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Dopamine D5 receptors in human peripheral blood lymphocytes: A radioligand binding study
Journal of Neuroimmunology
ELSEVIER
Journal of Neuroimmunology 53 (1994) 1-7
Dopamine D 5 receptors in human peripheral blood lymphocytes: A radioligand binding study A l b e r t o Ricci a,,, Francesco A m e n t a b a Dipartimento di Scienze Cardiovascolari e Respiratorie, Universit?l "La Sapienza", Via A. Borelli 50, 00161 Rome, Italy b Sezione diAnatomia Umana, Istituto di Farmacologia, Universith di Camerino, Camerino, Italy
Received 20 December 1993; revision received 1 March 1994; accepted 30 March 1994
Abstract
In the present study we have investigated, using radioligand binding techniques and the dopamine receptor antagonist [3H]SCH 23390 as a ligand, the existence of specific dopamine Dl-like receptors in human peripheral blood lymphocytes. [3H]SCH 23390 binding to human peripheral blood lymphocytes was time-, temperature-, concentration-dependent and of high affinity with a dissociation constant value (Kd) of 0.58 + 0.05 nM and a maximum binding density (Bmax) of 11.02 + 0.3 fmol/5 × 106 cells. The binding was also reversible. Pharmacological analysis of displacement curves of [3H]SCH 23390 binding with dopamine competing with the radioligand in the submicromolar range suggests that peripheral blood lymphocytes express dopamine D 5 receptors rather than dopamine D 1 receptors. These results, which are consistent with studies performed with molecular biology techniques, suggest that dopamine may modulate peripheral blood lymphocyte activity. Radioligand binding techniques, applied to lymphocyte receptor studies for their feasibility and flexibility may be used to investigate the possible relationship between the immune and dopaminergic systems. Moreover, they could be employed as a tool in Parkinson's disease, migraine, schizophrenia and hypertension research. Key words: Dopamine receptors; D 5 receptors; Lymphocytes; Humans; Radioligand binding
I. Introduction
Experimental evidence suggests the existence of interactions between the nervous and immune systems. Disorders of the nervous system (infections, cancer and autoimmune diseases), may have immunological pathogenesis or implications (SEar and Anisman, 1979; Plaut et al., 1981; Riley, 1981). Moreover it is known that stressing factors can influence morbidity from infections and tumors (Monjan, 1981; Palmblad, 1981; Keller et al., 1983; Laudenslager et al., 1983). These actions are probably mediated through neurohormone secretion a n d / o r the autonomic nervous system (Dunn, 1988). Lesions of the hypothalamus can induce immune responses (Rozman et al., 1985; Ader et al., 1990). Moreover, the existence of a sympathetic innervation of lymphoid organs, concentrated within lymphoid cells, has been documented (Felten et al., 1985).
* Corresponding author. Fax (06) 445 2349. 0165-5728/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 5 - 5 7 2 8 ( 9 4 ) 0 0 0 4 9 - T
Dopamine receptor agonists such as bromocriptine are able to modulate both humoral and cellular responses of the immune system. It is debated whether these effects are mediated through interaction with hypothalamic or hypophyseal function or through a direct activity of these compounds on immune organs (Clark, 1990). Peripheral blood lymphocytes may represent a useful tool for studying the interactions between the neuroendocrine and immune systems. It is known that peripheral blood lymphocytes express many classes of neuroreceptors, including those for classic (Bishopric et al., 1980; LeFur et al., 1980; Evans et al., 1986) and peptide neurotransmitters (Hazum et al., 1979; Wybran et al., 1986), releasing hormones (Smith et al., 1986) and immunotransmitters (Smith et al., 1987). Molecular biology techniques have demonstrated the expression of dopamine D 3 and D 5 receptors in human peripheral blood lymphocytes (Takahashi et al., 1992; Nagai et al., 1993), whereas the characterization of different subtypes of dopamine receptors using conven-
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A. Ricci, F. Amenta/Journal of Neuroimmunology 53 (1994) 1 7
tional radioligand binding techniques frequently gave conflicting results (Flemingher et al., 1982; Arnold et al., 1993; Santambrogio et al., 1993). The present study was designed to characterize the population of dopamine D 5 receptor sites in human peripheral blood lymphocytes using radioligand binding techniques.
2. Materials and methods
2.1. Human peripheral blood lymphocyte preparation
Human peripheral blood lymphocytes were obtained from healthy adult volunteers (20-40 years of age) (n = 10). 30 ml of venous blood was drawn and collected in plastic tubes containing 200 mg EDTA as anticoagulant. The blood was diluted with an equal volume of 0.9% NaCI. 6 ml of diluted blood were carefully layered over 3 ml Lymphoprep ~ (Nycomed Pharma, Oslo, Norway) in a graduated centrifuge tube, avoiding the mixture of blood and separation fluid. Samples were centrifuged at 800 x g for 20 min at 23°C in a swing-out rotor. After centrifugation the mononuclear cells forming a distinct band at the sample/medium interface were collected using a Pasteur pipette without removing the upper layer. The harvest fraction was diluted in a 0.9% NaCI solution to reduce its density and centrifuged for 10 min at 250 x g. The contamination of erythrocytes was usually less than 15%, whereas the washing procedure was sufficient to remove most platelets (data not shown). In order to deplete monocytes, cells were incubated for 1 h in plastic tissue culture flasks at 37°C under 5% CO 2 and 95% air. The population of non-adherent cells was resuspended in a 0.9% NaCI solution with a final concentration of 5 X 106 cells/ml.
of the incubation, cells were isolated onto Whatman GF-B glass fibre filters with a manifold filtration apparatus. Filters were washed rapidly twice with ice-cold incubation buffer (2 × 5 min) and transferred into scintillation vials. Radioactivity bound was measured using a Beckman liquid scintillation spectrometer at an efficiency of 40%. Moreover, in a series of preliminary experiments, binding of [3H]SCH 23390 to membrane particles of rat neostriatum was assessed according to the procedure detailed above. 2.3. Data analysis
Data from binding experiments were calculated by linear regression analysis of Scatchard plots of saturation isotherms. In competition experiments, drug concentrations producing 50% inhibition of [3H]SCH 23390 binding were determined by Hill plots (log (B 0 - B / v s . log concentrations)) where B 0 and B are specific binding in the absence and presence of competitor, respectively. Inhibition constant (K i) values were calculated by the method of Cheng and Prusoff (1973). Mean of the values and confidence limits were calculated from K i values obtained in 3-5 independent experiments. 2.4. Chemicals
[3H]SCH 23390 (specific activity 60-90 Ci/mmol) was purchased from Amersham Radiochemical Centre (Buckinghamshire, UK). SCH 23390 and isomers of sulpiride were obtained from Schering Plough Italy (Milan, Italy) and Ravizza (Milan, Italy), respectively. Other chemicals were purchased from Research Biochemicals, Inc. (Natick, MA).
3. Results
2.2. Radioligand binding analysis
300 ~1 of 5 x 106 lymphocyte suspension were incubated in triplicate with increasing concentrations of [3H]SCH 23390 (0.1-2 nM) for 60 min at 25°C in a 170 nM Tris" HCI buffer containing NaCI (120 mM), KC1 (5 mM) CaC12 (1.5 mM), MgC12 (4 mM) and EDTA (1 mM) (final pH 7.4). Non-specific binding was defined by the presence in the incubation medium of [3H]SCH 23390 plus 1/~M (+)-butaclamol. In a series of preliminary experiments incubation was done at different temperatures (4°C, 25°C and 37°C) and times (30, 45, 60, 90 and 120 min). The pharmacological profile of [3H]SCH 23390 binding to dopamine receptors was assessed by incubating cells as above in the presence of different concentrations of dopamine, adrenergic and serotonin receptor agonists and antagonists. At the end
[3H]SCH 23390 was specifically bound to human peripheral lymphocytes. The binding was time- (Fig. 1), temperature- (Fig. 1) and concentration-dependent (Fig. 2). Scatchard analysis of [3H]SCH 23390 binding revealed a single class of high affinity sites, with a dissociation constant value (K d) of 0.58 + 0.005 nM and a maximum binding capacity (Bmax) of 11.2 + 0.3 fmol/5 × 106 cells (Figs. 2 and 3). A similar pattern of radioligand binding was observed in membrane particles of rat neostriatum in which the K d was 0.65 + 0.04 nM and the Bmax value was 725 + 43 fmol/mg protein (data not shown). The number of [3H]SCH 23390 binding sites had a linear increase, with the cell number growing (Fig. 4). In subsequent experiments a standard concentration of 5 X 10 6 cells was used. This concentration allowed an
A. Ricci, F. Amenta /Journal of Neuroimmunology 53 (1994) 1-7
3
BOUND/FREE
fmol/5 x 106cells
25
14 20
12 10
15 8 1o
6 4
5
2 o
0~ 20
40
60
80
100
120
0.8
2.55
4.3
6,05
7.8
9.55
11.3
[3H]-SCH 2 3 3 9 0 bound
TIME (min) Fig. 1. Influence of different incubation times and temperatures (4°C, Q; 25°C o; and 37°C, • ) on specific [3H]SCH 23390 binding (abscissa) to human peripheral blood lymphocytes. The points are the means __.SEM of triplicate determinations.
easy determination of binding parameters with the advantage of being able to obtain enough material for several assays from a single subject. With a radioligand concentration corresponding to the K d value, approximately 50% [3H]SCH 23390 was bound specifically (Fig. 2). This radioligand concentration was therefore considered to be optimal for developing the highest specific: non-specific binding ratio and was used in the subsequent experiments. The results of the pharmacological analysis of [3H]SCH 23390 binding to peripheral human blood lymphocyte are shown in Figs. 5 and 6 and summarized
Fig. 3. Scatchard analysis of specific [3H]SCH 23390 binding to human peripheral blood lymphocytes. The Bmax value was 1i.02-I-0.3 fmol/5 × 106 tissue. Points are means of triplicate determinations.
in Table 1. From the analysis of these data it can be seen that the most powerful displacer of [3H]SCH 23390 was SCH 23390, followed by (+)-butaclamol, haloperidol, SKF 38393, dopamine and apomorphine. Serotonin and methysergide were weak displacers whereas the two isomers of sulpiride, spiperone and quinpirole were ineffective (Figs. 5, 6 and Table 1).
4. Discussion
Increasing experimental evidence indicates the occurrence of interactions between the immune and nervous systems although the functional correlates of these
f m o l / 5 x 106) 16
fmol/eell number 25
14 12
20
10 15
8 6
10 4
2 o 0
0.5
1
1.5
2
[3H]-SCH 23390 (nM) Fig. 2. Saturation curve of [3H]SCH 23390 binding to human peripheral blood lymphocytes. Cells were incubated with increasing concentrations of the radioligand alone (total binding, o ) or plus 1 p.M (+)-butaclamol to define non-specific binding ( • ) . Specific binding (12) was obtained by subtracting non-specific from total binding. Points are the mean + SEM of triplicate determinations.
i
0
1
2
3
4
5
6
7
8
9
10
,
11
CELLS (106 ) Fig. 4. Specific [3H]SCH 23390 binding as a function of human peripheral blood lymphocyte number: Cells were incubated with a 0.56 nM radioligand concentration. Binding experiments were performed as described in Materials and methods. Points are the mean + SEM of triplicate determinations.
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A. Ricci, F. Amenta/Journal of Neuroimmunology 53 (1994) 1-7
120
Table 1 K i values for [3H]SCH 23390 binding to dopamine D 5 receptors expressed in peripheral blood lymphocytes
100
Compound
Receptor specificity
K i (nM)
Apomorphine ( + )-Butaclamol Dopamine Haloperidol Methysergide Quinpirole SCH 23390 Serotonin SKF 38393 Spiperonc ( + )-Sulpiride ( - )-Sulpiride
D 2 / D 3/D4-1ike Dt/De-like D 1/D2-1ike D2-1ike 5HT D 2/D3-1ike D1-1ike 5HT D 1/Ds-like D2-1ike Dl-like De-like
350 + 21 180 + 15 39.10+_ 2.21 50 + 0.5 3 420 _+125 > 5 000 0..23+ 0.07 3 850 + 150 1.68+ 0.20 > 5 000 > 10 000 > 10 000
% Inhibition
80
60
40
20
0
8
7
6
5
4
3
Displacer (-log M) Fig. 5. Effect of increasing concentrations of dopamine receptor antagonists (haloperidol (11), (+)-sulpiride (e), (-)-suipiride (©), ( + )-butaclamol ( [] ), spiperone ( × ) and SCH 23390 ( , ) ) on [3H]SCH 23390 binding to human peripheral blood lymphocytes. Cells were incubated with a 0.56 nM radioligand concentration and processed as described in Materials and methods. Points are the mean_+ SEM of triplicate determinations.
interactions are still not clearly documented. In fact immune organs are richly supplied with an autonomic innervation (Felten et al., 1985; Zelten et al., 1991) and several classical neurotransmitters, neuropeptides and hormones are able to modify immune responses (Besedowsky et al., 1985; O'Dorisio et al., 1985; Payan and Goetzi, 1985a,b; Dunn, 1988; Ader et al., 1990; Bost and Pascual, 1992; Takahashi et al., 1992; Santambrogio et al., 1993). On the other hand, lymphoid cell % Inhibition 100 I
80
60
I
40
20
0
8
7
6
5
4
3
Displacer (-log M) Fig. 6. Effect of increasing concentrations of dopamine receptor agonists (apomorphine (rn), quinpirole (m), SKF 38393 (e) and dopamine (*)) and of compounds acting on serotonin receptors (methysergide ( x ) and serotonin (©)) on [3HISCH 23390 binding to human peripheral blood lymphocytes. Cells were incubated with a 0.56 nM radioligand concentration and processed as described in Materials and methods. Points are the m e a n + S E M of triplicate determinations.
[3H]SCH 23390 binding was assayed as described in the text. Values represent the competitor dissociation constant (K i) determined according to the method of Cheng and Prusoff (1973). Each figure is the mean _+SEM of three to five experiments performed in triplicate. For references on the pharmacological specificity of the compounds tested see Amenta (1990) and Gingrich and Caron (1993).
products (lymphokines) or peripheral lymphoid organ products (thymic peptides) may interact with nerve cells or have a role in the immune responses modified by stress agents (Martin, 1976; Besedowsky et al., 1985; Farrar et al., 1987; Dunn, 1988; Ader et al., 1990). Cellular immunity mechanisms and natural killer cell activities are conditioned in some experimental situations (Bovbjerg et al., 1982; Croiset et al., 1990; Hiramoto et al., 1993) and lesions of certain brain regions are accompanied by neuroendocrine or autonomic dysfunction (Biziere et al., 1985; Roszman et al., 1985). Peripheral blood lymphocytes express a variety of neurotransmitter receptors (LeFur, 1980; Meurs et al., 1982; Wiik et al., 1985; Calvo et al., 1986; Scicchitano et al., 1987; Stanisz et al., 1987; Takahashi et al., 1992; Santambrogio et al., 1993) and a possible autonomic supply of B lymphocytes of rat spleen and of T and B lymphocytes of avian Bursa of Fabricii has been suggested (Felten and Olschowka, 1987; Zentel et al., 1991). The catecholamine dopamine is probably involved in the modulation of immune function. In fact, dopamine receptor agonists are capable of interfering with a variety of humoral or cell-mediated reactions in rodents, contact sensitivity reactions and experimental allergic encephalopathy (Nagy et al., 1983). These immunosuppressive effects are considered to be due to dopamine's inhibitory activity on the release of anterior pituitary hormones stimulating immune function rather than to a direct action on the immune system (Clark, 1990). Studies on the presence of dopamine receptors in circulating immune cells before the introduction of molecular biology techniques have provided conflicting results. Some papers reported the occurrence of dopamine receptors labelled by [3H]spiroperidol (Le-
A. Ricci, F. Amenta /Journal of Neuroimmunology 53 (1994) 1-7
Fur et al., 1980, 1983) and [3H](-)-sulpiride (Santambrogio et al., 1993) in human lymphocytes, whereas others have suggested the occurrence of a passive uptake process of [3H]spiroperidol instead of a receptor binding by lymphocytes (Fleminger et al., 1982). These studies were centered primarily on the family of dopamine D2-1ike receptors (Gingrich and Caron, 1993), whereas only sparse information is available on the family of dopamine Dl-like sites (Takahashi et al., 1992). The characterization of subtypes of dopamine receptors performed in the last 10-15 years using several techniques has allowed the identification of two main subtypes of dopamine receptors, dopamine D a (DA1 at the periphery) and dopamine D 2 (DA 2 at the periphery) receptor sites (see Seeman and Grigoriadis, 1987; Amenta, 1990). More recently, the application of molecular biology techniques to receptor research has enlarged our knowledge on dopamine receptors and allowed the identification of at least 5 subtypes of dopamine receptors, named dopamine D1, D 2, D3, D 4 and D 5 sites, respectively (Dearry et al., 1990; Sokoloff et al., 1990; Zhou et al., 1990; Grandy et al., 1991; Sunahara et al., 1991; Takahashi et al., 1992; Nagai et al., 1993). Dopamine D 1 and D 5 receptors belong to the dopamine Dl-like receptor family, whereas dopamine D2, D 3 and D 4 receptors belong to the dopamine D2-1ike receptor family (Sibley and Monsma, 1992; Gingrich and Caron, 1993). Molecular biology techniques have demonstrated recently dopamine D 5 and D 3 receptor genes expressed by human peripheral blood lymphocytes (Takahashi et al., 1992; Nagai et al., 1993). This suggests that the pattern of dopamine receptors expression by lymphocyte is probably more complicated than considered in the past. The goal of the present study was to investigate the binding characteristics and the pharmacological profile of [3H]SCH 23390 binding to human peripheral blood lymphocytes which represent mainly a population of T lymphocytes. A comparison of data obtained in human peripheral blood lymphocytes and in membrane particles of neostriatum shows that the affinity of the radioligand for dopamine Dl-like receptors is similar, whereas the density of [3H]SCH 23390 binding sites is about 20 times lower in lymphocytes than in the neostriatum. Pharmacological analysis of [3H]SCH 23390 binding to human peripheral blood lymphocytes with dopamine competing with the radioligand in the submicromolar range suggests that site we have characterized belongs to the dopamine D 5 receptor subtype (Sibley and Monsma, 1992; Gingrich and Caron, 1993). Hence, our data are in agreement with molecular biology studies reporting the existence, in human blood lymphocyte, of dopamine D 5 receptor genes and their transcription in two pseudo genes as well as of [3H]SCH 23390 binding
5
to this kind of cells (Takahashi et al., 1992). The lower K d value of [3H]SCH 23390 binding observed in our study is probably due to technical reasons since Takahashi and co-workers (1992) have used longer incubation times and higher radioligand concentrations in their binding experiments. This may result in the development of non-optimal specific:non-specific binding ratio. On the other hand, [3H]SCH 23390 binding to lymphocytes was unaffected by quinpirole, which is a dopamine receptor agonist showing high affinity for dopamine D 3 receptors (Sokoloff et al., 1992). These results were espected in view of the lack of specificity of the radioligand used for dopamine D 3 receptors (Sokoloff et al., 1992). Hence further work is necessary to characterize with radioligand binding the population of lymphocyte dopamine D 3 receptors already demonstrated by molecular biology techniques (Nagai et al., 1993) Unlike the above authors (Takahashi et al., 1992), we have extended the characterization of [3H]SCH 23390 binding sites to human peripheral blood lymphocytes to the analysis of the pharmacological profile of the binding. Our data have shown that no dopamine D 1 receptor binding occurs in human peripheral blood lymphocytes and that the only labelled site belongs to the dopamine D 5 receptor. In fact, as expected for dopamine D 5 receptors, dopamine competed with [3H]SCH 23390 in the submicromolar range (Sibley and Monsma, 1992). In view of the feasibility and flexibility of radioligand binding studies applied to dopamine receptor research, our data suggest that these techniques may be extended to the analysis of dopamine's role in modulating immune functions or in neuroimmune interactions. A further application could be the study of human diseases in which probably changes in dopaminergic neurotransmission are involved. They include Parkinson's disease, neuropsychiatric disorders, migraine and hypertension.
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A. Ricci, F. Amenta /Journal of Neuroimmunology 53 (1994) 1-7 Sokoloff, P., Martres, M.P., Giros, B., Boithenet, M.L. and Schwartz, J.C. (1992) The third dopamine receptor (D3) as a novel target for antipsychotic. Biochem. Pharmacol. 43, 659-666. Stanisz, A.N., Scicchitano, R., Dazin, P., Bienenstock, J. and Payan, D.G. (1987) Distribution of substance P receptors on murine spleen and Peyer's patch T and B cells. J. Immunol. 139, 749-756. Sunahara, R.K., Guan, H.C., O'Dowd, B., Seeman, P., Laurier, L.G., Ng, G., George, S.R., Torchia, J., VanTol, H.H.M. and Niznik, H.B. (1991) Cloning of the gene for a human dopamine D5 receptor with higher affinity for dopamine than D1. Nature 350, 614-619. Takahashi, N, Nagai, Y., Ueno, S., Saeki, Y. and Yanagihara, T. (1992) Human peripheral blood lymphocytes express D5 dopamine receptor gene and transcribe the two pseudo genes. FEBS Lett. 314, 23-25.
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Wiik, P., Opstad, P.K. and Boyum, A. (1985) Binding of vasoactive intestinal polypeptide (VIP) by human blood monocytes: demonstration of specific binding sites. Reg. Pept. 12, 145-153. Wybran, J. (1986) Enkephalins as molecules of lymphocyte activation and modifiers of the biological response. In: N.P. Plotnikoff, R.E. Faith, A.J. Murgo and R.A. Good (Eds.), Enkephalins and Endorphins. Plenum, New York, NY, pp. 253-282. Zentel, H.J., Albrecht, D.N.R., Jeurissen, S.H.M., Vaino, O. and Weihe, P. (1991) Peptidergic innervation of the Bursa Fabricii: interrelation with T-lymphocyte subsets. Int. J. Neurosci. 59, 177-188. Zhou, Q.-Y., Grandy, D.K., Thambi, L., Kushner, J.A., VanTol, H.H.M., Cone, R., Pribnow, D., Salon, J., Bunzow, J.R. and Civelli, O. (1990) Cloning and expression of human and rat D1 dopamine receptors. Nature 347, 76-79.
ELSEVIER
Journal of Neuroimmunology 53 (1994) 1-7
Dopamine D 5 receptors in human peripheral blood lymphocytes: A radioligand binding study A l b e r t o Ricci a,,, Francesco A m e n t a b a Dipartimento di Scienze Cardiovascolari e Respiratorie, Universit?l "La Sapienza", Via A. Borelli 50, 00161 Rome, Italy b Sezione diAnatomia Umana, Istituto di Farmacologia, Universith di Camerino, Camerino, Italy
Received 20 December 1993; revision received 1 March 1994; accepted 30 March 1994
Abstract
In the present study we have investigated, using radioligand binding techniques and the dopamine receptor antagonist [3H]SCH 23390 as a ligand, the existence of specific dopamine Dl-like receptors in human peripheral blood lymphocytes. [3H]SCH 23390 binding to human peripheral blood lymphocytes was time-, temperature-, concentration-dependent and of high affinity with a dissociation constant value (Kd) of 0.58 + 0.05 nM and a maximum binding density (Bmax) of 11.02 + 0.3 fmol/5 × 106 cells. The binding was also reversible. Pharmacological analysis of displacement curves of [3H]SCH 23390 binding with dopamine competing with the radioligand in the submicromolar range suggests that peripheral blood lymphocytes express dopamine D 5 receptors rather than dopamine D 1 receptors. These results, which are consistent with studies performed with molecular biology techniques, suggest that dopamine may modulate peripheral blood lymphocyte activity. Radioligand binding techniques, applied to lymphocyte receptor studies for their feasibility and flexibility may be used to investigate the possible relationship between the immune and dopaminergic systems. Moreover, they could be employed as a tool in Parkinson's disease, migraine, schizophrenia and hypertension research. Key words: Dopamine receptors; D 5 receptors; Lymphocytes; Humans; Radioligand binding
I. Introduction
Experimental evidence suggests the existence of interactions between the nervous and immune systems. Disorders of the nervous system (infections, cancer and autoimmune diseases), may have immunological pathogenesis or implications (SEar and Anisman, 1979; Plaut et al., 1981; Riley, 1981). Moreover it is known that stressing factors can influence morbidity from infections and tumors (Monjan, 1981; Palmblad, 1981; Keller et al., 1983; Laudenslager et al., 1983). These actions are probably mediated through neurohormone secretion a n d / o r the autonomic nervous system (Dunn, 1988). Lesions of the hypothalamus can induce immune responses (Rozman et al., 1985; Ader et al., 1990). Moreover, the existence of a sympathetic innervation of lymphoid organs, concentrated within lymphoid cells, has been documented (Felten et al., 1985).
* Corresponding author. Fax (06) 445 2349. 0165-5728/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 5 - 5 7 2 8 ( 9 4 ) 0 0 0 4 9 - T
Dopamine receptor agonists such as bromocriptine are able to modulate both humoral and cellular responses of the immune system. It is debated whether these effects are mediated through interaction with hypothalamic or hypophyseal function or through a direct activity of these compounds on immune organs (Clark, 1990). Peripheral blood lymphocytes may represent a useful tool for studying the interactions between the neuroendocrine and immune systems. It is known that peripheral blood lymphocytes express many classes of neuroreceptors, including those for classic (Bishopric et al., 1980; LeFur et al., 1980; Evans et al., 1986) and peptide neurotransmitters (Hazum et al., 1979; Wybran et al., 1986), releasing hormones (Smith et al., 1986) and immunotransmitters (Smith et al., 1987). Molecular biology techniques have demonstrated the expression of dopamine D 3 and D 5 receptors in human peripheral blood lymphocytes (Takahashi et al., 1992; Nagai et al., 1993), whereas the characterization of different subtypes of dopamine receptors using conven-
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A. Ricci, F. Amenta/Journal of Neuroimmunology 53 (1994) 1 7
tional radioligand binding techniques frequently gave conflicting results (Flemingher et al., 1982; Arnold et al., 1993; Santambrogio et al., 1993). The present study was designed to characterize the population of dopamine D 5 receptor sites in human peripheral blood lymphocytes using radioligand binding techniques.
2. Materials and methods
2.1. Human peripheral blood lymphocyte preparation
Human peripheral blood lymphocytes were obtained from healthy adult volunteers (20-40 years of age) (n = 10). 30 ml of venous blood was drawn and collected in plastic tubes containing 200 mg EDTA as anticoagulant. The blood was diluted with an equal volume of 0.9% NaCI. 6 ml of diluted blood were carefully layered over 3 ml Lymphoprep ~ (Nycomed Pharma, Oslo, Norway) in a graduated centrifuge tube, avoiding the mixture of blood and separation fluid. Samples were centrifuged at 800 x g for 20 min at 23°C in a swing-out rotor. After centrifugation the mononuclear cells forming a distinct band at the sample/medium interface were collected using a Pasteur pipette without removing the upper layer. The harvest fraction was diluted in a 0.9% NaCI solution to reduce its density and centrifuged for 10 min at 250 x g. The contamination of erythrocytes was usually less than 15%, whereas the washing procedure was sufficient to remove most platelets (data not shown). In order to deplete monocytes, cells were incubated for 1 h in plastic tissue culture flasks at 37°C under 5% CO 2 and 95% air. The population of non-adherent cells was resuspended in a 0.9% NaCI solution with a final concentration of 5 X 106 cells/ml.
of the incubation, cells were isolated onto Whatman GF-B glass fibre filters with a manifold filtration apparatus. Filters were washed rapidly twice with ice-cold incubation buffer (2 × 5 min) and transferred into scintillation vials. Radioactivity bound was measured using a Beckman liquid scintillation spectrometer at an efficiency of 40%. Moreover, in a series of preliminary experiments, binding of [3H]SCH 23390 to membrane particles of rat neostriatum was assessed according to the procedure detailed above. 2.3. Data analysis
Data from binding experiments were calculated by linear regression analysis of Scatchard plots of saturation isotherms. In competition experiments, drug concentrations producing 50% inhibition of [3H]SCH 23390 binding were determined by Hill plots (log (B 0 - B / v s . log concentrations)) where B 0 and B are specific binding in the absence and presence of competitor, respectively. Inhibition constant (K i) values were calculated by the method of Cheng and Prusoff (1973). Mean of the values and confidence limits were calculated from K i values obtained in 3-5 independent experiments. 2.4. Chemicals
[3H]SCH 23390 (specific activity 60-90 Ci/mmol) was purchased from Amersham Radiochemical Centre (Buckinghamshire, UK). SCH 23390 and isomers of sulpiride were obtained from Schering Plough Italy (Milan, Italy) and Ravizza (Milan, Italy), respectively. Other chemicals were purchased from Research Biochemicals, Inc. (Natick, MA).
3. Results
2.2. Radioligand binding analysis
300 ~1 of 5 x 106 lymphocyte suspension were incubated in triplicate with increasing concentrations of [3H]SCH 23390 (0.1-2 nM) for 60 min at 25°C in a 170 nM Tris" HCI buffer containing NaCI (120 mM), KC1 (5 mM) CaC12 (1.5 mM), MgC12 (4 mM) and EDTA (1 mM) (final pH 7.4). Non-specific binding was defined by the presence in the incubation medium of [3H]SCH 23390 plus 1/~M (+)-butaclamol. In a series of preliminary experiments incubation was done at different temperatures (4°C, 25°C and 37°C) and times (30, 45, 60, 90 and 120 min). The pharmacological profile of [3H]SCH 23390 binding to dopamine receptors was assessed by incubating cells as above in the presence of different concentrations of dopamine, adrenergic and serotonin receptor agonists and antagonists. At the end
[3H]SCH 23390 was specifically bound to human peripheral lymphocytes. The binding was time- (Fig. 1), temperature- (Fig. 1) and concentration-dependent (Fig. 2). Scatchard analysis of [3H]SCH 23390 binding revealed a single class of high affinity sites, with a dissociation constant value (K d) of 0.58 + 0.005 nM and a maximum binding capacity (Bmax) of 11.2 + 0.3 fmol/5 × 106 cells (Figs. 2 and 3). A similar pattern of radioligand binding was observed in membrane particles of rat neostriatum in which the K d was 0.65 + 0.04 nM and the Bmax value was 725 + 43 fmol/mg protein (data not shown). The number of [3H]SCH 23390 binding sites had a linear increase, with the cell number growing (Fig. 4). In subsequent experiments a standard concentration of 5 X 10 6 cells was used. This concentration allowed an
A. Ricci, F. Amenta /Journal of Neuroimmunology 53 (1994) 1-7
3
BOUND/FREE
fmol/5 x 106cells
25
14 20
12 10
15 8 1o
6 4
5
2 o
0~ 20
40
60
80
100
120
0.8
2.55
4.3
6,05
7.8
9.55
11.3
[3H]-SCH 2 3 3 9 0 bound
TIME (min) Fig. 1. Influence of different incubation times and temperatures (4°C, Q; 25°C o; and 37°C, • ) on specific [3H]SCH 23390 binding (abscissa) to human peripheral blood lymphocytes. The points are the means __.SEM of triplicate determinations.
easy determination of binding parameters with the advantage of being able to obtain enough material for several assays from a single subject. With a radioligand concentration corresponding to the K d value, approximately 50% [3H]SCH 23390 was bound specifically (Fig. 2). This radioligand concentration was therefore considered to be optimal for developing the highest specific: non-specific binding ratio and was used in the subsequent experiments. The results of the pharmacological analysis of [3H]SCH 23390 binding to peripheral human blood lymphocyte are shown in Figs. 5 and 6 and summarized
Fig. 3. Scatchard analysis of specific [3H]SCH 23390 binding to human peripheral blood lymphocytes. The Bmax value was 1i.02-I-0.3 fmol/5 × 106 tissue. Points are means of triplicate determinations.
in Table 1. From the analysis of these data it can be seen that the most powerful displacer of [3H]SCH 23390 was SCH 23390, followed by (+)-butaclamol, haloperidol, SKF 38393, dopamine and apomorphine. Serotonin and methysergide were weak displacers whereas the two isomers of sulpiride, spiperone and quinpirole were ineffective (Figs. 5, 6 and Table 1).
4. Discussion
Increasing experimental evidence indicates the occurrence of interactions between the immune and nervous systems although the functional correlates of these
f m o l / 5 x 106) 16
fmol/eell number 25
14 12
20
10 15
8 6
10 4
2 o 0
0.5
1
1.5
2
[3H]-SCH 23390 (nM) Fig. 2. Saturation curve of [3H]SCH 23390 binding to human peripheral blood lymphocytes. Cells were incubated with increasing concentrations of the radioligand alone (total binding, o ) or plus 1 p.M (+)-butaclamol to define non-specific binding ( • ) . Specific binding (12) was obtained by subtracting non-specific from total binding. Points are the mean + SEM of triplicate determinations.
i
0
1
2
3
4
5
6
7
8
9
10
,
11
CELLS (106 ) Fig. 4. Specific [3H]SCH 23390 binding as a function of human peripheral blood lymphocyte number: Cells were incubated with a 0.56 nM radioligand concentration. Binding experiments were performed as described in Materials and methods. Points are the mean + SEM of triplicate determinations.
4
A. Ricci, F. Amenta/Journal of Neuroimmunology 53 (1994) 1-7
120
Table 1 K i values for [3H]SCH 23390 binding to dopamine D 5 receptors expressed in peripheral blood lymphocytes
100
Compound
Receptor specificity
K i (nM)
Apomorphine ( + )-Butaclamol Dopamine Haloperidol Methysergide Quinpirole SCH 23390 Serotonin SKF 38393 Spiperonc ( + )-Sulpiride ( - )-Sulpiride
D 2 / D 3/D4-1ike Dt/De-like D 1/D2-1ike D2-1ike 5HT D 2/D3-1ike D1-1ike 5HT D 1/Ds-like D2-1ike Dl-like De-like
350 + 21 180 + 15 39.10+_ 2.21 50 + 0.5 3 420 _+125 > 5 000 0..23+ 0.07 3 850 + 150 1.68+ 0.20 > 5 000 > 10 000 > 10 000
% Inhibition
80
60
40
20
0
8
7
6
5
4
3
Displacer (-log M) Fig. 5. Effect of increasing concentrations of dopamine receptor antagonists (haloperidol (11), (+)-sulpiride (e), (-)-suipiride (©), ( + )-butaclamol ( [] ), spiperone ( × ) and SCH 23390 ( , ) ) on [3H]SCH 23390 binding to human peripheral blood lymphocytes. Cells were incubated with a 0.56 nM radioligand concentration and processed as described in Materials and methods. Points are the mean_+ SEM of triplicate determinations.
interactions are still not clearly documented. In fact immune organs are richly supplied with an autonomic innervation (Felten et al., 1985; Zelten et al., 1991) and several classical neurotransmitters, neuropeptides and hormones are able to modify immune responses (Besedowsky et al., 1985; O'Dorisio et al., 1985; Payan and Goetzi, 1985a,b; Dunn, 1988; Ader et al., 1990; Bost and Pascual, 1992; Takahashi et al., 1992; Santambrogio et al., 1993). On the other hand, lymphoid cell % Inhibition 100 I
80
60
I
40
20
0
8
7
6
5
4
3
Displacer (-log M) Fig. 6. Effect of increasing concentrations of dopamine receptor agonists (apomorphine (rn), quinpirole (m), SKF 38393 (e) and dopamine (*)) and of compounds acting on serotonin receptors (methysergide ( x ) and serotonin (©)) on [3HISCH 23390 binding to human peripheral blood lymphocytes. Cells were incubated with a 0.56 nM radioligand concentration and processed as described in Materials and methods. Points are the m e a n + S E M of triplicate determinations.
[3H]SCH 23390 binding was assayed as described in the text. Values represent the competitor dissociation constant (K i) determined according to the method of Cheng and Prusoff (1973). Each figure is the mean _+SEM of three to five experiments performed in triplicate. For references on the pharmacological specificity of the compounds tested see Amenta (1990) and Gingrich and Caron (1993).
products (lymphokines) or peripheral lymphoid organ products (thymic peptides) may interact with nerve cells or have a role in the immune responses modified by stress agents (Martin, 1976; Besedowsky et al., 1985; Farrar et al., 1987; Dunn, 1988; Ader et al., 1990). Cellular immunity mechanisms and natural killer cell activities are conditioned in some experimental situations (Bovbjerg et al., 1982; Croiset et al., 1990; Hiramoto et al., 1993) and lesions of certain brain regions are accompanied by neuroendocrine or autonomic dysfunction (Biziere et al., 1985; Roszman et al., 1985). Peripheral blood lymphocytes express a variety of neurotransmitter receptors (LeFur, 1980; Meurs et al., 1982; Wiik et al., 1985; Calvo et al., 1986; Scicchitano et al., 1987; Stanisz et al., 1987; Takahashi et al., 1992; Santambrogio et al., 1993) and a possible autonomic supply of B lymphocytes of rat spleen and of T and B lymphocytes of avian Bursa of Fabricii has been suggested (Felten and Olschowka, 1987; Zentel et al., 1991). The catecholamine dopamine is probably involved in the modulation of immune function. In fact, dopamine receptor agonists are capable of interfering with a variety of humoral or cell-mediated reactions in rodents, contact sensitivity reactions and experimental allergic encephalopathy (Nagy et al., 1983). These immunosuppressive effects are considered to be due to dopamine's inhibitory activity on the release of anterior pituitary hormones stimulating immune function rather than to a direct action on the immune system (Clark, 1990). Studies on the presence of dopamine receptors in circulating immune cells before the introduction of molecular biology techniques have provided conflicting results. Some papers reported the occurrence of dopamine receptors labelled by [3H]spiroperidol (Le-
A. Ricci, F. Amenta /Journal of Neuroimmunology 53 (1994) 1-7
Fur et al., 1980, 1983) and [3H](-)-sulpiride (Santambrogio et al., 1993) in human lymphocytes, whereas others have suggested the occurrence of a passive uptake process of [3H]spiroperidol instead of a receptor binding by lymphocytes (Fleminger et al., 1982). These studies were centered primarily on the family of dopamine D2-1ike receptors (Gingrich and Caron, 1993), whereas only sparse information is available on the family of dopamine Dl-like sites (Takahashi et al., 1992). The characterization of subtypes of dopamine receptors performed in the last 10-15 years using several techniques has allowed the identification of two main subtypes of dopamine receptors, dopamine D a (DA1 at the periphery) and dopamine D 2 (DA 2 at the periphery) receptor sites (see Seeman and Grigoriadis, 1987; Amenta, 1990). More recently, the application of molecular biology techniques to receptor research has enlarged our knowledge on dopamine receptors and allowed the identification of at least 5 subtypes of dopamine receptors, named dopamine D1, D 2, D3, D 4 and D 5 sites, respectively (Dearry et al., 1990; Sokoloff et al., 1990; Zhou et al., 1990; Grandy et al., 1991; Sunahara et al., 1991; Takahashi et al., 1992; Nagai et al., 1993). Dopamine D 1 and D 5 receptors belong to the dopamine Dl-like receptor family, whereas dopamine D2, D 3 and D 4 receptors belong to the dopamine D2-1ike receptor family (Sibley and Monsma, 1992; Gingrich and Caron, 1993). Molecular biology techniques have demonstrated recently dopamine D 5 and D 3 receptor genes expressed by human peripheral blood lymphocytes (Takahashi et al., 1992; Nagai et al., 1993). This suggests that the pattern of dopamine receptors expression by lymphocyte is probably more complicated than considered in the past. The goal of the present study was to investigate the binding characteristics and the pharmacological profile of [3H]SCH 23390 binding to human peripheral blood lymphocytes which represent mainly a population of T lymphocytes. A comparison of data obtained in human peripheral blood lymphocytes and in membrane particles of neostriatum shows that the affinity of the radioligand for dopamine Dl-like receptors is similar, whereas the density of [3H]SCH 23390 binding sites is about 20 times lower in lymphocytes than in the neostriatum. Pharmacological analysis of [3H]SCH 23390 binding to human peripheral blood lymphocytes with dopamine competing with the radioligand in the submicromolar range suggests that site we have characterized belongs to the dopamine D 5 receptor subtype (Sibley and Monsma, 1992; Gingrich and Caron, 1993). Hence, our data are in agreement with molecular biology studies reporting the existence, in human blood lymphocyte, of dopamine D 5 receptor genes and their transcription in two pseudo genes as well as of [3H]SCH 23390 binding
5
to this kind of cells (Takahashi et al., 1992). The lower K d value of [3H]SCH 23390 binding observed in our study is probably due to technical reasons since Takahashi and co-workers (1992) have used longer incubation times and higher radioligand concentrations in their binding experiments. This may result in the development of non-optimal specific:non-specific binding ratio. On the other hand, [3H]SCH 23390 binding to lymphocytes was unaffected by quinpirole, which is a dopamine receptor agonist showing high affinity for dopamine D 3 receptors (Sokoloff et al., 1992). These results were espected in view of the lack of specificity of the radioligand used for dopamine D 3 receptors (Sokoloff et al., 1992). Hence further work is necessary to characterize with radioligand binding the population of lymphocyte dopamine D 3 receptors already demonstrated by molecular biology techniques (Nagai et al., 1993) Unlike the above authors (Takahashi et al., 1992), we have extended the characterization of [3H]SCH 23390 binding sites to human peripheral blood lymphocytes to the analysis of the pharmacological profile of the binding. Our data have shown that no dopamine D 1 receptor binding occurs in human peripheral blood lymphocytes and that the only labelled site belongs to the dopamine D 5 receptor. In fact, as expected for dopamine D 5 receptors, dopamine competed with [3H]SCH 23390 in the submicromolar range (Sibley and Monsma, 1992). In view of the feasibility and flexibility of radioligand binding studies applied to dopamine receptor research, our data suggest that these techniques may be extended to the analysis of dopamine's role in modulating immune functions or in neuroimmune interactions. A further application could be the study of human diseases in which probably changes in dopaminergic neurotransmission are involved. They include Parkinson's disease, neuropsychiatric disorders, migraine and hypertension.
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