD4+ cells (data not shown).

A2ARs are generally viewed as negative regulators of immune cells, including activated T cells. However, because AICD can be viewed as a process that terminates an immune response, the fact that A2AR activation prevents AICD indicates that the role of A2ARs in regulating immune responses is more complex then previously thought and that A2AR activation can actually prolong immune processes. Although it is not clear yet what type of immune responses and T-cell subsets are sustained after A2AR activation, it is possible that these are mostly suppressive or regulatory cells. To support such a scenario, it was recently reported that cAMP-elevating agents rescued cultures of T lymphocytes from anti-CD3/CD28 Ab-induced apoptosis by preventing acquisition of the CD45RO phenotype and leading to the generation of a new subpopulation of primed CD4+CD45RA+ effector cells (cAMP-primed CD45RA), which displayed prolonged survival compared with CD45RO effector cells, produced Th2-type cytokines, and had a phenotype characteristic of memory/effector T lymphocytes in terms of cell surface markers (63). Furthermore, Zarek et al.(20) have recently demonstrated that A2AR stimulation not only inhibits the proliferation of effector T cells but also promotes the generation of adaptive Foxp3+LAG3+ regulatory T cells and thus plays an important role in peripheral tolerance by inducing long-term T-cell anergy. Clearly, further studies will be necessary to dissect the precise role of the antiapoptotic effect of A2AR activation in regulating T-cell-mediated immune responses.

Acknowledgments

This work was supported by the U.S. National Institutes of Health (NIH; grant R01 GM66189), the Intramural Research Program of NIH, National Institute on Alcohol Abuse and Alcoholism, and the Hungarian Research Fund OTKA (73110 and 78275).

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