Serotonin, serotonin 5-HT1A receptors and dopamine in blood peripheral lymphocytes of major depression patients

International Immunopharmacology 3 (2003) 1345 – 1352 www.elsevier.com/locate/intimp

Preliminary report

Serotonin, serotonin 5-HT1A receptors and dopamine in blood peripheral lymphocytes of major depression patients O. Fajardo, J. Galeno, M. Urbina, I. Carreira, L. Lima * Laboratorio de Neuroquı´mica, Centro de Biofı´sica y Bioquı´mica, Instituto Venezolano de Investigaciones Cientı´ficas and Centro de Salud Mental del Este, El Pen˜o´n, Caracas, Venezuela Received 12 February 2003; received in revised form 15 March 2003; accepted 21 April 2003

Abstract There are increasing evidences of cell markers present in the immune and the nervous systems. These include neurotransmitter receptors and transporters. Serotonin receptor subtypes are related to depression and also have been shown to be present in certain cells of the immune system. In the present report, we determined the presence of 5-HT1A receptors by the binding of the selective agonist 8-hydroxy-2-(di-n-propyl-amino)tetralin in lymphocytes of peripheral blood isolated by Ficoll/ Hypaque gradients from controls and depressed patients. The capacity of these receptors was around 24 fmol/106 cells in both groups of subjects, without significant difference among them. The affinity was in the nM range and either differ between controls and patients. Serotonin, 5-hydroxyindoleacetic acid, dopamine and 3,4-dihydroxyphenylacetic acid were determined by HPLC with electrochemical detector. There were no significant differences between controls and major depression patients in the values obtained for rich and poor platelet plasma or in the isolated cells. However, there was a reduction in serotonin turnover rate indicated by an increase in the ratio serotonin/5-hydroxyindoleacetic acid, but not in that of dopamine, in lymphocytes of major depression patients. Thus, there is a serotonergic dysfunction in immune circulating cells of major depression patients, without changes in the number of 5-HT1A receptors, although the coupling of these receptors to transduction mechanisms could be affected and may be related to the alteration of 5-HT turnover rate. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Dopamine turnover rate; 5-HT1A receptors; Major depression; Lymphocytes; Serotonin turnover rate

1. Introduction Serotonin (5-HT) plays roles as a neurotransmitter and a neuromodulator with functions in brain and in peripheral tissues [1] by the mediation of a considerable number of 5-HT receptors, which participate

* Corresponding author. Tel.: +58-212-504-1213; fax: +58-212504-1295. E-mail address: [email protected] (L. Lima).

in the elaboration of adapted responses of the central nervous system to the external media [2]. Most studies on 5-HT receptors and affective disorders include 5-HT1A and 5-HT2A subtypes. For instance, an increase in binding sites for 5-HT2A receptors in platelets of depressed and suicidal patients [3], and in postmortem brain tissue, mainly in prefrontal cortical sites [4,5] have been reported. In addition, an increased number of 5-HT1A receptors, also in postmortem brain tissue of suicide victims, has been shown [6]. Although the great effort for investigating

1567-5769/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S1567-5769(03)00116-4

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5-HT receptors in affective disorders, most of the studies has not been conclusive. However, there is no doubt of the involvement of central serotonergic system in major depression and suicide. For instance, there is a diffuse reduction of 5-HT uptake sites in the dorsoventral area of the prefrontal cortex, and a localized reduction of this transporter in the ventral prefrontal cortex of suicide patients, indicating a reduced 5-HT input to that brain region [7]. Other changes include altered editing of 5-HT2C receptor mRNA in the prefrontal cortex of depressed suicide victims [8], circannual variation of platelet 5-HT2A binding sites in depression [9], variable changes of 5-HT2A receptors, reduction of 5-HT1A, and decreased 5-HT transporter in depression demonstrated by imaging studies [10]. 5-HT1A receptors have been shown in Jurkat cells and in mitogen-activated human T cells, which resemble those found in the brain [11]. These receptors are coupled to the regulation of adenylate cyclase [12], favor T cell proliferation [12,13], and upregulate B and T lymphocyte proliferation by activating mitogeninduced 5-HT1A receptors [14]. Recently, 5-HT1A receptors have been characterized in rat blood lymphocytes and are increased by in vivo administration of lipopolysaccharide, concanavalin A or immobilization stress [15]. 5-HT1A receptors suppress the immune response by decreasing the count of plaqueand rosette-forming cells with in vivo administration of the agonist 8-hydroxy-2-(di-n-propyl-amino)tetralin (DPAT) [16]. In contrast, 5-HT1A receptor antagonist pindobind mediate the suppression of natural killer cell activity [17]. Serotonergic, and also adrenergic receptors, mediate the activation of corticosterone secretion stimulated by mitogens [18]. Other receptors, such as 5-HT1B subtype, induce proliferation of human T lymphoblastic cells, an effect antagonized by the endogenous peptide 5-HT-moduline [19]. In the rat, mRNA expression of 5-HT receptors in immune cells indicate positive signals for 5-HT1B, 5-HT1F, 5-HT2A , 5-HT2B, 5-HT 6 and 5-HT7, in spleen, thymus and peripheral blood lymphocytes, but not for other subtypes [20]. This finding is in contrast to reports in which mRNA for 5-HT1A receptors has been reported in humans [12,13,21] and mice [22]. Binding studies reveal the presence of these receptors in immune cells of the rat [12, 15,23].

Since 5-HT1A receptors seem to be present in several immune cells and they might be related to major depression, the purposes of the present study were (i) to determine the presence of 5-HT1A receptors in blood peripheral lymphocytes of major depression patients and to compare them to a control group, (ii) to quantify 5-HT, 5-hydroxyindoleacetic acid (5HIAA), dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) in these immune cells, and (iii) to establish any correlation between these markers and the severity of the disorder.

2. Materials and methods 2.1. Subjects Thirty-one outpatients from 18 to 55 years of age, 38.81 F 1.92, 12 men, were selected according to criteria for major depression in the Diagnostic and Statistical Manual of the American Psychiatric Association (DSM-IV) [24]. The patients were free of treatment for at least two weeks prior to extraction of the sample, but most of them abandoned treatment for a few months. None of the patients presented other psychiatric illness of the Axis I or evident systemic disease, including those with compromise of the immune system. Severity of depression was determined by Hamilton Rating Scale for Depression (HAM-D), National Institute of Mental Health [25], lower limit of 18 points. Beck Depression Inventory (BDI) [26] was also used with scores of 15 or more. Controls were 35 subjects from 23 to 57 years of age, 36.83 F 1.62, 10 men, all apparently healthy and without family history of psychiatric disorders or immune system diseases. Both groups were not alcohol, coffee or cigarette abusers, although they could be social drinkers or occasional smokers. 2.2. Preparation of blood peripheral monocytes Blood samples were taken by venipuncture between 7 and 9 a.m. (50 + 0.6 ml of heparine, 1000 units/ml). The blood was centrifuged at 2000 rpm with a vasculant rotor for 10 min at room temperature. Plasma was taken and centrifuged at 38,000  g at 4– 8 jC for 30 min to obtain low platelet plasma for HPLC analysis. The layer of white cells plus some red

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the cells was resuspended in buffer solution for HPLC, homogenized, acidified with 20% sulfosalycilic acid, centrifuged and kept at 80 jC for monoamine and metabolite determinations. The rest of the lymphocytes were homogenized in 50 mM Tris – HCl buffer, 0.1% ascorbic acid pH 7.4, washed twice, and kept at 80 jC until performance of assays for 5HT1A binding sites. Fig. 1. Correlation between Hamilton Rating Scale for Depression and Beck Depression Inventory in patients with major depression.

2.3. Radioligand binding assay

blood cells were taken and transferred to tubes with 0.1 M sodium phosphate buffer pH 7.4 (PBS) and centrifuged at 1000 rpm for 10 min. The white layer was taken, completed to 10 ml of PBS, placed on 5 ml of Ficoll/Hypaque (1077 g/l), and after centrifugation at 2000 rpm for 30 min, the mononuclear cell layer (90 – 93% lymphocytes) was taken [27]. An aliquot of

[3H]DPAT was used as the specific ligand [28]. Preparation of membranes was only sufficient for incubation in the presence of three concentrations of ligand, 0.1, 1.5 and 4 nM, specific binding was defined with 200 AM buspirone, final volume was 500 Al. The mixture was incubated at 25 jC for 30 min. Number of sites (Bmax) and dissociation rate

Fig. 2. (A) Number of binding sites for [3H]8-hydroxy-2-(di-npropyl-amino)tetralin, and (B) affinity of the binding in monocytes of controls and major depression patients.

Fig. 3. (A) 5HT and 5HIAA levels in rich platelet plasma, (B) 5-HT and 5HIAA levels in poor platelet plasma in controls and major depressed patients.

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tained by curvilinear fitting with GraphPad Prism [29]. Comparison between means was done by Student’s t-test. Statistical significance was considered if P V 0.05 and number of subjects was estimated as previously described [30,31].

3. Results 3.1. Subjects The rates for HAM-D Scale were 24.45 F 0.56 and for BDI 25.32 F 1.37, there was a significant and positive correlation between the two evaluations of severity with a Pearson coefficient of 0.57, P < 0.05 (Fig. 1).

Fig. 4. (A) 5-HT and 5HIAA levels in blood peripheral lymphocytes of controls and major depression patients. (B) 5-HT/5HIAA ratio in monocytes of controls and major depression patients, *P < 0.05.

constant (Kd) are expressed in fmol/106 cells and in nM, respectively. 2.4. Monoamines determination 5-HT, 5HIAA, DA and DOPAC were determined as previously described [27] using reversed phase HPLC with electrochemical detection. The system consisted of a Waters system composed by a delivery pump 600 Controller, an injector 717 plus Autosampler, and a 464 Pulsed Electrochemical Detector. Calculation of levels was done by the external standard method using the program Millenium (Waters). Results were expressed as ng/ml for plasma and ng/106 cells for lymphocytes. 2.5. Statistical analysis Results are expressed as the mean F standard error of the mean (S.E.M.). Kinetic parameters were ob-

Fig. 5. (A) DA and DOPAC levels in rich platelet plasma, (B) DA and DOPAC levels in poor platelet plasma in controls and major depressed patient.

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3.2. [3H]DPAT binding sites to lymphocytes Specific binding of [3H]DPAT resulted in values of 24.87 F 7.77 and 23.86 F 5.92 fmol/106 cells for controls and depressed patients, respectively. There was no significant difference of Bmax values between the two groups (Fig. 2A). Concerning the affinity, the values of Kd were not statistically different between the two groups, patients and controls (Fig. 2B). 3.3. Serotonin, dopamine and metabolites levels in plasma and lymphocytes 5-HT and 5HIAA levels were not statistically different in rich or in poor platelet plasma of depressed patients respecting the control group (Fig. 3A,B), neither in the lymphocytes (Fig. 4A). The ratio 5-HT/5HIAA, as an index of 5-HT turnover rate, was higher in major depression patients than in controls ( P < 0.05), indicating a reduction of the metabolic turnover rate in lymphocytes of the patients (Fig. 4B). This measure-

Fig. 6. (A) DA and DOPAC levels in blood peripheral lymphocytes of controls and major depression patients. (B) DA/DOPAC ratio in monocytes of controls and major depression patients.

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ment presented a significant ( P < 0.05) and positive correlation with the severity of the illness as determined by HAM-D, Pearson coefficient 0.59. Plasma DA and DOPAC levels did not significantly differ between patients and controls (Fig. 5A,B), neither in the lymphocytes, either the ratio DA/DOPAC (Fig. 6A,B). There was no significant correlation between DA levels in poor platelet plasma and lymphocyte levels in controls or in major depression patients. 5-HT concentration in lymphocytes of controls presented a positive and significant correlation with poor plasma levels, although this was not significant in major depression patients.

4. Discussion There are a series of studies concerning 5-HT1A receptors on immune cells, for instance its presence has been demonstrated in human and murine T cells [12]. Moreover, its negative coupling to adenylate cyclase [12] and its higher expression in activated lymphocytes and in Jurkat cells respecting resting lymphocytes [11], have been also reported. In the rat, the presence of 5-HT1A receptors in lymphocytes and its positive modulation by mitogens or immobilization stress have being demonstrated [15]. Although, by the use of RT-PCR methods, the mRNA of 5-HT1A receptors is not detected in cells and immune tissues of the rat, but positive signals are obtained for 5-HT1B, 5-HT1F, 5-HT2A, 5-HT2B, 5-HT6 and 5-HT7 receptors [20]. It might be that the population obtained with Percoll gradients used in the latter study is slightly different from that obtained with Ficoll/Hypaque gradient, as done by Aune et al. [12] and Sempere et al. [15]. In addition, it is clear that human peripheral blood lymphocytes present 5HT1A receptors as it has been demonstrated by Aune et al. [11,12]. In the present study, these receptors were labeled by radioligand binding assays with DPAT, however, in this particular group of patients with major depression, there was no statistical difference as compared to controls. Although the coupling to transduction mechanisms or the intrinsic activity itself could be modified during major depression, since the proliferation of lymphocytes has been reported to change in this psychiatric illness [32 –35]. The role of 5-HT receptors in immunological dys-

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function, in stress situation or in depression is not known at the present. However, after immobilization stress in the rat there is an increase in the number of 5HT1A receptors in blood lymphocytes [15]. Despite the lack of difference in number or affinity for the ligand of 5-HT1A receptors used in this report, 5-HT transporter is reduced in blood peripheral lymphocytes of patients with major depression [27,36]. Platelet circannual variations of 5-HT2A receptors differ between patients with major depression and controls [9]. Although 5-HT systems are affected in major depression [37], the functionality of 5-HT1A receptors is not altered, but that of alpha2A-adrenoreceptors is affected in brains of suicide victims [38]. 5-HT plasma concentration represents the overall result of many compartments involving synthesis and storage of 5-HT, studies on 5-HT levels in plasma of major depression patients do not show any difference respecting controls [27,30]. In our hands, levels of 5HT in the plasma of depressed patients are decreased by antidepressant treatment, such as fluoxetine [27] or mirtazapine [36], but there is no difference in the basal levels. In the present report, no significant variations were observed in plasma 5-HT, 5HIAA, DA and DOPAC concentrations. In a recent report 5-HT and 5HIAA concentration in lymphocytes was determined in a control group and in patients with generalized anxiety disorder [30], with no difference between the two groups. In the present group of patients with major depression, the levels of 5-HT and 5HIAA in lymphocytes were not modified related to controls, although the turnover rate, as expressed by the ratio 5HT/5HIAA was lower in the patients than in the controls, indicating a slow serotonergic activity in these cells. This observation might be linked to the reduction of 5-HT transporter reported in previous studies [27,36], since this molecule could work in an inverse manner [39]. In addition, an increase in the synthesis, in the capacity of the storage mechanisms or a decrease in the release could be responsible for the observation on 5-HT turnover rate in this group of patients with major depression. Dopaminergic receptors has been also described in immune cells [40,41], but there is no report on DA or DOPAC concentrations in peripheral blood lymphocytes, as it is shown in this work, DA, DOPAC or DA/ DOPAC ratio did not differ between controls and major depression patients. In this sense, it seems that

the metabolic change is selective for 5-HT system in lymphocytes, as compared to DA system. Dopaminergic modifications are also related to depression, and central DA system is also involved in the neurocircuitry of major depression [42]. Although, this study, at least concerning DA turnover rate in this group of patients indicates that DA metabolism in immune cells does not seem to be affected. What are the meanings of these observations? Circulating cells of depressed patients, accumulating more 5-HT than controls, and also expressing 5-HT receptors, with a reduced 5-HT transporter, could have a different functional response to 5-HT as a modulator of cell proliferation. Rat blood lymphocytes respond to 5-HT and 5-HT agonists by increasing proliferation, also 5-HT antagonists reduce the proliferation [11,43]. Studies on transduction mechanisms or on proliferation of blood lymphocytes could be useful for understanding the role of the whole 5-HT system in lymphocytes of patients with major depression.

Acknowledgements This research was supported by the grant G-1387 from Fondo Nacional de Ciencia, Tecnologı´a e Innovacio´n (FONACIT), Venezuela. Mrs. Isabel Otaegui is acknowledged for secretarial assistance.

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