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The Journal of Immunology, 2004, 173: 932-944.
Copyright © 2004 by The American Association of Immunologists

Adenosine Acts through A2 Receptors to Inhibit IL-2-Induced Tyrosine Phosphorylation of STAT5 in T Lymphocytes: Role of Cyclic Adenosine 3',5'-Monophosphate and Phosphatases1

Hong Zhang*, David M. Conrad*, Jared J. Butler*, Chuanli Zhao*, Jonathan Blay{dagger} and David W. Hoskin2,*,{ddagger}

Departments of * Microbiology and Immunology, {dagger} Pharmacology, and {ddagger} Pathology, Dalhousie University, Halifax, Nova Scotia, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Adenosine is a purine nucleoside with immunosuppressive activity that acts through cell surface receptors (A1, A2a, A2b, A3) on responsive cells such as T lymphocytes. IL-2 is a major T cell growth and survival factor that is responsible for inducing Jak1, Jak3, and STAT5 phosphorylation, as well as causing STAT5 to translocate to the nucleus and bind regulatory elements in the genome. In this study, we show that adenosine suppressed IL-2-dependent proliferation of CTLL-2 T cells by inhibiting STAT5a/b tyrosine phosphorylation that is associated with IL-2R signaling without affecting IL-2-induced phosphorylation of Jak1 or Jak3. The inhibitory effect of adenosine on IL-2-induced STAT5a/b tyrosine phosphorylation was reversed by the protein tyrosine phosphatase inhibitors sodium orthovanadate and bpV(phen). Adenosine dramatically increased Src homology region 2 domain-containing phosphatase-2 (SHP-2) tyrosine phosphorylation and its association with STAT5 in IL-2-stimulated CTLL-2 T cells, implicating SHP-2 in adenosine-induced STAT5a/b dephosphorylation. The inhibitory effect of adenosine on IL-2-induced STAT5a/b tyrosine phosphorylation was reproduced by A2 receptor agonists and was blocked by selective A2a and A2b receptor antagonists, indicating that adenosine was mediating its effect through A2 receptors. Inhibition of STAT5a/b phosphorylation was reproduced with cell-permeable 8-bromo-cAMP or forskolin-induced activation of adenylyl cyclase, and blocked by the cAMP/protein kinase A inhibitor Rp-cAMP. Forskolin and 8-bromo-cAMP also induced SHP-2 tyrosine phosphorylation. Collectively, these findings suggest that adenosine acts through A2 receptors and associated cAMP/protein kinase A-dependent signaling pathways to activate SHP-2 and cause STAT5 dephosphorylation that results in reduced IL-2R signaling in T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Adenosine is a purine nucleoside that interacts with cell surface receptors to mediate both anti-inflammatory and immunosuppressive activities (1, 2). In addition to attenuating neutrophil and macrophage function (3, 4), adenosine is a potent inhibitor of T cell-mediated immune responses (5, 6). In this regard, adenosine may be an important mediator of tumor-associated immune suppression. Solid tumors contain local regions of hypoxia (7), which are associated with increased adenosine production and release through adenosine kinase inhibition and adenine nucleotide breakdown via the 5'-nucleotidase pathway (8, 9). Moreover, the level of adenosine (~10 µM) that is present in the extracellular fluid of mouse and human carcinomas grown in immune-deficient mice is more than sufficient to suppress T cell function (10). Elevated concentrations of adenosine within the tumor microenvironment may interfere with cell-mediated antitumor immune responses because comparable concentrations of adenosine suppress the activation of T lymphocytes to a killer phenotype (11), as well as the recognition/adhesion and effector phases of tumor cell destruction by cytotoxic lymphocytes (12, 13, 14). The inhibitory effects of adenosine on the induction and effector function of tumoricidal T lymphocytes may be the result of adenosine-induced alterations in T cell signaling pathways. In this regard, adenosine has been shown to inhibit TCR triggering (6, 13) and the activity of the protein tyrosine kinase 56lck (15), which is an important initiator of the TCR signaling cascade (16). However, the effect of adenosine on IL-2R-associated signaling pathways has not yet been explored in T lymphocytes.

IL-2 is an immunomodulatory cytokine that is essential for the growth and survival of activated T lymphocytes (17). The IL-2R consists of three subunits designated {alpha}, {beta}, and {gamma} (18). An IL-2R{beta} chain combines with an IL-2R{gamma} chain to yield an intermediate affinity IL-2R, whereas the addition of an IL-2R{alpha} chain results in the formation of a high affinity IL-2R. IL-2R signaling intermediates involved in the transduction of a proliferative signal include Jak1, Jak3, and STAT5 (19, 20). The tyrosine kinases Jak1 and Jak3 associate with membrane-proximal regions of IL-2R{beta} and IL-2R{gamma}, respectively, and undergo catalytic activation following ligand-induced heterodimerization of IL-2R{beta} and IL-2R{gamma} chains (21, 22). Jak1 and Jak3 activation leads to the phosphorylation of at least three tyrosine residues (Y338, Y392, and Y510) on IL-2R chains (23). Primed by these phosphorylation events, the IL-2R complex generates two major proliferative signals. One of these proliferative signals is mediated by the adaptor protein Shc (23, 24), which binds to phosphorylated Y338, undergoes tyrosine phosphorylation, and activates p42/p44-MAPK (also known as ERK) and PI3K signaling pathways. The Grb2/Sos complex is recruited to Shc, and Sos activates the ERK pathway that up-regulates genes such as c-fos and c-jun (25, 26). The PI3K signaling pathway, which includes the serine/threonine kinase Akt (27), is activated through recruitment of the adaptor protein GAB2 (28, 29). The second proliferative signal transduced by the IL-2R complex involves the transcription factor STAT5 (30, 31), which is recruited to phosphorylated Y510 Shc (23), becomes tyrosine phosphorylated, homo- and/or heterodimerizes with other STAT molecules, and then directly translocates to the nucleus, where the complex binds regulatory elements that control gene expression (32). The STAT5 family consists of highly homologous STAT5a and STAT5b proteins that are coded for by two different STAT5 genes (33). STAT5 plays a critical role in IL-2-driven T cell responses because T lymphocytes from mutant mice with targeted deletions of STAT5a and/or STAT5b exhibit impaired proliferative responses to IL-2 (34, 35).

In the present investigation, we demonstrated that adenosine inhibited IL-2-dependent proliferation of the CTLL-2 T cell line. The inhibitory effect was associated with a reduction in tyrosine phosphorylation of STAT5a and STAT5b that was mediated by the activation of a protein tyrosine phosphatase (PTP).3 The PTP Src homology region 2 domain-containing phosphatase-2 (SHP-2) was implicated in STAT5a/b dephosphorylation because adenosine strongly increased tyrosine phosphorylation of SHP-2 and the formation of complexes consisting of SHP-2 and STAT5 in IL-2-stimulated CTLL-2 T cells. In contrast, adenosine did not affect the phosphorylation status of the upstream kinases Jak1 or Jak3. The inhibitory effect of adenosine on STAT5a/b phosphorylation was mediated through cell surface A2a and A2b receptors and involved associated cAMP/protein kinase A (PKA)-dependent signaling pathways. Together, these data reveal a mechanism by which adenosine, at concentrations typically present in the microenvironment of solid tumors, might contribute to diminished IL-2 responsiveness and the progressive development of defective T lymphocyte responses in cancer patients.


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

Adult (6- to 8-wk-old) female C57BL/6 mice were purchased from Charles River Laboratories (Lasalle, Canada) and housed in the Carleton Animal Care Facility at Dalhousie University. Mice were maintained on standard laboratory chow and water supplied ad libitum.

Reagents

RPMI 1640 medium was purchased from Sigma-Aldrich (Oakville, Canada). L-glutamine, penicillin, streptomycin, FCS, and TRIzol reagent were purchased from Invitrogen Life Technologies (Burlington, Canada). Anti-phospho-STAT5a/b, anti-STAT5a, anti-phospho-Akt, anti-phosphotyrosine, and anti-ERK1/2 Ab were purchased from Upstate Biotechnology (Lake Placid, NY). Anti-phospho-ERK1/2 Ab, anti-SHP-1 Ab, anti-SHP-2 Ab, anti-PTP1B Ab, goat anti-mouse IgG-HRP, and goat anti-rabbit IgG-HRP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-Jak1 Ab and anti-phospho-Jak3 Ab were from BioSource International (Camarillo, CA). Coformycin, sodium orthovanadate, bpV(phen), and okadaic acid were purchased from Calbiochem-Novabiochem (La Jolla, CA). PMSF, leupeptin, pepstatin, aprotinin, adenosine, CGS 21680, alloxazine, 8-(3-chlorostyryl)caffeine (CSC), 5'-N-ethylcarboxamidoadenosine (NECA), N (6)-2-(4-aminophenyl)ethyladenosine (APNEA), 9-chloro-2-(2-furanyl)-5-([phenylacetyl]amino)[1,2,4]-triazolo[1,5-c]quinazoline (MRS-1220), 3,7-dimethyl-1-propargylxanthine (DMPX), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), Rp-cAMPS, forskolin, and 8-bromo-cAMP (8-Br-cAMP) were purchased from Sigma-Aldrich.

Cell culture

The IL-2-dependent CTLL-2 T cell line was obtained from the American Type Culture Collection (Manassas, VA). CTLL-2 cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (hereafter called complete RPMI 1640 medium), and 20 U/ml IL-2 (R&D Systems, Minneapolis, MN) in 250-ml culture flasks maintained at 37°C in a humidified 5% CO2 incubator. Spleen T cells were obtained from spleens of mice sacrificed by cervical dislocation. Spleens were excised using aseptic technique, and spleen cell suspensions were prepared in cold PBS (pH 7.4). Following erythrocyte removal by osmotic shock, lymphocytes were washed, resuspended in complete RPMI 1640 medium, and passaged through a nylon wool column (Cellular Products, Buffalo, NY) to enrich for T lymphocytes. T cells were placed in wells of a 24-well flat-bottom tissue culture plate (8 x 106 cells/well) and induced to up-regulate IL-2R by incubation for 24 h at 37°C in a humidified 5% CO2 incubator in the presence of anti-CD3 mAb (1/20 dilution of hybridoma 145-2C11 culture supernatant). CTLL-2 cells and activated spleen T cells were washed twice with PBS, resuspended in serum-free complete RPMI 1640 medium, and incubated for 4 h at 37°C in a humidified 5% CO2 incubator to remove residual IL-2 from IL-2R. CTLL-2 cells and activated spleen T cells were then washed with PBS and resuspended in complete RPMI 1640 medium before use in experiments. CTLL-2 and spleen T cell viability was verified by trypan blue dye exclusion to be at least 95%.

Cell proliferation assay

CTLL-2 cells were added in triplicate (104 cells/well) to a 96-well, flat-bottom microtiter plate and cultured in the absence or presence of the indicated concentrations of adenosine and/or coformycin for 30 min before the addition of 10 U/ml IL-2. Following 18 h of culture at 37°C in a 5% CO2, humidified incubator, each well was pulsed with 0.5 µCi of tritiated thymidine ([3H]TdR; sp. act., 60 Ci/mmol; ICN Biomedicals, Mississauga, Canada) and cultured for an additional 6 h. Samples were then harvested onto glass fiber filter mats (Skatron, Sterling, VA) using a Titer-Tek multiple sample harvester, and [3H]TdR incorporation was determined as a measure of DNA synthesis using a liquid scintillation counter. Data are a shown as mean cpm ± SD.

Flow cytometry

CTLL-2 cell surface expression of IL-2R {alpha}-, {beta}-, and {gamma}-chains was determined by flow cytometry using FITC-conjugated Ab (BD Pharmingen, Mississauga, Canada), as previously described (36).

RT-PCR analysis

Total RNA was isolated from CTLL-2 cells using TRIzol reagent, as recommended by the manufacturer (Invitrogen Life Technologies, Carlsbad, CA). Single-strand cDNA was synthesized from 0.5 µg of RNA with 200 U of Moloney murine leukemia virus-derived reverse transcriptase (Life Technologies) in the presence of 0.2 mM dNTPs, 1 µg of random hexamers, and 10 mM DTT. PCR was performed using an automatic DNA thermocycler (MJ Research, Watertown, MA). Each PCR used equal amounts of cDNA, 2.5 U of Taq polymerase (Life Technologies), 0.2 mM dNTPs, and 0.5 µM each primer pair in a 1/10 dilution of PCR buffer (2 M KCl, 1 M Tris-HCl, pH 8.4, 1 M MgCl2, and 1 mg/ml BSA). The primer pairs described below were used for PCR (amplicon size is given after the reverse primer). F, refers to forward primer; R, refers to reverse primer. All primers were designed to bind intron-bridging exons of the respective gene. A1 receptor (F), 5'-GTGATTTGGGCTGTGAAGGT-3'; A1 receptor (R), 5'-CAAGGGAGAGAATCCAGCAG-3' (product size: 322 bp); A2a receptor (F), 5'-CACGCAGAGAGTTCCATCTTCA-3'; A2a receptor (R), 5'-AGCAGTTGATGATGTGCAGG-3' (product size: 497 bp); A2b receptor (F), 5'-GCGAATAAAAGCTGCTGCTC-3'; A2b receptor (R), 5'-AAAATGCCCACGATCATAGC-3' (product size: 245 bp); A3 receptor (F), 5'-GACTGGCTTCAGAGAGACGC-3'; and A3 receptor (R), 5'-AGGGTTCATCATGGAGTTCG-3' (product size: 202 bp).

PCR mixtures were overlaid with 100 µl of mineral oil. All adenosine receptor cDNAs were amplified (35 cycles) by denaturation at 92°C for 30 s, annealing at 60°C for 30 s, and primer extension at 72°C for 1.5 min. PCR products were visualized by electrophoresis across an ethidium bromide-stained 1.5% Tris-acetate-EDTA agarose gel, and the detected PCR amplicon was compared with a 100-bp ladder (Promega, Madison, WI).

Preparation of cell lysates

CTLL-2 cells were adjusted to a density of 2.5 x 106 cells/ml and aliquoted into sample tubes before treatment for 15 min with adenosine or other reagents. CTLL-2 cells were then exposed to IL-2 (10 U/ml) for another 5 min. At the end of the incubation period, samples were chilled on ice and centrifuged at 400 x g and 4°C for 5 min. The cell pellet was washed once with ice-cold PBS, followed by lysis of the cell pellet with modified radioimmunoprecipitation buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 100 µM sodium orthovanadate, 1 mM PMSF, 5 µg/ml leupeptin, 5 µg/ml pepstatin, and 5 µg/ml aprotinin) for 30 min at 4°C. Insoluble cytoplasmic granules and nuclei were removed by centrifugation for 10 min at 12,000 x g and 4°C. The supernatants were collected and stored at –80°C before analysis by Western blotting. The protein concentrations in cellular protein preparations were determined by Bradford protein assay, according to the manufacturer’s instructions (Bio-Rad, Hercules, CA).

Immunoprecipitation and Western blotting

For immunoprecipitation, the cell lysates were precleared with normal rabbit IgG bound to protein A-Sepharose 4B beads by rotating overnight at 4°C. SHP-1, SHP-2, PTP1B, and STAT5 were immunoprecipitated from cell lysates with the appropriate Ab bound on protein A-Sepharose 4B beads for 2 h at 4°C. After four washes with lysis buffer, the beads were boiled in SDS sample buffer and analyzed by Western blotting. For Western blotting, cell lysates were boiled in SDS sample buffer, and 20 µg of total protein was loaded into each well of a SDS-PAGE gel for separation by electrophoresis. The protein bands were then transferred onto nitrocellulose membranes, and the resulting blots were blocked for 1 h with PBS-Tween (0.25 M Tris, pH 7.5, PBS, 150 mM sodium chloride, and 0.2% Tween 20) containing 5% powdered skim milk. Blots were probed overnight with the indicated Ab at a 1 µg/ml concentration. Blots were then washed three times with PBS-Tween and probed for 1 h with the appropriate HRP-conjugated secondary Ab (goat anti-rabbit IgG or anti-mouse IgG). Next, the blots were washed three times with PBS-Tween, and the protein bands on the blots were detected using an ECL Western blot detection system (Amersham Pharmacia Biotech, Kirkland, Canada). To confirm equal sample loading, the blots were stripped and reprobed with Ab against the appropriate nonphosphorylated protein. Membrane stripping and reprobing were conducted according to the instructions provided by the manufacturer of the ECL kit.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Adenosine inhibits IL-2-induced CTLL-2 proliferation

The effect of adenosine on IL-2-dependent T cell proliferation was determined by culturing IL-2-dependent CTLL-2 cells in medium containing IL-2 without or with various concentrations of adenosine (plus 2.5 µM coformycin to prevent adenosine degradation by endogenous adenosine deaminase). After 24 h of culture, IL-2-induced CTLL-2 proliferation was assessed by [3H]TdR incorporation. As seen in Fig. 1A, the proliferation of CTLL-2 cells in response to IL-2 was significantly inhibited in a dose-dependent fashion by 5 and 10 µM adenosine (p = 0.0009 and p < 0.0001, respectively). Higher concentrations of adenosine (25 and 50 µM) did not have a substantially greater inhibitory effect (Fig. 1B). Unless otherwise noted, all subsequent experiments used 10 µM adenosine because this adenosine concentration had a maximal inhibitory effect on CTLL-2 proliferation. Coulter counting of CTLL-2 cells cultured over a 120-h time course in the presence of IL-2 without or with adenosine (plus coformycin) revealed that adenosine reduced total cell accumulation (Fig. 1C). No inhibition of CTLL-2 DNA synthesis or cell accumulation was observed in the presence of coformycin alone. To ensure that adenosine did not cause nonspecific cytotoxicity in CTLL-2 cell cultures, the viability of CTLL-2 cells following culture in the presence of adenosine was determined by trypan blue dye exclusion. No effect of adenosine at the concentrations used in these studies was observed on CTLL-2 cell viability, indicating that reduced IL-2-driven proliferation of CTLL-2 cells in the presence of adenosine was not due to diminished cell viability (data not shown). In addition, flow cytometric analysis revealed that adenosine did not significantly affect IL-2R expression by CTLL-2 cells (data not shown), indicating that adenosine-treated CTLL-2 cells retained the capacity to bind IL-2.



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  		FIGURE 1. Adenosine inhibits the IL-2-dependent proliferation of CTLL-2 T cells. A and B, CTLL-2 cells were treated with medium alone, 2.5 µM coformycin (Cof) alone, or the indicated concentrations of adenosine (Ado) plus 2.5 µM coformycin, and then stimulated with IL-2 (10 U/ml). After 24 h of culture, [3H]TdR incorporation assay was assessed. Data are shown as mean cpm ± SD of [3H]TdR incorporation in triplicate samples. Background proliferation of CTLL-2 cells in the absence of IL-2 was 840 ± 627 (A) and 535 ± 129 (B). Results are representative of nine different experiments. Statistical significance was determined by one-way ANOVA and Bonferroni multiple comparisons test. C, CTLL-2 cells were treated with medium alone ({diamondsuit}), 2.5 µM coformycin (Cof) alone ({blacksquare}), or 50 µM adenosine (Ado) plus 2.5 µM coformycin ({blacktriangleup}), and then stimulated with IL-2 (10 U/ml). After 48, 72, 96, and 120 h of culture, CTLL-2 cell numbers were determined by Coulter counting. Results are representative of two independent experiments.

 
Adenosine inhibits IL-2-induced tyrosine phosphorylation of STAT5

We hypothesized that the inhibitory effect of adenosine on IL-2-dependent CTLL-2 proliferation might be due to decreased phosphorylation of tyrosine residues on STAT5a and/or STAT5b, because this is an essential step in IL-2R signaling (30, 31). To test this hypothesis, CTLL-2 cells were treated with 10 µM adenosine (plus coformycin) for 15 min, followed by exposure to different concentrations of IL-2 (1, 5, or 10 U/ml) for different lengths of time (5 min-12 h). Cell lysates were prepared, and Western blotting with anti-phospho-STAT5a/b or anti-STAT5a Ab was performed to determine STAT5a/b tyrosine phosphorylation and confirm equal protein loading, respectively. The percentage of OD ratios was calculated by comparing the density of individual phospho-STAT5a bands with the corresponding STAT5a band. As shown in Fig. 2, adenosine dramatically decreased dose-dependent STAT5a/b tyrosine phosphorylation in response to IL-2 over the entire 12-h time course, suggesting that adenosine was acting at or before the level of STAT5 to inhibit IL-2-dependent CTLL-2 proliferation. Fig. 3 shows that adenosine, as well as the adenosine analog NECA, had similar inhibitory effects on IL-2-induced tyrosine phosphorylation of STAT5a/b in mouse spleen T cells, demonstrating the physiological relevance of our findings using CTLL-2 cells.



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  		FIGURE 2. Adenosine inhibits IL-2-induced STAT5 tyrosine phosphorylation in CTLL-2 cells. CTLL-2 cells were treated with medium alone or 10 µM adenosine (Ado; plus 2.5 µM coformycin) for 15 min at 37°C and then stimulated with IL-2 (10 U/ml) for the indicated periods of time (A) or for 5 min with the indicated concentrations of IL-2 (B). Control consisted of cells that were exposed to only medium. Cell lysates were prepared, and Western blotting was performed with Ab to phospho-STAT5a/b or STAT5a (both 1 µg/ml) to detect STAT5 tyrosine phosphorylation and protein loading, respectively. The percentage of OD ratios was calculated by comparing the density of individual phospho-STAT5a bands with the corresponding STAT5a band. Results are representative of four different experiments.

 


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  		FIGURE 3. Adenosine inhibits IL-2-induced STAT5 tyrosine phosphorylation in mouse spleen T cells. T cells isolated from mouse spleen were cultured for 24 h in the presence of soluble anti-CD3 mAb, washed extensively, and then treated with medium alone, 10 µM adenosine (Ado; plus 2.5 µM coformycin), or 1 µM NECA for 15 min at 37°C, and then stimulated with exogenous IL-2 (10 U/ml) for 5 min. Control consisted of anti-CD3-treated T cells that were not stimulated with exogenous IL-2. Unstimulated T cells were cultured in the absence of anti-CD3 mAb and were not exposed to exogenous IL-2. Cell lysates were prepared, and Western blotting was performed with Ab to phospho-STAT5a/b or STAT5a (both 1 µg/ml) to detect STAT5 tyrosine phosphorylation and protein loading, respectively. The percentage of OD ratios was calculated by comparing the density of individual phospho-STAT5a bands with the corresponding STAT5a band. Results are representative of two independent experiments.

 
Adenosine does not affect IL-2-induced Jak1 or Jak3 phosphorylation

To determine whether the inhibitory effect of adenosine on STAT5 tyrosine phosphorylation might be due to decreased activity of Jak1 and/or Jak3, which are upstream kinases required for STAT5 induction (37, 38), CTLL-2 cells were treated with 10 µM adenosine (plus coformycin) or the adenosine analogues NECA or CGS 21680 (both at 1 µM) for 15 min before stimulation for 5 min with IL-2. Cell lysates were then prepared, and Western blotting with anti-phospho-Jak1 or anti-phospho-Jak3 Ab was performed to detect Jak1 or Jak3 tyrosine phosphorylation, respectively. The same blot was then stripped and reprobed with anti-phospho-STAT5a/b Ab for comparison with phospho-Jak1 and phospho-Jak3 or with anti-STAT5a Ab to confirm equal protein loading. In contrast to the marked inhibitory effect of adenosine or adenosine analogues (NECA, CGS 21680) on tyrosine phosphorylation of STAT5a/b, neither adenosine nor the adenosine agonists affected IL-2-induced Jak1 or Jak3 tyrosine phosphorylation (Fig. 4). These data indicate that adenosine was not mediating its inhibitory effect on IL-2R signal transduction by acting upstream of STAT5.



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  		FIGURE 4. Adenosine does not prevent Jak1 or Jak3 phosphorylation. CTLL-2 cells were treated with medium alone or 10 µM adenosine (Ado; plus 2.5 µM coformycin), 1 µM NECA, or 1 µM CGS 21680 for 15 min at 37°C, followed by stimulation with IL-2 (10 U/ml) for 5 min. Control consisted of cells that were exposed to only medium. Cell lysates were prepared, and Western blotting with Ab to phospho-Jak1 (1 µg/ml) or phospho-Jak3 (10 µg/ml) was used to detect Jak1 and Jak3 tyrosine phosphorylation, respectively. The same blot was stripped and reprobed with anti-phospho-STAT5a/b Ab for comparison with Jak1/Jak3 and with anti-STAT5a Ab to confirm equal protein loading. Results are representative of four individual experiments.

 
PTP inhibitors rescue STAT5 tyrosine phosphorylation in the presence of adenosine

We next determined whether the inhibitory effect of adenosine on tyrosine phosphorylation of STAT5 might involve activation of a PTP. CTLL-2 cells were treated for 15 min with sodium orthovanadate (1 mM) or bpV(phen) (100 µM) to inhibit PTP (39, 40), or with 50 nM okadaic acid, which inhibits protein phosphatase 2A, but not PTPs (41), and were then exposed to 10 µM adenosine (plus coformycin) for another 15 min, followed by stimulation for 5 min with IL-2. Cell lysates were then prepared, and Western blotting was performed using anti-phospho-STAT5a/b Ab. Fig. 5A shows that the inhibitory effect of adenosine on IL-2-induced STAT5a/b tyrosine phosphorylation was dramatically reversed in the presence of both sodium orthovanadate and bpV(phen), but not by okadaic acid. Equal protein loading was confirmed by stripping and reprobing the blot with anti-STAT5a Ab. These data indicate that exposure of CTLL-2 cells to adenosine induced a PTP that targeted STAT5a/b.



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  		FIGURE 5. PTP inhibitors rescue STAT5 phosphorylation in the presence of adenosine. CTLL-2 cells were treated with 100 µM bpV(phen), 1 mM sodium orthovanadate (Na3VO4), or 50 nM okadaic acid (OA) for 15 min at 37°C before treatment with medium alone or 10 µM adenosine (Ado) plus 2.5 µM coformycin for an additional 15 min, followed by stimulation with IL-2 (10 U/ml) for 5 min (A). CTLL-2 cells were treated with medium alone or 10 µM adenosine (Ado) plus 2.5 µM coformycin for 15 min at 37°C, followed by stimulation with IL-2 (20 U/ml) for 5 min (B). Control (A and B) consisted of cells that were exposed to only medium. Cell lysates were prepared, and Western blotting with Ab to phospho-STAT5a/b, phospho-ERK1/2, STAT5a, or ERK1/2 (all 1 µg/ml) was used to detect STAT5a/b and ERK1/2 phosphorylation and confirm equal protein loading, respectively. The percentage of OD ratios was calculated by comparing the density of individual phospho-STAT5a bands with the corresponding STAT5a band. Results are representative of four different experiments.

 
The blot was then stripped and reprobed with anti-phospho-ERK1/2 Ab to determine whether adenosine affected IL-2-driven ERK1/2 phosphorylation under our experimental conditions. The blot was also probed with anti-ERK1/2 Ab to confirm equal protein loading. Surprisingly, the concentration of IL-2 (10 U/ml) that we used to stimulate the Jak/STAT5 pathway of CTLL-2 cells failed to induce ERK1/2 phosphorylation (Fig. 5A), ruling out a role for the ERK pathway in proliferative signaling induced by the concentration of IL-2 that was used in our studies. ERK1/2 phosphorylation was apparent in CTLL-2 cells when bpV(phen) was present, suggesting that constitutive phosphorylation of ERK1/2 is negatively regulated by a PTP. Higher concentrations of IL-2 (20 U/ml or greater) induced ERK1/2 phosphorylation in CTLL-2 cells that was not inhibited by adenosine (Fig. 5B).

Adenosine-induced tyrosine phosphorylation of SHP-2 and complex formation with STAT5

We next determined the effect of adenosine on the phosphorylation status of SHP-1, SHP-2, and PTP1B because these PTPs have been shown to dephosphorylate and deactivate growth factor receptor-activated STAT5a and STAT5b in other experimental systems (42, 43, 44). Phosphorylation of tyrosine residues on SHP-1, SHP-2, and PTP1B results in the activation of these PTPs (45, 46, 47). Fig. 6A shows that treatment of IL-2-stimulated CTLL-2 cells with 10 µM adenosine (plus coformycin) caused a dramatic increase in SHP-2 tyrosine phosphorylation in comparison with the level of tyrosine phosphorylation induced by IL-2 alone (2.19 vs 1.04 OD ratio of phospho-SHP-2/SHP-2), suggesting that adenosine increased the phosphatase activity of SHP-2. In contrast, there was little difference between the effect of IL-2 alone or IL-2 in combination with adenosine on tyrosine phosphorylation of SHP-1 (0.82 vs 0.95 OD ratio of phospho-SHP-1/SHP-1), indicating that SHP-1 is not activated by adenosine under these conditions. Although tyrosine phosphorylation of PTP1B in CTLL-2 cells was increased in the presence of IL-2, PTP1B tyrosine phosphorylation was only slightly increased following treatment with IL-2 plus adenosine in comparison with treatment with IL-2 alone (1.14 vs 0.87 OD ratio of phospho-PTP1B/PTP1B), suggesting that adenosine failed to substantially increase the phosphatase activity of PTP1B.



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  		FIGURE 6. Adenosine induces SHP-2 phosphorylation and SHP-2 association with STAT5 in IL-2-stimulated CTLL-2 cells. A, CTLL-2 cells were treated with medium alone or 10 µM adenosine (Ado; plus 2.5 µM coformycin) for 15 min at 37°C, followed by stimulation with IL-2 (10 U/ml) for 5 min. Control consisted of cells that were exposed to only medium. Cell lysates were prepared; immunoprecipitation with anti-SHP-1, anti-SHP-2, or anti-PTP1B Ab (2 µg/sample) and Western blotting with anti-phosphotyrosine Ab (1 µg/ml) were used to detect SHP-1, SHP-2, and PTP1B tyrosine phosphorylation, respectively. Blots were stripped and reprobed with Ab to SHP-1, SHP-2 (both 1 µg/ml), or PTP1B (2 µg/ml) to confirm equal protein loading. B, CTLL-2 cells were treated with medium alone or 10 µM adenosine (Ado; plus 2.5 µM coformycin) for 15 min at 37°C, followed by stimulation with IL-2 (10 U/ml) for 5 min. Control consisted of cells that were exposed to only medium. Cell lysates were prepared, and immunoprecipitation with anti-STAT5a Ab (2 µg/sample) and Western blotting with anti-SHP-2 Ab (1 µg/ml) were performed. Blots were stripped and reprobed with Ab to STAT5a (1 µg/ml) to confirm equal protein loading. The percentage of OD ratios was calculated by comparing the density of individual SHP-2 bands with the corresponding STAT5a band. Results are representative of two independent experiments.

 
Because a substrate-trapping mutant of SHP-2 has recently been shown to physically interact with and form a complex with cytosolic tyrosine-phosphorylated STAT5 from IL-2-stimulated CTLL-20 mouse T cells (43), it was important to determine whether adenosine also increased the association of SHP-2 with STAT5 in our system. CTLL-2 cells were exposed to 10 µM adenosine (plus coformycin) for 15 min, followed by stimulation with IL-2 for 5 min. Immunoprecipitation of cell lysates with anti-STAT5a Ab, followed by Western blotting with anti-SHP-2 Ab, revealed that adenosine and NECA both caused at least a 2-fold increase in the amount of SHP-2 that coprecipitated with STAT5 relative to the IL-2 control (Fig. 6B), indicating that adenosine promotes the formation of SHP-2-STAT5 complexes.

Adenosine receptor expression by CTLL-2 cells

Adenosine mediates its effects through four receptor subtypes designated A1, A2a, A2b, and A3, which are G protein-coupled membrane-assocated molecules (48). We used RT-PCR analysis to determine the adenosine receptor subtypes expressed by CTLL-2 cells. Mouse brain was used as a positive control because this tissue expresses all known adenosine receptor subtypes (49). Fig. 7 shows that CTLL-2 cells expressed mRNA coding for A2a, A2b, and A3 receptors, but little detectable mRNA coding for A1 receptors.



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  		FIGURE 7. Adenosine receptor expression by CTLL-2 cells. Total RNA was isolated from mouse brain (lanes 2, 5, 8, and 11) or from CTLL-2 cells (lanes 3, 6, 9, and 12), and A1, A2a, A2b, and A3 adenosine receptor expression was determined by RT-PCR.

 
A2 receptor agonists inhibit IL-2-induced STAT5a/b tyrosine phosphorylation

To determine whether the inhibitory effect of adenosine on tyrosine phosphorylation of STAT5 was mediated through a particular cell surface adenosine receptor, we compared the effect of treating CTLL-2 cells for 15 min with 10 µM adenosine (plus coformycin) with that of treatment either with the stable adenosine analog NECA at a concentration able to stimulate both A2a and A2b receptors, or with the A2a receptor-selective agonist CGS 21680, or with the A1/A3 receptor-selective agonist APNEA (all at 1 µM) before stimulation with IL-2 for 5 min. Fig. 8 shows that both NECA and CGS 21680 mimicked the inhibitory effect of adenosine on STAT5a/b phosphorylation, suggesting that the effect was mediated through A2a and, possibly, A2b receptors. In contrast, the A1/A3 receptor-selective agonist APNEA did not prevent IL-2-induced STAT5a/b tyrosine phosphorylation, ruling out a role for A1 or A3 receptors. As before, the inhibitory effect of adenosine, NECA, and CGS 21680 was reversed in the presence of the PTP inhibitor bpV(phen).



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  		FIGURE 8. A2 adenosine receptor agonists inhibit IL-2-induced STAT5 phosphorylation. CTLL-2 cells were treated with medium alone, 10 µM adenosine (Ado; plus 2.5 µM coformycin), 1 µM NECA (A2a/A2b receptor agonist), CGS 21680 (A2a receptor agonist), or APNEA (A3 receptor agonist) for 15 min at 37°C, followed by stimulation for 5 min with IL-2 (10 U/ml). Control consisted of cells that were exposed to only medium. Cell lysates were prepared, and Western blotting with anti-phospho-STAT5a/b and anti-STAT5a Ab (both 1 µg/ml) was used to detect STAT5a/b tyrosine phosphorylation and confirm equal protein loading, respectively. The percentage of OD ratios was calculated by comparing the density of individual phospho-STAT5a bands with the corresponding STAT5a band. Results are representative of four different experiments.

 
A2 receptor antagonists block the inhibitory effect of adenosine and adenosine receptor agonists on IL-2-induced tyrosine phosphorylation of STAT5a/b

To confirm that A2 receptors mediated the inhibitory effect of adenosine on STAT5a/b tyrosine phosphorylation, we treated CTLL-2 cells for 15 min with the A1 receptor antagonist DPCPX (20 µM), the A2 receptor antagonist DMPX (20 µM), the A2a receptor-selective antagonist CSC (2 µM), the A2b receptor-selective antagonist alloxazine (2 µM), CSC in combination with alloxazine (both at 2 µM), or the A3 receptor antagonist MRS-1220 (20 µM) before the addition of 10 µM adenosine (plus coformycin). Fifteen minutes later, the cells were stimulated with IL-2 for 5 min, and cell lysates were prepared and Western blotting was performed using anti-phospho-STAT5a/b Ab. As shown in Fig. 9A, both the A2 receptor antagonist DMPX and the combination of the A2a receptor-selective antagonist CSC and the A2b receptor-selective antagonist alloxazine completely blocked the inhibitory effect of adenosine on IL-2-induced tyrosine phosphorylation of STAT5a/b. CSC or alloxazine alone showed partial blocking activity, consistent with the involvement of both A2a and A2b adenosine receptors. As expected, neither the A1 receptor antagonist DPCPX nor the A3 receptor antagonist MRS-1220 reduced the inhibitory effect of adenosine on IL-2-induced STAT5a/b tyrosine phosphorylation. To confirm the selectivity of CSC and alloxazine for A2a and A2b adenosine receptors, respectively, we treated CTLL-2 cells with CSC or alloxazine (both at 2 µM) for 15 min before the addition of the A2a receptor-selective agonist CGS 21680 (1 µM) and subsequent stimulation with IL-2. Fig. 9B shows that CSC completely blocked the inhibitory effect of CGS 21680 on IL-2-induced tyrosine phosphorylation of STAT5a/b, while alloxazine was without effect. Taken together, these data indicate that the inhibitory effect of adenosine on IL-2-induced STAT5a/b tyrosine phosphorylation is mediated through A2a and A2b adenosine receptors.



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  		FIGURE 9. A2 adenosine receptor signaling mediates the inhibitory effect of adenosine on IL-2-induced STAT5 phosphorylation. A, CTLL-2 cells were treated with medium alone, 20 µM DPCPX (A1 receptor antagonist), 20 µM DMPX (A2 receptor antagonist), 2 µM CSC (A2a receptor-selective antagonist), 2 µM alloxazine (A2b receptor-selective antagonist), 2 µM CSC + 2 µM alloxazine, or 20 µM MRS-1220 (A3 receptor antagonist) for 15 min at 37°C, followed by treatment with medium alone or 10 µM adenosine (Ado; plus 2.5 µM coformycin) for an additional 15 min. B, CTLL-2 cells were treated with medium alone, 2 µM CSC, or 2 µM alloxazine for 15 min at 37°C, followed by treatment with medium alone or 1 µM CGS 21680. CTLL-2 cells were then stimulated with IL-2 (10 U/ml) for 5 min. Control consisted of cells that were exposed to only medium. Cell lysates were prepared, and Western blotting with anti-phospho-STAT5a/b and anti-STAT5a Ab (both 1 µg/ml) was used to detect STAT5 tyrosine phosphorylation and confirm equal protein loading, respectively. The percentage of OD ratios was calculated by comparing the density of individual phospho-STAT5a bands with the corresponding STAT5a band. Results are representative of four separate experiments.

 
cAMP/PKA-dependent signaling pathways regulate SHP-2 and STAT5 tyrosine phosphorylation

Because adenosine-mediated inhibition of some T cell functions has been linked to increased intracellular cAMP accumulation caused by the activation of adenylyl cyclase that is coupled to A2a and A2b adenosine receptors (13, 50), we examined the effect of both adenosine receptor ligands and indirect methods of eliciting cAMP on concurrent STAT5a/b and SHP-2 phosphorylation in CTLL-2 cells. Stimulation of adenylyl cyclase activity with forskolin (25 µM) or exposure to the stable, cell-permeable cAMP analog 8-Br-cAMP (100 µM) inhibited IL-2-induced tyrosine phosphorylation of STAT5a/b to an extent that was similar to that caused by adenosine (plus coformycin) or NECA (Fig. 10). Forskolin and 8-Br-cAMP also caused a marked increase in SHP-2 tyrosine phosphorylation in IL-2-stimulated CTLL-2 cells that was well above that caused by IL-2 alone, and was comparable to that caused by adenosine (plus coformycin) or NECA. The increase in tyrosine phosphorylation of SHP-2 closely paralleled the inhibitory effect of adenosine or NECA on STAT5a/b tyrosine phosphorylation, consistent with a role for SHP-2 in mediating STAT5 dephosphorylation. To determine whether cAMP-dependent activation of PKA was linked to the inhibitory effect of adenosine on tyrosine phosphorylation of STAT5a/b, we treated CTLL-2 cells with Rp-cAMPS (100 µM), which is a specific membrane-permeable inhibitor of cAMP-dependent protein kinase I and II (51), before exposure to adenosine (plus coformycin) and subsequent stimulation with IL-2. Fig. 11 shows that Rp-cAMPS almost completely prevented adenosine-mediated inhibition of STAT5a/b tyrosine phosphorylation, suggesting that PKA induces SHP-2-mediated dephosphorylation of STAT5a/b. In addition, adenosine-induced tyrosine phosphorylation of SHP-2 was dramatically reduced in the presence of Rp-cAMPS (data not shown). Collectively, these data implicate cAMP/PKA-dependent signaling pathways coupled to A2a and A2b adenosine receptors in the activation of SHP-2 and the subsequent dephosphorylation of STAT5.



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  		FIGURE 10. Adenosine and cAMP signaling pathways promote SHP-2 phosphorylation and inhibit STAT5 phosphorylation. CTLL-2 cells were treated with medium alone, 10 µM adenosine (Ado; plus 2.5 µM coformycin), 1 µM NECA, 25 µM forskolin, or 100 µM 8-Br-cAMP for 15 min at 37°C, followed by stimulation with IL-2 (10 U/ml) for 5 min. Control consisted of cells that were exposed to only medium. Cell lysates were prepared, and immunoprecipitation with anti-SHP-2 Ab (2 µg/sample) and Western blotting with anti-phosphotyrosine Ab (1 µg/ml) were used to detect SHP-2 tyrosine phosphorylation. STAT5a/b tyrosine phosphorylation was determined by Western blotting parallel samples of cell lysate with anti-phospho-STAT5a/b Ab. Blots were stripped and reprobed with Ab to SHP-2 or STAT5a (both 1 µg/ml) to confirm equal protein loading. The percentage of OD ratios was calculated by comparing the density of individual phospho-SHP-2 bands with the corresponding SHP-2 band, and phospho-STAT5a bands with the corresponding STAT5a band. Results are representative of three individual experiments.

 


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  		FIGURE 11. Rp-cAMPS rescue STAT5 phosphorylation in the presence of adenosine. CTLL-2 cells were treated with medium alone or 100 µM RP-cAMPS for 15 min at 37°C, followed by treatment with medium alone or 10 µM adenosine (Ado; plus 2.5 µM coformycin) for an additional 15 min, followed by stimulation with IL-2 (10 U/ml) for 5 min. Control consisted of cells that were exposed to only medium. Cell lysates were prepared, and Western blotting with Ab to phospho-STAT5a/b or STAT5a (both 1 µg/ml) was performed to detect STAT5 tyrosine phosphorylation and confirm equal protein loading, respectively. The percentage of OD ratios was calculated by comparing the density of individual phospho-STAT5a bands with the corresponding STAT5a band. Results are representative of two independent experiments.

 
Adenosine does not affect IL-2-induced phosphorylation of Akt

Finally, we determined whether altered signaling through the PI3K pathway might also contribute to the inhibitory effect of adenosine on IL-2-dependent growth of CTLL-2 cells. CTLL-2 cells were treated for 15 min with 10 µM adenosine (plus coformycin) or 1 µM NECA or CGS 21680 before stimulation with IL-2 for 5 min. Cell lysates were then prepared, and a Western blot was probed with anti-phospho-Akt Ab to detect tyrosine phosphorylation of Akt, a serine/threonine kinase that is activated by phosphorylation in a PI3K-dependent fashion (27). The same blot was then stripped and reprobed with anti-phospho-STAT5a/b Ab for comparison with phospho-Akt, as well as with anti-STAT5a Ab to confirm equal protein loading. In comparison with the inhibitory effect of adenosine on STAT5a/b tyrosine phosphorylation, neither adenosine nor adenosine analogues affected IL-2-induced Akt phosphorylation (Fig. 12). Note that coformycin alone did not affect IL-2-induced tyrosine phosphorylation of STAT5a/b in CTLL-2 cells.



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  		FIGURE 12. Adenosine and adenosine analogues fail to affect IL-2-induced Akt phosphorylation. CTLL-2 cells were treated with medium alone, 10 µM adenosine (Ado) plus 2.5 µM coformycin (Cof), 1 µM NECA, 1 µM CGS 21680, or 1 µM APNEA for 15 min at 37°C. In some samples, 10 µM bpV(phen) was added 15 min before treatment with Ado or Ado receptor agonists. CTLL-2 cells were then stimulated with IL-2 (10 U/ml) for an additional 5 min. Control consisted of cells that were exposed to only medium. Cell lysates were prepared, and Western blotting with anti-phospho-Akt Ab (1 µg/ml) was used to detect the Akt phosphorylation. The same blot was stripped and reprobed with anti-phospho-STAT5a/b Ab (1 µg/ml) for comparison or with anti-STAT5a Ab (1 µg/ml) to confirm equal protein loading. The percentage of OD ratios was calculated by comparing the density of individual phospho-STAT5a bands with the corresponding STAT5a band. Results are representative of four individual experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present investigation, we show that adenosine inhibited IL-2-driven DNA synthesis and accumulation of mouse CTLL-2 T cells (Fig. 1). This observation is consistent with our earlier finding that adenosine interferes with the proliferation of mouse primary T cells in response to TCR stimulation with anti-CD3 mAb (11). Cell viability and flow cytometric analysis established that the inhibitory effect on CTLL-2 cells was not due to nonspecific toxicity by adenosine or adenosine-induced down-regulation of IL-2R (data not shown). Adenosine-mediated inhibition of CTLL-2 cell proliferation was in sharp contrast to a previous study in which adenosine was reported to enhance IL-2-dependent proliferation of CTLL-2 cells. Antonysamy et al. (52) based their conclusion on the observation that elimination of endogenous adenosine with exogenous adenosine deaminase inhibits the IL-2-dependent growth of CTLL-2 cells, which can then be restored by the addition of the stable adenosine analog R-phenylisopropyladenosine. However, there are several possible explanations for the apparent contrast with our results. First, the concentration of R-phenylisopropyladenosine that augments IL-2-driven CTLL-2 cell proliferation in the presence of exogenous adenosine deaminase is 3 logs lower than the adenosine concentration that we showed inhibited IL-2-dependent growth of CTLL-2 cells (10 nM vs 10 µM). Adenosine would most likely affect CTLL-2 cells differently at these concentrations because the A1 adenosine receptor subtype (which is detectable, although not abundant on our CTLL-2 cells) that transduces a stimulatory signal has a higher affinity for ligand than the A2b adenosine receptor that transduces an inhibitory signal (49). Consistent with this hypothesis, Antonysamy et al. (52) report low A1, A2a, and A3 adenosine receptor expression by CTLL-2 cells, while our own data also indicated A2b adenosine receptor expression by CTLL-2 cells (Fig. 7). Moreover, Antonysamy et al. (52) exclude any growth-enhancing role for A2 adenosine receptors on CTLL-2 cells. In addition, commercial preparations of adenosine deaminase are variably contaminated with superoxide dismutase (53), which is known to protect T cells from activation-induced cell death (54). It is therefore possible that CTLL-2 cell numbers might be increased in the presence of exogenous adenosine deaminase due to the protective effect of contaminating superoxide dismutase. Finally, adenosine deaminase facilitates R-phenylisopropyladenosine binding to A1 adenosine receptors and subsequent signaling through a mechanism that involves the physical interaction of adenosine deaminase with A1 adenosine receptors (55). The addition of exogenous adenosine deaminase plus low concentrations of R-phenylisopropyladenosine to IL-2-stimulated CTLL-2 cell cultures may, therefore, have favored stimulatory A1 adenosine receptor signaling over inhibitory A2 adenosine receptor signaling in the experiments performed by Antonysamy et al. (52).

Proliferative signaling through the IL-2R can occur via two distinct routes, one involving STAT5 (30, 31) and the other using the adaptor protein Shc that activates ERK and PI3K signaling pathways (56). STAT transcription factors are cytoplasmic proteins that are tyrosine phosphorylated and then translocate to the nucleus following activation of associated kinases (32). Our studies revealed that adenosine exerted a potent inhibitory effect on IL-2-induced tyrosine phosphorylation and activation of STAT5a and STAT5b in both CTLL-2 and primary T cells (Figs. 2 and 3), which was not due to inhibition of IL-2-induced phosphorylation and activation of the upstream kinases Jak1 or Jak3 (Fig. 4). This finding is consistent with a recent report that UV radiation, which suppresses cellular immune responses (57), also inhibits IL-2-induced tyrosine phosphorylation and activation of STAT5 in T cells without affecting IL-2-induced tyrosine phosphorylation of Jak1 or Jak3 (58).

We also examined the effect of adenosine on ERK1/2 and Akt phosphorylation in IL-2-stimulated CTLL-2 cells. The PI3K signaling pathway (which includes the serine/threonine kinase Akt) and the ERK signaling pathway are both activated in response to IL-2R signaling (56). Surprisingly, we found that the concentration of IL-2 (10 U/ml) that was used to induce CTLL-2 proliferation and STAT5 phosphorylation in our experiments failed to induce phosphorylation of ERK1/2 (Fig. 5A), suggesting that activation of the ERK signaling pathway is not essential for IL-2-driven T cell proliferation. This result is contrary to recent reports that IL-2 induces ERK1/2 phosphorylation in CTLL-2 cells (56, 59). However, in both cases, the investigators used a concentration of IL-2 that was 10-fold higher (100 U/ml) than the concentration of IL-2 (10 U/ml) that we used in our experiments. Consistent with these earlier reports, we observed that at least 20 U/ml IL-2 was required to induce ERK1/2 phosphorylation in CTLL-2 cells (Fig. 5B). However, adenosine failed to inhibit ERK1/2 phosphorylation induced by a higher concentration of IL-2. We also observed that ERK1/2 is phosphorylated in the presence of the PTP inhibitor bpV(phen) (Fig. 5A), suggesting that constitutive ERK1/2 phosphorylation in CTLL-2 cells is regulated by a PTP. Our findings also indicate that the Jak/STAT5 pathway of CTLL-2 cells is more sensitive than the ERK pathway to IL-2R signaling. In addition, adenosine did not affect IL-2-induced tyrosine phosphorylation of Akt (Fig. 12). Because phosphorylation plays a major role in Akt activation (60) and Akt is an important intermediate in the PI3K signaling pathway (27), this finding suggested that IL-2-induced PI3K signaling in CTLL-2 cells was not impaired in the presence of adenosine. Collectively, these results indicate that adenosine-mediated inhibition of IL-2-induced CTLL-2 proliferation was due to impaired activation of the IL-2-induced Jak/STAT5 pathway and did not involve perturbation of ERK or PI3K signaling pathways.

Protein tyrosine phosphorylation events that are crucial for the control of T cell activation are closely regulated by the opposing actions of protein tyrosine kinases and PTPs (16). PTPs have been shown to participate as either positive or negative regulators of signal transduction in a wide range of physiological processes, which include cellular growth and proliferation, migration, differentiation, and survival (16, 61). Despite the important roles played by PTPs in a wide range of physiological and pathological processes, the mechanism by which PTPs exert their regulatory effects is not well understood. Because adenosine did not inhibit IL-2-induced Jak1 or Jak3 phosphorylation in CTLL-2 cells, we considered the possibility that adenosine-mediated inhibition of STAT5a/b tyrosine phosphorylation might result from the activation of a PTP that targets STAT5. PTPs are known to closely regulate kinases that are associated with various cytokine signal transduction pathways to maintain cellular homeostasis (62). More recently, a PTP associated with the nucleus was shown to specifically dephosphorylate and deactivate STAT5a and STAT5b in COS-7 cells, as well as in mammary epithelial COMMA-1D cells (63). Our results show that the inhibitory effect of adenosine on STAT5a/b tyrosine phosphorylation in CTLL-2 cells was abrogated in the presence of the PTP inhibitors bpV(phen) and sodium orthovanadate (Fig. 5A), suggesting that adenosine activated a PTP that dephosphorylated STAT5.

SHP-1, SHP-2, and PTP1B were all possible candidates to mediate adenosine-induced PTP activity that targeted IL-2R-associated STAT5 in CTLL-2 cells because these PTPs have been reported to dephosphorylate and deactivate STAT5a and STAT5b associated with growth factor receptors in other experimental systems (42, 43, 44). Activation of SHP-1, SHP-2, and PTP1B requires phosphorylation of PTP tyrosine residues (45, 46, 47). Tyrosine phosphorylation of SHP-2, but not SHP-1, was strongly enhanced in IL-2-stimulated CTLL-2 cells that were exposed to adenosine, indicating that exposure to adenosine activated SHP-2, but not SHP-1, in CTLL-2 cells (Fig. 6A). This finding is in good agreement with a recent report that SHP-2, rather than SHP-1, mediates tyrosine dephosphorylation of IL-2R-associated STAT5 in the cytosolic compartment of mouse T cells, thereby maintaining STAT5 activity at a steady state basal level (43). SHP-2, but not SHP-1, has also been shown to specifically interact with tyrosine-phosphorylated STAT5a and accelerate STAT5a dephosphorylation in IL-3-stimulated myeloid cells (64). Importantly, the same study showed that dephosphorylation of STAT5a is dramatically delayed in SHP-2-deficient mouse fibroblast cells, providing direct evidence that SHP-2 is an important mediator of STAT5a dephosphorylation. Although IL-2 caused a modest increase in tyrosine phosphorylation of PTP1B, there was no substantial change in phosphorylation following treatment with adenosine (Fig. 6A), which argued against a role for PTP1B in adenosine-induced STAT5a/b dephosphorylation in IL-2-stimulated CTLL-2 cells. It is also important to note that the effect of tyrosine phosphorylation on the phosphatase activity of PTP1B is still somewhat controversial because phosphorylation of PTP1B tyrosine residues has been reported by different groups to increase or inhibit phosphatase activity (47, 65). Protein phosphatase 2A is also able to dephosphorylate STAT5a following IL-3 stimulation of myeloid progenitor cells (66). However, the inhibitory effect of adenosine on STAT5a/b phosphorylation in IL-2-stimulated CTLL-2 cells was not diminished in the presence of okadaic acid, a well-known inhibitor of protein phosphatase 2A (Fig. 5A). This observation excluded a role for protein phosphatase 2A in our experimental system. We also showed that treatment with adenosine or the adenosine analog NECA resulted in a substantial increase in the amount of SHP-2 that coimmunoprecipitated with STAT5 from IL-2-stimulated CTLL-2 cells (Fig. 6B), suggesting that adenosine promoted the formation of SHP-2-STAT5 complexes that have been implicated in the efficient dephosphorylation and deactivation of STAT5 by SHP-2. This result is consistent with the ability of a substrate-trapping mutant of SHP-2, but not SHP-1, to form a stable complex with tyrosine-phosphorylated cytosolic STAT5 from IL-2-stimulated mouse CTLL-20 cells, thereby showing that STAT5 can serve as a direct substrate of SHP-2 (43). STAT5 tyrosine dephosphorylation and deactivation do not require nuclear translocation of the SHP-2-STAT5 complex because SHP-2 cannot be detected in the nucleus of IL-2-stimulated CTLL-20 cells. Taken together, our findings provide strong evidence that adenosine-induced dephosphorylation of STAT5a/b in IL-2-stimulated CTLL-2 T cells was mediated by SHP-2. However, we cannot at the present time rule out a possible contribution by one or more as yet unidentified STAT5-reactive phosphatases to this process. It may be possible to use SHP-2 deletion/inactivation approaches (e.g., dominant-negative mutant of SHP-2, small interfering RNA knockdown) to resolve this issue, although the compound signaling functions of SHP-2 may complicate the interpretation of results obtained from such experiments (67).

Adenosine mediates its effects on responsive cells through cell surface receptors that are divided into A1, A2a, A2b, and A3 subtypes (48). Activation of A1 or A3 adenosine receptors inhibits adenylyl cyclase activity, while activation of A2 adenosine receptors stimulates adenylyl cyclase and promotes the accumulation of intracellular cAMP. A3 adenosine receptor stimulation also activates the phospholipase C/inositol trisphosphate/diacylglycerol pathway (68). Our analysis showed that CTLL-2 cells predominantly expressed A2a, A2b, and A3 adenosine receptor subtypes, although a low level of A1 adenosine receptor expression was also present (Fig. 7). We used specific adenosine receptor agonists and antagonists to demonstrate that the inhibitory effect of adenosine on IL-2-induced STAT5 tyrosine phosphorylation was mediated through A2a and A2b adenosine receptors. This conclusion was based on the finding that A2a/A2b adenosine receptor agonist NECA and the A2a adenosine receptor agonist CGS 21680) mimicked the effect of adenosine (Fig. 8), whereas the A2 receptor antagonist DMPX completely blocked the effect of adenosine (Fig. 9A). In addition, the A2a adenosine receptor antagonist CSC and the A2b adenosine receptor antagonist alloxazine each only partially blocked the adenosine effect, whereas a combination of the two antagonists completely blocked the adenosine effect. The selectivity of CSC and alloxazine was confirmed by the observation that CSC blocked the inhibitory effect of A2a adenosine receptor agonist CGS 21680 on STAT5a and STAT5b tyrosine phosphorylation, while alloxazine was without effect (Fig. 9B). Furthermore, the A1/A3 adenosine receptor agonist APNEA failed to inhibit STAT5 phosphorylation, and neither the A1 adenosine receptor antagonist DPCPX nor the A3 adenosine receptor antagonist MRS-1220 reversed the inhibitory effect of adenosine on STAT5 phosphorylation.

Because A2a and A2b adenosine receptors are coupled to adenylyl cyclase and receptor stimulation leads to increased levels of intracellular cAMP (13, 50), it was likely that adenosine-induced SHP-2 activation was mediated through a cAMP-dependent mechanism. As expected, both 8-Br-cAMP and forskolin treatment of IL-2-stimulated CTLL-2 cells increased SHP-2 phosphorylation and, at the same time, decreased STAT5 phosphorylation (Fig. 10). Similar results were obtained when IL-2-stimulated CTLL-2 cells were treated with adenosine or NECA. Taken together, these results suggested that adenosine-induced dephosphorylation of STAT5 was due to adenylyl cyclase activation in response to A2a and A2b adenosine receptor stimulation, which resulted in the accumulation of intracellular cAMP and activation of SHP-2, leading to STAT5a/b dephosphorylation. Moreover, the inhibitory effect of adenosine on tyrosine phosphorylation of STAT5a/b involved the cAMP-dependent activation of PKA because inhibition was lost when CTLL-2 cells were treated with Rp-cAMPS, which is a specific membrane-permeable inhibitor of PKA (51), before exposure to adenosine (Fig. 11). In addition, treatment of CTLL-2 cells with Rp-cAMPS prevented adenosine-induced phosphorylation of SHP-2 tyrosine residues (data not shown). Our findings are therefore in good agreement with a recent report that SHP-2 in bovine adrenocortical cells is phosphorylated and activated by cAMP-dependent PKA in response to stimulation with adrenocorticotrophic hormone (69).

In conclusion, we have demonstrated for the first time that adenosine mediated an inhibitory effect on IL-2-induced tyrosine phosphorylation of STAT5a and STAT5b in CTLL-2 T lymphocytes, as well as in primary T cells. This interruption of a key proliferative signaling pathway that is associated with the IL-2R most likely accounts for the reduced capacity of adenosine-treated CTLL-2 T cells to proliferate in response to IL-2. In addition, we showed that the inhibitory effect of adenosine on tyrosine phosphorylation and activation of STAT5a and STAT5b was not due to a reduction in Jak1 or Jak3 phosphorylation, but rather was the result of adenosine-induced activation of a PTP. Increased SHP-2 activation and enhanced formation of SHP-2-STAT5 complexes in the presence of adenosine strongly suggested that the adenosine-induced PTP responsible for the inhibitory effect was SHP-2. We have previously reported that adenosine is an important immunosuppressive constituent of the microenvironment of solid carcinomas (10). Because T cells play a major role in antitumor immunity (70), the adenosine-induced defect in IL-2-stimulated proliferative signaling through STAT5 caused by PTP activation would be expected to result in impaired T cell-mediated antitumor immunity. The inhibitory effect of adenosine on STAT5a/b tyrosine phosphorylation was mediated via cAMP/PKA-dependent signaling pathways coupled to A2a and A2b adenosine receptors. This finding opens up the possibility of pharmacological intervention with appropriate adenosine receptor antagonists that would be expected to prevent adenosine inhibition of IL-2R signal transduction and, thereby, potentiate T cell-based immunotherapy of solid cancers.


   Acknowledgments
 
We thank A. Bennett for expert technical assistance.


   Footnotes
 
1 This work was supported by operating grants from the Natural Sciences and Engineering Research Council of Canada and Cancer Research and Education Nova Scotia. H.Z. was the recipient of a fellowship from the Canadian Institutes for Health Research and the Nova Scotia Health Research Foundation. D.M.C. was the recipient of a postgraduate scholarship from Natural Sciences and Engineering Research Council of Canada. J.J.B. was supported by a Cancer Research and Education Trainee Award with funding from the Faculty of Medicine, Dalhousie University. C.Z. was supported by a fellowship from the China Scholarship Council. Back

2 Address correspondence and reprint requests to Dr. David W. Hoskin, Department of Microbiology and Immunology, Dalhousie University Halifax, Nova Scotia, Canada B3H 1X5. E-mail address: d.w.hoskin{at}dal.ca Back

3 Abbreviations used in this paper: PTP, protein tyrosine phosphatase; APNEA, N (6)-2-(4-aminophenyl)ethyladenosine; 8-Br-cAMP, 8-bromo-cAMP; CSC, 8-(3-chlorostyryl)caffeine; DMPX, 3,7-dimethyl-1-propargylxanthine; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine; MRS-1220, 9-chloro-2-(2-furanyl)-5-([phenylacetyl]amino)[1,2,4]-triazolo[1,5-c]quinazoline; NECA, 5'-N-ethylcarboxamidoadenosine; PKA, protein kinase A; SHP-2, Src homology region 2 domain-containing phosphatase-2. Back

Received for publication April 11, 2003. Accepted for publication May 5, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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