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Department of Clinical and Experimental Medicine, Pharmacology Unit (S.G., K.V., S.M., E.C., A.A., P.A.B.) and Department of Biochemistry and Molecular Biology (R.G., C.F.), University of Ferrara, Ferrara, Italy; "Centro Nazionale di Eccellenza per lo Sviluppo di Metodologie Innovative per lo Studio ed il Trattamento delle Patologie Infiammatorie" Ferrara, Ferrara, Italy (S.G., K.V., S.M., E.C., A.A., P.A.B.); and King Pharmaceuticals, Cary, North Carolina (E.L., S.M).
Received July 14, 2003; accepted December 3, 2003
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Abstract |
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). These adenosine subtypes have been classified as such because of differences at the molecular, biochemical, and pharmacological levels (Linden, 2001; Klinger et al., 2002). It is well recognized that adenosine can exert powerful effects on the immune system; this molecule has been identified as an important endogenous immunosuppressing regulator; it has been demonstrated that the lack of the enzyme adenosine deaminase is responsible for severe immunodeficient disease (Apasov et al., 1995; Hirschhorn, 1995). A number of effects ascribed to adenosine on lymphocyte function, such as inhibition of interleukin-2 production, cell proliferation, and major histocompatibility complex-restricted cytotoxicity, seem to be mediated through Gs-coupled A2A receptors (Polmar, 1990). Indeed, in human lymphocytes, the A2A receptor has been thoroughly characterized based on binding (Varani et al., 1997; Murphree et al., 2002), immunohistochemistry (Koshiba et al., 1997), flow cytometry (Koshiba et al., 1999), and functional studies taking advantage of the wealth of pharmacological tools, including radioligands, available (Gessi et al., 1999; Sullivan et al., 1999, 2001; Ohta and Sitkovsky, 2001; Thiel et al., 2003). In the case of the Gi-coupled A3 subtype, the data are less extensive. It has been shown in a murine model that adenosine, through an A3 receptor, can interfere with the tumor cell recognition and the cytolytic activity of cytotoxic lymphocytes thus leading to the hypothesis that A3 antagonists might be used to relieve tumor-associated immunosuppression and facilitate adoptive immunotherapy (MacKenzie et al., 1994; Hoskin et al., 2002). As for human models, we have previously demonstrated that this adenosine subtype is highly expressed in Jurkat cells, a human tumor cell line originating from the immune system (Gessi et al., 2001), whereas a coherent picture of this subtype in human lymphocytes has not yet emerged. A considerable body of evidence has accumulated suggesting that the A3 receptor may regulate various functions in lymphoid cells by inducing both pro- and anti-inflammatory effects. Its activation results in proinflammatory activities by facilitating release of allergic mediators in mast cells (Ramkumar et al., 1993) and by initiating actin polymerization and migration in immature dendritic cells (Panther et al., 2001). In contrast, its stimulation induces anti-inflammatory effects by inhibiting pro-inflammatory cytokine release, formyl-Met-Leu-Phe–triggered respiratory burst, and tissue factor expression in monocytic cells (Sajjadi et al., 1996; Broussas et al., 1999, 2002). Moreover, anti-inflammatory effects are evoked by reduction of superoxide anion generation and degranulation in eosinophils and neutrophils (Bouma et al., 1997; Ezeamuzie and Philips, 1999; Gessi et al., 2002) and by induction of apoptosis in blood mononuclear cells (Barbieri et al., 1998). However, in the case of human lymphocytes, novel roles for the A3 receptor must be considered with caution until the ligand binding profile of this subtype has been properly defined. Prompted by these observations, we undertook an in-depth investigation using receptor-binding, western blotting studies, quantitative real-time RT-PCR and cAMP assays of A3 receptors in resting and phytohemagglutinin (PHA)-activated T cells. Our results demonstrate that activated human lymphocytes undergo to a rapid induction of both transcript and protein of A3 receptors and suggest that A3 subtypes may be involved in the immunological responses mediated by adenosine in T cells. |
Materials and Methods |
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Lymphocyte Isolation. Lymphocytes were isolated from buffy coats kindly provided by the Blood Bank of the University Hospital of Ferrara, according with the methods reported by Varani et al. (1997). Briefly, the blood was centrifuged on Ficoll-Hypaque density gradients. The human peripheral blood mononuclear cells were isolated and removed from the Ficoll-Hypaque gradients. Subsequently, they were washed in 0.02 M phosphate-buffered saline at pH 7.2 containing 5 mM MgCl2 and 0.15 mM CaCl2. Finally, they were decanted into a culture flask and placed in a humidified incubator (5% CO2) for 2 h at 37°C. This procedure, aimed at removing monocytes, which adhere to the culture flask, resulted in a purified lymphocyte preparation containing at least 99% small lymphocytes identified by morphological criteria (PBL). Then, T cells were activated with 2 µg/ml PHA and, for Western blot experiments, CD8+ and CD4+-enriched T cells were purified from the PBL using dynabeads coated with an anti-CD8 mAb (Dynal ASA, Oslo, Norway).
Membrane Preparation. After centrifugation at 400g for 15 min at 4°C, the mononuclear cell pellets were resuspended in ice-cold, glass-distilled water and allowed to stand on ice for 60 min to ensure adequate lysis. The preparation was centrifuged at 20,000g for 15 min, the resulting pellet was resuspended in 50 mM Tris-HCl, 10 mM MgCl2, 1 mM EDTA, pH 7.4 at 25°C, at a concentration of 60 µg protein/100 µl, and this suspension was incubated with 3 IU/ml of adenosine deaminase for 30 min at 37°C before use for binding studies.
[3H]MRE 3008F20 Binding Assay. Binding assays were carried out according to Gessi et al. (2002). Association kinetic studies were performed incubating lymphocyte membranes with 2 nM [3H]MRE 3008F20 in a thermostatic bath at 4°C. For the measurement of the association rate, the reaction was terminated at time points ranging from 5 to 200 min by rapid filtration under vacuum, followed by four washes with ice-cold buffer. For dissociation experiments, membranes were preincubated with [3H]MRE 3008F20 at 4°C for 120 min. Specific binding was then evaluated at 5 to 100 min after the addition of 1 µM MRE 3008F20. The binding of [3H]MRE 3008F20 to human lymphocytes was carried out in a 96-well microplate in a total volume of 250 µl containing 50 mM Tris HCl buffer, 10 mM MgCl2, and 1 mM EDTA, pH 7.4. In saturation experiments, 100 µl of membrane homogenate (60 µg protein/assay) were incubated in duplicate with 10 to 12 different concentrations of [3H]MRE 3008F20 in the range of 0.1 to 20 nM. In competition experiments, 2 nM [3H]MRE 3008F20 was incubated in duplicate with at least 12 to 14 different concentrations of typical adenosine receptor agonists and antagonists. Analogous experiments were performed in the presence of 100 µM GTP. Nonspecific binding, defined as binding in the presence of 1 µM MRE 3008F20 at the KD value for the radioligand was 
35% of total binding. Incubation time was 120 min at 4°C to allow equilibrium to be reached. This temperature was chosen in consideration of the fact that A3 antagonist binding is prevalently enthalpy-driven (Varani et al., 2000). Bound and free radioactivity were separated by filtering the assay mixture through Whatman GF/B glass-fiber filters using a Micro-Mate 196 cell harvester (PerkinElmer Life and Analytical Sciences, Boston, MA). The filter bound radioactivity was counted on a TopCount microplate scintillation counter (efficiency 57%) with MicroScint 20 (both from PerkinElmer). The protein concentration was determined according to a Bio Rad method (Bradford, 1976) with bovine albumin as a standard reference.
Thermodynamic Analysis. For a generic binding equilibrium the affinity association constant KA = 1/KD is directly related to the standard free energy 
G° (G° = -RT lnKA, where R is the gas constant and T is the absolute temperature) that can be separated in its enthalpic and entropic contributions according to the Gibbs equation: G° = H°-TS°. The standard free energy was calculated as G° = -RTlnKA at 298.15 K, the standard enthalpy, H°, from the van't Hoff plot lnKA versus (1/T) (the slope of which is -H°/R) and the standard entropy as S° = (H° - G°)/T, with T = 298.15 K and r = 8.314 J/K/mol. KA values were obtained from saturation experiments of [3H]MRE 3008F20 binding to the A3 adenosine receptors in human lymphocytes performed at 4, 10, 15, 20, 25, and 30°C.
Western Blotting. T cells (5 x 106) were washed with ice-cold PBS and centrifuged. The pellets were resuspended in buffer solution (200 µl of 2% SDS, 10% glycerol, and 60 mM Tris-HCl, pH 6.8) and lysed for 7 min at 100°C. Protein concentration was determined using a bicinchoninic acid protocol (Pierce Chemical, Rockford, IL). Equal amounts of proteins were loaded on a 12% SDS-polyacrylamide gel and electroblotted to Protran nitrocellulose membranes (Schleicher and Schuell, Keene, NH). The blots were probed with the appropriate dilution of primary antibody in fresh blocking buffer (5% skim milk powder in phosphate-buffered saline containing 0.1% Tween 20) overnight at 4°C. Filters were washed and incubated for 1 h at room temperature with peroxidase-conjugated anti-rabbit immunoglobulin G (1:5000). Specific reactions were revealed with the enhanced chemiluminescence Western blotting detection reagent (Amersham Biosciences).
Real-Time RT-PCR Experiments. Total cytoplasmic RNA was extracted by the acid guanidinium-thiocyanate-phenol method (Chomczynski and Sacchi, 1987). Quantitative real-time RT-PCR assay (Higuchi et al., 1993) of A3 mRNA transcript was carried out using gene-specific double fluorescently labeled TaqMan MGB probe (minor groove binder) in a ABI Prism 7700 Sequence Detection System (Applied Biosystems). The following primer and probe sequences were used for real-time RT-PCR: A3 forward primer, 5'-ATGCCTTTGGCCATTGTTG-3'; A3 reverse primer, 5'-ACAATCCACTTCTACAGCTGCCT-3'; A3 MGB probe, 5'-FAM-TCAGCCTGGGCATC-TAMRA-3' (in which the fluorescent reporter FAM and the quencher TAMRA are 6-carboxy fluorescein and 6-carboxy-N,N,N',N'-tetramethylrhodamine, respectively). For the real-time RT-PCR of the reference gene, the endogenous control human GAPDH kit was used, and the probe was fluorescence-labeled with VICTM (Applied Biosystems, Monza, Italy).
Measurement of Cyclic AMP Levels. Human lymphocytes (5 x 106 cells/assay) were suspended in 0.5 ml of Krebs Ringer phosphate buffer (136 mM NaCl, 5 mM KCl, 0.67 mM Na2HPO4, 0.2 mM KH2PO4, 3 mM NaHCO3, 1 mM CaCl2, 5 mM glucose, 5 mM HEPES, and 10 mM MgCl2, at pH 7.45), containing 0.5 mM Ro 20-1724, water solution, as phosphodiesterase inhibitor, and 2.0 IU/ml adenosine deaminase and preincubated for 10 min in a shaking bath at 37°. Then the A3 adenosine agonist Cl-IB-MECA plus forskolin (10 µM) was added to the mixture and incubated for a further 5 min. The effect of increasing concentrations of MRE 3008F20 in the range 0.1 to 100 nM was determined by antagonism of the inhibition of cyclic AMP production induced by Cl-IB-MECA (100 nM). Analogous experiments were performed by using the nonselective agonist NECA in the range 10 nM to 10 µM. The effect of a fixed concentration of NECA (1 µM) was antagonized by increasing doses of SCH 58261 (0.1–100 nM), in the absence of forskolin. The reaction was terminated by the addition of ice-cold 6% trichloroacetic acid. The trichloroacetic acid suspension was centrifuged at 2000g for 10 min at 4°C and the supernatant was extracted four times with water-saturated diethyl ether. The final aqueous solution was tested for cyclic AMP levels by a competition protein binding assay carried out essentially according to Varani et al. (1997). Samples of cyclic AMP standards (0–10 pmol) were added to each test tube containing 0.1 M Trizma base, 8.0 mM aminophylline, 6.0 mM 2-mercaptoethanol, pH 7.4, and [3H]cyclic AMP in a total volume of 0.5 ml. The binding protein, previously prepared from bovine adrenal glands, was added to the samples and incubated at 4°C for 150 min. After the addition of charcoal, samples were centrifuged at 2000g for 10 min. The clear supernatant (0.2 ml) was mixed with 4 ml of Atomlight (PerkinElmer Life and Analytical Sciences) and counted in a liquid scintillation counter (LS-1800; Beckman Coulter, Fullerton, CA).
Data Analysis. All binding studies (kinetics, saturation, competition) were analyzed with the program LIGAND (Cambridge, UK) (Munson and Rodbard, 1980). EC50 and IC50 values in the cyclic AMP and Ca2+ assays were calculated with the nonlinear least-squares curve fitting program Prism (GraphPad Software, San Diego, CA). Statistical analyses were performed using Student's t test.
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Results |
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). The expression of A3 receptors was analyzed in activated lymphocytes. One-hour treatment with PHA, a T cell mitogen, caused an increase of binding sites to A3 receptors (Bmax of 225 ± 23 fmol/mg of protein, P < 0.01), whithout changes in KD value (1.85 ± 0.25 nM) (Fig. 1A). Incubation for 1 h of the cells with PHA at different concentrations caused an increase of A3 receptors, which reached a maximal expression at 2 µg/ml, as demonstrated by binding studies (Fig. 1B). Comparable results were obtained by activating T cells with PMA (range, 0.1–100 ng/ml) plus 1 µg/ml ionomycin for 1 h; in this case, the maximal expression of A3 receptor was obtained at 25 ng/ml PMA plus 1 µg/ml ionomycin (data not shown). Therefore, the concentration of 2 µg/ml PHA was chosen to perform a more detailed kinetic analysis of the increase in expression of the A3 receptor protein. The time course of A3 receptor up-regulation was followed at 15, 30, 60, 180, 480, and 1440 min. Saturation experiments revealed a rapid increase of binding to the A3 subtype starting within 15 min, with a Bmax value of 175 ± 14 fmol/mg of protein (P < 0.05), and reaching a plateau at 30 min (Bmax values of 228 ± 22, 225 ± 23, 233 ± 26, 230 ± 25, and 220 ± 20 fmol/mg of proteins at 30, 60, 180, 480, and 1440 min, respectively, P < 0.01) (Fig. 1C); the respective equilibrium dissociation constants were not significantly different.
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Kinetic Experiments. Association studies showed that binding equilibrium was reached after approximately 60 min and was stable for at least 5 h (Fig. 2A). [3H]MRE 3008F20 binding was rapidly reversed by the addition of 1 µM MRE 3008F20 (Fig. 2B). A two-site fit of association and dissociation curves was not significantly better than a one-site fit (P < 0.05). The rate constants were: kobs = 0.049 ± 0.001 min-1 and k-1 = 0.024 ± 0.001 min-1. The apparent equilibrium dissociation constant (KD) was calculated from the k+1 (k+1 = 0.0125 min-1 nM-1) and k-1 values to be 1.92 nM. Similar results were obtained in activated T cells (data not shown).
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Competition Experiments. Adenosine agonists were found to inhibit [3H]MRE 3008F20 binding, in human lymphocytes, in a manner consistent with the labeling of the A3 adenosine receptor, as shown in Fig. 3A. The order of potency in [3H]MRE 3008F20 inhibition assays was Cl-IB-MECA > NECA > R-PIA > S-PIA. R-PIA was approximately six to seven times more active (KH = 38 nM; KL = 2100 nM) than its stereoisomer, S-PIA (KH = 260 nM; KL = 19,000 nM) showing the stereoselectivity of agonist binding. The competition curves of all agonists were best fit assuming a two-state model corresponding to high- and low-affinity states of the A3 adenosine receptor (Fig. 3A), a phenomenon expected for a G-protein-coupled receptor. Affinities are reported in Table 1 as Ki, KH, and KL values, and the percentage of receptors in the high-affinity state (RH) is also shown. To test whether the high-affinity state of the A3 receptors was linked to a guanine nucleotide regulatory protein, the effect of GTP on the affinity states was examined. The addition of 100 µM GTP shifted the competition curves with agonists from a biphasic to a monophasic shape (LIGAND, P < 0.01), with a Ki value near that of the low affinity sites, as shown in Table 1. In contrast, the above treatment did not change the shape of the competition curves with antagonists. Antagonist competition curves for [3H]MRE 3008F20 binding showed a rank order of potency of MRE3105F20 > MRE3008F20 > CGS 15943 > DPCPX, each of which exhibited Hill slopes near unity (Fig. 3B; Table 1). SCH 58261 showed a Ki value >10 µM.
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Thermodynamic Experiments. Saturation experiments of [3H]MRE 3008F20 binding, performed at the six temperatures selected revealed that, in resting cells, KD and Bmax values were in the range 1.8 to 4.3 nM and 110 to 130 fmol/mg of proteins, respectively. Whereas the dissociation constant (KD) changed with temperature, Bmax values obtained from [3H]MRE 3008F20 saturation experiments seemed to be largely independent. The van't Hoff plot of lnKA versus 1/T for [3H]MRE 3008F20 binding to the A3 adenosine receptor in human lymphocytes gives the following final equilibrium thermodynamic parameters (expressed as mean values ± S.E. of three independent determinations): G° = -47.76 ± 0.16 kJ/mol; H° = -19.78 ± 1.50 kJ/mol; S° = 94 ± 7 J/mol/K and indicates that the binding was both enthalpy- and entropy-driven. Similar results were obtained after PHA-activation as shown in Fig. 4 (G° = -48.17 ± 0.17 kJ/mol; H° = -19.37 ± 1.75 kJ/mol; S° = 97 ± 8 J/mol/K).
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Western Blotting Studies
To compare the expression of A3 receptors in the most representative subset of human T lymphocytes, resting and PHA-activated PBL from different human volunteers were separated into CD8+ and enriched CD4+ fractions. The resting and activated cells of these two fractions and an aliquot of the total unseparated PBL were then evaluated for the expression of the specific A3 receptor by Western blot analysis. A significant change in the expression of the A3 subtype within the total unseparated population was observed between resting and activated cells confirming our binding experiments (Fig. 5 A, lines 1 and 2). The increase in A3 receptor expression after PHA activation was attributed to the CD4+ enriched fraction (Fig. 5A, lines 3 and 4) whereas CD8+ cells, which do express A3 receptors, failed to show any significant change after T cell activation (Fig. 5A, lines 5 and 6); CHO cells transfected with human A3 receptors were chosen as positive controls (line 7). When earlier time points were investigated in separated CD4+ and CD8+ cells, we observed that the increase of protein in CD4+ cells started at 15 min and was maximal at 30 min (Fig. 5 B). As for CD8+ cells, the density of A3 subtype showed no change after T cell activation (data not shown).
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Real-Time RT-PCR Experiments
To determine whether the increase in A3 density was caused by an increase in mRNA encoding the A3 receptor, mRNA content was investigated from resting and PHA-stimulated human lymphocytes by use of quantitative real-time RT-PCR. The expression level of A3 receptors was normalized to the expression level of the endogenous reference (GAPDH) in each sample. Activation of T cells by the addition of PHA for 1 h produced a 5.2 ± 0.7-fold increase of A3 receptor mRNA accumulation in activated T cells with respect to the corresponding resting ones, as obtained from different human volunteers. A representative example of the real-time RT-PCR quantitation of A3 receptors and GAPDH mRNAs in one individual case is shown in Fig. 6, A and B. When investigating the time-dependence of mRNA accumulation in separated CD4+ and CD8+ cells, a rapid increase was observed starting within 15 min (3.5 ± 0.4 fold, P < 0.01) in CD4+ subset; this increase was highly reproducible and reached a plateau at 30 min (5.5 ± 0.8 fold, P < 0.01) (Fig. 6 C). Conversely, PHA treatment did not change A3 receptor mRNA in CD8+ cells (data not shown).
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Cyclic AMP Assays
To demonstrate the existence of functional A3 receptors in human lymphocytes and to evaluate whether changes of receptor density under activating conditions were reflected at a functional level, we determined the potency of the most selective A3 agonist Cl-IB-MECA in the inhibition of cAMP levels. Cl-IB-MECA was able to inhibit forskolin-stimulated cAMP levels with an IC50 value of 2.7 ± 0.3 nM, according to the affinity determined in radioligand binding assays (Fig. 7A); interestingly, after PHA treatment, there was a shift to the left of the agonist IC50 value (1.5 ± 0.4 nM). In resting lymphocytes, the selective A3 ligand MRE 3008F20 antagonized 100 nM Cl-IB-MECA–induced inhibition of cAMP levels with an IC50 of 5.0 ± 0.3 nM (Fig. 7B), suggesting the involvement of A3 receptors. A low-affinity analog of MRE 3008F20, MRE 3016F20 (hA3CHO cells; Ki = 800 nM) (Baraldi et al., 2000), was chosen to ascertain that the block of cAMP levels was caused not by the structure of this family of compounds but by the activity toward A3 receptors. As shown in Fig. 7B, the failure of this ligand to antagonize Cl-IB-MECA effects on cAMP levels confirms the involvement of A3 receptors in this response. DPCPX (100 nM), a selective blocker of A1 subtypes, did not significantly modify 100 nM Cl-IB-MECA-mediated response (65 ± 5 and 62 ± 4% in the absence and in the presence of DPCPX, respectively), thus ruling out the involvement of A1 receptors. The nonselective agonist NECA was able to induce a stimulation of cyclic AMP levels, with an EC50 value of 215 ± 30 nM. Because of the stimulatory effect observed in the presence of NECA, we antagonized a fixed concentration of this agonist (1 µM) by increasing doses of the selective A2A blocker SCH 58261 (0.1–100 nM) in the absence of forskolin. Under these experimental conditions, SCH 58261 antagonized the rise in cyclic AMP levels with an IC50 value of 15 ± 3 nM, suggesting the involvement of A2A subtype.
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Discussion |
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; Hoskin et al., 2002). Therefore, even though many pharmacological works rely on the selectivity of adenosine agonists and antagonists, we are convinced that before establishing a role for the A3 subtype in the regulation of human immune system, it is important to identify the leukocytes that express functional A3 receptors. Prompted by these considerations and after the pharmacological characterization by means of receptor-binding of the A3 subtype in human eosinophils (Kohno et al., 1996), neutrophils (Gessi et al., 2002), and recently dendritic cells (Fossetta et al., 2003), in the present study, we provide an analysis of the A3 adenosine receptor in human lymphocytes.
Different experimental approaches attest to the existence of adenosine A3 receptors on these peripheral blood cells and indicate an up-regulation after T cell activation. First, their presence on plasma membrane was detected through saturation experiments of [3H]MRE 3008F20 binding, revealing a single class of binding sites with a KD value of 1.79 ± 0.30 nM, comparable with that determined by kinetic experiments (1.92 nM). The binding was rapid, reversible, and saturable, with a capacity of 125 ± 15 fmol/mg of protein, indicating that the number of A3 receptors in resting human lymphocytes was similar to the number of A2A subtypes (Varani et al., 1997) but lower than the number of A3 receptors found in Jurkat cells, a human tumor leukemia line (Gessi et al., 2001).
Interestingly, after PHA activation, we observed a rapid increase of A3 receptor density, starting at 15 min, that was maximal within 30 min of treatment and was stable when evaluated at 3, 8, or 24 h of PHA treatment. Analog results were obtained by stimulating lymphocytes with other activators such as PMA plus ionomycin, suggesting that A3 receptors may play a role in activated T cells.
Competition experiments of various adenosine receptor ligands, performed to characterize the A3 subtype in T cells, showed a rank order of potency typical of the A3 subtype (Gessi et al., 2002; Fossetta et al., 2003). All agonist competition curves were best described by the existence of one high-affinity (KH) and one low-affinity (KL) agonist receptor binding state. Approximately 25 to 28% of the A3 receptor existed in the high-affinity state under the present assay conditions. The addition of GTP, and subsequent uncoupling of receptors from G-proteins, converted the agonist displacement curves from biphasic to monophasic, suggesting a guanine nucleotide-mediated shift of the high-affinity binding sites to a low-affinity form, in agreement with that reported for human A3 receptors (Varani et al., 2000). On the contrary, competition binding curves with antagonists, including substituted pyrazolo triazolo pyrimidine compounds (Baraldi and Borea, 2000) were monophasic and did not change upon addition of GTP. Thermodynamic parameters obtained from the van't Hoff plot indicate that [3H]MRE 3008F20 binding to A3 adenosine receptors is enthalpy- and entropy-driven in agreement with data obtained in all systems in which human A3 receptors have been studied from this point of view (Varani et al., 2000; Gessi et al., 2001, 2002; Merighi et al., 2001). A similar thermodynamic behavior was found in activated T cells, suggesting that the activation process does not involve, at a molecular level, receptor alterations, leading to modifications in the A3-related binding mechanisms.
In addition to the A3 receptors, PHA has been shown to alter the expression of other adenosine subtypes. For example, it has been reported that A2B receptors are also up-regulated by PHA in both CD4+ and CD8+ cells (Mirabet et al., 1999); as for A2A subtypes, it has been observed that the activation process increases their expression predominantly in CD8+ T cells (Koshiba et al., 1999). Therefore, we evaluated whether activation-dependent changes in A3 expression were attributable to CD8+ or CD4+ subsets of human lymphocytes, the two major T cell subsets involved in the recognition of peptide antigens presented by class I and II major histocompatibility complex, respectively. Our results allow us to conclude that CD8+ fraction does express A3 receptors but is not responsible for their increase under activating conditions, which is instead caused by an enriched CD4+ cell fraction. When we investigated the kinetic of this up-regulation, we found that even at earlier time points, the increase was present only in CD4+ cell fractions, whereas it was not changed in CD8+ cells. Therefore, it is possible that in humans, as in the murine model (Hoskin et al., 2002), A3 receptors play an immunosuppressive role in CD8+ T cells, but their up-regulation in CD4+ fraction strongly suggests that they might also be implicated in T helper cell activities. One method for increasing the amount of A3 receptors on the cell membrane is to increase the accumulation of mRNA encoding the A3 subtypes. As evaluated by means of real-time RT-PCR experiments, activation of T cells with PHA rapidly increased the level of A3 message in the CD4+ subset, but not in the CD8+ cells. This increase in A3 receptor mRNA, which could occur as a result of an increase in transcription and/or an increase in mRNA stability, is likely to be responsible for the increased synthesis of receptor proteins as detected by means of binding and Western blotting studies. Interestingly, it has been reported that A2A mRNA is also regulated after activating conditions, suggesting that adenosine receptor expression is of greater significance in the modulation of the functions of these cells (Thiel et al., 2003). Numerous data attest to the importance of A2A subtype in the modulation of immune response mediated by adenosine, whereas less impressive information is actually available on the effects exerted through A3 receptor. To verify whether the increase in receptor density was reflected in the regulation of second messengers activated by A3 adenosine receptors in human lymphocytes, we investigated the ability of Cl-IB-MECA to modulate adenylyl cyclase activity. Forskolin-stimulated cAMP levels were inhibited by Cl-IB-MECA, and this effect was potently antagonized by MRE 3008F20. The agonist IC50 value was shifted to the left under stimulating conditions, suggesting that A3 receptors may activate this pathway during T cell activation and may affect the T cell-mediated immune response. As for NECA, this compound, even if able to compete for [3H]MRE 3008F20 binding, did not inhibit cAMP levels, as already observed in Jurkat cells (Gessi et al., 2001). Most probably, its stimulatory effect on cAMP levels, raised through the activation of A2A receptors, prevailed over the A3-mediated inhibitory action, suggesting a stronger potency of A2A receptors in signal transduction over the A3 receptors. This is not surprising, because a similar behavior has already been reported in doubly Gs/Gi-coupled chimeric A1/A2A adenosine receptors expressed in human embryonic kidney 293 cells (Tucker et al., 2000). Therefore, future studies will help to reconcile the complicated effects of adenosine through Gi protein-coupled A3 adenosine receptors in the modulation of immune and inflammatory processes and to clarify the relevance of the differential activation by adenosine of Gs/Gi coupled receptor subtypes to reach a balance between destruction of pathogen agents and tissue protection from excessive damage (Sitkovsky, 2003).
At variance with adenylyl cyclase activity, only high micromolar doses of Cl-IB-MECA induced Ca2+ increase in both resting and activated conditions. The low potency of the agonist in the stimulation of Ca2+ increase indicates that Cl-IB-MECA elevated calcium by an unknown mechanism that cannot clearly be related to A3 receptor activation (data not shown). However, there are other effector systems that may be activated by A3 subtypes to modulate immune functions (Butler et al., 2003), and ongoing studies in our laboratory are aimed at evaluating the A3 receptor involvement in cytokine release and its role in the expression of T cells activation markers.
In conclusion, all the data reported in this study indicate that human lymphocytes present A3 adenosine receptors with a pharmacological, biochemical, and thermodynamic profile typical of the human A3 subtype. The rapid up-regulation of A3 receptors functionally coupled with adenylyl cyclase in activated T cells may indicate another potential candidate of biological significance for adenosine-mediated responses in the immune system.
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Acknowledgements |
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Footnotes |
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Address correspondence to: Prof. Dr. Pier Andrea Borea, Chair of Pharmacology, Faculty of Medicine, University of Ferrara, Department of Clinical and Experimental Medicine, Pharmacology Unit, Via Fossato di Mortara 17-19, 44100 Ferrara, Italy. E-mail: bpa{at}dns.unife.it
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) and activated conditions (
); KD values were 1.79 ± 0.30 and 1.85 ± 0.25 nM and Bmax values were 125 ± 15 and 225 ± 23 fmol/mg of protein, respectively. Experiments were performed as described under Materials and Methods. Insets, Scatchard plots of the same data. B, effect of increasing concentrations of PHA on A3 receptor density in human lymphocytes. C, time course of A3 receptor density in human lymphocytes stimulated with PHA, assayed by means of [3H]MRE 3008F20 saturation binding experiments. Analysis was by Student's t test. #, P < 0.05; *, P < 0.01 versus resting cells (0 h). Values are the means and vertical lines are S.E.M. of five separate experiments performed in triplicate.
), an analog of MRE 3008F20, with low affinity to A3 receptors on the Cl-IB-MECA (100 nM) inhibition of forskolin-stimulated cAMP levels in human resting lymphocytes.