Dopamine receptors on human T- and B-lymphocytes

Journal of Neuroimmunology, 45 (1993) 113-120

113

Elsevier SciencePublishers B.V. JNI 02375

Dopamine receptors on human T- and B-lymphocytes L a u r a Santambrogio b, Maria Lipartiti a, Alessandro Bruni c and R o b e r t o Dal Toso a a Fidia Research Laboratories, Abano Terme (PD), Italy, b H Department of Internal Medicine, University of Perugia, Perugia, Italy, and c Department of Pharrnacology, University ofPadova, Padova, Italy

(Received 19 January1993) (Accepted 19 January1993)

Key words." Humanlymphocyte;Dopamine receptor; Sulpiride; cAMP; Neuroimmunomodulation

Summary The presence of functional dopamine receptors on differentiated cells of the mammalian immune system is still under discussion. This study has utilized (-)-[3H]sulpiride as a ligand, to detect the presence of recognition sites of the dopamine D 2 receptor family on human T- and B-lymphocytes. The (-)-[3H]sulpiride binding was of high affinity (K d 0.9 nM + 0.2 nM), specific, saturable (Bmax 10.2 + 1.4 fmol/106 cells) and reversible. The pharmacological characterization of the recognition site suggests similarities mainly with the D 2 and D 4 rather than D 3 subtype of dopamine receptor. Furthermore, dopamine treatment was able to reduce the intracellular cAMP levels of lymphocytes stimulated with forskolin, thus suggesting a potential functional significance of this dopamine receptor in mediating neural-immune interactions.

Introduction Increasing evidence indicates the existence of neurally mediated immunomodulatory mechanisms (Roszman et al., 1985). Neuro-immune interactions may occur through the hypothalamic-pituitary axis and through the sympathetic and parasympathetic innervation of primary and secondary lymphoid organs (Bullock et al., 1985; Livnat et al., 1985). The molecular basis for the modulation of immune responses by neurotransmitters or neurohormones depends on the presence of specific receptors on the surface of immunocytes. Previous studies have shown that human lymphocytes bear surface/3-adrenergic receptors modulating a large number of immune functions such as antibody synthesis (Besedovsky et al., 1979), mitogen-induced lymphocyte transformation (Hadden et al., 1970), Tlymphocyte-mediated cytolytic activity (Livnat et al., 1985), inhibition of the synthesis of complement components (Lappin et al., 1982) and alteration in the total number of lymphocytes and T cell subsets (Crary et al.,

Correspondence to: Maria Lipartiti,Fidia ResearchLaboratories,Via

Ponte della Fabbrica 3/A, 35031 Abano Terme (PD), Italy.

1983; Miles et al., 1985). Furthermore, also muscarinic receptors have been recently demonstrated and pharmacologically characterized in human blood lymphocytes (Zaleman et al., 1981), with their activation by cholinergic agonists inducing proliferative activity of murine thymocytes (Rossi et al., 1989). However, the existence of dopamine receptors (DA-R) on lymphocytes is still an open question. Various radioligand binding techniques have been used to label D2-DA-R on murine and human lymphocytes and thymocytes. Although a two component binding curve has been described for [3H]spiperone in mammalian lymphocytes (Lefur et al., 1980; Shaskan et al., 1984), [3H]spiperone may provide a doubtful saturability and low stereospecificity with respect to the isomers of butaclamol or flupenthixol. Furthermore, there is no correlation between the potency of neuroleptics in displacing [3H]spiperone binding from stria~al binding sites as compared to the binding sites present on lymphocytes (Bloxham et al., 1981; Maloteau et al., 1983). Using [3H]-DA it has been shown that rat lymphocytes and mouse thymocytes have a specific, saturable and high-affinity binding which is not inhibited by sulpiride, bromocriptine and haloperidol. Lymphocytes have therefore been suggested to possess a recep-

114 tor site for dopamine that might be a catecholaminergic receptor (Ovadia et al., 1987; Stefano et al., 1989). Recently, the human brain and pituitary D2-DA-R were isolated and cloned, with two isoforms of this receptor being generated by alternative splicing (D2A and D2B) (Dal Toso et al., 1989; Grandy et al., 1989). On the basis of the homology with D 2 receptor, two other DA-R have been cloned. A D3-DA-R which has high amino acid identity with the D2A form but which differs in its tissue distribution, specificity for neuroleptics (Sokoloff et al., 1990) and a D4-DA-R which also shares high amino-acid sequence identity with both the D 2 and D 3 receptor subtypes (Van Tol et al., 1991). Our laboratory has investigated the presence of D2-DA receptors on human T- and B-lymphocytes, using both binding techniques and functional coupling to adenylate cyclase activity. Binding studies were performed with (-)-[3H]sulpiride which is a highly specific ligand for Dopamine receptors of the D 2 type (Martres et al., 1985; Sokoloff et al., 1990; Van Tol et al., 1991). Using an improved procedure, suitable for detecting specific binding sites in tissues with low DA-R density (Stefanini et al., 1987), we demonstrate high-affinity sulpiride binding in both T- and B- human lymphocytes. Furthermore, we also demonstrate that DA provokes a decrease in cAMP content of human lymphocytes. This provides evidence that this receptor has the pharmacological properties of the D2-R type (Onali et al., 1985) and might be functionally important in the regulation of lymphocyte responses.

Materials and Methods

T- and B-lymphocyte preparation Blood was drawn from healthy volunteers, aged 2050 yr. The blood was collected in heparinized plastic tubes, diluted 1:3 with phosphate-buffered saline (PBS) (Eurobio Paris, France), and mononuclear leukocytes (MNL) isolated by density gradient centrifugation using Ficoll-Paque (Pharmacia, Uppsala, Sweden) (Boyum, 1968). The MNL fraction was washed twice with RPMI 1640 Medium (Flow Laboratories), counted and resuspended at 2 x 106 cells per ml in complete RPMI 1640 Medium plus 5% fetal calf serum (FCS) (Gibco). Cells were incubated for 1 h in plastic tissue culture flasks at 37°C under 5% CO 2 95% air to deplete monocytes. The population of non-adherent cells was suspended in Earl's balanced salt solution (EBSS, GIBCO) (incubation medium) at a final concentration of 20 x 106 cells/ml. In all experimental conditions cell viability, as assessed by trypan blue dye exclusion, was greater than 95%. T- and B-lymphocytes were obtained by negative selection using the comple-

ment-dependent lysis with monoclonal antibodies (mAbs) OKT3 and OKB7 (Ortho Diagnostic Systems) plus rabbit complement (Cederlane Hornby, Ontario, Canada) (Laski et al., 1983). Briefly, aliquots of 5 x 106 cells were incubated with saturating amounts of antibodies and a 1:50 dilution of non cytotoxic rabbit complement for 1 h at 37°C. After lysis with mAb OKT3 and complement, over 95% of the remaining cells were CD20÷; using mAb OKB7 and complement, over 95% of the remaining cells were CD3 ÷.

DA-receptor binding assay The optimal conditions for (-)-[3H]sulpiride binding to intact human lymphocytes were found after testing different incubation temperatures (0-37°C), incubation volumes (O.5-1 ml), cell densities (2.5-30 × 105/ml), volume of the buffer added to the tube to stop incubation (1.5-5 ml) and number (1-3) and volumes (2-5 ml) of filter washings. In standard experiments, reactions were started by adding 2 x 106 cells to incubation medium containing (-)-[3H]sulpiride (specific activity 80 Ci/mmol, New England, Nuclear Boston, MA) in a final volume of 0.5 ml. For saturation experiments six to nine concentrations of ( - ) [3H]sulpiride were used, ranging from 0.25 to 20 nM. Incubations were carried out in triplicate and nonspecific binding measured in the presence of 0.5 ~M non-radioactive sulpiride. After 1 h at 0°C, reactions were stopped by the addition of 5 ml ice-cold EBSS followed by rapid filtration through Whatman G F / B glass fiber filters (Whatman Inc., Clifton, NJ) under vacuum. Filters were presoaked in buffer and rinsed with two additional 5 ml washes. The radioactivity trapped on the filters was counted after the addition of 5 ml of FILTER COUNT (Packard), by liquid scintillation spectrometry. Saturation binding isotherms were analyzed either by Scatchard analysis (Scatchard, 1949) or a nonlinear regression computer program (Sacchi et al., 1983). For displacement assays cells were allowed to equilibrate for 20 min at 0°C with increasing concentrations of drugs, (10-9-10 -3 M) and then incubated with 2.5 nM of (-)-[3H]sulpiride for 45 min at 0°C. All subsequent steps were performed as above. IC50 was defined as the concentration of drug that elicited half-maximal inhibition of specific binding. The inhibition constant (K i) was calculated with the Cheng and Prusoff equation, g i = IC50/(1 + L / K d) where L is the concentration of free labelled ligand and K a the equilibrium dissociation constant of the labelled ligand (Cheng and Prusoff, 1973). Bromocriptine, apomorphine, dopamine, noradrenaline, adrenaline, serotonin, haloperidol, chlorpromazine and fluphenazine were purchased from Sigma, St, Louis, MO. Quinpirole, (-)-sulpiride, (+)-sulpiride and (+)-butaclamol were obtained from RBI, Natick, MA. Clozapine was kindly gifted by San-

115

doz, Basel, Switzerland, pergolide and fenoldopam by Dr. C. Missale, University of Brescia, Italy. The effect of temperature on binding was investigated by incubating purified human lymphocytes at a final concentration of 2 × 106 cells in the presence of 2.5 nM (-)-[3H]sulpiride at different temperatures. Incubations were stopped after 10 min or 1 h and specific (-)-[3H]sulpiride binding obtained at each temperature was measured. For association studies, lymphocytes (2 × 106 cells) were incubated with 2.5 nM (-)-[3H]sulpiride at 0°C and the reaction stopped at different times by rapid filtration and two additional 5 ml washes. For dissociation studies, after (-)-[3H]sulpiride equilibrium binding (1 h at 0°C), lymphocytes were rapidly diluted with 5 ml ice-cold EBSS buffer. After 5, 10, 15, 30, 60, 90 and 120 min the reaction was stopped by rapid filtration. Non-specific binding was determined in the presence of 0.5/~M non-radioactive sulpiride in both association and dissociation studies.

cAMP assay The samples containing MNL (2 × 106 cells in 1 ml DMEM) were preincubated at 37°C for 15 min with 1 mM isobutylmethylxanthine (IBMX) to inhibit phosphodiesterase activity. The reaction was started by adding 10/xl of forskolin (10/zM) to cell suspension in the presence or absence of different concentrations of DA (from 10 - 9 t o 1 0 - 4 M ) . The incubation was terminated 5 min thereafter by centrifuging at 10.000 × g for 30 s in a Beckman microfuge. The supernatant was quickly removed, and the pellet was resuspended in 150 /~1 of ice-chilled TrisEDTA (50 mM) buffer at pH 7.5. The samples were kept at 4°C, sonicated, and then placed in a boiling water bath for 5 min, after which they were frozen. After thawing and pelletting the protein precipitate, two aliquots were assayed for cAMP using cAMP assay kits from Radiochemical Centre (Amersham, UK).

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[3HI SULPIRIOE(riM)

Fig. 1. (-)-[3H]sulpiride binding to human T- and B-lymphocytes. Binding of (-)-[3H]sulpiride (0.25-20 nM) was performed under standard conditions as described in Materials and Methods. Results are the mean (+SD) of at least 20 observations. The inset shows a Scatchard plot of the binding data. The affinity constant (K d) was 0.9 +0.2 nM and the estimated maximum binding (Bmax) was 10.2+ 1.4 fmol/10 6 lymphocytes.

tested. Furthermore, the number of (-)-[3H]sulpiride binding sites was found to increase linearly with cell densities, between 0.25 × 105 and 30 × 105 cells per tube (Fig. 2). Standard assays were then conducted at a concentration of 2 × 106 cells/tube. (-)-[3H]Sulpiride binding was temperature-dependent, with a lower number of binding sites at 37°C. However, in parallel experiments specific binding was the same when performed either at 37°C for 10 min or at 0°C for 1 h (Fig. 3). (-)-[3H]Sulpiride binding at 0°C was rapid and fully reversible within 180 min (Fig. 4). The reaction reached equilibrium after 30 min with no further binding up to 180 min. The calculated association rate constant (K 1) was 3.03 min-l nmol-1. The dissociation rate constant

Results

Binding characteristics of (-)-[3H]sulpiride to intact human T- and B-lymphocytes (-)-[3H]Sulpiride binding to both T- and B-lymphocytes, evaluated as a function of radioactive ligand concentration, indicated the occurrence of high-affinity, specific, and saturable binding sites (Fig. 1). Nonlinear regression and Scatchard analysis of the data was compatible with the presence of a single population of receptor sites, with a Bm~x of 10.2_+ 1.4 fmol/106 cells and a K d of 0.9 _+0.2 nM, respectively. Similar values for Bmax (9.90 _+ 1.1; 11.95 _+ 1.6 fmol/ 106 cells) and g d (0.8 _+ 0.2; 1 _+ 0.3 nM) were found in T- and B-lymphocytes, respectively, when separatedly

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CELLS(xl0 5 ) Fig. 2. Specific (-)-[3H]sulpiride binding as a function of cell density. Binding of (-)-[3H]sulpffide (2.5 nM) to human T- and Blymphocytes was performed as described in Materials and Methods. Results are the mean ( _+SD) of at least triplicate determinations.

116 TABLE 1

INCUBATION TIME 1h INCUBATION TIME 10 min

---e---

Inhibition of (-)-[3H]sulpiride binding to h u m a n lymphocytes by dopaminergic ligands and comparison with drug dissociation constants ( K i) of the known dopamine receptors.

% x

Agents

4"

2"

0

25

37

TEMPERATURE(°C)

Fig. 3. Specific (-)-[3H]sulpiride binding as a function of temperature. Binding of (-)-[3H]sulpiride (2.5 nM) to h u m a n T- and Blymphocytes was performed as described in Materials and Methods. Results are the mean ( 5: SD) of at least triplicate determinations.

of the radioactive ligand was measured at various times by the addition of ice-cold EBSS after ( - ) [3H]sulpiride binding had reached equilibrium. The dissociation rate constant (K_ 1) thus obtained was 4.76 min -1. The K d value, calculated according to the formula K a = ( K _ J K l ), was 0.6 nM. The binding of (-)-[3H]sulpiride to human lymphocytes was specifically inhibited by the major dopaminergic agonists and antagonists (Table 1). Among dopamine agonists, bromocriptine was the most potent with a K i of about 12 nM, pergolide was also an effective inhibitor at nanomolar concentrations, followed by apomorphine, dopamine, quinpirole and fenoldopam. Adrenaline and noradrenaline were inactive, as well as serotonin, as displacing agents. Of the dopaminergic antagonists tested, the most effective in displacement of (-)-[3H]sulpiride were (-)-sulpiride, haloperidol, butaclamol and chlorpromazine followed,

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300 "2OO 100 0

0

30

60

90 TIME Imin)

Antagonists ( - )Sulpiride Haloperidol Chlorpromazine (+)Butaclamol Fluphenazine Clozapine ( + )Sulpiride

Dlym

Dz

12 5 : 1 . 8 395:7 8 7 + 13 115 5:_12 150 5:27 502 5:69 > 15 000 > 15 000 > 15 000

5.3 21 24 576 474 2.8 6 000 10000

1.5 5 : 0 . 2 2.1 5 : 0 . 3 3.25:0.6 315:6 36 5 : 0 . 5 41- 7 85 - 11

D3

9.2 0.45 2.8 0.8 0.5 56 85

7.4 0.26 20 5.1 5.1 -

25 9.8 6.1 180 422

D4 340 4.1 46 28 321 1760 4180

52 5.1 37 40 46 9 -

Displacement of (-)-[3H]sulpiride by various agents was performed under standard conditions of binding assays as described in Materials and Methods. Serial dilutions ( 1 0 - 9 - 1 0 - 3 M ) were added to the binding assay (in triplicate) to estimate IC50 values (see Materials and Methods). Each curve consisted of at least 6 points each performed in triplicate. K i values for lymphocyte recognition site are the m e a n (5: SD) from three to six independent determinations. The K i values for the D 2 and D 3 receptors were taken from Sokoloff et al. (1990) and the D 4 from V a n Tol et al. (1991).

with a rank order of potency of almost 10 times less, by fluphenazine, clozapine and (-)-sulpiride. (-)-[3H] sulpiride appeared to label a stereospecific dopaminergic receptor, since its steroisomer (+)-sulpiride appears to be 10 to 15 times less potent as binding agent. Reduction of intracellular cAMP levels by dopamine

~0

400 r,

Agonist Bromocriptine Pergolide Apomorphine Quinpirole Dopamine Fenoldopam Noradrenaline Adrenaline Serotonin

K i value (nM)

120

150

180

Fig. 4. Kinetics of (-)-[3H]sulpiride binding to h u m a n lymphocytes. Association of (-)-[3H]sulpiride (2.5 nM) ( 0 ) to T- and B-lymphocytes was measured as a function of time. Specific binding was determined in the presence of 0.5 /zM sulpiride. For dissociation studies (e), 5 ml of EBSS were added at the time indicated by the arrow (60 min) and the samples incubated for the indicated times. Each point is the m e a n ( 5: SD) of three to five determinations.

Activation of D2-DA-R is known to inhibit adenylate cyclase activity through receptor coupling to inhibitory G-proteins. Thus a reduction of the intracellular cAMP levels in human lymphocytes induced by dopamine would provide further information on the dopamine receptor subtype present on human lymphocytes and also evidence of a potentially functional relevance of the identified dopamine receptors. The effects of different concentrations of DA on basal and forskolin-stimulated intracellular cAMP levels were thus investigated. As shown in Fig. 5, forskolin induced a 2.26-fold increase in intracellular cAMP content of human lymphocytes. DA, per se, was unable to modify basal cAMP level at all tested doses (Fig. 5). However when added together with forskolin, DA re-

117

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Fig. 5. Concentration-dependent inhibition by dopamine of forskolin stimulated cAMP levels in human lymphocytes. Intracellular cAMP content was determined as described in Materials and Methods. Each point is the mean of Three determinations. S.D. of each point was within 10% of mean value. • forskolin stimulated cAMP levels o basal cAMP levels

duced cAMP content to almost basal level. This effect was reproducible and dose-dependent (Fig. 5).

Discussion

In our experimental conditions, binding of (-)-[3H] sulpiride to intact human T- and B-lymphocytes fulfilled the expected criteria for ligand interaction with dopaminergic D 2 receptors. Binding was of high-affinity, saturable in a range of 2.5-20 riM, reversible, and stereoselective. Previous studies with [3H] spiperone (Lefur et al., 1980; Shaskan et al., 1984) on rat and human lymphocytes showed a two-component binding curve suggestive of a heterogeneous population of binding sites (List and Seeman, 1981). Binding of ( - ) [3H]sulpiride to lymphocyte preparations showed a single component with a K d of 0.9 nM, similar to the dissociation constant of the high-affinity component of the [3H]spiperone biphasic curve (K d 1.9 nM), thus confirming the high selectivity of (-)-[3H]sulpiride for D2-DA-R (Martres et al., 1985). Furthermore, nanomolar concentrations of dopamine agonists and antagonists efficiently displaced specific (-)-[3H]sulpiride binding, while serotonin, adrenaline and noradrenaline were ineffective, suggesting that (-)-[3H]sulpiride binds only to DA receptors, and not to adrenergic or serotoninergic sites which are also present on lymphocytes (Williams et al., 1976; Bonnet et al., 1987). ( - ) [sH]Sulpiride displacement curves thus indicate the existence of a DA-R and not a general receptor site for catecholamines, as suggested mice and rat lymphocytes (Ovadia and Abramsky, 1987). The binding of (-)-[3H]sulpiride in human lymphocytes revealed the presence of DA binding sites with features that appeared similar to those of the family of cerebral D2, D 3 (Sokoloff et al., 1990) and D 4 (Van Tol

et al., 1991) receptor subtypes. However (-)-[3H]sulpiride binding features cannot clearly distinguish among the various DA-receptor subtypes. D2, D 3 and D 4 receptors can be pharmacologically classified by analysing the binding characteristics of several DA agonists and antagonists, dopamine itself is approximately 20 times more potent on the D 3 and D 4 than at the D 2 receptors. Among dopamine agonists apomorphine displays similar binding efficiency on the members of the D 2 receptor family (D2, 9 3 and D 4) whereas quinpirole presents higher affinity for D 3 and D 4 receptors than D 2. Furthermore, bromocriptine and fenoldopam appear to have higher affinity for the D 2 receptor than the D 4 (see Table 1). In the present study most of the DA agonists tested were able to displace (-)-[3H]sulpiride from the lymphocyte DA-R in the nanomolar range defining for this receptor a pharmacological profile more similar to both D 2 and D 4 DA receptors than to the D3-R. Furthermore, the binding features of fenoldopam suggest a stronger similarity of the DA-R under study with the Da-DA-R. Also among antagonists, the displacement constants of (+)-butaclamol and fluphenazine to the lymphocyte DA receptor resemble the potencies determined for the cerebral Da-R. Whereas, on the other hand, the values of the dissociation constants measured for ( - ) sulpiride, (+)-sulpiride, chlorpromazine and clozapine appears to be very similar to those of the dopamine D2-R. Thus, all together ligand binding studies suggest that the lymphocyte DA receptor shares several pharmacological features mainly with D 2 but also with D 4 receptors, whereas similarities with the D3-R appear to be less evident. Further support to this conclusion is also given by the effects of dopamine on the forskolin stimulated levels of intracellular cAMP. Our results show a consistent and concentration-dependent decrease in cAMP content of human lymphocytes. This observation indicates that the lymphocyte DA receptor is negatively coupled to the adenylate cyclase activity which, on this basis, acts similarly to the D2-R, since no coupling with adenylate cyclase has been yet found for the D 3 and D 4 receptors (Sokoloff et al., 1990; Van Tol et al., 1991). The decrease in cAMP concentration contrasts with the results of other authors describing a moderate (Kovassi et al., 1987) or high (Stepien et al., 1981) increase in cAMP levels in mouse and rat lymphocytes after stimulation with DA. These differences are presently difficult to explain but may be related to species differences or to the presence of a general catecholaminergic receptor as reported in mice and rats (Ovadia and Abramsky, 1987) and to a specific dopaminergic receptor in humans. However, further investigations are necessary to clarify this point. The presence of DA receptors on both human Tand B-lymphocytes further supports and extends the

118

possibility of neuro-immune interactions. In this context, adrenergic compounds have inhibitory effects on lymphocyte proliferation (Hadden et al., 1970) and antibody secretion (Besedovsky et al., 1979), whereas agents such as cholinergic agonists enhance lymphocyte proliferation in vitro (Rossi et al., 1989). Previous studies have also shown that the activation of lymphocytes by concanavalin-A causes an increase in specific binding of [3H]DA, thus suggesting a possible physiological role of the DA-R in lymphocytes (Ovadia and Abramsky, 1987). Furthermore, the results reported here, demonstrating the presence of sulpiride binding sites in lymphocytes, raise the question of their functional rela-: tionship to D2-DA-R present in other tissues. If such a relation will be proven, lymphocytes could be a useful and readily accessible source for analyzing dopaminergic transduction mechanisms in humans. Further studies are also necessary to determine if DA lymphocyte receptors change in number or affinity in human pathological states, including Parkinson's disease and schizophrenia, and, furthermore, if they could contribute to an understanding of the fluctuations in response to levodopa or antipsychotic treatments in these pathologies.

Acknowledgments The authors thank Dr. Guido Vantini for his insightful discussions and suggestions during these studies and Miss Laura Schena and Gianna Crivellaro for their expert secretarial assistance.

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