- Email: [email protected]
Serotonin, 5-hydroxyindoleacetic acid and serotonin transporter in blood peripheral lymphocytes of patients with generalized anxiety disorder
International Immunopharmacology 2 (2002) 893 – 900 www.elsevier.com/locate/intimp
Serotonin, 5-hydroxyindoleacetic acid and serotonin transporter in blood peripheral lymphocytes of patients with generalized anxiety disorder E. Herna´ndez, S. Lastra, 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, Apdo. 21827, Caracas 1020-A, Venezuela Received 19 October 2001; received in revised form 26 February 2002; accepted 27 February 2002
Abstract Several immune system modifications have been reported in pathological anxiety, such as generalized anxiety, panic and obsessive-compulsive disorders. Since serotonin transporter is a marker of peripheral blood lymphocytes and it is modified in major depression, the aim of the present work was to evaluate this transporter by the binding of [3H]paroxetine to membrane preparations of blood peripheral lymphocytes from control subjects and patients with generalized anxiety disorder. The number of transporters and the affinity for the ligand did not differ among the two groups. Serotonin and 5-hydroxyindoleacetic acid (5HIAA) were determined in platelet-rich and -poor plasma, and in lymphocytes. Nonsignificant changes were found in the patients as compared to controls. However, there was a significant positive correlation between serotonin concentration in platelet-poor plasma and in lymphocytes in the patients, but not in the controls. This finding might be an indication of a poor regulation of the transporter function by which serotonin plasma concentration might influence lymphocyte serotonin concentration. Previous results indicate that serotonin transporter is reduced in these cells in major depression disorder; however, in generalized anxiety disorder, the number of transporters was not modified, although the functional efficiency of serotonin transporter might be altered. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Serotonin; 5-Hydroxyindoleacetic acid; Lymphocytes
1. Introduction The interaction between the central nervous system and the immune system has been documented at many levels, such as direct nervous connection in lymphoid organs [1], modulation of the hypothalamic – pitui-
*
Corresponding author. Tel.: +58-212-504-1213; fax: +58-212504-1295. E-mail address: [email protected] (L. Lima).
tary –adrenal axis by neurotransmitters [2] or cytokines [3], by the production of cytokines in the central nervous system [4], and also by the expression of neural markers in a variety of cells of the immune system. For instance, several neurotransmitter receptors have been documented in this type of cells, such as dopaminergic [5], cholinergic [6], GABAergic [7], adrenergic [8,9] and serotonergic [10,11]. The transport of [3H]serotonin (5HT) [12] and the presence 5HT transporter determined by the binding of [3H]paroxetine to membranes [13] have been demon-
1567-5769/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 7 - 5 7 6 9 ( 0 2 ) 0 0 0 2 5 - 5
894
E. Herna´ndez et al. / International Immunopharmacology 2 (2002) 893–900
strated in human circulating lymphocytes; moreover, the latter report also showed the decrease of the number of binding sites in patients with major depression and the partial recovery close to control values after treatment with fluoxetine. Several clinical psychopathological alterations have a cardinal symptom anxiety, which was even considered as an ontogenic symptom [14]. Certain degree of anxiety is maybe necessary to face daily challenges, so anxiety could be normal or could be pathological if it goes further the limits [15]. Generalized anxiety disorder includes the main symptoms of anxiety and of excessive worries, and the presence of psychological and somatic symptoms [16]. Research for many years has linked stress situations with adverse health changes, although the mechanisms of specific interactions remain poorly understood. For instance, self-reported life change stress and psychiatric symptoms have been negatively correlated to the activity of natural killer cells [17]. Moreover, a series of benzodiazepines produces an inhibition of lymphocyte proliferation acting on peripheral sites in the mouse [18,19,20]. Also, a decrease in the density of lymphocyte and monocyte benzodiazepine receptors and their function have been described in anxiety disorders [7,21,22,23]. In addition, a variety of modifications of the immunological system has been described, such as enrichment in cells expressing surface antigens of immune cells in panic [24] and in other anxiety disorder patients [25]. The relation between anxiety and poor immune control and of emotional stability and immunoenhancement were documented [26], and the psychological state in a normal population, measured by the Profile of Mood States and Spielberger-Trait Anxiety Inventory, indicates a negative correlation between beta-adrenergic receptors in lymphocytes with age and with tension– anxiety, depression – dejection, and anger – hostility [27]. We have previously measured the transporter of 5HT in human lymphocytes by the binding of [3H]paroxetine [13], and we reported that the number of sites was reduced in patients with major depression. Similar experiments were conducted to determine the possible modification of 5HT uptake sites in lymphocytes of patients with generalized anxiety disorder. In addition, turnover rate of 5HT was determined in this type of cells by relating 5HT
and 5-hydroxyindoleacetic acid (5HIAA) concentrations.
2. Materials and methods 2.1. Subjects Twenty outpatients (more than estimated), range of age 26 – 61 years (40.75F1.89), 10 women (42.20F2.84) and 10 men (39.30F2.58), were diagnosed according to DSM-IV [16] criteria for generalized anxiety. They had no other major psychiatric disorder and did not have somatic illness. All patients were drug-free for at least 2 weeks before participating in the study. During this period, they visited the physician twice a week. The severity of the anxiety was determined by Hamilton Rating Scale for Anxiety (HAM-A, National Institute of Mental Health) [28]. Severity was as follows: mild 14 –27, moderate 28– 41, and severe 42 –56. The control group was composed by 20 subjects, range of age 28 – 59 years (38.00F2.27), 12 women (36.58F2.78) and 8 men (40.12F3.96), free of medical illness and without personal or family history of psychiatric disorders. The same physicians interviewed patients and controls. 2.2. Preparation of peripheral blood lymphocytes Blood samples (50 ml+0.6 ml of heparine, 1000 units/ml) were taken by venipuncture between 7:00 and 8:00 a.m., and placed in 50-ml centrifuge tubes. A first centrifugation was performed at 2000 rpm with a vasculant rotor in a Damon/IEC Division centrifuge, Model HN-S for 10 min at room temperature. A sample of plasma was taken and centrifuged at 38,000g during 30 min to obtain low platelets plasma for 5HT and 5HIAA determination. The rest of the plasma was discarded and the interphase layer plus some of the red blood cells was taken and transferred to tubes with isotonic saline 0.1 M sodium phosphate buffer pH 7.4 (PBS) and centrifuged at 1000 rpm for 10 min. The white layer was taken and placed in tubes in which volume was completed to 10 ml with PBS. This preparation was carefully placed in 15-ml tubes in which 5 ml Ficoll/Hypaque (1077 g/l) was previ-
E. Herna´ndez et al. / International Immunopharmacology 2 (2002) 893–900
ously added. Centrifugation was performed at 2000 rpm for 30 min. After this period, the supernatant was discarded and the lymphocyte enriched layer was taken and placed in 15-ml tubes plus PBS, centrifuged at 1200 rpm for 10 min, cells were recovered and the procedure was repeated. The pellet was resuspended in 50 mM Tris – HCl buffer pH 7.4, an aliquot was taken for counting cells in a hemocytometer and determining viability by the exclusion of Trypan Blue (70 – 98%). The contamination with platelets was b 1% if detected. After homogenization with a Tissumizer (Tekmar, Cincinnati, OH), centrifugation was done in a Sorvall RC5 refrigerated centrifuge at 38,000g for 30 min. Membrane preparations were washed three times. The final pellet was resuspended in 1 ml of 50 mM Tris –HCl buffer pH 7.4 and kept at 800 jC until the binding assay was performed. 2.3. [3H]Paroxetine binding assays The binding of [3H]paroxetine (NEN, 17.1 Ci/ mmol), a marker of 5HT carrier, was performed according to Urbina et al. [13]. Saturation experiments (final volume 500 Al) was performed with 80 Al of membranes (0.74 mg of protein/ml) and with concentrations of [3H]paroxetine of 0.07, 1 and 2 nM, specific binding was defined with 100 AM imipramine. The tubes were prepared in duplicates and incubation was done at 250 jC for 90 min. The reaction was stopped by adding cold buffer and placing the tubes on ice. Ligand – receptor complex was separated from free ligand by filtration through Whatman GF/A glass fiber filters, which were washed, placed on vials, dried and counted in a Beckman LS 5000 TD scintillation counter (efficiency 60%). 2.4. Serotonin and 5-hydroxyindoleacetic acid determination 5HT and 5HIAA were determined by reversed phase HPLC with electrochemical detection [29]. The system consisted of a Varian HPLC pump Model 2510, a Rheodyne manual injector Model 7125, a Varian integrator Model 4400, a Supelco LC-18 column (15 cm4.6 mm inside diameter, 5 Am average particle), and an amperometric detector from
895
Bioanalytical Systems Model LC-4B (+0.7 V, 0.2 nA/ V). The limit of detection was 3 pg. The mobile phase was made of 0.02 M sodium acetate, 0.0125 M citric acid, 1 mM EDTA, 1.52 mM octanylsulfonate, and 10% acetonitrile, at a pH of 3.9. After centrifugation of plasma, a solution of 20% sulfosalicylic acid was added, giving a final concentration of 5%. The samples were then centrifuged at 34,000g, 4 –80 jC, for 10 min, and the supernatant was kept at 80 jC until analysis. The amount present in the plasma was calculated from the area under the curve of samples and external standards. 2.5. Statistical analysis Results are meanFstandard error of the mean (S.E.M.) [46]. Bmax and Kd were calculated by curvilinear fitting with the program PRISMA [30]. Student’s t-test was carried out. Significance level a was set to 0.05 or lower. The number of subjects was estimated by using previous standard deviation (S.D.) of Bmax (17%) and Kd (25%) [13] and precision ( P) of 10% for Bmax and 15% for Kd, according to the equation Nf4(S.D./P)2 [31].
3. Results 3.1. Subjects According to the HAM-A Scale, the patients presented mild (three males), moderate (three males and seven females), and severe anxiety (four males and three females). The rates in the control group were much lower than 14. The psychiatric and somatic symptoms were separated for males and females. The total rate of symptoms, psychiatric or somatic, was not different between males and females (Table 1). Total time of the illness course was similar in
Table 1 Psychiatric and somatic symptoms from the Hamilton Anxiety Scale in patients with generalized anxiety Symptoms
Males
Females
Total
Psychiatric Somatic Total
15.50F1.55 18.00F1.90 33.50F3.33
19.40F1.33 19.60F1.20 39.00F1.71
17.45F1.24 18.80F1.11 36.25F1.92
896
E. Herna´ndez et al. / International Immunopharmacology 2 (2002) 893–900
males (24.90F9.66) and females (29.70F10.71), and the total average value was 27.30F7.04 months. Fourteen of the 20 patients received benzodiazepine
Table 2 Serotonin levels in plasma and lymphocytes of patients with generalized anxiety Sample
Males
Females
Plasma rich in platelets Plasma poor in platelets Lymphocytes
111.65F26.13 3.83F1.05 4.06F1.26
104.69F21.31 5.84F1.05 3.67F1.25
Plasma values in nanograms per milliliter. Lymphocytes values in nanograms per 106 cells.
treatment before taking the blood sample; however, all of them went into 2 weeks washout of any drug always under a physician’s or psychologist’s supervision. 3.2. Serotonin and 5-hydroxyindoleacetic acid levels in plasma and lymphocytes Platelet-rich and -poor plasma obtained as described in Materials and Methods were used to determine the levels of 5HT and 5HIAA. The values of the monoamine or the metabolite in both types of plasma and in lymphocytes were not different between controls and patients with generalized anxiety (Fig. 1), and did not differ in males and females (Table 2). 5HT turnover rate in lymphocytes, calculated as the ratio 5HT/5HIAA for each subject, neither presented significant changes between patients and controls (Fig. 2). Several correlations were performed between 5HT and 5HIAA in plasma-rich or -poor platelets as well
Fig. 1. 5HT and 5HIAA concentration in (A) platelet-rich plasma, (B) platelet-poor plasma, and (C) lymphocytes of control subjects and patients with generalized anxiety disorder.
Fig. 2. 5HT turnover rate expressed as the index 5HIAA/5HT in control subjects and in patients with generalized anxiety disorder.
E. Herna´ndez et al. / International Immunopharmacology 2 (2002) 893–900
897
and in lymphocytes, and between these measurements and the severity of the anxiety, resulting in nonsignificant differences. However, the correlation between 5HT levels in plasma-poor platelets and those in the lymphocytes was positive and significant in the patients, but not in the controls (Fig. 3). 3.3. [3H]Paroxetine binding sites in lymphocytes [3H]Paroxetine number and affinity of binding sites were not significantly different between controls and patients. Kd values were 2.96F1.01 and 4.40F1.27
Fig. 4. Number of maximal binding sites for [3H]paroxetine to lymphocyte membrane preparation of (A) control subjects and patients with generalized anxiety disorder, and (B) male and female patients with generalized anxiety disorder.
nM for controls and patients, respectively ( P=0.379). Bmax is reported in Fig. 4. Male and female results in the patients were separated and Bmax compared; there was no significant difference in the number of the transporter sites.
4. Discussion
Fig. 3. Correlation between 5HT concentration in platelet-poor plasma and 5HT concentration in lymphocytes in (A) control subjects, and (B) patients with generalized anxiety disorder, F=6.063, P=0.0248.
In a previous report, we demonstrated that in a group of patients with major depression disorder, there was a decreased number of 5HT transporters in blood peripheral lymphocytes and an increase in their number after treatment, as determined by the binding of [ 3 H]paroxetine to membrane preparations [13]. [3H]5HT transport into these cells was reported earlier [12] and an increase of it occurs in abstinent alcoholics [32]. These evidences indicate the possibility of modulation of 5HT transporter in lymphocytes during the course of different medical conditions.
898
E. Herna´ndez et al. / International Immunopharmacology 2 (2002) 893–900
Blood cells, such as platelets, are known to accumulate 5HT [33], and several reports indicate the presence and modulation of 5HT carrier in these cells [34,35]. The high concentration of 5HT as compared to 5HIAA in blood peripheral lymphocytes of controls and patients might indicate a low turnover rate, a high level of synthesis or a specialized storage system [36]. Synthesis of 5HT from tryptophan has been reported in rat lymphocytes [37]; however, such a demonstration is lacking in humans. Despite the lack of significant differences of 5HT and 5HIAA concentrations in platelet-rich or -poor plasma among the two groups of subjects, a clear difference was observed between the two plasma preparations, indicating the capacity of accumulating 5HT by lymphocytes and other blood cells, as shown by the relatively high level of 5HT, also taking place in the rat [37]. The significant and positive correlation between 5HT in platelet-poor plasma and 5HT in lymphocytes occurring in the patients could reflect a lack of regulation of the transporter in blood peripheral lymphocytes in generalized anxiety disorder, being unable to maintain endogenous levels by modulating the transport. By contrast, in control subjects, the endogenous levels were independent of the circulating free 5HT, probably indicating a stronger role of synthesis and storage for preserving 5HT concentration in lymphocytes. The number of [3H]paroxetine binding sites to lymphocyte membrane preparations did not show significant differences between controls and patients with generalized anxiety disorder, although in major depression disorder, a decrease in this parameter was reported [13], which might be a differential index between both psychiatric illnesses. However, in the present group of subjects, nonsignificant variations for all determined parameters were observed between genders; in addition, this disorder is as frequent in men as in women, also being different from depression, which it is known to be higher in women than in men (DSM-IV). Various immune system parameters, such as expression of CD62L, have been related to severity of anxiety disorders like panic disorder, which might be related to diminished cell activation in vivo [24]. Although the intent to find a correlation between 5HT or 5HIAA concentrations or [3H]paroxetine binding to severity of the anxiety was not successful, related to cell proliferation is the fact that beta-adrenergic recep-
tor responsiveness is altered in patients with anxiety or depression disorders [27]. Moreover, there is a considerable number of reports concerning cytokine levels [38], lymphocyte beta-adrenergic receptor function [39,40,41] and other parameters [25,42] in anxiety disorders. There is no report studying 5HT transporter function or modulation, despite that dysregulation of this carrier might be related to certain psychiatric disorders, such as those of the effect [43], although 5HT transporter expression in immortalized B lymphocytes is modulated by proinflammatory and anti-inflammatory cytokines [44]. In addition, functional promoter analysis and screening for length differences revealed polymorphism of 5HT human transporter, variations related to anxiety-behavioural traits [43], and differential initial response to selective 5HT uptake inhibitors [45]. The above quoted results indicate that some markers in the immune system, related to the central nervous system, might be altered in anxiety disorder patients; however, certain studies do not show significant changes. The fact that [3H]paroxetine binding to lymphocyte membrane preparations of patients with anxiety disorder was not significantly modified respecting values in a control group makes a difference with major depression in which case a reduction of this transporter was reported [13] or in abstinent alcoholics, who present high 5HT uptake sites [32]. Although there was no variation in [3H]paroxetine binding sites, the fact that the correlation of lymphocyte versus plasma concentration of 5HT is only significant in the group of patients suggests that functional differences in 5HT influx might be taking place in patients with generalized anxiety disorder. Acknowledgements This work was supported by the Grant G-1387 from Fondo Nacional de Ciencia, Tecnologı´a e Innovacio´n (FONACIT). We appreciate the secretarial assistance of Mrs. Isabel Otaegui. References [1] Felten DL, Felten SY, Bellinger DL, Carlsson SL, Ackerman KD, Madden KS, Olschowka JA, Livnat S. Noradrenergic sympathetic neuronal interactions with the immune system: structure and function. Immuno Rev 1987;100:225 – 60.
E. Herna´ndez et al. / International Immunopharmacology 2 (2002) 893–900 [2] Solomon GF. Psychoneuroimmunology: interactions between central nervous system and immune system. J Neurosci Res 1987;18:1 – 9. [3] Merrill JE, Jonakait GM. Interactions of the nervous and immune systems in development, normal brain homeostasis, and disease. FASEB J 1995;9:611 – 8. [4] Koenig JI. Presence of cytokines in the hypothalamic pituitary axis. Prog Neuroendocrinol Immunol 1991;4:143 – 53. [5] Amenta F, Bronzetti E, Fellici L, Ricci A, Tayebati SK. Dopamine D2-like receptors on human peripheral blood lymphocytes: a radioligand binding assay and immunocytochemical study. J Auton Pharmacol 1999;19:151 – 9. [6] Strom BT, Lane AM, George K. The parallel, time – time, bimodal change in lymphocyte cholinergic binding activity and cholinergic influence upon lymphocyte-mediated cytotoxicity. J Immunol 1981;127:705 – 10. [7] Rocca P, Ferrero P, Gualerzi A, Zanalda E, Maina G, Bergamasco B, Ravizza L. Peripheral type of benzodiazepine receptors in anxiety disorders. Acta Psychiatr Scand 1991;84:537 – 44. [8] Khan MMP, Sansoni P, Silverman EG, Engleman EG, Melmon KL. Beta-adrenergic receptors on human suppressor, helper, and cytolytic lymphocytes. Biochem Pharmacol 1986;35:1137 – 42. [9] Williams LT, Snyderman R, Lefkowitz RJ. Identification of beta-adrenergic receptors in human lymphocytes by [3H]alprenolol binding. J Clin Invest 1976;57:149 – 52. [10] Abdouh M, Storring JM, Riad M, Paquette Y, Albert PR, Drobetsky E, Kouassi E. Transcriptional mechanisms for induction of 5HT1A receptor mRNA and protein in activated B and T lymphocytes. J Biol Chem 2001;276:4382 – 8. [11] Aune TM, McGrath KM, Sarr T, Bombara MO, Kelley KA. Expression of 5HT1A receptors on activated human T cells: regulation of cAMP levels and T cell proliferation by 5HT. J Immunol 1993;151:1175 – 80. [12] Faraj BA, Olkowski L, Jackson RT. Expression of high-affinity serotonin transporter in human lymphocytes. Int J Immunopharmacol 1994;16:561 – 7. [13] Urbina M, Pineda S, Pin˜ango L, Carreira I, Lima L. [3H]Paroxetine binding to human peripheral lymphocyte membranes of patients with major depression before and after treatment with fluoxetine. Int J Immunopharmacol 1999;21:631 – 46. [14] Cyrlnik B. Ethology and anxiety in phylogeny and ontogeny. Acta Psychiatr Scand 1998;98:48 – 9. [15] Rosen JB, Schulkin J. From normal fear to pathological anxiety. Psychol Rev 1998;105:326 – 7. [16] American Psychiatry Association. DSM-IV: Diagnostic and Statistical Manual for Mental Disorders. 3rd edn. Washington, DC: APA, 1994. [17] Locke SE, Kraus L, Leserman J, Hurst MW, Heisel JS, Williams RM. Life change stress, psychiatric symptoms, and natural killer cell activity. Psychosom Med 1984;46:441 – 53. [18] Covelli V, Maffione AB, Nacci C, Tato E, Jirillo E. Stress, neuropsychiatric disorders and immunological effects exerted by benzodiazepines. Immunopharmacol Immunotoxicol 1998; 20:199 – 209. [19] Pawlikowski M, Lyson K, Kunter-Radek J, Stepien H. Effect of benzodiazepines on the proliferation of mouse spleen lymphocytes in vitro. J Neural Transm 1988;73:161 – 6.
899
[20] Zavala F. Benzodiazepines, anxiety and immunity. Pharmacol Ther 1997;75:100 – 216. [21] Ferrarese C, Appollonio I, Frigo M, Perego M, Pioti R, Trabucchi M, Frattola L. Decreased density of benzodiazepine receptors in lymphocytes of anxious patients: reversal after chronic diazepam treatment. Acta Psychiatr Scand 1990;82: 169 – 73. [22] Rocca P, Beoni AM, Eva C, Ferrero P, Zanalda E, Ravizza L. Peripheral benzodiazepine receptor messenger RNA is decreased in lymphocytes of generalized anxiety disorder patients. Biol Psychiatry 1998;43:767 – 73. [23] Sacerdote P, Panerai AE, Frattola L, Ferrarese C. Benzodiazepine-induced chemotaxis is impaired in monocytes from patients with generalized anxiety disorder. Psychoneuroendocrinology 1999;24:243 – 9. [24] Manfro GG, Pollack MH, Otto MW, Wothington JJ, Rosenbaun JF, Scott EL, Kradin RL. Cell-surface expression of Lselectin (CD62L) by blood lymphocytes: correlates with affective parameters and severity of panic disorder. Depression Anxiety 2000;11:31 – 7. [25] Rapaport MH. Circulating lymphocyte phenotypic surface markers in anxiety disorder patients and normal volunteers. Biol Psychiatry 1998;43:458 – 63. [26] Borella P, Bargellini A, Rovesti S, Pinelli M, Vivoli R, Solfrini G, Vivoli G. Emotional stability, anxiety and natural killer activity under examination stress. Psychoneuroendocrinology 1999;24:613 – 27. [27] Yu BH, Dimsdale JE, Mills PJ. Psychological states and lymphocyte betaadrenergic receptor responsiveness. Neuropsychopharmacology 1999;21:147 – 52. [28] Hamilton MA. The assessment of anxiety states by rating. Br Med Psychol 1959;32:35. [29] Lima L, Schmeer C. Characterization of serotonin transporter in goldfish retina by binding of [3H]paroxetine and the uptake of [3H]serotonin: modulation by light. J Neurochem 1994;62: 528 – 35. [30] GraphPad Prism, Version 2.0, San Diego, CA, 1998. [31] Motulsky H. Intuitive Biostatistics. New York: Oxford Univ. Press, 1995. [32] Faraj BA, Olkowski L, Jackson RT. Prevalence of high serotonin uptake in lymphocytes of abstinent alcoholics. Biochem Pharmacol 1997;53:53 – 7. [33] Sarrias MJ, Artigas F, Martı´nez E, Gelpi E, Udina C, Casas M. Decreased plasma serotonin in melancholic patients: a study with clomipramine. Biol Psychiatry 1987;22:1429 – 38. [34] Fozard J. Peripheral actions of 5-hydroxytryptamine. New York: Oxford Univ. Press, 1989. [35] Mellerup ET, Plenge P, Engelstoft M. High affinity binding of 3 H-paroxetine and 3H-imipramine to human platelet membranes. Eur J Pharmacol 1983;96:303 – 9. [36] Shannon NJ, Gunnet JW, Moore KE. A comparison of biochemical indices of 5-hydroxytryptaminergic neuronal activity following electrical stimulation of the dorsal raphe nucleus. J Neurochem 1986;47:958 – 65. [37] Ceden˜o N, Lima L, Obrego´n F, Urbina M. Synthesis of serotonin in peripheral blood lymphocytes of rats. Scand J Immunol 2001;54:1325.
900
E. Herna´ndez et al. / International Immunopharmacology 2 (2002) 893–900
[38] Zorrilla EP, Redel E, DeRubeis RJ. Reduced cytokine levels and T-cell function in healthy males: relation to individual differences in subclinical anxiety. Brain Behav Immunol 1994; 8:293 – 312. [39] Brambilla F, Bellodi L, Perna G, Garberi A, Panerai A, Sacerdote P. Lymphocyte cholecystokinin concentrations in panic disorder. Am J Psychiatry 1993;150:1111 – 3. [40] Brown SL, Charney DS, Woods SW, Heninger GR, Tallman J. Lymphocyte beta-adrenergic receptor binding in panic disorder. Psychopharmacology (Berlin) 1988;94:24 – 8. [41] Maddock RJ, Carter CS, Magliozi JR, Gietzen DW. Evidence that decreased function of lymphocyte beta adrenoreceptors reflects regulatory and adaptive processes in panic disorder with agoraphobia. Am J Psychiatry 1993;150: 1219 – 25. [42] Brambilla F, Bellodi L, Perna G, Battaglia M, Sciuto G, Diaferia G, Petraglia F, Panerai A, Sacerdote P. Psychoimmunoen-
[43]
[44]
[45]
[46]
docrine aspects of panic disorder. Neuropsychobiology 1992; 25:94 – 8. Heils A, Mossner R, Lesch KP. The human serotonin transporter gene polymorphism—basic research and clinical implications. J Neural Transm 1997;104:1005 – 14. Mossner R, Daniel S, Schmitt A, Albert D, Lesch KP. Modulation of serotonin transporter function by interleukine-4. Life Sci 2001;68:873 – 80. Pollock BG, Ferrell RE, Mulsant BH, Mazzumdar S, Miller M, Sweet RA, Davis S, Kirshner MA, Houck PR, Stack JA, Reynolds CF, Kupfer DJ. Allelic variation in the serotonin transporter promoter affects onset of paroxetine treatment response in late-life depression. Neuropsychopharmacology 2000;23: 587 – 90. Sokal R, Rohlf F. In: Blume H, editor. Biometrı´a. Madrid; 1989.
Serotonin, 5-hydroxyindoleacetic acid and serotonin transporter in blood peripheral lymphocytes of patients with generalized anxiety disorder E. Herna´ndez, S. Lastra, 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, Apdo. 21827, Caracas 1020-A, Venezuela Received 19 October 2001; received in revised form 26 February 2002; accepted 27 February 2002
Abstract Several immune system modifications have been reported in pathological anxiety, such as generalized anxiety, panic and obsessive-compulsive disorders. Since serotonin transporter is a marker of peripheral blood lymphocytes and it is modified in major depression, the aim of the present work was to evaluate this transporter by the binding of [3H]paroxetine to membrane preparations of blood peripheral lymphocytes from control subjects and patients with generalized anxiety disorder. The number of transporters and the affinity for the ligand did not differ among the two groups. Serotonin and 5-hydroxyindoleacetic acid (5HIAA) were determined in platelet-rich and -poor plasma, and in lymphocytes. Nonsignificant changes were found in the patients as compared to controls. However, there was a significant positive correlation between serotonin concentration in platelet-poor plasma and in lymphocytes in the patients, but not in the controls. This finding might be an indication of a poor regulation of the transporter function by which serotonin plasma concentration might influence lymphocyte serotonin concentration. Previous results indicate that serotonin transporter is reduced in these cells in major depression disorder; however, in generalized anxiety disorder, the number of transporters was not modified, although the functional efficiency of serotonin transporter might be altered. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Serotonin; 5-Hydroxyindoleacetic acid; Lymphocytes
1. Introduction The interaction between the central nervous system and the immune system has been documented at many levels, such as direct nervous connection in lymphoid organs [1], modulation of the hypothalamic – pitui-
*
Corresponding author. Tel.: +58-212-504-1213; fax: +58-212504-1295. E-mail address: [email protected] (L. Lima).
tary –adrenal axis by neurotransmitters [2] or cytokines [3], by the production of cytokines in the central nervous system [4], and also by the expression of neural markers in a variety of cells of the immune system. For instance, several neurotransmitter receptors have been documented in this type of cells, such as dopaminergic [5], cholinergic [6], GABAergic [7], adrenergic [8,9] and serotonergic [10,11]. The transport of [3H]serotonin (5HT) [12] and the presence 5HT transporter determined by the binding of [3H]paroxetine to membranes [13] have been demon-
1567-5769/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 7 - 5 7 6 9 ( 0 2 ) 0 0 0 2 5 - 5
894
E. Herna´ndez et al. / International Immunopharmacology 2 (2002) 893–900
strated in human circulating lymphocytes; moreover, the latter report also showed the decrease of the number of binding sites in patients with major depression and the partial recovery close to control values after treatment with fluoxetine. Several clinical psychopathological alterations have a cardinal symptom anxiety, which was even considered as an ontogenic symptom [14]. Certain degree of anxiety is maybe necessary to face daily challenges, so anxiety could be normal or could be pathological if it goes further the limits [15]. Generalized anxiety disorder includes the main symptoms of anxiety and of excessive worries, and the presence of psychological and somatic symptoms [16]. Research for many years has linked stress situations with adverse health changes, although the mechanisms of specific interactions remain poorly understood. For instance, self-reported life change stress and psychiatric symptoms have been negatively correlated to the activity of natural killer cells [17]. Moreover, a series of benzodiazepines produces an inhibition of lymphocyte proliferation acting on peripheral sites in the mouse [18,19,20]. Also, a decrease in the density of lymphocyte and monocyte benzodiazepine receptors and their function have been described in anxiety disorders [7,21,22,23]. In addition, a variety of modifications of the immunological system has been described, such as enrichment in cells expressing surface antigens of immune cells in panic [24] and in other anxiety disorder patients [25]. The relation between anxiety and poor immune control and of emotional stability and immunoenhancement were documented [26], and the psychological state in a normal population, measured by the Profile of Mood States and Spielberger-Trait Anxiety Inventory, indicates a negative correlation between beta-adrenergic receptors in lymphocytes with age and with tension– anxiety, depression – dejection, and anger – hostility [27]. We have previously measured the transporter of 5HT in human lymphocytes by the binding of [3H]paroxetine [13], and we reported that the number of sites was reduced in patients with major depression. Similar experiments were conducted to determine the possible modification of 5HT uptake sites in lymphocytes of patients with generalized anxiety disorder. In addition, turnover rate of 5HT was determined in this type of cells by relating 5HT
and 5-hydroxyindoleacetic acid (5HIAA) concentrations.
2. Materials and methods 2.1. Subjects Twenty outpatients (more than estimated), range of age 26 – 61 years (40.75F1.89), 10 women (42.20F2.84) and 10 men (39.30F2.58), were diagnosed according to DSM-IV [16] criteria for generalized anxiety. They had no other major psychiatric disorder and did not have somatic illness. All patients were drug-free for at least 2 weeks before participating in the study. During this period, they visited the physician twice a week. The severity of the anxiety was determined by Hamilton Rating Scale for Anxiety (HAM-A, National Institute of Mental Health) [28]. Severity was as follows: mild 14 –27, moderate 28– 41, and severe 42 –56. The control group was composed by 20 subjects, range of age 28 – 59 years (38.00F2.27), 12 women (36.58F2.78) and 8 men (40.12F3.96), free of medical illness and without personal or family history of psychiatric disorders. The same physicians interviewed patients and controls. 2.2. Preparation of peripheral blood lymphocytes Blood samples (50 ml+0.6 ml of heparine, 1000 units/ml) were taken by venipuncture between 7:00 and 8:00 a.m., and placed in 50-ml centrifuge tubes. A first centrifugation was performed at 2000 rpm with a vasculant rotor in a Damon/IEC Division centrifuge, Model HN-S for 10 min at room temperature. A sample of plasma was taken and centrifuged at 38,000g during 30 min to obtain low platelets plasma for 5HT and 5HIAA determination. The rest of the plasma was discarded and the interphase layer plus some of the red blood cells was taken and transferred to tubes with isotonic saline 0.1 M sodium phosphate buffer pH 7.4 (PBS) and centrifuged at 1000 rpm for 10 min. The white layer was taken and placed in tubes in which volume was completed to 10 ml with PBS. This preparation was carefully placed in 15-ml tubes in which 5 ml Ficoll/Hypaque (1077 g/l) was previ-
E. Herna´ndez et al. / International Immunopharmacology 2 (2002) 893–900
ously added. Centrifugation was performed at 2000 rpm for 30 min. After this period, the supernatant was discarded and the lymphocyte enriched layer was taken and placed in 15-ml tubes plus PBS, centrifuged at 1200 rpm for 10 min, cells were recovered and the procedure was repeated. The pellet was resuspended in 50 mM Tris – HCl buffer pH 7.4, an aliquot was taken for counting cells in a hemocytometer and determining viability by the exclusion of Trypan Blue (70 – 98%). The contamination with platelets was b 1% if detected. After homogenization with a Tissumizer (Tekmar, Cincinnati, OH), centrifugation was done in a Sorvall RC5 refrigerated centrifuge at 38,000g for 30 min. Membrane preparations were washed three times. The final pellet was resuspended in 1 ml of 50 mM Tris –HCl buffer pH 7.4 and kept at 800 jC until the binding assay was performed. 2.3. [3H]Paroxetine binding assays The binding of [3H]paroxetine (NEN, 17.1 Ci/ mmol), a marker of 5HT carrier, was performed according to Urbina et al. [13]. Saturation experiments (final volume 500 Al) was performed with 80 Al of membranes (0.74 mg of protein/ml) and with concentrations of [3H]paroxetine of 0.07, 1 and 2 nM, specific binding was defined with 100 AM imipramine. The tubes were prepared in duplicates and incubation was done at 250 jC for 90 min. The reaction was stopped by adding cold buffer and placing the tubes on ice. Ligand – receptor complex was separated from free ligand by filtration through Whatman GF/A glass fiber filters, which were washed, placed on vials, dried and counted in a Beckman LS 5000 TD scintillation counter (efficiency 60%). 2.4. Serotonin and 5-hydroxyindoleacetic acid determination 5HT and 5HIAA were determined by reversed phase HPLC with electrochemical detection [29]. The system consisted of a Varian HPLC pump Model 2510, a Rheodyne manual injector Model 7125, a Varian integrator Model 4400, a Supelco LC-18 column (15 cm4.6 mm inside diameter, 5 Am average particle), and an amperometric detector from
895
Bioanalytical Systems Model LC-4B (+0.7 V, 0.2 nA/ V). The limit of detection was 3 pg. The mobile phase was made of 0.02 M sodium acetate, 0.0125 M citric acid, 1 mM EDTA, 1.52 mM octanylsulfonate, and 10% acetonitrile, at a pH of 3.9. After centrifugation of plasma, a solution of 20% sulfosalicylic acid was added, giving a final concentration of 5%. The samples were then centrifuged at 34,000g, 4 –80 jC, for 10 min, and the supernatant was kept at 80 jC until analysis. The amount present in the plasma was calculated from the area under the curve of samples and external standards. 2.5. Statistical analysis Results are meanFstandard error of the mean (S.E.M.) [46]. Bmax and Kd were calculated by curvilinear fitting with the program PRISMA [30]. Student’s t-test was carried out. Significance level a was set to 0.05 or lower. The number of subjects was estimated by using previous standard deviation (S.D.) of Bmax (17%) and Kd (25%) [13] and precision ( P) of 10% for Bmax and 15% for Kd, according to the equation Nf4(S.D./P)2 [31].
3. Results 3.1. Subjects According to the HAM-A Scale, the patients presented mild (three males), moderate (three males and seven females), and severe anxiety (four males and three females). The rates in the control group were much lower than 14. The psychiatric and somatic symptoms were separated for males and females. The total rate of symptoms, psychiatric or somatic, was not different between males and females (Table 1). Total time of the illness course was similar in
Table 1 Psychiatric and somatic symptoms from the Hamilton Anxiety Scale in patients with generalized anxiety Symptoms
Males
Females
Total
Psychiatric Somatic Total
15.50F1.55 18.00F1.90 33.50F3.33
19.40F1.33 19.60F1.20 39.00F1.71
17.45F1.24 18.80F1.11 36.25F1.92
896
E. Herna´ndez et al. / International Immunopharmacology 2 (2002) 893–900
males (24.90F9.66) and females (29.70F10.71), and the total average value was 27.30F7.04 months. Fourteen of the 20 patients received benzodiazepine
Table 2 Serotonin levels in plasma and lymphocytes of patients with generalized anxiety Sample
Males
Females
Plasma rich in platelets Plasma poor in platelets Lymphocytes
111.65F26.13 3.83F1.05 4.06F1.26
104.69F21.31 5.84F1.05 3.67F1.25
Plasma values in nanograms per milliliter. Lymphocytes values in nanograms per 106 cells.
treatment before taking the blood sample; however, all of them went into 2 weeks washout of any drug always under a physician’s or psychologist’s supervision. 3.2. Serotonin and 5-hydroxyindoleacetic acid levels in plasma and lymphocytes Platelet-rich and -poor plasma obtained as described in Materials and Methods were used to determine the levels of 5HT and 5HIAA. The values of the monoamine or the metabolite in both types of plasma and in lymphocytes were not different between controls and patients with generalized anxiety (Fig. 1), and did not differ in males and females (Table 2). 5HT turnover rate in lymphocytes, calculated as the ratio 5HT/5HIAA for each subject, neither presented significant changes between patients and controls (Fig. 2). Several correlations were performed between 5HT and 5HIAA in plasma-rich or -poor platelets as well
Fig. 1. 5HT and 5HIAA concentration in (A) platelet-rich plasma, (B) platelet-poor plasma, and (C) lymphocytes of control subjects and patients with generalized anxiety disorder.
Fig. 2. 5HT turnover rate expressed as the index 5HIAA/5HT in control subjects and in patients with generalized anxiety disorder.
E. Herna´ndez et al. / International Immunopharmacology 2 (2002) 893–900
897
and in lymphocytes, and between these measurements and the severity of the anxiety, resulting in nonsignificant differences. However, the correlation between 5HT levels in plasma-poor platelets and those in the lymphocytes was positive and significant in the patients, but not in the controls (Fig. 3). 3.3. [3H]Paroxetine binding sites in lymphocytes [3H]Paroxetine number and affinity of binding sites were not significantly different between controls and patients. Kd values were 2.96F1.01 and 4.40F1.27
Fig. 4. Number of maximal binding sites for [3H]paroxetine to lymphocyte membrane preparation of (A) control subjects and patients with generalized anxiety disorder, and (B) male and female patients with generalized anxiety disorder.
nM for controls and patients, respectively ( P=0.379). Bmax is reported in Fig. 4. Male and female results in the patients were separated and Bmax compared; there was no significant difference in the number of the transporter sites.
4. Discussion
Fig. 3. Correlation between 5HT concentration in platelet-poor plasma and 5HT concentration in lymphocytes in (A) control subjects, and (B) patients with generalized anxiety disorder, F=6.063, P=0.0248.
In a previous report, we demonstrated that in a group of patients with major depression disorder, there was a decreased number of 5HT transporters in blood peripheral lymphocytes and an increase in their number after treatment, as determined by the binding of [ 3 H]paroxetine to membrane preparations [13]. [3H]5HT transport into these cells was reported earlier [12] and an increase of it occurs in abstinent alcoholics [32]. These evidences indicate the possibility of modulation of 5HT transporter in lymphocytes during the course of different medical conditions.
898
E. Herna´ndez et al. / International Immunopharmacology 2 (2002) 893–900
Blood cells, such as platelets, are known to accumulate 5HT [33], and several reports indicate the presence and modulation of 5HT carrier in these cells [34,35]. The high concentration of 5HT as compared to 5HIAA in blood peripheral lymphocytes of controls and patients might indicate a low turnover rate, a high level of synthesis or a specialized storage system [36]. Synthesis of 5HT from tryptophan has been reported in rat lymphocytes [37]; however, such a demonstration is lacking in humans. Despite the lack of significant differences of 5HT and 5HIAA concentrations in platelet-rich or -poor plasma among the two groups of subjects, a clear difference was observed between the two plasma preparations, indicating the capacity of accumulating 5HT by lymphocytes and other blood cells, as shown by the relatively high level of 5HT, also taking place in the rat [37]. The significant and positive correlation between 5HT in platelet-poor plasma and 5HT in lymphocytes occurring in the patients could reflect a lack of regulation of the transporter in blood peripheral lymphocytes in generalized anxiety disorder, being unable to maintain endogenous levels by modulating the transport. By contrast, in control subjects, the endogenous levels were independent of the circulating free 5HT, probably indicating a stronger role of synthesis and storage for preserving 5HT concentration in lymphocytes. The number of [3H]paroxetine binding sites to lymphocyte membrane preparations did not show significant differences between controls and patients with generalized anxiety disorder, although in major depression disorder, a decrease in this parameter was reported [13], which might be a differential index between both psychiatric illnesses. However, in the present group of subjects, nonsignificant variations for all determined parameters were observed between genders; in addition, this disorder is as frequent in men as in women, also being different from depression, which it is known to be higher in women than in men (DSM-IV). Various immune system parameters, such as expression of CD62L, have been related to severity of anxiety disorders like panic disorder, which might be related to diminished cell activation in vivo [24]. Although the intent to find a correlation between 5HT or 5HIAA concentrations or [3H]paroxetine binding to severity of the anxiety was not successful, related to cell proliferation is the fact that beta-adrenergic recep-
tor responsiveness is altered in patients with anxiety or depression disorders [27]. Moreover, there is a considerable number of reports concerning cytokine levels [38], lymphocyte beta-adrenergic receptor function [39,40,41] and other parameters [25,42] in anxiety disorders. There is no report studying 5HT transporter function or modulation, despite that dysregulation of this carrier might be related to certain psychiatric disorders, such as those of the effect [43], although 5HT transporter expression in immortalized B lymphocytes is modulated by proinflammatory and anti-inflammatory cytokines [44]. In addition, functional promoter analysis and screening for length differences revealed polymorphism of 5HT human transporter, variations related to anxiety-behavioural traits [43], and differential initial response to selective 5HT uptake inhibitors [45]. The above quoted results indicate that some markers in the immune system, related to the central nervous system, might be altered in anxiety disorder patients; however, certain studies do not show significant changes. The fact that [3H]paroxetine binding to lymphocyte membrane preparations of patients with anxiety disorder was not significantly modified respecting values in a control group makes a difference with major depression in which case a reduction of this transporter was reported [13] or in abstinent alcoholics, who present high 5HT uptake sites [32]. Although there was no variation in [3H]paroxetine binding sites, the fact that the correlation of lymphocyte versus plasma concentration of 5HT is only significant in the group of patients suggests that functional differences in 5HT influx might be taking place in patients with generalized anxiety disorder. Acknowledgements This work was supported by the Grant G-1387 from Fondo Nacional de Ciencia, Tecnologı´a e Innovacio´n (FONACIT). We appreciate the secretarial assistance of Mrs. Isabel Otaegui. References [1] Felten DL, Felten SY, Bellinger DL, Carlsson SL, Ackerman KD, Madden KS, Olschowka JA, Livnat S. Noradrenergic sympathetic neuronal interactions with the immune system: structure and function. Immuno Rev 1987;100:225 – 60.
E. Herna´ndez et al. / International Immunopharmacology 2 (2002) 893–900 [2] Solomon GF. Psychoneuroimmunology: interactions between central nervous system and immune system. J Neurosci Res 1987;18:1 – 9. [3] Merrill JE, Jonakait GM. Interactions of the nervous and immune systems in development, normal brain homeostasis, and disease. FASEB J 1995;9:611 – 8. [4] Koenig JI. Presence of cytokines in the hypothalamic pituitary axis. Prog Neuroendocrinol Immunol 1991;4:143 – 53. [5] Amenta F, Bronzetti E, Fellici L, Ricci A, Tayebati SK. Dopamine D2-like receptors on human peripheral blood lymphocytes: a radioligand binding assay and immunocytochemical study. J Auton Pharmacol 1999;19:151 – 9. [6] Strom BT, Lane AM, George K. The parallel, time – time, bimodal change in lymphocyte cholinergic binding activity and cholinergic influence upon lymphocyte-mediated cytotoxicity. J Immunol 1981;127:705 – 10. [7] Rocca P, Ferrero P, Gualerzi A, Zanalda E, Maina G, Bergamasco B, Ravizza L. Peripheral type of benzodiazepine receptors in anxiety disorders. Acta Psychiatr Scand 1991;84:537 – 44. [8] Khan MMP, Sansoni P, Silverman EG, Engleman EG, Melmon KL. Beta-adrenergic receptors on human suppressor, helper, and cytolytic lymphocytes. Biochem Pharmacol 1986;35:1137 – 42. [9] Williams LT, Snyderman R, Lefkowitz RJ. Identification of beta-adrenergic receptors in human lymphocytes by [3H]alprenolol binding. J Clin Invest 1976;57:149 – 52. [10] Abdouh M, Storring JM, Riad M, Paquette Y, Albert PR, Drobetsky E, Kouassi E. Transcriptional mechanisms for induction of 5HT1A receptor mRNA and protein in activated B and T lymphocytes. J Biol Chem 2001;276:4382 – 8. [11] Aune TM, McGrath KM, Sarr T, Bombara MO, Kelley KA. Expression of 5HT1A receptors on activated human T cells: regulation of cAMP levels and T cell proliferation by 5HT. J Immunol 1993;151:1175 – 80. [12] Faraj BA, Olkowski L, Jackson RT. Expression of high-affinity serotonin transporter in human lymphocytes. Int J Immunopharmacol 1994;16:561 – 7. [13] Urbina M, Pineda S, Pin˜ango L, Carreira I, Lima L. [3H]Paroxetine binding to human peripheral lymphocyte membranes of patients with major depression before and after treatment with fluoxetine. Int J Immunopharmacol 1999;21:631 – 46. [14] Cyrlnik B. Ethology and anxiety in phylogeny and ontogeny. Acta Psychiatr Scand 1998;98:48 – 9. [15] Rosen JB, Schulkin J. From normal fear to pathological anxiety. Psychol Rev 1998;105:326 – 7. [16] American Psychiatry Association. DSM-IV: Diagnostic and Statistical Manual for Mental Disorders. 3rd edn. Washington, DC: APA, 1994. [17] Locke SE, Kraus L, Leserman J, Hurst MW, Heisel JS, Williams RM. Life change stress, psychiatric symptoms, and natural killer cell activity. Psychosom Med 1984;46:441 – 53. [18] Covelli V, Maffione AB, Nacci C, Tato E, Jirillo E. Stress, neuropsychiatric disorders and immunological effects exerted by benzodiazepines. Immunopharmacol Immunotoxicol 1998; 20:199 – 209. [19] Pawlikowski M, Lyson K, Kunter-Radek J, Stepien H. Effect of benzodiazepines on the proliferation of mouse spleen lymphocytes in vitro. J Neural Transm 1988;73:161 – 6.
899
[20] Zavala F. Benzodiazepines, anxiety and immunity. Pharmacol Ther 1997;75:100 – 216. [21] Ferrarese C, Appollonio I, Frigo M, Perego M, Pioti R, Trabucchi M, Frattola L. Decreased density of benzodiazepine receptors in lymphocytes of anxious patients: reversal after chronic diazepam treatment. Acta Psychiatr Scand 1990;82: 169 – 73. [22] Rocca P, Beoni AM, Eva C, Ferrero P, Zanalda E, Ravizza L. Peripheral benzodiazepine receptor messenger RNA is decreased in lymphocytes of generalized anxiety disorder patients. Biol Psychiatry 1998;43:767 – 73. [23] Sacerdote P, Panerai AE, Frattola L, Ferrarese C. Benzodiazepine-induced chemotaxis is impaired in monocytes from patients with generalized anxiety disorder. Psychoneuroendocrinology 1999;24:243 – 9. [24] Manfro GG, Pollack MH, Otto MW, Wothington JJ, Rosenbaun JF, Scott EL, Kradin RL. Cell-surface expression of Lselectin (CD62L) by blood lymphocytes: correlates with affective parameters and severity of panic disorder. Depression Anxiety 2000;11:31 – 7. [25] Rapaport MH. Circulating lymphocyte phenotypic surface markers in anxiety disorder patients and normal volunteers. Biol Psychiatry 1998;43:458 – 63. [26] Borella P, Bargellini A, Rovesti S, Pinelli M, Vivoli R, Solfrini G, Vivoli G. Emotional stability, anxiety and natural killer activity under examination stress. Psychoneuroendocrinology 1999;24:613 – 27. [27] Yu BH, Dimsdale JE, Mills PJ. Psychological states and lymphocyte betaadrenergic receptor responsiveness. Neuropsychopharmacology 1999;21:147 – 52. [28] Hamilton MA. The assessment of anxiety states by rating. Br Med Psychol 1959;32:35. [29] Lima L, Schmeer C. Characterization of serotonin transporter in goldfish retina by binding of [3H]paroxetine and the uptake of [3H]serotonin: modulation by light. J Neurochem 1994;62: 528 – 35. [30] GraphPad Prism, Version 2.0, San Diego, CA, 1998. [31] Motulsky H. Intuitive Biostatistics. New York: Oxford Univ. Press, 1995. [32] Faraj BA, Olkowski L, Jackson RT. Prevalence of high serotonin uptake in lymphocytes of abstinent alcoholics. Biochem Pharmacol 1997;53:53 – 7. [33] Sarrias MJ, Artigas F, Martı´nez E, Gelpi E, Udina C, Casas M. Decreased plasma serotonin in melancholic patients: a study with clomipramine. Biol Psychiatry 1987;22:1429 – 38. [34] Fozard J. Peripheral actions of 5-hydroxytryptamine. New York: Oxford Univ. Press, 1989. [35] Mellerup ET, Plenge P, Engelstoft M. High affinity binding of 3 H-paroxetine and 3H-imipramine to human platelet membranes. Eur J Pharmacol 1983;96:303 – 9. [36] Shannon NJ, Gunnet JW, Moore KE. A comparison of biochemical indices of 5-hydroxytryptaminergic neuronal activity following electrical stimulation of the dorsal raphe nucleus. J Neurochem 1986;47:958 – 65. [37] Ceden˜o N, Lima L, Obrego´n F, Urbina M. Synthesis of serotonin in peripheral blood lymphocytes of rats. Scand J Immunol 2001;54:1325.
900
E. Herna´ndez et al. / International Immunopharmacology 2 (2002) 893–900
[38] Zorrilla EP, Redel E, DeRubeis RJ. Reduced cytokine levels and T-cell function in healthy males: relation to individual differences in subclinical anxiety. Brain Behav Immunol 1994; 8:293 – 312. [39] Brambilla F, Bellodi L, Perna G, Garberi A, Panerai A, Sacerdote P. Lymphocyte cholecystokinin concentrations in panic disorder. Am J Psychiatry 1993;150:1111 – 3. [40] Brown SL, Charney DS, Woods SW, Heninger GR, Tallman J. Lymphocyte beta-adrenergic receptor binding in panic disorder. Psychopharmacology (Berlin) 1988;94:24 – 8. [41] Maddock RJ, Carter CS, Magliozi JR, Gietzen DW. Evidence that decreased function of lymphocyte beta adrenoreceptors reflects regulatory and adaptive processes in panic disorder with agoraphobia. Am J Psychiatry 1993;150: 1219 – 25. [42] Brambilla F, Bellodi L, Perna G, Battaglia M, Sciuto G, Diaferia G, Petraglia F, Panerai A, Sacerdote P. Psychoimmunoen-
[43]
[44]
[45]
[46]
docrine aspects of panic disorder. Neuropsychobiology 1992; 25:94 – 8. Heils A, Mossner R, Lesch KP. The human serotonin transporter gene polymorphism—basic research and clinical implications. J Neural Transm 1997;104:1005 – 14. Mossner R, Daniel S, Schmitt A, Albert D, Lesch KP. Modulation of serotonin transporter function by interleukine-4. Life Sci 2001;68:873 – 80. Pollock BG, Ferrell RE, Mulsant BH, Mazzumdar S, Miller M, Sweet RA, Davis S, Kirshner MA, Houck PR, Stack JA, Reynolds CF, Kupfer DJ. Allelic variation in the serotonin transporter promoter affects onset of paroxetine treatment response in late-life depression. Neuropsychopharmacology 2000;23: 587 – 90. Sokal R, Rohlf F. In: Blume H, editor. Biometrı´a. Madrid; 1989.