Original Paper

Neuroimmunomodulation 2001;9:23–33

Physiological Concentrations of DopamineInhibit the Proliferation and Cytotoxicityof Human CD4+ and CD8+ T Cells in vitro:A Receptor-Mediated Mechanism

Baisakhi Sahaa Amal Chandra Mondala Jahar Majumderb Sujit Basua,c

Partha Sarathi Dasguptaa

aSignal Transduction and Biogenic Amines Lab, and Departments of bSurgical and cMedical Oncology,Chittaranjan National Cancer Institute, Calcutta, India

Received: August 7, 2000Accepted: December 13, 2000

Dr. Partha Sarathi Dasgupta, Signal Transduction and Biogenic Amines LaboratoryChittaranjan National Cancer Institute37, S.P. Mukherjee Road, 700026 Calcutta (India)Tel. +91 33 476 5101/02/04, ext. 324, Fax +91 33 475 7606E-Mail partha2@cal3.vsnl.net.in

ABCFax + 41 61 306 12 34E-Mail karger@karger.chwww.karger.com

© 2001 S. Karger AG, Basel1021–7401/01/0091–0023$17.50/0

Accessible online at:www.karger.com/journals/nim

Key WordsCytotoxicity W Dopamine W Lymphocytes, human W

Proliferation

AbstractObjective: Dopamine, a catecholamine neurotransmit-ter, influences growth and proliferation of lymphocytes.Pharmacological doses of dopamine have been shownto modulate T cell functions significantly, but no informa-tion is available on the effect of physiological concentra-tions of circulating dopamine on CD4+ and CD8+ T cellfunctions. This information may be of importance sincesignificantly elevated plasma dopamine levels were ob-served in humans during uncoping stress, and suppres-sion of T cell functions during stress is a well-known phe-nomenon. However, the mechanism inducing the sup-pression of T cell functions during stress is not yet clear.In the present investigation, we evaluated the effect ofthe dopamine level attained in the plasma of individualswith uncoping stress on the proliferation and cytotoxicityof CD4+ and CD8+ T cells in vitro. Methods: T cell sub-populations were separated by panning. The effect ofdopamine on IL-2-induced cell proliferation in vitro wasevaluated by [3H]thymidine incorporation and cytotoxici-

ty by 51Cr release, receptors by radioligand binding,cAMP by an assay kit and apoptosis by DNA fragmenta-tion. Results: At these elevated physiological concentra-tions, dopamine was found to inhibit significantly theproliferation and cytotoxicity of CD4+ and CD8+ T cells invitro. This dopamine-mediated inhibition of proliferationwas more marked on CD8+ T cells than on CD4+ T cells.The underlying mechanism was found to be D1 class ofdopamine-receptor-mediated stimulation of intracellularcAMP. Conclusion: Results may be of significance tounderstand the role of peripheral dopamine in humanneuroimmune communication in terms of physiologicalhomeostasis in health and disease.

Copyright © 2001 S. Karger AG, Basel

The regulation of functional activity of the differenteffector cells of the immune system by the sympatheticnervous system is a manifestation of neural-immune com-munication [1]. This unique endogenous biological com-munication for maintaining homeostasis is mediated byneurotransmitters [2], neuropeptides [3], hormones [4]and cytokines [5]. Among these neural mediators ofhomeostasis, the role of dopamine (DA), as a neurotrans-mitter, in the regulation of functional activity of lympho-

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24 Neuroimmunomodulation 2001;9:23–33 Saha/Mondal/Majumder/Basu/Dasgupta

cytes is of current interest due to the identification of DAreceptors on lymphocytes [6, 7] and a DA transport sys-tem in lymphocytes [8]. Several in vitro and in vivo exper-iments were carried out to evaluate the effect of DA onT lymphocytes, but results were contradictory [9–11].Moreover, in all these experiments the concentration ofDA used had been within the range of 10–3 to 10–6 M or athigher pharmacological doses, and experiments were car-ried out mostly in murine T cells [9, 10]. However, infor-mation regarding the effect of physiological concentra-tions of DA on the functional activity of human CD4+and CD8+ T lymphocytes is not available. These T cellsubpopulations play a significant role in cell-mediatedimmune response against different diseases. In somecases, CD8+ cytolytic T lymphocytes are the chief effectorcells of the immune system while in other cases CD4+ Thelper cells act as major effector cells amplifying cytotoxicT cells [12, 13]. In addition, these cells also act in consortwith other accessory cells of the immune system. Duringuncoping stress, the plasma level of DA has been shown tobe significantly increased compared to normal levels inmurine [14, 15] and human hosts [16–18]. Concomitantwith this elevation in plasma DA, functional abnormali-ties of circulating T cells were also found to be evidentduring stress [19, 20]. Although some mechanisms of thisstress-mediated inhibition have been suggested [19, 20], acorrelation between elevated plasma DA levels and func-tional activity of CD4+ and CD8+ T cells has not beenestablished. Therefore, the present in vitro studies havebeen carried out to evaluate the effect of these physiologi-cal concentrations of DA on the proliferation and cytotox-icity of human CD4+ and CD8+ T cells.

Materials and Methods

Selection of SubjectsVolunteers were healthy individuals without any known medical

or psychiatric illness. They did not receive any treatment or medica-tion during this course of the study. Subjects were selected from bothsexes and were 20–55 years old. The uncoping stress phenomenonwas reported to be common among individuals suffering from anacute disease [14–18], with the stress phenomenon being mostmarked in individuals suffering from a malignancy [17], which wasdetermined by standard protocols of biochemical criteria used for thediagnosis of uncoping stress (increased plasma cortisol, catechol-amine and hyperresponsiveness to the intramuscular clonidine test[17, 18]). Moreover, the biochemical criteria of these individualpatients correlated well with their psychiatric assessment whichstrongly suggested the presence of uncoping stress. Therefore, in thepresent investigation, individuals from both sexes, who were diag-nosed to have oral malignancy, one of the most common malignan-cies in India, were screened for symptoms of uncoping stress. Most of

the cancer patients were found to be significantly positive for all thestandard biochemical criteria of uncoping stress and were thus select-ed. These patients were devoid of any major neurological disorderlike schizophrenia or Parkinson’s disease, which might otherwisealter the dopaminergic profile.

Isolation of CD4+ and CD8+ T Cells from Peripheral BloodMononuclear cells were isolated from peripheral blood by Ficoll-

Hypaque density gradient centrifugation. To purify T cells, panningmethods on anti-human IgG-antibody-coated plates were used [21].Further separation of T cell subpopulations was achieved by incubat-ing T cell populations on anti-CD16, anti-CD19 and anti-CD14mAbs and finally separated on either anti-CD4- or anti-CD8-coatedplates. CD4+ and CD8+ cells separated by this technique were 96%of the specific T cell subpopulation as determined by immunoperoxi-dase staining [21]. The nature of the isolated CD4+ or CD8+ T cellswas further documented by assaying the profile of the cytokines (IL-2and IFNÁ) released by these cell populations in culture by ELISA(Biotrak assay system, Amersham, UK).

Proliferation AssayAnti-CD3 mAb (5 Ìg/ml) were cross-linked to the well surface of

96-well plates at 37°C in 0.1 M sodium bicarbonate buffer (pH 9.6)for 3 h. Subsequently, incubation plates were washed with PBS. Ei-ther CD4+ or CD8+ peripheral blood T lymphocytes from normalvolunteers were loaded in each well in culture medium (1 ! 106 cells/well) and incubated for 72 h with 1,000 U/ml of human recombinantIL-2 (Amersham) in the presence or absence of serum from therespective individual. The final concentration of the serum wasmaintained at 5% of the total culture volume. Immediately beforeIL-2, DA was added at concentrations ranging from 10 to 80 pg/mlsince the normal level of plasma DA (10 pg/ml) increases up to80 pg/ml during stressful conditions [16, 18]. Antagonists were addedin temporal order of antagonist-agonist-IL-2. Also, similar experi-ments were carried out with cells from individuals with uncopingstress. DA concentration was maintained similar to the respectiveplasma concentration of these patients. In other experiments, cellsfrom normal individuals were preincubated for 30 min with the ade-nylate cyclase inhibitor SQ22536, the phosphodiesterase inhibitortheophylline and with dibutyryl cAMP, an agonist of cAMP, alongwith theophylline. The proliferative capacity of T cell populationswas determined by [3H]thymidine uptake added 24 h before the endof the culture [22].

Cytotoxicity AssayCytotoxic T lymphocytes (CTL) were generated by culturing T

cell populations from normal volunteers with anti-CD3 antibody(5 Ìg/ml) and 2,000 U/ml of human recombinant IL-2 for 96 h, in thepresence or absence of their respective serum (final concentrationmaintained at 5% of the total culture volume). K562 target cells werelabeled with 51Cr (BARC, India) and used as target cells (2 ! 104

cells/ml). The effect of different physiological concentrations of DA(ranging from 10 to 80 pg/ml) on CTL cytotoxicity was evaluated.The effect of DA, simulating plasma concentration of individualsexperiencing uncoping stress, on cytotoxic functions of their T cellsin the presence or absence of their respective serum, was also evaluat-ed in similar studies. The effects of the adenylate cyclase inhibitorSQ22536, the phosphodiesterase inhibitor theophylline and cAMPagonist dibutyryl cAMP on the cytotoxicity of T cells from normalindividuals were analyzed. The percentage of specific cytotoxicity

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CD4+

Dopamine Inhibits Human CD4+ andCD8+ T Cell Functions

Neuroimmunomodulation 2001;9:23–33 25

Table 1. CD4+ and CD8+ T cellpopulations in CD3+ peripheral bloodT cells and their cytokine profile innormal volunteers and individuals withuncoping stress

Normal controls

cell type cytokine (pg/ml)/105 cells/well

IL-2 IFN-Á

Uncoping stress (cancer patients)

cell type cytokine (pg/ml)/105 cells/well

IL-2 IFN-Á

67B8 48B6 23B4 71B1 21B3 14B3CD8+ 32B3 not

detected29B3 28B5 not

detected12B0.5

CD3+ T cells were separated by panning. Phenotype determined by immunoperoxidasestaining showed more than 96% purity. Cytokine profiles of the cell populations were deter-mined in anti-CD3-stimulated culture by ELISA using the Biotrak assay system from Amers-ham.

was determined according to the formula: [experimental meanrelease (cpm) – spontaneous mean release (cpm)] ! 100 = percentageof cytotoxicity [23].

DA Receptor Assay[3H]DA was used as radioligand. The method of Ricci et al. [7]

was followed. The nonspecific binding was determined by the pres-ence of [3H]DA +1 ÌM butaclamol. Data from the binding experi-ments were analyzed by Scatchard plot to determine the maximumbinding sites (Bmax) and affinity of the binding sites (Kd).

DNA Fragmentation Assay (Apoptosis)DNA was isolated from cells after 12 h of treatment with DA

and electrophoresed in 1.5% agarose gel in TBE buffer. DNA (72-1,353 bp; Boehringer Mannheim, Germany) was used as marker [24].DNA from apoptotic cells (cells treated with 1 mM DA which hasbeen reported to induce apoptosis in lymphocytes [24]) was used as apositive control.

cAMP DeterminationDA-treated cells were kept in a boiling water bath and cAMP was

assayed using a cAMP assay kit according to the instructions of themanufacturer (Amersham).

Statistical AnalysisResults were analyzed by Student’s t test. p ! 0.05 has been con-

sidered to be statistically significant.

Results

T Cell Subpopulations and Their Cytokine ProfilesIn the present investigation, no significant difference

in CD4+ and CD8+ cell populations was observed be-tween normal and stressed individuals. This corroborateswell with the findings of Choi et al. [25]. The cytokineprofile (IL-2 and IFN-Á) of these cell populations from thetwo groups of individuals is also in line with the observa-tion of Holden et al. [26] (table 1) who showed decreased

Fig. 1. Plasma concentration of DA in normal volunteers and indi-viduals with uncoping stress (oral cancer patients). * p ! 0.05.

IL-2 and IFN-Á release from CD4+ and CD8+ T cells instressed individuals.

Plasma Level of DA in Individuals with UncopingStressDue to the disease process of oral malignancy, the DA

plasma level in individuals with uncoping stress has beenfound to be 46.6 B 3.9 pg/ml, which was significantlyhigher than the plasma DA level of normal controls (10 B2.7 pg/ml; fig. 1). This elevated level of DA corroborateswell with the reported range of plasma DA levels in indi-viduals with uncoping stress during other diseases [16–18].

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26 Neuroimmunomodulation 2001;9:23–33 Saha/Mondal/Majumder/Basu/Dasgupta

Fig. 2. Effect of DA on proliferation of IL-2-induced CD4+ andCD8+ T cells from normal volunteers. T cell subsets were separatedby panning. Cells were pulsed with [3H]thymidine for the last 8 h ofculture. Results are means B SEM of each experiment in triplicate.* p ! 0.05. a Cultures without serum. b Cultures with serum (5%concentration) from the respective individual.

Fig. 3. Effect of DA on IL-2-induced proliferation of CD4+ andCD8+ T cell subsets from individuals with uncoping stress (oral can-cer patients) in the presence or absence of their respective serum (5%concentration). DA was used in culture simulating the respectiveplasma concentration of this monoamine (mean value 46.6 B 3.9pg/ml) in stressed individuals. SCH23390, a D1-class-specific antag-onist, was added in the temporal order of antagonist, DA and IL-2.Results are means B SEM of five separate experiments. * p ! 0.05.a Effect on CD4+ cells. b Effect on CD8+ cells.

Effect of DA on the Proliferation of T CellsIn normal individuals, the plasma DA level has been

reported to be around 10 pg/ml [17, 27] whereas in indi-viduals exposed to uncoping stress, plasma DA is elevatedto 40–80 pg/ml [16–18]. Therefore CD4+ and CD8+ Tcells were exposed in vitro to concentrations of DA rang-ing from 10 to 80 pg/ml, and the effects of respective con-centrations of DA on the proliferation of CD4+ andCD8+ T cells were evaluated. In vitro, 10–20 pg/ml DAdid not show any significant inhibitory effect on the pro-liferation of CD4+ and CD8+ T cells. However, concen-trations of DA ranging from 40 to 80 pg/ml significantly

inhibited the proliferation of CD4+ and CD8+ T cells invitro, both in the absence and in the presence of normalcontrol serum. Furthermore, the results clearly indicatethat the CD8+ T cells are more vulnerable to DA-mediated inhibition than CD4+ T cells. This DA-me-diated inhibition of proliferation of these T cell subpopu-lations was dose dependent. However, saturation of theinhibition was nearly achieved at the higher levels of thesephysiological concentrations (fig. 2a, b).

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Dopamine Inhibits Human CD4+ andCD8+ T Cell Functions

Neuroimmunomodulation 2001;9:23–33 27

Fig. 4. Inhibitory effect of DA on the functional activity of CTL fromnormal volunteers. CTL were generated by anti-CD3 mAb and IL-2(2,000 U/ml) in 96-hour culture. K562 were labeled with 51Cr andused as target cells at an effector:target cell ratio of 1:10. Cultureswere carried out either in the presence or absence of serum (5% con-centration) from the same individual. Results are means B SEM of5–8 experiments, each in triplicate. * p ! 0.05 in comparison to con-trol (same culture without DA).

Fig. 5. Inhibitory effect of DA on the functional activity of CTL frompatients with uncoping stress (oral cancer patients) in the presence orabsence of their respective serum (5% concentration). DA was usedin culture simulating the respective plasma concentration of thismonoamine (mean value 46.6 B 3.9 pg/ml) in a stressed individual.SCH23390, a D1-class-specific antagonist, was added in the tempo-ral order of antagonist, DA and IL-2. Results are means B SEM offive separate experiments. * p ! 0.05.

Experiments with DA, simulating its plasma level inindividuals with uncoping stress (46.6 B 3.9 pg/ml),showed significant inhibition of proliferation of CD4+and CD8+ T cells from the respective individual, both inthe presence and in the absence of serum from the sameindividual. However, in the presence of serum, only someinsignificant abrogation of DA-mediated inhibition wasnoted (fig. 3a, b).

Effect of DA on the Cytotoxic Activity of T CellsLike proliferation inhibition, in vitro DA inhibited T-

cell-mediated cytotoxicity in a dose-dependent manner innormal volunteers. In vitro, concentrations of DA at 40,60 and 80 pg/ml showed significant inhibition of the cyto-toxic effect (46.2, 49.6 and 51.7% in the absence of serumand 43.0, 47.2 and 48.7% in the presence of serum) ofthese CTL, respectively, in comparison to controls (sameculture without DA) (fig. 4).

Exposure of CTL from uncoping-stressed individualsto their respective plasma concentrations of DA alsoshowed cytotoxic inhibition in comparison to their re-spective control (same cell culture without DA). No signif-icant differences were obtained between cultures withserum and without serum (fig. 5).

Effect of DA Receptor Antagonists on the Proliferationand Cytotoxicity of T CellsTo evaluate the mechanism underlying DA-mediated

inhibition of T cell proliferation and cytotoxicity, differ-ent DA receptor-class-specific antagonists were screenedfor their effect on proliferation and cytotoxicity of CD4+and CD8+ T cell subpopulations. Among different DAreceptor antagonists, only SCH23390, which is specificfor the D1 class of DA receptors, showed significant abro-gation of DA-induced inhibition of proliferation of CD4+and CD8+ T cells (91.2 and 96.0%, respectively). Similar-ly, this D1-class-specific antagonist was able to abrogateup to 96.9% of the DA-induced inhibition of cytotoxicityof CTL in vitro. The other DA-receptor-specific antago-nists did not show any effect on DA-mediated inhibition(table 2).

Similarly, T cell subsets from patients with uncopingstress showed abrogation of DA-mediated proliferativeand cytotoxic inhibitions when treated with the D1 DAreceptor antagonist SCH23390 (fig. 3a, b, 5) in the pres-ence or absence of their respective serum.

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SCH23390

12.8B1.6 SCH23390

28 Neuroimmunomodulation 2001;9:23–33 Saha/Mondal/Majumder/Basu/Dasgupta

Fig. 6. Saturation binding of [3H]DA (0.25–1.0 nM ) showing satura-bility of DA binding sites to circulating CD4+ and CD8+ T cells fromnormal volunteers. Results are means B SEM of five determinationsand representative of five such experiments.

Fig. 7. Influence of time on specific binding of [3H]DA to circulatingCD4+ and CD8+ T cells from normal volunteers. Results are meansB SEM of five determinations and representative of five such experi-ments.

Table 2. Effect of DA receptorantagonists on DA-induced inhibition ofproliferation and cytotoxicity of circulatingT cells

Antagonist Receptorspecificity

Inhibition of proliferation, %

CD4+ CD8+

Inhibition ofcytotoxicity, %(CTL)

D1/D5 8.8B1.9* 4.0B1.2* 3.1B0.1*Raclopride D2 98.2B11 96B18 95.7B11.2U99194A D3 88.0B1.4 81B0.4 86.7B14.2Clozapine D4 81.5B0.9 85.4B12 88.0B16

DA was added at a concentration of 40 pg/ml. Antagonists (4 ng/ml) were added immedi-ately before DA. Results are means B SEM. * p ! 0.05.

Table 3. Radioligand [3H]DA binding sites on human peripheral Tcell populations

CD4+

Bmax, fmol/106 cells Kd, nM

CD8+

Bmax, fmol/106 cells Kd, nM

0.56B0.08 16.2B1.1* 0.57B0.08

Cells were isolated by panning and incubated with different con-centrations of [3H]DA. Nonspecific binding was determined in thepresence of 1 ÌM butaclamol. Specific binding was determined bysubtracting nonspecific binding from total binding. Bmax and Kd val-ues were determined from Scatchard analysis of the specific binding.Results are means B SEM. * p ! 0.05.

Table 4. Ki values for [3H]DA binding to its receptors in humancirculating T cells

Antagonists Receptorspecificity

Ki values, nM

CD4+ CD8+

D1/D5 0.18 0.22Raclopride D2 4,000 14,500U99194A D3 45 55Clozapine D4 30 28

Values represent the competitor dissociation constant (Ki) deter-mined according to the formula Ki = IC50/(1 + S/Kd of DA binding).S is the concentration [3H]DA used in the present experiment (2 nM ).Results are means B SEM of five experiments, each in triplicate.

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Dopamine Inhibits Human CD4+ andCD8+ T Cell Functions

Neuroimmunomodulation 2001;9:23–33 29

Fig. 8. Influence of temperature on specific binding of [3H]DA tocirculating CD4+ and CD8+ T cells from normal volunteers. Resultsare means B SEM of five determinations and representative of fivesuch experiments.

Fig. 9. DNA analysis of DA-treated human circulating T lympho-cytes from normal volunteers after DA treatment for 12 h. Cell con-centration was maintained at 2 ! 106 cells/ml. Cells were treatedwith different concentrations of DA and the isolated DNA was ana-lyzed by agarose gel electrophoresis. DNA from phi X 174 cut withHaeIII was used as a molecular weight marker (72–1,353 bp). Lane 1= Molecular weight marker; 2 = positive control [cells treated with anapoptotic dose of DA (1 mM )]; 3 = 10 pg/ml DA; 4 = 20 pg/ml DA;5 = 40 pg/ml DA; 6 = 60 pg/ml DA; 7 = 80 pg/ml DA; 8 = 100 pg/mlDA.

[3H]DA Binding on CD4+ and CD8+ T CellsSCH23390, a DA receptor antagonist specific for the

D1 class, significantly abrogated the DA-induced inhibi-tion in vitro, suggesting the involvement of DA receptors.Therefore, the status of DA binding sites has been evalu-ated. [3H]DA binding to both CD4+ and CD8+ T cellswere saturable and time and temperature dependent(fig. 6–8). The maximum DA binding sites (Bmax) ofCD8+ cells were higher than those of CD4+ cells (table 3).Pharmacological characterization of the DA receptorswas determined by evaluating the dissociation constant of[3H]DA binding by various DA-receptor-class-specific an-tagonists (table 4). From the analysis of the Ki valuesobtained from the present results, it has been observedthat various DA receptor antagonists showed affinityin the following rank order of potency SCH23390 1

Clozapine 1 U 99194A 1 Raclopride in both T cell subsets(table 4). This is in conformity with our observation thatonly the D1-class-specific antagonist SCH23390 signifi-cantly abrogates the DA-mediated inhibitory effect on Tcell functions.

Effect of DA on Apoptosis of T CellsOne of the distinguishing features of apoptosis is the

onset of specific endonuclease-mediated cleavage of cellu-lar DNA into the nucleosome ladder. Therefore, DNAfragmentation analysis was performed following incuba-tion of T cells with different concentrations of DA (10–

80 pg/ml) in order to evaluate whether this neurotrans-mitter-mediated inhibition of T cell proliferation was dueto their apoptotic death. In the present investigation, noDNA fragmentation was evident in an unfractioned T cellpopulation (fig. 9).

Effect of DA-Induced cAMP Accumulation in T Cellsand Its Correlation with Cell Proliferation andCytotoxicityFrom the present experiments, it is evident that DA-

mediated inhibition of proliferation and cytotoxicity of Tcells involves the D1 class of receptors which has beenknown to be coupled to Gs membrane protein. Therefore,to evaluate the underlying mechanism of this receptor-mediated inhibition, the intracellular accumulation ofsecond messenger cAMP following DA treatment wasassayed. Results showed a distinct parallelism betweenDA-induced increased intracellular cAMP level in CD4+and CD8+ T cells and the inhibitory effect of this neuro-transmitter on proliferation and cytotoxicity of these im-mune effector cells. The differences in the degree of DA-mediated inhibition between CD4+ and CD8+ T cells is

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–

DA (40 pg/ml)

30 Neuroimmunomodulation 2001;9:23–33 Saha/Mondal/Majumder/Basu/Dasgupta

Table 5. Effect of DA on intracellular cAMP accumulation in T cells and its correlation with inhibition of proliferation and cytotoxicity

DA (pg/ml) CD4+

proliferationinhibition, %

cAMPpmol/106 cells

CD8+

proliferationinhibition, %

cAMPpmol/106 cells

CTL

cytotoxicityinhibition, %

cAMPpmol/106 cells

– 1.3B0.02 – 1.5B0.03 – 1.8B0.0440 35.1B3.4** 7.1B1.1** 48.6B4.3** 10.0B1.2* 46.2B3.5** 12.3B1.3**60 36.4B2.3** 7.9B1.1** 50.8B4.4** 11.0B1.8* 49.6B4.5** 13.1B1.4**80 38.9B9.8** 8.5B2.3** 51.4B5.4** 11.9B1.9* 51.7B7.2** 14.2B1.1**

Results are means B SEM of 5–8 experiments. cAMP was assayed according to the protocol supplied with the assay kit from Amersham.Control experiments were w/o DA treatment. * p ! 0.05 and ** p ! 0.01 vs. control.

Table 6. Effect of pharmacological modulation of intracellular cAMP on DA-induced inhibition of IL-2-mediatedproliferation and cytotoxicity of T cells

Drug Inhibition of proliferation, %

CD4+ CD8+

Cytotoxic inhibition, %(CTL)

35.1* 48.5* 46.2*DA (40 pg/ml) + SQ22536 (100 ÌM ) 1.2B0.04 1.1B0.02 2.1B0.08DA (40 pg/ml) + theophylline (1 mM ) 44.4B2.1* 55.4B3.7* 57.4B5.7*dcAMP (100 ÌM ) + theophylline (1 mM ) 67.2B6.8** 66.1B6.1** 68.4B7.8**

Results are means B SEM. Cells were preincubated for 30 min with SQ22536, an adenylate cyclase inhibitor, andwith theophylline, a phosphodiesterase inhibitor. Similarly, cells were incubated with dcAMP and theophylline for30 min before addition of IL-2 to the culture. DA was added immediately before IL-2. * p ! 0.05 vs. control (sameculture without DA). ** p ! 0.05 vs. control (same culture without dcAMP + theophylline).

also well correlated with the level of cAMP accumulatedin these two T cell subpopulations (table 5).

Effect of Adenylate Cyclase and PhosphodiesteraseInhibitors and Exogenous dcAMP on IL-2-InducedCD4+ and CD8+ T Cell Proliferation and CytotoxicityTo evaluate if a correlation exists between DA-induced

increased cAMP level in CD4+ and CD8+ T cells and inCTL and inhibition of their proliferation and cytotoxici-ty, further experiments were carried out by modulatingthe intracellular cAMP pool of these cell populations.When the cells were preincubated with SQ22536, an ade-nylate cyclase inhibitor, the inhibitory effect of DA wassignificantly abrogated. Contrarily, when cells were ex-posed before DA treatment to theophylline, a phospho-diesterase inhibitor, DA-induced inhibition of IL-2-me-diated proliferation and cytotoxicity of cells was en-hanced. Similarly when cells were incubated with

dcAMP, a cAMP agonist, and theophylline, significantinhibition of IL-2-induced proliferation and cytotoxicityof T cells was observed (table 6).

Discussion

Neural-immune communication plays an importantrole in the regulation of responses to environmental stressand diseases [28]. The in-depth mechanism of the interac-tion between the nervous and immune systems is stillobscure. Neurotransmitters have been suggested as poten-tial signaling molecules linking the two major systems formaintaining the homeostatic condition. In the presentinvestigation we have discussed the in vitro effect of aphysiological concentration of DA on the functional ac-tivity of human CD4+ and CD8+ T cells to evaluate thepossible role of peripheral DA as an immunomodulator.

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Dopamine Inhibits Human CD4+ andCD8+ T Cell Functions

Neuroimmunomodulation 2001;9:23–33 31

In the present in vitro experiments, DA concentrationswere between 10 and 80 pg/ml. From our experiments, itwas evident that when both T cell subsets were exposed invitro to a DA concentration around 10 pg/ml, simulatingthe plasma DA level of normal individuals [17, 27], nosignificant alteration in functional activity of T cell sub-sets occurred. But when cells were exposed to a DA levelof 40–80 pg/ml, similar to the plasma DA level duringuncoping stress [16–18, 29], significant inhibition of IL-2-induced proliferation and cytotoxicity of CD4+ andCD8+ cells was observed. Although other in vitro obser-vations [9, 11] also showed DA-mediated inhibition ofConA-induced murine or human unfractioned T cell pro-liferation by DA, the concentrations used were muchhigher (10–3–10–6 M). Moreover, an effect of this neuro-transmitter on specific T cell subpopulations was notavailable from those results. Interestingly, our experi-ments have evidenced that DA, at physiological concen-trations (40–80 pg/ml), can be inhibitory to IL-2-inducedproliferation and cytotoxicity of CD4+ and CD8+ T cellsin vitro. Furthermore, in another separate experiment onthe basis of biochemical criteria like increased plasma cat-echolamines, cortisol levels and hyperresponsiveness tointramuscular clonidine [17], some oral cancer patientswere selected as individuals suffering from uncopingstress. They showed significantly elevated plasma DA lev-els, which is in agreement with increased plasma DA lev-els of individuals with uncoping stress during other patho-physiological conditions [18]. In experiments with thesepatients experiencing uncoping stress, DA concentration,simulating its plasma level in the same individual,showed significant inhibition of proliferation and cyto-toxicity of their T cells in comparison to the respectivecontrols (same cell culture without DA). To simulatemore closely the complex in vivo situation and in order toevaluate the possible role of other positive and negativefactors interacting with DA, serum of the same individualwas added to the culture with cells from normal volun-teers and from respective patients with uncoping stress. Inthe presence of serum, DA-mediated inhibition was alsosignificant. It has also been evident that T cell subpopula-tions respond differently to the inhibitory effect of DA.This neurotransmitter inhibits the proliferation of CD8+T cells more markedly than CD4+ T cells. Some of therecent in vitro experiments showed that DA-mediatedinhibition of lymphocyte proliferation was due to apo-ptotic death [24, 30]. However, in those experiments, DAconcentrations were in pharmacological doses. These re-sults are not in line with our experiment in which noapoptotic death of lymphocytes was observed following

exposure to different concentrations of DA. The reasonmay be that in our experiment, the concentration of DAhad been adjusted within the picomolar range to simulateplasma concentrations, and it appears that this concentra-tion of DA was too low to cause apoptotic death of theseimmune effector cells though their proliferation was sig-nificantly inhibited. However, at higher concentration(1 mM), DA has been reported to induce apoptosis oflymphocytes [24] and has also been shown in the presentexperiment as a positive control. It has also been suggest-ed that the biological response of T cells to DA is consider-ably influenced by the concentration of DA present [31].In the present investigation, while evaluating the mecha-nism underlying this DA-mediated action, SCH23390, aDA receptor antagonist specific for the D1 class, wasfound to abrogate this inhibitory effect significantly, notonly in cells from normal volunteers but also in cells fromindividuals with uncoping stress in the presence of theirrespective serum. Therefore, it appears that DA at thesephysiological concentrations exerts its inhibitory effect onboth of the T cell subpopulations by its D1 class of recep-tors. This has further been confirmed by our presentresults showing the presence of [3H]DA binding sites onboth T cell subpopulations, pharmacologically whichwere shown to be of the D1 class of DA receptors. Pres-ence of D1 class of DA receptors in human lymphocyteshas also been documented from the observation of Ricciet al. [7]. The differences in the degree of inhibition ofproliferation of CD4+ and CD8+ cells by DA may berelated with the number of DA receptors expressed onthese two cell types. More marked DA-mediated inhibi-tion of CD8+ cells may be correlated with the increasednumber of DA receptors expressed on these cells. Theinhibition was dose dependent, but at doses of DA rang-ing from 40 to 80 pg/ml, the inhibition appeared to reacha saturation level which was associated with a patternsimilar to second messenger cAMP accumulation in T cellpopulations.

The D1 class of DA receptors is Gs membrane proteincoupled [32], and binding of ligands to this receptor leadsto increased synthesis of the second messenger cAMP[32]. In the present experiment, DA significantly in-creased intracellular cAMP in both T cell types. However,the cAMP level was higher in CD8+ T cells than in CD4+T cells. This may be due to more availability of DA bind-ing sites in CD8+ T cells. This second messenger cAMP isa well-known regulator of cell proliferation. Evidence isindicating that increased cAMP in lymphocytes inhibitsdifferent mitogen- and cytokine-induced proliferation[33–35]. Our results have shown that the DA-induced

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32 Neuroimmunomodulation 2001;9:23–33 Saha/Mondal/Majumder/Basu/Dasgupta

intracellular cAMP level correlates well with the degree ofinhibition of IL-2-induced cell proliferation. Therefore,the more marked inhibitory effect of DA on functionalactivity of CD8+ T cells may be correlated with a highercAMP level in CD8+ T cells than in CD4+ T cells. Ourfindings are in line with the observation of Johnson et al.[33], who showed that cAMP exerted an antagonizingeffect on IL-2-promoted T cell cycle progression. Further-more, the present observation also demonstrated thatDA-induced intracellular accumulation of cAMP corre-lated well with the inhibition of cytotoxic functions of Tlymphocytes. The present results also indicate that pre-treatment of cells with SQ22536, an adenylate cyclaseinhibitor, significantly abrogated DA-induced inhibitionof IL-2-induced proliferation and cytotoxicity of T cells.Parallel to this observation, pretreatment of these cellswith the phosphodiesterase inhibitor theophylline en-hanced the DA-mediated inhibition. Similarly, cells incu-bated with the cAMP agonist dcAMP concomitant withtheophylline also showed significant inhibition of IL-2-induced proliferation of T cells. These observations em-phasize convincingly the significance of DA-induced in-creased intracellular cAMP in the regulation of functionalactivity of T cells. Though in the present in vitro experi-ment, the mechanism by which the DA-induced increasedcAMP level inhibits the IL-2-mediated T cell prolifera-tion or cytotoxic functions is not known, some recentexperimental evidence suggests that elevated cAMP sup-presses the JAK3 level which plays an important role in

IL-2-induced signaling for T cell proliferation [36]. How-ever, in vivo, the mechanism may be more complex, sincethe available experimental results are contradictory,showing both DA-mediated inhibition [37] as well asstimulation of T cell proliferation [31] in murine hosts.Therefore, the knowledge regarding the interaction of thismonoamine neurotransmitter with other neurotransmit-ters, neuropeptides and cytokines with T cell surfacereceptors and its effect on the functional activity of otheraccessory cells of the immune system may be of signifi-cance in the understanding of the DA-mediated regula-tion of T cell functions in vivo. However, the present invitro results showing inhibition of proliferation and cyto-toxicity of human CD4+ and CD8+ T cells followingexposure to 40–80 pg/ml of DA, even in the presence ofserum from the same individual, may be of clinical signif-icance regarding the role of peripheral DA as an immuno-modulator in view of the similar or higher plasma DA lev-els following uncoping stress [16–18, 29] during diseaseprocesses. Further studies may enhance the understand-ing of the integratory functions of the nervous system inimmunological surveillance and the molecular mecha-nism underlying stress-induced disorders of physiologicalhomeostasis.

Acknowledgment

This study was supported by a research grant [(0081)/97-EMRII]from the Council of Scientific and Industrial Research (India).

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