Characterization of serotonin transporter in blood lymphocytes of rats. Modulation by in vivo...

www.elsevier.com/locate/jneuroim

Journal of Neuroimmunolo

Characterization of serotonin transporter in blood lymphocytes of rats.

Modulation by in vivo administration of mitogens

Nemesio Cedeno1, Mary Urbina, Francisco Obregon, Lucimey Lima*

Laboratorio de Neuroquımica, Centro de Biofısica y Bioquımica, Instituto Venezolano de Investigaciones Cientıficas,

Apdo. 21827, Caracas 1020-A, Venezuela

Received 18 June 2004; received in revised form 16 September 2004; accepted 17 September 2004

Abstract

Serotonin transporter sites were characterized in blood lymphocytes of rats. Pharmacological characteristics of drug interactions were in

concordance with recent studies in nervous and human immune cells. The potency order of inhibition of [3H]paroxetine binding was

imipramineNcitalopramNalaproclateNserotonin. Selective inhibitors of dopamine or noradrenaline transporters did not inhibit it. The specific

binding of [3H]paroxetine was higher at intermediate than at low concentrations, and the plot of free vs. specific binding had a sigmoid shape.

The affinity constant or Kd, 1.77 nM, was in close agreement with data obtained from kinetic studies (Kd=1.33 nM), which evidences that the

equilibrium was reached. In addition, serotonin transporter was evaluated by lipopolysaccharide or concanavalin A administration in vivo

(0.1 mg/kg, i.p., 18 h). After the treatment with lipopolysaccharide, no changes were observed in the numbers of sites or Bmax or in the

affinity, Kd. The treatment with concanavalin A showed a significant reduction in Bmax and reduction in Kd. Additionally, serotonin and 5-

hydroxyindoleacetic acid levels were determined in plasma and lymphocytes by high-performance liquid chromatography. Treatment with

lipopolysaccharide produced a significant increased of serotonin levels in lymphocytes without changes in 5-hydroxyindoleacetic acid level;

in plasma, it produced an increase in serotonin and 5-hydroxyindolacetic acid levels. In addition, serotonin synthesis was evaluated by adding

300 AM of tryptophan in the medium, which significantly increased serotonin levels in control lymphocytes. Moreover, the concentrations of

5-hydroxyindoleacetic acid was enhanced significantly, both in plasma and lymphocytes in the presence of tryptophan after treatment with

lipopolysaccharide. The administration of concanavalin A significantly decreased plasma levels of serotonin, as well as the concentrations of

serotonin and 5-hydroxyindoleacetic acid in lymphocytes. These results demonstrate the presence of serotonin transporter in lymphocytes of

rat blood, the capacity for serotonin synthesis in lymphocytes, and the modulation of these parameters by systemic administration of

mitogens. The findings of this work contribute to understanding the immunological role of serotonin and the communication of immune and

nervous systems.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Concanavalin A; Lipopolysaccharide; Lymphocytes; Serotonin; Serotonin synthesis; Serotonin transporter

1. Introduction

Evidences for communication between immune and

nervous systems have been described, in which pro-

0165-5728/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.jneuroim.2004.09.010

* Corresponding author. Tel.: +58 212 504 1213; fax: +58 212 504

1295.

E-mail address: [email protected] (L. Lima).1 Present address: Universidad Romulo Gallegos, Departamento de

Ciencias Morfologicas y Funcionales, San Juan de los Morros, Guarico

2301, Venezuela.

nounced effects of neurotransmitters on immune functions

are associated (Haas and Schauenstein, 1997). Serotonin

(5-HT) is one of those neurotransmitters affecting the

immune system (Jackson et al., 1985).

During the last 20 years, 5-HT has been reported to

play an inhibitory role in the regulation of the immune

system (Jackson et al., 1985; Slauson et al., 1984;

Sternberg et al., 1986; Pellegrino and Bayer, 2000). On

the other hand, new proofs suggest that 5-HT up-regulates

natural killer (NK), T and B cells proliferation (Hellstrand

and Hermmodsson, 1990; Iken et al., 1995; Laberge et al.,

gy 159 (2005) 31–40

N. Cedeno et al. / Journal of Neuroimmunology 159 (2005) 31–4032

1996; Eugen-Olsen et al., 1997). Recently, inhibition of

fish T-cell proliferation by p-clorophenylalanine, an

inhibitor of 5-HT synthesis, and its reversal by exogenous

5-HT has been showed, which suggest that 5-HT is

implicated in T cell proliferation stimulated by phytohe-

maglutinin (PHA) (Ferriere et al., 1999). Nonetheless, a

biphasic dose–response effect of 5-HT on immune cells

activity has been described (Kut et al., 1992). Low dose

of 5-HT has a stimulatory effect whereas high dose has a

suppressive effect (Stefulj et al., 2001).

High concentrations of 5-HT are associated with capital

immune functions. For example, 5-HT induces release of

interleukine-16 (IL-16), cellular recruitment and release of

chemotactic factors and cytokines, as observed in the

inflammatory response (Laberge et al., 1996), and defends

NK cells from injure at inflammatory sites (Betten et al.,

2001). Additional evidences suggest that 5-HT might be

potentially participating in the development of cell-

mediated immune responses (Askenase, 1988; Matsuda

et al., 1997). Moreover, various reports further document

a wide range of 5-HT immune functions mediated through

interaction with 5-HT1A receptor subtype (Iken et al.,

1995; Eugen-Olsen et al., 1997), which has been

pharmacologically demonstrated on activated human T

cells (Aune et al., 1993), in rainbow trout peripheral

blood lymphocytes (Ferriere et al., 1996), after mitogenic

stimulation, in activated B and T lymphocytes of mice

(Abdouh et al., 2001), rats (Sempere et al., 2003), and

humans (Urbina et al., 1999) resting lymphocytes.

Thus, 5-HT is capable of producing different effects on

the immune system; however, a bewildering number of

signals are required in order to stimulate immune cells

(Mossner et al., 2001). One of the important character-

istics in 5-HT signaling is the precise function of 5-HT

transporter (5-HTT) that modulates the signaling and

determines the magnitude and length of serotonergic

responses. In fact, 5-HTT is present in the central nervous

system and in non-neuronal cells. For instance, 5-HTT

has been reported in human blood platelets (Rudnick,

1997), goldfish retina (Lima and Schmeer, 1994), adrenal

chromaffin cells (Blakely et al., 1995), and in the crypt

epithelial cells in the gastrointestinal tract (Jackson et al.,

1985; Wade et al., 1996). Similarly, 5-HTT has been

reported in immune cells; for example, murine (Bonnet et

al., 1984), rainbow trout (Ferriere et al., 1999), and

human lymphocytes (Faraj et al., 1997; Urbina et al.,

1999; Lima and Urbina, 2002). This emphasizes the

potential importance of 5-HT as immunomodulator. More-

over, 5-HTT was reported as a biochemical and pharma-

cological-modulated marker in lymphocytes of depressed

patients under pharmacological treatment (Lima and

Urbina, 2002; Urbina et al., 1999).

Due to the potential importance of 5-HT as an

immunomodulator, the aim of this work was the character-

ization of 5-HTT in blood lymphocytes of rats, which is an

unchallenged field. In addition, other objectives were to

determine 5-HT and 5-hydroxyindolacetic acid (5-HIAA)

concentrations in lymphocytes and in plasma, to study the in

vivo modulation of 5-HTT by mitogens, such as lip-

opolysaccharide (LPS) from E. coli serotype 0111:B4 and

concanavalin A (Con A), and to evaluate 5-HT synthesis

from tryptophan in lymphocytes of the rats.

2. Materials and methods

2.1. Chemicals

[3H]Paroxetine (17.1 Ci/mmol) was purchased from New

England Nuclear (Boston, MA, USA). The following

compounds were obtained from Sigma (St. Louis, MO,

USA): 5-HT creatine sulfate, desipramine HCl, imipramine

HCl. Alaproclate, GBR 12909, and maleate of nomifensine

were purchased from Research Biochemical. NycoPrep

gradient lymphocyte medium was obtained from Nycomed

Pharma (Oslo, Norway). All other chemicals were standard

laboratory reagents of analytical grade.

2.2. Experimental animals

Male Sprague–Dawley rats (Rattus norvegicus) ranging

in weight from 200 to 250 g were obtained from the

Instituto Venezolano de Investigaciones Cientıficas (IVIC)

hatchery. The animals were housed individually and were

kept under conventional conditions with commercial rat

food and tap water ad libitum.

2.3. Isolation of lymphocytes

Blood was taken by intracardial puncture, collected in

tubes and centrifuged at 500�g for 10 min in order to

separate white and red blood cells from the plasma. Next,

the white cell layer were withdrawn with a Pasteur pipette

into a 50-ml polypropylene graduated conical tube with

sodium phosphate saline buffer 0.1 M, pH 7.4 (PBS).

This preparation was centrifuged at 500�g for 30 min at

room temperature over a density gradient of NycoPrep.

Mononuclear cell layer was obtained by gradient centri-

fugation, harvested, washed twice with PBS, and centri-

fuged at 500�g for 10 min. To achieve a purified

lymphocyte preparation with a minimal monocyte con-

tamination, the resulting pellet was diluted with Roswell

Park Memorial Institute Medium 1640 (RPMI) free of

fetal bovine albumin and incubated into a culture flask for

45 min at 37 8C and 5% of CO2. After the incubation,

lymphocytes, which are non-adherent cells (~80% of

cells), were dislodged from adherent monocytes (~20%

of cells) by gentle agitation, transferred to plastic tubes

and washed twice. Finally, the pellet was resuspended in

50 mM Tris–HCl, pH 7.4, and the cell viability of

isolated lymphocytes was determined by Trypan blue

exclusion test, and was greater than 90%.

N. Cedeno et al. / Journal of Neuroimmunology 159 (2005) 31–40 33

2.4. Lymphocyte membrane preparation

Lymphocytes pellet (80–90% CD3+) were resuspended

in Tris–HCl 50 mM, pH 7.4, and homogenized in a

Tissumizer (Tekmar, Cincinnati, OH). Then, the preparation

was centrifuged in a refrigerated Sorvall RC-5 at 38,000�g

for 10 min at 4 8C. This step was repeated three times, and

the final pellet was resuspended in 50 mM Tris–HCl, 120

mM NaCl, 5 mM KCl, pH 7.4, and stored at �80 8C.Proteins were measured by the method of Lowry et al.

(1951) using albumin as standard.

2.5. Radioligand binding assays

In order to determine the proper cell concentration, an

experiment with various dilutions of membranes was

performed. Inhibition experiments were carried out with

the aim of determining concentration of drug producing

50% inhibition of binding (IC50). The selectivity of binding

inhibition interaction was performed by incubation of 100 Alof membrane preparation (106 cell/ml) in a final volume of

0.5 ml, with a concentration of 2 nM of [3H]paroxetine, in

the presence of 15 concentrations (0.1–1 mM) of the

following drugs: imipramine, alaproclate and citalopram,

and 5-HT, serotonin reuptake inhibitors (SSRIs); nomifen-

sine and GBR12090, selective inhibitors of dopamine

transporter; and desipramine, inhibitor of noradrenaline

uptake. The reaction was stopped by placing the tubes on

ice and adding cold buffer solution. Separation of the

ligand-binding site complex from the free ligand was carried

out by rapid filtration over Whatman GF/A glass fiber

filters. The filters were washed twice with 5 ml of cold

buffer using a vacuum equipment system (Brandel M-48),

which took less than 20 s, placed on vials, and dried.

Toluene/Omnifluor (0.4%), 4 ml, was added and radio-

activity was measured in a liquid scintillation counter

(Packard Tricarb Model 1900 CR, USA), efficiency 65%.

Saturation experiments were performed and the follow-

ing parameters were determined: the transporter density

(Bmax), the affinity, which is usually expressed as the

dissociation constant at equilibrium (Kd), and the Hill

coefficient (nH) (Bylun and Yamamura, 1990). Saturation

experiments were done with 100 Al of membrane prepara-

tion (106 cells/ml), and in the presence of increasing

concentrations of [3H]paroxetine (0.1–7 nM). The specific

binding was determined with 100 AM imipramine, a

tricyclic antidepressant and inhibitor of 5-HT uptake. The

tubes were prepared in duplicates and were incubated at 25

8C for 90 min since the equilibrium was reached during this

period.

Kinetic experiments were performed in order to deter-

mine the apparent association constant (kobs), and to

measure when the equilibrium was reached (Bylun and

Yamamura, 1990). Lymphocyte membranes were incubated

with 2 nM [3H]paroxetine; specific binding was defined in

the presence of 100 AM alaproclate. Four tubes were used

for each point, two for total binding and two for nonspecific

binding. Incubation times were 5, 10, 15, 20, 30, 60, and 90

min, respectively, at 25 8C. To determine dissociation rate

constant (k2), the membrane preparation was incubated for

90 min and the displacement of ligand bound at equilibrium

was assessed by the addition of 100 AM imipramine in 1 ml

of buffer solution (100 AM).

2.6. Determination of serotonin and 5-hydroxyindoleacetic

acid in plasma and lymphocytes

5-HT and 5-HIAA were determined by reversed-phase

high-performance liquid chromatography (HPLC) with

electrochemical detection. Lymphocytes were collected as

described above, resuspended and homogenized in 500 Al ofmobile phase, which was 0.02 M sodium acetate, 0.0125 M

citric acid, 1 mM ethilendiamine tetra acetic acid (EDTA),

1.52 mM octanylsulfonate, pH 3.9, plus 10% acetonitrile.

After centrifugation, 40 Al of the supernatant was injected

into the chromatographic system. The quantity of 5-HT and

5-HIAA in the lymphocytes was calculated from the area

under the curve of samples and external standards, and

expressed as ng/ml or ng/106 cells, respectively.

2.7. Modulation of serotonin transporter by lipopolysac-

charide and concanavalin A

Rats were treated with LPS 0.1 mg/kg (Linthorst and

Reul, 1998) or with Con A (Canavalia ensiforme, Jack

Bean, Sigma), which were administrated intraperitoneally

(i.p.) diluted in PBS. Controls received the vehicle. After 18

h, the blood samples were taken and lymphocyte mem-

branes were prepared according to the methodology

previously described. The transporter capacity was calcu-

lated by saturation experiments.

2.8. Serotonin synthesis in lymphocytes of rats treated with

lipopolysaccharide by high-performance liquid

chromatography

5-HT synthesis in lymphocytes was evaluated. Previ-

ously, a curve time vs. tryptophan concentration was

performed in order to obtain the optimal incubation time

and tryptophan concentration. Lymphocytes were incu-

bated for 10, 20, 30, and 60 min in the presence or

absence of 100, 200, 300, and 500 AM tryptophan at 37

8C. Then, lymphocytes of rats were incubated in RPMI

free of fetal bovine serum for 20 min at 37 8C in the

presence of 300 AM tryptophan and incubated into a

culture flask for 20 min at 37 8C and 5% of CO2. After

the incubation, lymphocytes were transferred to plastic

tube and washed twice. Finally, the pellet was resus-

pended and homogenized as described to determine 5-HT

intracellular concentration by HPLC. In order to evaluate

the effect of LPS on 5-HT synthesis in lymphocytes, rats

were treated i.p. with LPS, 0.1 mg/kg, as described above.

Fig. 1. Inhibition of the binding of [3H]paroxetine (2 nM) to rat

lymphocyte membranes in the presence of unlabeled drug concen-

trations from 0.1 to 1 nM, as described in Materials and methods.

Mean of duplicated determinations from one representative experiment.

R2=0.9577.


Finally, lymphocytes were obtained and the intracellular

concentration of 5-HT and 5-HIAA was determined

according to the indicated procedure.

2.9. Analysis of data

The software Biodata Handling with microcomputer

was used to calculate the concentration to produce 50%

inhibition (IC50), Hill coefficient (nH), and pseudocoeffi-

cient (pseudo-nH) (Hill, 1910; Barlow, 1983). The

software PRISM version 2.0 (GraphPad Software, 2003)

was used for the saturation experiments and calculation of

Bmax. Kd was calculated by the antilog of the log

concentration of ligand giving 50% of occupation in the

Hill plot and by biometric experiments (Hill, 1910), and

also by kinetic experiments, using k1 and k2 values. The

data are expressed as the meanFstandard error of the

mean (S.E.M.) from different and independent experi-

ments. Analysis of variance (ANOVA) and Student’s t-test

were used for statistical comparisons. The probability of

the differences between means was considered to be

significant if pb0.05.

3. Results

3.1. Inhibition experiments

The inhibition of [3H]paroxetine binding by the 5-HTT

inhibitors is shown in Table 1 and Fig. 1. The potency order

of inhibition was imipramineNcitalopramNalaproclateN5-HT.

The pseudo nH was less than 1 for selective inhibitory drugs.

Desipramine, nomifensine, and GBR-12909 did not inhibit

[3H]paroxetine binding.

3.2. Saturation experiments

The values of Bmax and Kd for the binding of [3H]pa-

roxetine to membranes of peripheral blood lymphocytes of

Table 1

Inhibition of [3H]paroxetine binding to membranes of lymphocytes from

blood of rats

Drugs IC50 (nM) CCM Pseudo nH

Imipramine 2.16F0.04 97F0.3 �0.91F0.16

Citalopram 15.70F1.3 92F5.0 �0.29F0.10

Alaproclate 26.40F1.2 95F1.2 �0.26F0.09

5-HT 398.45F102 88F1.8 �0.19F0.03

Desimipramine N1000

GBR12090 N1000

Nomifensine N1000

The potency of inhibition was determined by incubating the membrane

preparation with 2 nM [3H]paroxetine in the presence of 15 concentrations

of the corresponding drug as described in Materials and methods. Mean-

FS.E.M., n=6. IC50 was calculated by interactive analysis INHIBITION

(Barlow, 1983). CMC is coefficient of multiple correlation, nH is pseudo-

Hill coefficient.

rats were 6156F1714 fmol/106 cells, and 1.77F0.30 nM,

respectively. The analysis of the saturation curves showed

that they did not correspond to a hyperbolic curve

according to the Michaelis–Menten equation, but were

sigmoid, and the Scatchard plot consisted of an inverted

concave curve (Fig. 2A,B). The values of nH were larger

than 1 and the analysis of Klotz showed that the work

was done under saturation conditions (data not shown).

Fig. 2. Saturation experiments for the binding of [3H]paroxetine to

lymphocyte membrane. (A) Sigmoid curve representative of saturation

binding, 100 Al of membrane preparation were incubated in the presence

of 0.2–2 nM concentration of the ligand. Specific binding was defined

with imipramine. Incubation was carried out at 25 8C for 90 min.

MeanFS.E.M. of duplicate determination from one representative

experiment. (B) Scatchard plot obtained by the Bound/Free vs. Bound

data transformation.

Fig. 3. Association and dissociation kinetics of specific binding of [3H]paroxetine to membranes of blood lymphocytes. Each point is the meanFS.E.M. of

duplicate from one representative experiment. (A) The association constant was determined by incubating the membrane preparation with 2 nM of the ligand. The

specific binding was defined with alaproclate. kobs=0.070F0.014 min�1. (B) The dissociation constant was determined by the displacement of ligand bound with

100 AM of imipramine in 1 ml of buffer solution. Displacement was greater than 90% of specific binding. k1=0.019F0.007 min�1, Kd=1.33F0.02 nM.


3.3. Kinetic experiments

The association tests indicate that the status of equili-

brium reached up to 90 min. No degradation of the ligand

used was observed (Fig. 3A), kobs was 0.070F0.014 min�1.

The dissociation was fast and more than 90% of the specific

binding was displaced at the maximum time studied (Fig.

3B). k1 was 0.019F0.007 min�1. The value of Kd (k2/k1)

was 1.33F0.02 nM.

3.4. Modulation of serotonin transporter by mitogens

3.4.1. Binding of [3H]paroxetine to serotonin transporter in

membranes of lymphocytes from rats treated with

lipopolysaccharide

The results obtained in these experiments did not show

significant differences between Bmax and the Kd of specific

binding of [3H]paroxetine to membranes of lymphocytes of

rats treated with LPS when compared to the controls (Table

2). The curves were sigmoidal, the value of nH was larger

than 1, and the Scatchard was concave and inverted.

Table 2

Saturation parameters of the binding of [3H]paroxetine to membranes from

lymphocytes of rats treated with lipopolysaccharide

Bmax (fmol/106 cells) Kd (nM) nH

Control 6156F1714 1.27F0.33 2.37F0.21

LPS 5460F1572 2.27F0.32 1.59F0.19

Saturation experiments were performed with 100 Al of membrane

preparation in the presence of concentrations of 0.1–2 nM of the ligand.

Specific binding was defined with imipramine. MeanFS.E.M., n=6. Kd

was calculated by antilog of the log concentration of ligand giving 50% of

occupation in Hill plot (Hill, 1910; Barlow, 1983).

3.4.2. Determination of serotonin and 5-hydroxyindole-

acetic acid by high-performance liquid chromatography in

plasma and peripheral blood lymphocytes of rats treated

with lipopolysaccharide

The concentration of 5-HT in lymphocytes increased

after LPS treatment, and was significantly higher in the

animals treated as compared to the control group

( pb0.01). 5-HIAA in lymphocytes was not changed in

the animals after LPS administration (Fig. 4). The levels

of 5-HT in plasma increased after treatment with LPS,

Fig. 4. Serotonin and 5-hydroxyindoleacetic acid concentrations in

lymphocytes from controls and rats treated with lipopolysaccharide.

Lymphocytes were obtained as described in Materials and methods. The

concentrations were determined by reversed-phase HPLC with electro-

chemical detection and quantified from the area under the curve of sample

and external standards. MeanFS.E.M., n=6. *pb0.01 with respect to

control.

Fig. 7. Effect of tryptophan on 5-hydroxyindolacetic acid levels in

lymphocytes from control and rats treated with lipopolysaccharide.

Lymphocytes were incubated for 20 min at 25 8C in the absence (�) or

in the presence (+) of 300 AM tryptophan. Each value represents

meanFS.E.M., n=6, *pb0.05 with respect in the absence of tryptophan.

Fig. 5. Levels of serotonin and 5-hydroxyindoleacetic acid in plasma from

controls and rats treated with lipopolysaccharide. The concentrations were

determined by reversed-phase HPLC with electrochemical detection. The

amount present was calculated from the area under the curve of samples and

external standard. MeanFS.E.M., n=6. *pb0.01 with respect to control.


with significant differences between the controls and the

treated group. Plasma levels of 5-HIAA increased signi-

ficantly after the injection with LPS with respect to the

control ( pb0.01) (Fig. 5).

3.4.3. Evaluation of the synthesis of serotonin from

tryptophan in peripheral blood lymphocytes of rats treated

with lipopolysaccharide

In the LPS-treated group, a significant increase of the 5-

HT levels was observed with respect to the control group

( pb0.05) while a significant reduction of 5-HT levels was

observed in the presence of tryptophan (Fig. 6). No

significant difference was observed in 5-HIAA levels in

the controls with respect to those treated in the absence of

tryptophan. When the cells were incubated with tryptophan,

Fig. 6. Effect of tryptophan on serotonin levels in lymphocytes from

controls and rats treated with lipopolysaccharide. Lymphocytes were

incubated for 20 min at 25 8C in the absence (�) or in the presence (+)

of 300 AM tryptophan. MeanFS.E.M., n=6, *pb0.05 with respect to

control in the absence of tryptophan, **pb0.05 with respect to lip-

opolysaccharide in the presence of tryptophan.

there was a significant increase of this metabolite, both in

the control group as well as in the treated group (Fig. 7).

3.4.4. Saturation binding of [3H]paroxetine to serotonin

transporter in lymphocyte membranes from rats treated with

concanavalin A

The results obtained in these experiments indicate a

significant reduction ( pb0.01) in Bmax and the Kd of

[3H]paroxetine binding to membranes of lymphocytes from

rats treated with Con A in comparison to controls (Table 3).

The saturation curves showed sigmoid behavior, both in the

controls and in the treated animals, and a Hill coefficient

larger than 1.

3.4.5. Determination of serotonin and 5-hydroxyindole-

acetic acid by high-performance liquid chromatography in

plasma and peripheral blood lymphocytes of rats treated

with concanavalin A

The results of these tests are shown in Figs. 8 and 9. A

significant reduction of the 5-HT concentration was

observed in treated animals with respect to controls

( pb0.01). 5-HIAA also decreased significantly 18 h after

the administration of the mitogen ( pb0.01). In plasma, the

5-HT levels decreased significantly in the treated animals

Table 3

Saturation parameters of the binding of [3H]paroxetine to membranes of rat

lymphocytes treated with concanavalin A

Bmax (fmol/106 cells) Kd (nM) nH

Control 2490F473 2.68F0.25 1.35F0.23

Con A 1362F415* 0.73F0.24* 2.07F0.43

Saturation experiments were performed with 100 Al of membrane

preparation in the presence of 0.1–2 nM of ligand. Specific binding was

defined with imipramine. MeanFS.E.M., n=6. Kd was calculated by antilog

of the log concentration of ligand giving 50% of occupation in Hill plot.

* pb0.005. (Hill, 1910; Barlow, 1983).

Fig. 8. Serotonin and 5-hydroxyindolacetic acid concentrations in

lymphocytes from control and rats treated with Con A. Lymphocytes were

obtained as described in Materials and methods. The concentrations were

determined by reversed-phase HPLC with electrochemical detection. The

amount present was calculated from the area under the curve of samples and

external standard. Mean S.E.M., n=6. *pb0.01 with respect to control.


( pb0.01). In relation to 5-HIAA, no significant differences

were observed in the treated group with respect to the

controls.

Fig. 9. Levels of serotonin and 5-hydroxyindoleacetic acid in plasma from

control and rats treated with Con A. The concentrations were determined by

reversed-phase HPLC with electrochemical detection and calculated as

described before. MeanFS.E.M., n=6. *pb0.01 with respect to control.

4. Discussion

5-HTT has been demonstrated in human immune cells, in

which might have important physiological and pathological

implications (Faraj et al., 1994; Urbina et al., 1999;

Marazziti et al., 2001). The present study shows, for the

first time, the presence of 5-HTT in peripheral blood

lymphocytes of rats. The general characteristics of this

transporter are similar to those of the 5-HTT in neuronal

(Lima et al., 1995) and non-neural tissues (Urbina et al.,

1999; Ferriere et al., 1999). For example, dependence of

time, temperature, and membrane concentrations, as well as

stability, reversibility, saturability, specificity, and high

affinity were similar to the mentioned preparations. The

specific binding of [3H]paroxetine to rat lymphocytes in the

present report was defined with imipramine, according to

the inhibition obtained with various other drugs (Fig. 1,

Table 1). [3H]Paroxetine has been used to characterize 5-

HTT because is a powerful and highly specific 5-HT re-

uptake inhibitor (Schloss and Williams, 1998). Also, it has

been widely used to characterize 5-HTT in different tissues

as a specific marker (Rotondo et al., 1996; Brust et al.,

1996; Marazziti et al., 1998). In our experiments, 5-HTT in

rat lymphocytes was labeled with the same affinity and

specificity as described for other tissues. Differences in Bmax

of controls, although capacity and affinity were in the same

range in all experiments, could be due to lots of animals and

to unspecific activation during the processes, reasons for

including own controls in each experiment.

The specific binding of this ligand to rat lymphocytes

was higher at intermediate than at low concentrations, and

the plot of free vs. specific binding had a sigmoid shape

(Fig. 2). The Scatchard plot provided a concave curve, and

the nH value was greater than 1, indicating positive

cooperativity. These results suggest an allosteric regulation

with a modulatory site in 5-HTT of rat lymphocytes. Similar

results were showed in goldfish retina (Lima and Schmeer,

1994) and in rainbow trout peripheral blood lymphocytes

(Ferriere et al., 1999). The kinetic parameters, apparent

association and dissociation rate constants, evidenced that

the equilibrium was reached with no ligand degradation.

Moreover, the values of those constants were in the same

range as those reported for neuronal tissues (Lima and

Schmeer, 1994; Lima et al., 1995; Brust et al., 1996).

The pharmacological and biochemical characteristics of

5-HTT in lymphocytes of rats are in concordance with

recent studies in nervous cells (Inazu et al., 2001). But,

probably, there are different sites of interaction that are

reflected in the order of inhibitory potency obtained for the

tested inhibitors. In the present study, imipramine, which is

a potent inhibitor of 5-HT uptake (Chen et al., 1983), was a

powerful inhibitor of [3H]paroxetine binding to lymphocyte

membranes of rats, more than citalopram and alaproclate,

whereas 5-HT had a lower potency. Similar inhibitory

results with imipramine have been reported in goldfish

retina (Lima and Schmeer, 1994) and rat astrocytes (Inazu et

al., 2001). Another important factor supporting the presence

of 5-HTT in lymphocytes of rats with similarities to

neuronal cells is the distinction of 5-HTT system from

norepinephrine and dopamine transporters. For example,

desimipramine, a norepinephrine selective uptake inhibitor,

was unable to inhibit [3H]paroxetine binding to lymphocyte

membranes. Similarly, neither nomifensine nor GBR 12909,

selective inhibitors of dopamine transporter (Anderson,


1989; Tella and Goldberg, 2001; Moron et al., 2002),

blocked the binding of the radioligand.

5-HTT in lymphocytes of rat might be under same

genetic control as in other tissues. For instance, in human

brain and platelets, 5-HTT is codified by the same gene,

human chromosome 17 (Blakely et al., 1991; Lesch et al.,

1994, 1996), which was also cloned and evaluated in human

placenta (Ramamoorthy et al., 1993).

LPS (Hancock, 1991) induces a variety of inflammatory

and toxic effects (Mathew, 1998). When evaluating the

capacity of 5-HTT in lymphocytes from blood of rats after

treatment with LPS, no changes were observed in the

number of sites or the affinity of the ligand for 5-HTT

(Table 1), even though changes of the serotonergic system in

the brain by peripheral administration of LPS have been

shown (Linthorst and Reul, 1998).

Treatment with LPS produced a significant increase of

5-HT levels in lymphocytes in peripheral blood of rats

(Fig. 4). It might be that high concentrations of 5-HT in

lymphocytes could mediate some of the effects in

response to the administered dose of LPS. In immune

system cells, the immunoregulatory effect of 5-HT seems

to be partially mediated through 5-HT1A receptors

(Hellstrand and Hermmodsson, 1990; Aune et al., 1993;

Sempere et al., 2003; Eugen-Olsen et al., 1997). In rats,

5-HT and the 5HT1A agonist, 8-hydroxy-2(di-n-propilami-

no)tetralin, produce an increase in the proliferation of

mitogen-stimulated B cells (Iken et al., 1995). These

mechanisms, among others, could be taking place in

lymphocytes of LPS-treated rats.

The high 5-HT concentration observed in rat lympho-

cytes may be due to the activation of its synthesis through

stimulation with LPS, like that exercised by INF-g in

human mononuclear cells (Arzt et al., 1988). This

reaffirms the property of immune system cells of

producing not only cytokines, but also neuropeptides,

hormones, and neurotransmitters (Blalock et al., 1992;

Blalock, 1994). When evaluating the synthesis of this

indolamine, a significant increase in 5-HT levels was

observed in lymphocytes of rats that were treated with

LPS. This might be influenced by an increase in the

synthesis of 5-HT, or by affection of 5-HT recapturing

mechanisms, even though the number of carriers was not

significantly changed.

When adding tryptophan to the medium, a lowering of

the 5-HT levels was observed in the lymphocytes of rats

treated with LPS. Possibly this decrease in the intra-

cellular concentration of 5-HT is the result of a greater

release of 5-HT because of the effect of LPS and the use

of tryptophan available in the medium for 5-HT synthesis.

Probably, once the synthesis of 5-HT is activated and

released to the extracellular medium, it will favor either

clonal expansion (Finocchiario et al., 1988; Jackson et al.,

1985; Kut et al., 1992) or the presence of other immune

cell lines that may mediate a greater release of 5-HT

(Matsuda et al., 1997). 5-HIAA levels in lymphocytes

were not changed after the injection of LPS, and this

supports the idea of an increase in the origin of 5-HT, as

well as a possible reduction of catabolism. The concen-

trations of 5-HIAA increased significantly both in plasma

and lymphocytes in the presence of tryptophan after

treatment with LPS, although the intracellular 5-HIAA

levels did not change after administering LPS in the

absence of tryptophan. These results reflect a higher

metabolic replacement of 5-HT or increased turnover rate,

produced by LPS, evidenced when supplying the substrate

to the cells.

The general effects of LPS on T cells does not appear

to be direct, because it is not thymus-dependent, while

the proliferative response of this cell stock increases after

stimulation with Con A (Chen et al., 1995). Increased

expression of the 5-HT1A receptor has been shown in T

cells activated with PHA, which could be related to the

lymphoproliferative effect of 5-HT (Aune et al., 1993;

Iken et al., 1995). In our results, the number of binding

sites showed a significant reduction; however, the affinity

was increased by Con A, which might suggest the

presence of an autocrine mechanism through which

lymphocytes regulate their own activity. This autocrine

regulation mechanism by lectins has been described for

different subpopulations of human immune cells (Beuth

et al., 1992). In addition to this mechanism, possibly the

populations of lymphocytes modulated by the various

treatments are not the same, and the obtained results

correspond to the conjoint interaction of many factors.

Moreover, the administration of Con A significantly

decreased plasma levels of 5-HT, as well as the

intracellular concentrations of 5-HT and 5-HIAA in

lymphocytes. These results might be favoring lymphocyte

proliferation, the induction of the expression of surface

receptors that are closely related to the activation of

immune system cells, such as IL-2 and 5-HT1A receptors

(Waldmann, 1986; Oshima et al., 1991; Aune et al.,

1993; Sempere et al., 2003). Both receptors would play

a critical role in the evolution of specialized T cell

populations, necessary to establish the normal response

of those cells. If the administration of Con A favors

cellular proliferation per se, lymphocytes would regulate

their activity via an autocrine way involving 5-HT, in

which the expression of the number of 5-HTT could

play a modulatory role.

The observations reported in the literature when

combined with the results obtained in this work

support the idea of a strong immunomodulatory role

of 5-HT through producing certain specific actions

that promote and regulate immune responses.

Acknowledgements

This work was supported by Grant S1-3490 from

Fondo Nacional de Investigacion, Tecnologıa e Innova-


cion (FONACIT). We appreciate the secretarial assistance

of Mrs. Isabel Otaegui. Nemesio Cedeno was a Graduate

Student of Centro de Estudios Avanzados (CEA) from

Instituto Venezolano de Investigaciones Cientıficas

(IVIC).

References

Abdouh, M., Storring, J.M., Riad, M., Paquette, Y., Albert, P.R., Drobetsky,

E., Kouassi, E., 2001. Transcriptional mechanisms for induction of 5-

HT1A receptor mRNA and protein in activated B and T lymphocytes.

J. Biol. Chem. 276, 4382–4388.

Anderson, P.H., 1989. The dopamine uptake inhibitor GBR 12909:

selective and molecular mechanism of action. Eur. J. Pharmacol. 166,

493–504.

Arzt, E.S., Fernandez-Castelo, S., Finocchiaro, L.M., Criscuolo, M.E.,

Dıaz, A., Askenase, P.W., 1988. Effector and regulatory mechanism in

delayed-type hypersensitivity. In: Middleton Jr., E., Reed, C.E., Ellis,

E.F., Atkinson, N.F. (Eds.), En Allergy: Principles and Practice (2nd

ed.). C.V. Mosby, St. Lluis.

Askenase, P.W., 1988. Effector and regulatory mechanism in delayed-type

hypersensitivity. In: Middleton Jr., E., Reed, C.E., Ellis, E.F., Atkinson,

N.F. (Eds.), En Allergy: Principles and Practice (2nd ed.). C.V. Mosby,

St. Lluis.

Aune, T.M., McGrath, K.M., Sarr, T., Bombara, M.P., Kelley, K.A.,

1993. Expression of 5-HT1A receptors on activated human T cells.

Regulation of cAMP levels and T cell proliferation by 5-hydroxy-

tryptamine. J. Immunol. 151, 1175–1183.

Barlow, R.W., 1983. Biodata Handling with Microcomputers. Elsevier

Science Publishers, Amsterdam, pp. 125–143.

Betten, A., Dahigren, C., Hermodsson, S., Hellstrand, K., 2001. Serotonin

protects NK cells against oxidatively induced functional inhibition and

apoptosis. J. Leukoc. Biol. 70, 65–72.

Beuth, J., Ko, H.L., Gabius, H.J., Burrichter, H., Oette, K., Pulverer, G.,

1992. Behavior of lymphocyte subsets and expression of activation

markers in response to immunotherapy with galactoside-specific lectin

from mistletoe in breast cancer patients. Clin. Investig. 70, 658–661.

Blakely, R., Berson, H.E., Fremeau, R.T., Caron, M.G., Peek, M.M., Prince,

H.K., Bradley, C.C., 1991. Cloning and expression of a functional

serotonin transporter from rat brain. Nature 354, 66–70.

Blakely, R.D., Wise, K.R., Schroeter, S., 1995. Localization of the

serotonin transporter to rodent adrenal chromaffin cells: support for a

role of 5-HT in adrenal physiology. Abstr.-Soc. Neurosci. 21, 316.1.

Blalock, J.E., 1994. The syntax of immune-neuroendocrine communication.

Immunol. Today 15, 504–511.

Blalock, J.E., Eds, K., Ishizaka, Lachman, P.J., Waskman, B.H., 1992.

Production of peptide hormones and neurotransmitters by the immune

system. Chem. Immunol. 52, 1–24.

Bonnet, M., Lespinats, G., Burtin, C., 1984. Histamine and serotonin

suppression on lymphocyte response phytohemagglutinin and alloge-

neic cells. Cell. Immunol. 83, 280–291.

Brust, P., Bergmann, R., Johannsen, B., 1996. High-affinity binding of

[3H]paroxetine to caudate nucleus and microvessels from porcine brain.

NeuroReport 31, 1405–1408.

Bylun, D.V., Yamamura, H.I., 1990. Methods in Neurotransmitter Receptor

Analysis. Reaven Press, New York.

Chen, S.S., Tung, J.S., Good, R.A., Hadden, J.W., 1983. Changes in surface

antigens of immature thymocytes under the influence of T cell growth

and thymic factors. Proc. Natl. Acad. Sci. 80, 5980–5985.

Chen, C., David, A., Nunnerley, H., Michel, M., Dawson, L., Berry, H.,

Dobbs, J., Fahy, T., 1995. Adverse life events and breast cancer: case-

control study. Med. J. 311, 1527–1530.

Eugen-Olsen, J., Afzelius, P., Andersen, L., Iversen, J., Kronborg, G.A.,

Aebech, P., Nielsen, J.O., Hofmann, B., 1997. Serotonin modulates

immune function in T cells from HIV-seropositive subjects. Clin.

Immunol. Immunopharmacol. 84, 115–121.

Faraj, B.A., Olkowski, Z.L., Jackson, R.T., 1994. Expression of high

affinity serotonin transporter in human lymphocytes. Int. J. Immuno-

pharmacol. 16, 561–567.

Faraj, B.A., Olkowski, Z.L., Jackson, R.T., 1997. Prevalence of high

serotonin uptake in lymphocytes of abstinent alcoholics. Biochem.

Pharmacol. 53, 53–57.

Ferriere, F., Khan, N.A., Troutaud, D., Deschaux, P., 1996. Serotonin

modulation of lymphocyte proliferation via 5-HT1A receptors in

rainbow trout (Oncorhynchus mykiss). Dev. Comp. Immunol. 20,

273–283.

Ferriere, F., Khan, N.A., Meyniel, J.P., Deschaux, P., 1999. 5-Hydroxytrypt-

amine-induce calcium-channel gating in rainbow trout (Oncorhynchus

mykiss) peripheral blood lymphocytes. Biochem. J. 323, 251–258.

Finocchiario, L.M., Artz, S., Fernandez, M., Criscuolo, A., Dıaz, S.,

Finkielman, S., Nahrod, V.E., 1988. Serotonin and melatonin synthesis

in peripheral blood mononuclear cells stimulated by interferon-gamma

as part of immunomodulatory pathway. J. Interf. Res. 8, 705–711.

Haas, H.S., Schauenstein, K., 1997. Neuroimmunomodulation via limbic

structures—The neuroanatomy of psychoimmunology. Prog. Neurobiol.

51, 195–222.

Hancock, R.E.W., 1991. Bacterial outer membranes: evolving concepts.

Specific structures provide gram-negative bacteria with several unique

advantages. ASM News 57, 175.

Hellstrand, K., Hermmodsson, S., 1990. Enhancement of human natural

killer cell cytotoxicity by serotonin: role of non-T/CD16+ NK cells,

accessory monocytes, and 5HT1A receptors. Cell. Immunol. 127,

199–214.

Hill, A.V., 1910. The possible effects of the aggregation of molecules of

hemoglobin on its dissociation curves. J. Physiol. 1, iv–vii.

Iken, K., Chheng, S., Fargin, A., Goulet, A., Kouassi, E., 1995. Serotonin

upregulates mitogen-stimulated B lymphocyte proliferation through 5-

HT1A receptors. Cell. Immunol. 163, 1–9.

Inazu, M., Takeda, H., Ikoshi, H., Sugisawa, M., Uchida, Y., Matsumiya, T.,

2001. Pharmacological characterization and visualization of the glial

serotonin transporter. Neurochem. Int. 39, 39–49.

Jackson, J.C., Cross, R.J., Walker, R.F., Markesbery, W.R., Brooks, W.H.,

Roszman, T.L., 1985. Influence of serotonin on the immune response.

Immunology 54, 503–511.

Kut, J.P., Young, R.M., Crayton, J.W., Wright, M.A., Young, M.E.,

1992. Regulation of murine T-lymphocyte function by spleen cell-

derived and exogenous serotonin. Immunopharmacol. Immunotoxicol.

14, 783–796.

Laberge, S., Cruikshank, W.W., Beer, D.J., Center, D.M., 1996. Secretion

of IL-16 (lymphocyte chemottractant factor) from serotonin-stimulated

CD8+ T cells in vitro. J. Immunol. 156, 310–315.

Lesch, K.-P., Balling, U., Gross, J., Strauss, K., Wolozin, B.L., Murphy,

D.L., Riederer, P., 1994. Organization of the human serotonin

transporter gene. J. Neural Transm. 95, 157–162.

Lesch, K.P., Bengel, D., Heils, A., Sabol, S.Z., Greenberg, B.D.,

Petri, S., Benjamin, J., Muller, C.R., Hamer, D.H., Murphy, D.L.,

1996. Association of anxiety-related traits with a polymorphism in

the serotonin transporter gene regulatory region. Science 274,

1527–2530.

Lima, L., Schmeer, C., 1994. Characterization of serotonin transporter in

goldfish retina by binding of [3H]paroxetine in the uptake of

[3H]serotonin: modulation by light. J. Neurochem. 62, 528–535.

Lima, L., Urbina, M., 2002. Serotonin transporter modulation in blood

lymphocytes from patients with major depression. Cell. Mol. Neurobiol.

22, 797–804.

Lima, L., Trejo, E., Urbina, M., 1995. Serotonin turnover rate, [3H]Parox-

etine binding sites, and 5-HT1A receptors in the hippocampus of rats

subchronically treated with clonazepam. Neuropharmacology 34,

1327–1333.

Linthorst, A., Reul, J.M.H., 1998. Brain neurotransmission during

peripheral inflammation. Ann. N.Y. Acad. Sci. 840, 139–152.


Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randal, R.J., 1951. Protein

measurement with folin phenol reagent. J. Biol. Chem. 193, 265–275.

Marazziti, D., Rossi, A., Giannaccini, G., Baroni, S., Lucacchini, A.,

Cassano, G., 1998. Presence and characterization of the serotonin

transporter in human resting lymphocytes. Neuropsychopharmacology

19, 154–159.

Marazziti, D., Ori, M., Nardini, M., Rossi, A., Nardi, I., Cassano, G.B.,

2001. mRNA expression of serotonin receptors of type 2C and 5A in

human resting lymphocytes. Neuropsychobiology 43, 123–126.

Mathew, P., 1998. Specificity and function of lipopolysaccaride antibodies.

In: Szentivanyi, A., Friedman, H.Y., Nowotny, A. (Eds.), En Immuno-

pharmacol. and Pathophysiol. of Bacterial Endotoxins, vol. II. Plenum

Press, New York, pp. 347–374.

Matsuda, H., Ushio, H., Geba, G.P., Askenase, P., 1997. Human platelets

can initiate T cell-dependent contact sensitivity through local serotonin

release mediated by IgE antibodies. J. Immunol. 158, 2891–2897.

Moron, J.A., Brockington, A., Wise, R.A., Rocha, B.A., Hope, B.T., 2002.

Dopamine uptake through the norepinephrine transporter in brain

regions with low levels of the dopamine transporter: evidence from

knock-out mouse lines. J. Neurosci. 22, 389–395.

Mossner, R., Daniel, S., Schmitt, A., Albert, D., Lesch, K.P., 2001.

Modulation of serotonin transporter function by interleukin-4. Life Sci.

68, 873–880.

Oshima, Y., Ko, H.L., Beuth, J., Burrichter, H., Oette, K., Pulverer, G.,

1991. Activation of mononuclear immune cells in response to

staphylococcal lipoteichoic acid. Zentralbl. Bakteriol. 275, 374–381.

Pellegrino, T.C., Bayer, B.M., 2000. Specific serotonin reuptake inhibitor-

induced decreases in lymphocyte activity require endogenous serotonin

release. Neuroimmunomodulation 8, 179–187.

Ramamoorthy, J.D., Leibach, F.H., Maeshh, V.B., Ganapathy, V., 1993.

Partial purification and characterization of the human placental

serotonin transporter. Placenta 14, 449–461.

Rotondo, A., Giannaccini, G., Betti, L., Chiellini, G., Marazziti, D., Martin,

C., Lucacchini, A., Cassano, G.B., 1996. The serotonin transporter from

human brain: purification and partial characterization. Neurochem. Int.

28, 299–307.

Rudnick, G., 1997. Active transporter of 5-hydroxytryptamine by plasma

membrane vesicles isolated from human blood platelets. J. Biol. Chem.

252, 2170–2174.

Schloss, P., Williams, D.C., 1998. The serotonin transporter: a primary

target for antidepressant drugs. J. Psychopharmacol. 12, 115–121.

Sempere, T., Cedeno, N., Urbina, M., Lima, L., 2003. 8-[3H]-hydroxy-2-

(di-n-propylamino)tetralin binding sites in blood lymphocytes of rats

and the modulation by mitogens and immobilization. J. Neuroimmunol.

138, 8–16.

Slauson, D.O., Walker, C., Kristensen, F., Wang, Y., De Weck, A.L., 1984.

Mechanisms of serotonin-induced lymphocyte proliferation inhibition.

Cell. Immunol. 84, 240–252.

Stefulj, J., Cicin-Sain, L., Schauenstein, K., Jernej, B., 2001. Serotonin and

immune response: effect of the amine on in vitro proliferation of rat

lymphocytes. Neuroimmunomodulation 9, 103–108.

Sternberg, E.M., Trial, J., Parker, C.W., 1986. Effect of serotonin on

murine macrophages: suppression of Ia expression by serotonin and its

reversal by 5-HT2 serotononergic receptor antagonists. J. Immunol.

137, 276–282.

Tella, S.R., Goldberg, S.R., 2001. Subtle differences in the discriminative

stimulus effects of cocaine and GBR-12909. Prog. Neuro-psychophar-

macol. Biol. Psychiatry 25, 639–656.

Urbina, M., Pineda, S., Pinango, L., Carreira, I., Lima, L., 1999.

[3H]Paroxetine binding to human peripheral lymphocyte membranes

of patients with major depression before and after treatment with

fluoxetine. Int. J. Immunopharmacol. 21, 631–646.

Wade, P.R., Chen, J., Jaffe, B., Kassem, I.S., Blakely, R.D., Gershon, M.D.,

1996. Localization and function of a 5-HT transporter in crypt epithelia

of the gastrointestinal tract. J. Neurosci. 16, 2352–2364.

Waldmann, T.M., 1986. The structure, function and expression of

interleukin-2 receptor on normal and malignant lymphocytes. Science

232, 727–732.

Characterization of serotonin transporter in blood lymphocytes of rats. Modulation by in vivo...

Documents

Transcript of Characterization of serotonin transporter in blood lymphocytes of rats. Modulation by in vivo...