A2B Adenosine Receptors and T Cell Activation 493

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Journal of Cell Science 112, 491-502 (1999) 491 Printed in Great Britain © The Company of Biologists Limited 1999 JCS0069

Expression of A2B adenosine receptors in human lymphocytes: their role in T cell activation

Maribel Mirabet1, Carolina Herrera1, Oscar J. Cordero2, Josefa Mallol1, Carmen Lluis1 and Rafael Franco1,* 1Department of Biochemistry and Molecular Biology, Faculty of Chemistry, University of Barcelona, Barcelona, Catalonia, Spain 2Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Santiago de Compostela, Spain *Author for correspondence (e-mail: [email protected]; homepage: www.bq.ub.es/recep/franco.html)

Accepted 9 December 1998; published on WWW 25 January 1999

SUMMARY

Extracellular adenosine has a key role in the development A2BRs but not of A2A receptors in these human cells. The and function of the cells of the immune system. Many of percentage of A2BR-expressing cells was similar in the the adenosine actions seem to be mediated by specific CD4+ or CD8+ T cell subpopulations. Interestingly surface receptors positively coupled to adenylate cyclase: activation signals delivered by either phytohemagglutinin A2A and A2B. Despite the fact that A2A receptors (A2ARs) or anti-T cell receptor/CD3 complex antibodies led to a can be easily studied due to the availability of the specific significant increase in both the percentage of cells agonist CGS21680, a pharmacological and physiological expressing the receptor and the intensity of the labeling. characterization of adenosine A2B receptors (A2BRs) in These receptors are functional since interleukin-2 lymphocytes has not been possible due to the lack of production in these cells is reduced by NECA but not by R- suitable reagents. Here we report the generation and PIA or CGS21680. These results show that A2BR characterization of a polyclonal antipeptide antibody expression is regulated in T cell activation and suggest that raised against the third extracellular loop of the A2BR the role of adenosine in lymphocyte deactivation is human clone which is useful for immunocytochemical mediated by A2BRs. + studies. This antibody has permitted the detection of A2BR cells in lymphocyte samples isolated from human peripheral blood. The pharmacology of cAMP-producing Key words: Adenosine receptor, T cell activation, Antipeptide compounds is consistent with the presence of functional antibody

INTRODUCTION 1992; Linden et al., 1993; Salvatore et al., 1993; Tucker and Linden, 1993). All these P1 purinoceptors belong to the Adenosine, acting outside the cell, exerts potent actions on a superfamily of G protein-coupled receptors having seven wide variety of physiological systems, including the nervous, transmembrane domains. cardiovascular, gastrointestinal, urogenital, respiratory, and The relevance of adenosine for the development and lymphatic systems. Many of these actions are mediated via function of the immune system is deduced from the severe specific receptors named P1 purinoceptors (Daly et al., 1983). combined immunodeficiency syndrome (SCID) associated From studies on the variation of adenylate cyclase activity with the congenital defect of adenosine deaminase, the produced by adenosine analogues, and on the rank order of enzyme which degrades the nucleoside. In adenosine potency of agonists, P1 purinoceptors were subdivided into deaminase-deficient children, elevated levels of adenosine two classes: A1, which mediate decreases in cAMP levels, (and 2′-deoxyadenosine) are found in body fluids. Several and A2, which mediate increases in cAMP levels (Van mechanisms whereby adenosine deaminase deficiency may Calcker et al., 1979; Londos et al., 1980). The affect the development and function of lymphoid cells have pharmacological characterization of the A2 subtype in been suggested but none can satisfactorily explain all the different tissues led to the finding of two different populations relationships found in SCID. Most of the proposed which were named A2A and A2B, and had very different mechanisms (see Hershfield and Mitchell, 1989, for review) affinities for the agonist CGS21680. It is now becoming clear have rested upon the assumption that accumulation of the two that the subtypes of adenosine receptors can be linked to a physiological substrates of adenosine deaminase, adenosine variety of signal transduction systems apart from adenylate and 2′-deoxyadenosine, are toxic for lymphocytes (Franco et cyclase (Stiles, 1992; Linden, 1994; Schubert et al., 1994; al., 1998, and references therein). There is recent data Fredholm, 1995). These receptor subtypes (A1, A2A and A2B) suggesting that signaling through purinergic receptors in as well as another member of the family, which was lymphocyte and lymphocyte precursors may contribute to the discovered more recently (A3), have been cloned (Zhou et al., pathogenesis of adenosine deaminase-related SCID (Apasov 492 M. Mirabet and others et al., 1997; Resta and Thompson, 1997). On the other hand, Cells and culture conditions adenosine receptors seem to be involved in the regulation by Chinese hamster ovary (CHO) cells transfected with cDNA coding for adenosine of T-cell receptor-triggered activation-related the human A2B adenosine receptor (Pierce et al., 1992) were kindly events, such as antibody production, cell proliferation, IL-2 provided by Dr Peter R. Schofield from the Garvan Institute of production, upregulation of IL-2 receptor α-chain (CD25) Medical Research at St Vincent’s Hospital in Sidney, Australia. and lymphocyte-mediated cytolysis (Wolberg et al., 1975; Chinese hamster ovary (CHO) cells transfected with a cDNA coding Dos Reis et al., 1986; Antonysamy et al., 1995; Huang et al., for a recombinant protein composed of the human A2A adenosine 1997). Adenosine receptors regulating these different aspects receptor containing the M2 flag in its C-terminal end (Rivkees et al., 1995) were kindly provided by Dr Scott A. Rivkees from Yale of lymphoid function are those leading to increases in cAMP University, New Haven, CT, USA. Transfected CHO cells were grown levels, i.e. A2AR and/or A2BR (Dos Reis et al., 1986). In fact, in Dulbecco’s modified Eagle’s medium/Hams F12 nutrient mixture adenosine and 2-chloroadenosine lead to increases in human (1:1) containing 10% (v:v) fetal calf serum, 2 mM L-glutamine, lymphocyte cAMP levels (Marone et al., 1978; Dinjens et al., antibiotics (100 i.u./ml penicillin, 100 µg/ml streptomycin and 0.25 1986). Recently, Huang et al. (1997) have shown that A2ARs µg/ml fungizone and 1.6 mg/ml of the neomycin analog G-418), at are involved in adenosine-mediated inhibition of murine T- 37°C in an humid atmosphere of 5% CO2. Wild-type CHO cells were cell activation and expansion. In these events the role of cultured in the same conditions described for transfected cells but in A2BRs has not been established due to the lack of suitable the absence of G-418. Jurkat cells were maintained in RPMI 1640 tools for their study. In this paper we describe the generation medium supplemented with 10% (v:v) fetal bovine serum, 2 mM L- and characterization of two polyclonal antipeptide antibodies glutamine and antibiotics at 37°C under a 5% CO2 atmosphere. HEP- G2 cells were cultured in DMEM supplemented with 4.5 g/l glucose raised against different regions of the A2BR human clone. and 10% fetal serum. All the culture reagents were from Gibco (Grand One of these antibodies has proven to be inestimable for the Island, NY, USA). identification of A2BR-expressing human blood lymphocytes. Peripheral blood mononuclear cells (PBMC) from healthy donors Further analysis of these cells have shown that the expression were isolated from buffy coats (kindly provided by Dr Hernández of A2BR is regulated in T cell activation events thus indicating from the Banco de Sangre of the Hospital General Vall d’Hebrón de that A2BRs are involved in the regulation by adenosine of Barcelona) using the Ficoll gradient method described by Boyum signaling processes occurring in human lymphocytes. (1968). Further purification of lymphocytes (PBL) from PBMC was Furthermore, upregulated A2BRs are functional and elicit performed by depletion of contaminating cells by adherence to plastic significant reductions in interleukin-2 production. These plates. An homogeneous population corresponding to total results are similar to those found in murine macrophages in lymphocytes was observed according to forward and side light scatter γ parameters by flow cytometry analysis with less than 7% of which A2BRs are upregulated in response to interferon- . As contaminating monocytes. PBMC or PBL were split at 106 cells/ml we described here for T lymphocytes, adenosine, acting and cultured in the same conditions as described above for Jurkat through A2BRs, contributes to the deactivation of cells. In vitro activation of cells was carried out with 1 µg/ml macrophages since it reduces the upregulation of MHC phytohemagglutinin (PHA; Difco Laboratories, Detroit, MI, USA) or class II molecules, the activity of nitric oxid synthase or was conducted in well plates coated with OKT3 antibody. For OKT3 the production of pro-inflammatory cytokines (Xaus et al., antibody immobilization, 300 µl of 10 mM PBS, pH 7.4, containing 1999). 2.5 µg/ml purified OKT3 antibody were placed for 3 hours at 37°C in a 24-well flat bottom plate. After 2 washes with 2 ml of cold PBS, cells were added to wells at a density of 106 cells/ml to begin the MATERIALS AND METHODS activation experiments. Anti-A2B adenosine receptor antibody generation and Materials purification 5′-N-ethylcarboxamidoadenosine (NECA), N6-(R)-phenylisopropyl- The procedure for obtaining the antibodies is patent pending. adenosine (R-PIA), fluorescein isothiocyanate (FITC), Antipeptide antisera were generated in New Zealand white rabbits tetramethylrhodamine isothiocyanate (TRITC), saponin, by Biokit, S.A. (Barcelona). The peptides used for immunization paraformaldehyde, Indo-1/AM, Triton X-100, EGTA and bovine correspond to the deduced amino acid sequence from the putative serum albumin (BSA) were purchased from Sigma Chemical Co. (St third extracellular loop (FQPAQGKNKPKWA) and from a portion of Louis, MO, USA). Ficoll was from Biochrom KG (Berlin, Germany) the C-terminal domain (AYRNRDFRYTFHKI) of the cloned human and 2-(p-[2-carboxyethyl]phenylethylamino)-5′-N-ethylcarboxamido- A2B adenosine receptor (Pierce et al., 1992) (Fig. 1). They were adenosine (CGS21680) from Research Biochemicals International selected on the basis of hydropathy analysis (Kyte and Doolitte, 1982) (Natick, MA, USA). Keyhole limpet hemocyanin was purchased from and, whenever possible, low homology with the A1, A2A and A3 Calbiochem (La Jolla, CA, USA), glycine and IL-2 were from adenosine receptors. The peptides were synthesized by the Peptide Boehringer Mannheim (Barcelona, Spain) and Immuno-Fluore Synthesis Service at the University of Barcelona with an extra Mounting Medium was from ICN Biomedical Inc. (Costa Mesa, CA, carboxy-terminal cysteine residue, to facilitate conjugation with USA). Thiol-Sepharose 4B and Sephadex G-25 fine grade were keyhole limpet hemocyanin via m-maleimidobenzoyl-N- obtained from Pharmacia (Uppsala, Sweden) and OKT3 antibody was hydroxysuccinimide coupling (Kitagawa and Aikawa, 1976). The kindly provided by Dr Terhorst (Beth Israel-Deaconess Medical composition of each peptide was verified by high performance liquid Center, Boston, MA, USA). The anti-M2 flag antibody was purchased chromatography. Each keyhole limpet hemocyanin-coupled peptide from Eastman Kodak company (New Haven, USA). Phycoerythrin was injected into two rabbits using the immunization protocol method (PE)-anti CD4 (T4), PE-anti CD8 (T8) and PE-anti CD3 (CD3, clone of Tanaka et al. (1985). The reactivity of the resulting antisera, ME HIT3A) mAbs were from Coulter (Hialeah, FL, USA). All other in the case of the extracellular peptide and MI in the case of the products were of the best grade available and were purchased from intracellular peptide (Fig. 1), against the appropriate peptide was Merck (Darmstad, Germany) and Sigma. Deionized water further tested by enzyme-linked immunosorbent assay (ELISA). For this purified with a Millipore Milli-Q system (Bedford, MA, USA) was purpose, 96-well Maxisorp microtiter plates (Nunc) were coated with used throughout. the peptides (2.5 µM), blocked, and incubated with various dilutions A2B adenosine receptors and T cell activation 493

EXTRACELLULAR

G T E P D W T T C N N E N S M NH2 Antiserum: ME T C L A C Purified antibody: MPE1 L S L E Y G D V T F C K K N K P D L S C K Q L K D T F N G W W E F A C L G V V N Q A L F A L P A M Y V A L I S L G C F V T P F M S Y M L F Q P N M A E L V P F A I T L V I G L V Y F V N C V T I L I A A L L F A I I T Q A F G N F F G P V H A L S H S V A D V A V G S S I F L W V L C V L A L C W L A N S G N V L L A A A S L L I A V P P L L V G I F V V N P V C A A F L V S A V A R G V I M L V S L A M I I V Y V Y V A I K A N R Y A G D A H Y T Y R T I I T L G K E R P T A A I R N T V Q N T L Q I F R C L L L Fig. 1. Sequence and predicted membrane- S T D V V P L R Y K T F spanning topography of the human A A 2B R R adenosine receptor. The amino acid sequences C S Y R T E D H used for antibody generation are indicated Q L Q R L M T L G V G G A Q N K H F with dark circles. The figure is adapted from HOOC L A P Q V G G S K V D A Q C L L Y R S I I the amino acid sequence reported by Pierce et al. (1992) and from the receptor structure Antiserum: MI proposed by Stehle et al. (1992). Consensus Purified antibody: MPI1 sequences for glycosylation (Y) and acylation ٙٙր) are indicated. INTRACELLULAR) of the ME and the MI antisera. The antibodies were detected with (buffer A) and were incubated for 15 minutes at room temperature peroxidase-conjugated sheep anti-rabbit IgG antibodies (Boehringer with buffer A containing 1% BSA and 0.05% NaN3 (buffer B) before Mannheim). Reactivity could be detected in the ELISA down to a 105- addition of the antibodies. For indirect immunostaining, samples were fold dilution for both antisera (data not shown). incubated for 45 minutes at 37°C with 50 µg/ml MPE1 in buffer B Purification of antibodies from whole rabbit serum was achieved (50 ml), washed 3 times with the same buffer and stained with FITC- by affinity chromatography using the appropriate immunizing peptide conjugated anti-rabbit IgG antibody (dilution 1:50; Boehringer coupled to Thiol-Sepharose 4B. Columns were prepared using 3 mg Mannheim). For direct immunostaining, incubation (45 minutes at of peptide/ml of swollen activated Thiol-Sepharose 4B, in accordance 37°C) was performed with 100 µg/ml FITC-MPE1 and/or 100 µg/ml with the manufacturer’s instructions. For antibody purification, 8 ml TRITC-MPI1. For staining of lymphocytic populations, samples were of antiserum was diluted (3-fold) in 10 mM PBS, pH 7.4, and this incubated (30 minutes in ice) with PE-anti CD3, PE-anti CD4 or PE- material was recirculated for 2 hours at room temperature or overnight anti CD8 mAb and washed before adding FITC-MPE1 antibody (30 at 4°C through 3 ml of the peptide-Sepharose column. The column minutes in ice). After 3 washes with buffer B, stained cells were was then washed with PBS and antibodies were eluted by addition of mounted in Immuno-Fluore Mounting Medium for confocal 50 mM glycine/HCl, pH 2.3. Fractions (1 ml) were collected in tubes microscopy analysis. Alternatively, cell suspensions were containing 20 µl of 1 M Tris, to give a final pH of 7.4. Protein- resuspended in buffer B for flow cytometry analysis. containing fractions were pooled, dialyzed against 2000 volumes of Stained cell suspensions were analyzed using an Epics Elite flow PBS and aliquoted for storage at −80°C. The affinity purified cytometer (Coulter Corporation; Hialeah, FL, USA). FITC and PE antibodies obtained from ME and MI antisera were called MPE1 and excitation was obtained by a 488-nm Argon laser lamp and their MPI1, respectively. fluorescences were collected using 525 nm and 575 nm band-pass filters, respectively. The parameters used to select cell populations for Antibody conjugation to fluorocromes analysis were forward and side light scatter. Purified antibodies were dialyzed against labeling buffer (50 mM Confocal microscope observations were made with a Leica TCS 4D H3BO3, 200 mM NaCl, pH 9.2) at 4°C. Subsequently, 20 µl of 5 mg/ml confocal scanning laser microscope adapted to an inverted Leitz FITC (for MPE1) or TRITC (for MPI1) in DMSO were added for each DMIRBE microscope (Leica Lasertechnik GmbH, Heidelberg, milligram of antibody. After 2 hours at room temperature, unbound Germany). The light source was an Argon Krypton laser and FITC fluorocrome was separated by gel filtration using a Sephadex G-25 and TRITC were respectively excited at 488 and 568 nm. In this column. Fluorochrome-conjugated antibodies were stored at −80°C. system a 580 nm short-pass filter directs the emitted green (FITC) and red (TRITC) fluorescences towards two photomultipliers, one with a Immunostaining and immunofluorescence assays 513-527 nm band-pass filter and the other with a 590 nm long-pass Transfected and wild-type CHO cells grown onto glass coverslips and filter. The colocalization analysis was made by means of the Multi peripheral blood lymphocytes and Jurkat cells (4×106) were washed Color software (version 2.0; Leica Lasertechnik GmbH). with PBS and fixed in 2% paraformaldehyde, 60 mM sucrose in PBS, pH 7.4, for 15 minutes at room temperature. In some experiments cells Analysis of intracellular Ca2+ by double wavelength were permeabilized by adding 0.05% saponin to the fixation solution. fluorimetry Then cells were washed twice with PBS containing 20 mM glycine Cells (5×106/ml) were loaded with 5 µM Indo-1/AM in HBSS buffer 494 M. Mirabet and others A2B adenosine receptors and T cell activation 495

Fig. 2. Specificity of the anti-A2BR antibodies. (A) Confocal images of monitored with a RF-5000 Shimadzu spectrofluorimeter in cuvettes FITC-MPE1 labeling in fixed wild-type CHO cells (left) and TRITC- thermostatically controlled at 37°C and continuously stirred. The cell MPI1 labeling in permeabilized wild-type CHO cells (right). suspension was excited at 355 nm and fluorescence emissions were 2+ 2+ (B) Confocal images of fixed A2BR-transfected CHO cells labeled detected at 405 (Ca -bound dye) and 485 nm (Ca -free dye). Final 2+ with FITC-MPE1 antibody (left) or with TRITC-MPI1 antibody [Ca ]i values were calculated from the ratio of emission (right). (C) Confocal images of permeabilized A2B-R-transfected CHO fluorescences (405/485 nm) using the equation described by cells labeled as in B. The superposition of FITC and TRITC labeling Grynkiewickz et al. (38), with a Kd value of 250 nM for Indo-1. The shows a high degree of colocalization (in yellow), as it can be seen also Rmax value was obtained by lysing the cells with 0.1% Triton X-100, in the cytofluorogram. (D) Confocal image of fixed HEP-G2 cells followed by an addition of excess EGTA for Rmin. stained with FITC-MPE1 antibody. (E) Confocal image of fixed and permeabilized CHO cells overexpressing A2A-M2 flag receptors Determination of cAMP stained using 15 µg/ml of anti-M2 flag antibody. (F) Fixed and Peripheral blood lymphocytes (PBL) were preincubated at 2×106 permeabilized CHO cells overexpressing A2A-M2 flag receptors cells/ml in HBSS, pH 7.2, containing 30 µM RO-20-1724 stained using the MPE1 antibody (top) or the MPI1 antibody (bottom). (Calbiochem) for 10 minutes at 37°C. 250 µl of the cell suspension Note the lack of crossreactivity of the MPE1 antibody against the A2A were added to tubes containing 2.5 µl of the appropriate stimuli and, receptor and some crossreactivity of the MPI1 antibody. after a 10 minute incubation at 37°C, 500 µl of ice-cold ethanol were used to stop the reactions. Samples were centrifuged at 2000 g for 20 minutes at 4°C and supernatants were lyophilized. Quantification of (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM MgSO4, 1.2 mM cAMP levels was determined using an enzyme-immunoassay kit from CaCl2, 10 mM Hepes, 5 mM glucose, 0.3 mM KH2PO4 and 2 mM Amersham (Amersham Iberica, Madrid, Spain). Na2HPO4), pH 7.0, for 30 minutes at 37°C. Then, an equal volume of HBSS, pH 7.4, containing 10% heat inactivated fetal calf serum, Interleukin-2 determination was added and the cell suspension was incubated for 30 minutes. After Peripheral blood lymphocytes were incubated in 96-well plates at 106 washing with HBSS, pH 7.2, containing 5% heat inactivated fetal calf cells/ml in the presence or absence of the adenosine receptor agonists. serum and 10 µg/ml bovine pancreatic deoxyribonuclease I (Sigma), Wells were precoated with 2.5 µg/ml OKT3 and the incubation was cells were resuspended at 5×106 cells/ml and maintained at room performed for 72 hours at 37°C. Samples were centrifuged and the temperature in the dark until use. Just before the analysis, cells were supernatants were tested for interleukin-2 content using a human diluted to 1×106 cells/ml and warmed to 37°C. Fluorescence was interleukin-2 ELISA kit from Endogen (Woburn, USA).

Fig. 3. Phenotype analysis + of A2BR lymphocytes from PBMC by two color immunofluorescence. The upper dot plot (left) represents side versus forward scatter distribution of total PBMC and B1 in red represents the gated population. Quadrants were placed according to the nonspecific labeling obtained using FITC-goat anti-mouse and PE-goat anti-mouse treated cells. X- axis represents the green fluorescence (log scale) using FITC-MPE1; Y-axis represents the red fluorescence (log scale) using the respective PE- conjugated anti subset marker mAb. The percentage of cells in each quadrant is indicated. Data correspond to a representative experiment. 496 M. Mirabet and others

Protein determination permeabilized cells (Fig. 2B,C). Due to the specificity of MPI1 Protein was determined by the bicinchoninic acid method (Pierce) as towards an intracellular region of the receptor, this antibody described by Sorensen and Brodbeck (1986) and using BSA as only detected A2BRs in permeabilized cells (Fig. 2C). Liver is standard. a tissue where the level of A2BR expression is very low (Stehle et al., 1992). For this reason, the human hepatocellular carcinoma-derived cell line, HEP-G2, was used to test whether RESULTS MPE1 led to some cell surface labeling. In these cells where A2BRs were barely detectable by pharmacological techniques, Specificity of the anti-A2BR antibodies MPE1 did not lead to specific labeling, thus indicating that the The specificity of MPE1 and MPI1, which are antipeptide antibodies did not recognize molecules different from A2BR antibodies directed against the third extracellular loop and a (Fig. 2D). Moreover this antibody did not recognize A1 portion of the C-terminal domain of the human A2BR, adenosine receptors expressed in neurons (not shown). The respectively (see Fig. 1 and Materials and Methods), has been high degree of colocalization of the label in permeabilized assessed by using CHO cells transfected with the cDNA transfected CHO cells stained using MPE1 or MPI1 indicate encoding for human A2BR. Untransfected cells were not that these two antibodies recognize the same molecule. labeled whereas transfected cells showed a very high level of Whereas MPE1 did not recognize A2ARs receptors labeling (Fig. 2), which is consistent with overexpression of overexpressed in CHO cells (Fig. 2E), MPI1 lead to some faint functional A2BRs positively coupled to adenylate cyclase labeling in permeabilized cells overexpressing A2ARs (Fig. (Pierce et al., 1992). As expected, MPE1 antibody, which is 2F). None of these antibodies did detect by immunoblotting directed against an extracellular epitope of A2BR was able to any band, which could be readily considered specific for detect A2BR molecules in either nonpermeabilized or A2BRs. All these results indicate that MPE1 antibody works

Fig. 4. Immunodetection of A2BR in resting peripheral blood lymphocytes (A) and Jurkat T cells (B). Cells were fixed and labeled with MPE1 antibody and a FITC-conjugated secondary antibody as described in Materials and Methods. Fluorescence of labeled cells was analyzed by flow cytometry (specific label is in bold solid line) and confocal microscopy. A2B adenosine receptors and T cell activation 497 well for immunocytochemistry and is specific for A2BRs 5 whereas MPI1 antibody presents some cross-reactivity with A2ARs. All subsequent immunocytochemical studies were 4 performed with the MPE1 antibody. cells 6 Expression of A2BR in resting lymphocytes 3 The MPE1 antibody, which specifically recognizes an extracellular epitope of A2BR, was used to analyze the 2 expression of these receptors in lymphocytes. The percentage of peripheral blood lymphocytes expressing A2BRs was 58±13 pmol cAMP/10 1 (mean ± s.d., n=13), with individual values ranging from 29 to 74. Double labeling experiments were performed to analyze 0 the expression of the receptor in different subsets of T cells. CONTROL NECA R-PIA CGS 21680 According to Fig. 3 no significant changes in the percentage + + + of A2BR cells were found among CD4 or CD8 T-cell Fig. 5. Cyclic AMP production via A2B adenosine receptors. PBL subpopulations. In general terms, the intensity of the labeling were incubated with the cAMP phosphodiesterase inhibitor RO-20- + µ in A2BR cells was moderate (Fig. 4A). In the T cell line Jurkat, 1724 for 10 minutes. Then 25 M of each adenosine receptor agonist the number of cells expressing the receptor (>95%) and the was added and cells were stimulated for 10 minutes at 37°C. cAMP level of expression was higher than that found in blood levels were determined as described in Materials and Methods. lymphocytes (Fig. 4B). A2BRs in peripheral blood lymphocytes are functional since the agonist NECA led to increases in cAMP levels (Fig. 5). The calcium levels was investigated in peripheral blood relative potency of the different agonists at a concentration of lymphocytes. The compound neither had any effect by itself nor 25 µM (NECA»R-PIA>CGS21680) is consistent with the it altered the effect exerted by OKT3, a mAb directed against pharmacology of A2BR. Due to the fact that NECA, via A2BR, the T cell receptor/CD3 complex which mobilizes calcium via leads to intracellular calcium mobilization in Jurkat cells inositol phosphate-sensitive stores (Fig. 6). The same failure to (Mirabet et al., 1997), the possible effect of NECA upon detect NECA-induced changes in calcium levels was evidenced

A 400 400 NECA OKT3

300 NECA 300 OKT3 (nM) (nM) i i

] 200 ] 200 2+ 2+ [Ca 100 100

0 0 1 min

B 600 600 NECA

500 500 OKT3

400 NECA 400 OKT3 (nM) (nM) [Ca i i ] 300 ] 300 2+ 2+ [Ca [Ca 200 200

100 100

0 0 1 min

Fig. 6. Lack of calcium mobilization in PBL via A2BR. A fraction of PBL was maintained in the culture conditions described in Materials and Methods until the Ca2+ determination assay (cells in A). Another fraction was activated with 1 µg/ml PHA for 72 hours, washed and maintained for an additional 24 hour-period in complete medium containing 10 units/ml IL-2 (cells in B). Then resting (A) and activated (B) PBL were loaded with Indo-1 and the effect of 100 µM NECA on intracellular calcium levels was analyzed, both in the absence of a previous Ca2+- mobilizing stimulus or after the addition of 1 µg/ml OKT3. Traces correspond to a representative experiment performed with PBL obtained from a donor. 498 M. Mirabet and others irrespective of the blood donor. On the other hand, NECA was activation signals were used, a relatively unspecific, triggered unable to mobilize intracellular calcium in T cells expanded by by PHA and one specific, triggered by the anti-T cell chronic activation with phytohemagglutinin (PHA) followed by receptor/CD3 complex mAb, OKT3. Irrespective of the IL-2 treatment (Fig. 6). stimulus, the percentage of lymphocytes expressing A2BRs was significantly higher (72±10 with PHA and 79±13 with OKT3) Expression and function of A2BR in T cell activation than that found in resting cells. events When OKT3-activated cells were analyzed by forward and The expression of A2BR was analyzed in activated cells. Two side scatter criteria, two different populations (B1 and B2)

Fig. 7. Expression of A2BR on the cell surface of activated lymphocytes. Isolated mononuclear cells were incubated in flasks precoated or not with purified OKT3 mAb. (A) Dot plot representing the side (properties of cytoplasmic and nuclear components) versus forward scatter (cell size) distribution of activated lymphocytes. B1, in red, represents the gated population remaining in the same region than nonstimulated lymphocytes (not shown), whereas B2, in green, represents the lymphocyte population with higher size and cellular complexity generated upon activation. (B) Dot plots representing the forward scatter versus the fluorescence intensity of cell surface A2BR in the above described two populations, B1 (red) and B2 (green). The patterns for unstimulated and stimulated cells are represented in the upper part and the lower part of the panel, respectively. Unspecific labeling is shown in the images on the left. Note that although in this experiment there are no major differences on fluorescence intensity between stimulated and unstimulated cells in the B1 population, we found significant differences in 5 out of 9 experiments performed with blood from different donors. A2B adenosine receptors and T cell activation 499 appeared as described elsewhere (Martín et al., 1995). B1 population contains cells structurally similar to the nonstimulated controls, whereas larger, more complex cells are included within B2 population (Fig. 7A). The percentage of cells expressing A2BR in B1 population was similar (Fig. 7B) or slightly higher than in resting cells. Following activation with OKT3, the majority of cells (>91±7%, n=9, range: 81- 99%) in the B2 population expressed enhanced levels of A2BRs (Fig. 7B). Variations in the intensity of labeling were monitored by flow cytometry (Fig. 7B) and by confocal microscopy (Fig. 8). The level of cell surface A2BR expression in small cells, which correspond to the B1 population of OKT3-activated cells, was slightly higher than that found in resting cells (see legend to Fig. 7), whereas the expression of the receptor in cells from the B2 population (cells of higher size) was markedly enhanced. Similar results were obtained when cells were activated with PHA although the degree of labeling enhancement was not as marked as in the case of lymphocytes activated via the T cell receptor (results not shown). These results provide evidence that the expression of A2BR increases upon lymphocyte activation. The role of A2BRs present in activated lymphocytes was investigated by measuring interleukin-2 production in cells activated using OKT3. The presence of NECA, at a concentration which activates A2BRs (5 µM), led to a significant reduction of interleukin-2 levels produced by activated T cells. In contrast, similar concentrations of either R-PIA or CGS21680 did not exert any effect (Fig. 9). These results indicate that adenosine, via A2BRs, mediates a reduction in the production of interleukin-2. This probably constitutes a feed-back mechanism for lymphocyte deactivation.

DISCUSSION

The results presented in this paper indicate that the antipeptide antibody MPE1 is speciﬁc for human A2BRs in immunolabeling assays. The speciﬁcity is based on the results obtained with wild-type and A2BR-transfected CHO cells, on the lack of label when using a hepatoma-derived human cell Fig. 8. Confocal microscopy images of resting and OKT3-activated line in which A2BRs are not detectable by pharmacological PBL labeled with MPE1 antibody. Note that the intensity of labeling procedures, and on the failure to recognize other adenosine in activated cells is enhanced in cells of higher size. Small cells (B1 receptor subtypes such as the A1 or A2A receptors. population) are marked with an arrowhead. Bar, 10 µm.

800

600 ) *

pg 400

IL-2 (

Fig. 9. Effect of adenosine analogs on interleukin-2 200 production. PBLs were activated using 2.5 µg OKT3 in the presence or the absence of NECA, R-PIA or 0 CGS21680. Interleukin-2 secreted to the medium in 72 hours (37ºC), was measured as indicated in Materials and 2.5 µg/ml OKT3 -+-+-+-+ Methods. Data are the mean ± s.e.m. from a representative Untreated 5 µM NECA 5µM CGS 21680 5 µM R-PIA experiment in triplicates. *P<0.01. 500 M. Mirabet and others

This is the first report showing direct evidence of A2BR by engagement of A2BRs. In fact, a reduction in interleukin-2 expression on the cell surface of blood cells. Based upon the production is observed when OKT3-activated T lymphocytes binding of the nonselective agonist NECA to human T were treated with NECA, at a concentration that ensures lymphocytes, Schultz et al. (1988) suggested that A2 but not activation of A2BRs. Other adenosine analogs, R-PIA or A1, receptors were present in membranes from these cells. A CGS21680, which are more selective for A2A receptors, did not similar conclusion was obtained by the agonist-induced have any effect. This indicates that upregulation of A2BRs is a increases in cAMP levels observed in human T lymphocytes mechanism for lymphocyte deactivation. A similar role has and thymocytes (Dinjens et al., 1986). The establishment of been devised for A2BR expressed in murine macrophages. In which of the two A2 receptor subtypes, A2AR or A2BR, is these cells A2BR are upregulated in response to interferon-γ operating in human T lymphocytes has remained elusive. The and they mediate an inhibition of the interferon-γ-induced fact that adenosine and NECA are low-potency stimulators of expression of either MHC class II genes, nitric oxide synthase cAMP production, has led to the suggestion that A2B receptors or pro-inflammatory cytokines (Xaus et al., 1999). Therefore, are those mainly found in the different T lymphocytes and T up-regulation of A2BR expression induced by activation signals cell lines assayed (Fredholm et al., 1991). The results of flow can be a feed-back mechanism for deactivation. This cytometry (Figs 3 and 4) indicate that the A2BR is expressed, mechanism seems to operate in a variety of immunocompetent but at a relatively low level, in a percentage of resting cells and can be important for the design of new lymphocytes (58±13). In comparative terms, the level of A2BRs immunoregulatory and antiinflammatory drugs. on the cell surface of Jurkat T cells was higher than in Using murine lymphocytes Huang et al. (1997) have shown peripheral blood cells (Fig. 4). The functionality of A2BRs in that agonists of A2ARs modulate the T cell receptor-mediated these samples isolated from the blood of human donors is CD25 upregulation. In a murine lymphoblastoid cell line the proven by the agonist-induced increase in cAMP levels. The A2BR is also expressed (data not shown) and, therefore, A2BR different capacity for producing cAMP exerted by NECA, are probably expressed on activated murine T cells. This would CGS21680 and R-PIA (Fig. 5) is consistent with a stimulation suggest that in the murine model adenosine likely regulates T of adenylate cyclase activity via A2BR and not via A2AR. cell activation events via both A2A and A2B receptors. Besides Working with lymphocyte membranes we have failed to detect murine lymphocytes, a variety of cell types from different 3 specific [ H]CGS21680 binding (data not shown), which is sources coexpress A2A and A2B receptors. Activation of A2AR suggestive of the absence of A2ARs in these blood cells. The and A2BR in the same cell may lead to synergy in cAMP absence of functional A2ARs and the presence of A2BRs production. In addition, adenosine may deliver divergent signals coupled to adenylate cyclase has been shown in Jurkat cells if each of these receptors subtypes are linked to different (Mirabet et al., 1997). It should be noted that activation of effector molecules. In nonlymphoid cell systems (Windscheif, A2BRs in Jurkat cells leads to calcium mobilization by inositol 1996) and in Jurkat (Mirabet et al., 1997) or CEM T cells (data phosphate-insensitive stores (Mirabet et al., 1997). not shown) adenosine receptors can be linked to a variety of Interestingly, A2BRs in either resting or activated human signaling pathways in addition to that of adenylate cyclase. lymphocytes are not coupled to calcium channels (Fig. 6) in The expression analysis in the two structurally different cell contrast to what happens in the leukemia-derived T cell line. populations found in T lymphocytes triggered via the T cell Other cAMP-raising agents such as prostaglandins, also receptor/CD3 complex (B1 and B2) suggests that the up- produce increases in intracellular calcium by inositol regulation of A2BR is higher in the population of cells phosphate-independent pools in Jurkat cells (Kelley et al., responding better to the stimulus (B2), which present a higher 1990) but not in peripheral blood lymphocytes (Chouaib et al., intracellular complexity (Martín et al., 1995). In the case of 1987). This suggests that different G protein-coupled receptor- mitogen-stimulated cells it is also found that the population of mediated signaling pathways operate depending upon the stage structurally more complex cells express more surface A2BR. In of T cell development. a previous study we have analyzed the pattern of expression of In this report we also provide evidence that the expression ecto-adenosine deaminase and CD26 in B1 and B2 populations of A2BR in lymphocytes changes upon activation. Activation from activated T cells (Martín et al., 1995). The pattern found of T cells via PHA or TCR/CD3 leads to a moderate but was very similar to that presented here for A2BR, which indicates significant increase in the percentage of cells expressing A2BR. that expression of the three cell surface molecules, A2BR, ecto- Moreover, according to flow cytometric data and micrographs adenosine deaminase and CD26, is analogously regulated in shown in Figs 7 and 8, there was a marked increase in activated T lymphocytes. It is difficult to reconcile the fluorescence intensity, thus indicating that the number of coordinated expression ecto-adenosine deaminase/adenosine molecules on the plasma membrane of activated T cells receptor with the fact that the enzyme degrades the ligand of the increased significantly. This behavior is similar to that receptor. This apparent contradiction can be circumvented if observed for CD26, which is expressed at relatively low some kind of regulation of the enzyme activity occurs or if intensity in about half of resting lymphocytes, whereas its adenosine deaminase has an extraenzymatic role. In a cell model expression is markedly enhanced upon activation (Martín et al., in which A1 adenosine receptors and ecto-adenosine deaminase 1995). CD26, which is considered a T cell activation antigen, are expressed, it has been demonstrated that both proteins is able to bind ecto-adenosine deaminase, the extracellular interact on the cell surface (Ciruela et al., 1996) and, in response adenosine-consuming enzyme, to the surface of lymphocytes. to receptor agonists, they are internalized together using the The lack of functional A2AR (at least in resting lymphocytes), same endocytic pathway (Saura et al., 1998). Interestingly, in the demonstration of A2BRs in human lymphocytes and the up- such a cellular model, the interaction ecto-adenosine regulation of A2BR in T cell activation strongly indicates that deaminase/A1 adenosine receptor is necessary for efficient receptor-mediated adenosine effects are elicited in these cells signaling via A1 receptors. Actually, catalytically inactive ADA A2B adenosine receptors and T cell activation 501 can deliver signals through its interaction with A1 adenosine Ca2+ indicators with greatly improved fluorescence properties. J. Biol. receptors in nonlymphoid cells (Ciruela et al., 1996; Saura et al., Chem. 260, 3440-3450. 1996, 1998) or with CD26 in T lymphocytes (Martín et al., Hershfield, M. S. and Mitchell, B. S. (1989). Immunodeficiency diseases caused by adenosine deaminase deficiency and purine nucleoside 1995). Thus, coordinated upregulation of A2BRs and ecto- phosphorylase deficiency. In The Metabolic and Molecular Bases of adenosine deaminase in T cell activation events may reflect a Inherited Disease, 7th edition (ed. C. R. Scriver, A. L. Beaudet, W. S. Sly functional interaction not necessarily related to the catalytic and D. Valle), pp. 1725-1768. McGraw-Hill, New York. activity of the enzyme. This possibility may lead to new insights Huang, S., Apasov, S., Koshiba, M. and Sitkovsky, M. V. (1997). Role of A2a extracellular adenosine receptor-mediated signaling in adenosine-mediated into the molecular basis of the pathophysiology of adenosine inhibition of T-cell activation and expansion. Blood 90, 1600-1610. deaminase-related SCID. Kelley, L. L., Blackmore, P. F., Graber, S. E. and Stewart S. T. (1990). Agents that raise cAMP in human T lymphocytes release an intracellular We acknowledge the technical help received from Jaume Comas pool of calcium in the absence of inositol phosphate production. J. 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