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5-HT7 Receptors and Tryptophan Hydroxylase in Lymphocytes of Rats:

Mitogen Activation, Physical Restraint or Treatment with Reserpine

Article  in  NeuroImmunoModulation · March 2014

DOI: 10.1159/000357148 · Source: PubMed

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 Original Paper
Neuroimmunomodulation 2014;21:240–249
DOI: 10.1159/000357148
5-HT7 Receptors and Tryptophan Hydroxylase
in Lymphoctytes of Rats: Mitogen Activation,
Physical Restraint or Treatment with Reserpine
Mary Urbina Rubén Arroyo Lucimey Lima 
Laboratorio de Neuroquímica, Centro de Biofísica y Bioquímica, Instituto Venezolano de Investigaciones
Científicas (IVIC), Caracas, Venezuela
Key Words
Lymphocytes · Serotonin 5-HT7 receptors · Stress ·
Tryptophan hydroxylase
Abstract
Objectives: Serotonin (5-HT)7 receptors in lymphocytes play
a relevant role as modulators of T cell functions and might
be modified by stress protocols. The aims of this work were
to evaluate: (i) the presence of 5-HT7 receptors in specific
lymphocyte populations, (ii) the probable modifications of
them by inflammatory stress with mitogen and (iii) the ef-
fects of physical and pharmacological stress. Methods: Blood
lymphocytes were isolated by density gradients and differ-
ential adhesion to plastic. Concanavalin A (Con A) was sys-
temically administered (500 μg/kg) or added to lymphocyte
cultures (2.5 μg/ml, final volume 200 μl). Physical restraint
was performed in Plexiglass boxes for 5 h per day for 5 days.
Reserpine administration was 2.5 mg/kg for 3 days. Immuno-
cytochemical labeling of CD4+, CD8+ and 5-HT7 receptors,
and also tryptophan hydroxylase cells was performed. mRNA
of 5-HT7 receptors was evaluated by RT-PCR. Controls were
included for each protocol. Results: Con A treatment or cul-
ture exposure increased the number of lymphocytes ex-
pressing 5-HT7 receptors or tryptophan hydroxylase, as com-
pared to absence of the mitogen. Receptors were present in
12–16% of total rat lymphocytes, in ∼10% of CD4+ and in
© 2014 S. Karger AG, Basel
1021–7401/14/0215–0240$39.50/0
E-Mail karger@karger.com
www.karger.com/nim
Received: August 16, 2013
Accepted after revision: November 6, 2013
Published online: March 1, 2014
∼5% of CD8+ cells from control rats. CD4+ decreased, and
CD8+ and 5-HT7 cells increased after physical restraint. Re-
serpine treatment elevated CD8+ and 5-HT7 cells. Con A and
physical restraint, but not reserpine treatment, significantly
augmented 5-HT7 receptor mRNA in lymphocytes. Conclu-
sions: Rat lymphocytes, expressing tryptophan hydroxylase,
could synthesize 5-HT, functioning as a direct autocrine
modulator. The modifications of CD4+, CD8+ and 5-HT7 re-
ceptors in lymphocytes by three stress protocols could have
an impact on immune responses. In addition, the differential
distribution of 5-HT7 receptors indicates potential specific
physiopathological roles. © 2014 S. Karger AG, Basel
Introduction
Monoamines are known regulators of the immune
system [1, 2]. In addition, serotonin (5-HT)1A receptors
in lymphocytes are modified by stress protocols, such as
physical restraint, or by mitogen activation [3, 4] and re-
serpine treatment [4]. For instance concanavalin A (Con
A) increases 5-HT1A receptors in lymphocytes [3] and af-
fects the effect of fluoxetine on proliferation [4]. Reser-
pine, acting on the sympathetic system as an inhibitor of
vesicular transport of monoamines, constitutes a docu-
mented pharmacological stress [5] and alters the 5-HT
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Humbold-Universität zu Berlin
Dr. Lucimey Lima
Laboratorio de Neuroquímica, Centro de Biofísica y Bioquímica
Instituto Venezolano de Investigaciones Científicas, Altos de Pipe
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Carretera Panamericana Km 11, Apartado 21827, Caracas 1020A (Venezuela)
E-Mail lucimeylima @ gmail.com
transporter in lymphocytes [6]. Thus, this evidence stim-
ulates the exploration of other 5-HT receptors involved
in basic lymphocyte physiopathology by its presence and
its susceptibility to stimulus, such as inflammatory, phys-
ical or pharmacological stress.
5-HT7 metabotropic receptors, which are coupled to
adenylate cyclase, have been cloned by different groups,
and present a homology between species greater than
90% [7–10]. In the central nervous system, 5-HT7 recep-
tors are involved in frontal-like cognitive disturbance
[11, 12] since antagonists enhance procognitive efficacy.
In addition, 5-HT7 receptor antagonists may function as
analgesics in nerve injury pain states [13]. On the other
hand, agonists possess analgesic properties in neuropath-
ic pain [14]. Moreover, these receptors are related to
memory consolidation [15], cognitive deficits, anxiety
and depression [16]. In the periphery, 5-HT7 receptors
regulate cardiovascular function in relation to their role
on vasopressin release [17], and are involved in the patho-
genesis of irritable bowel syndrome [18]. 5-HT7 receptor
mRNA is expressed in spleen, thymus and peripheral
blood lymphocytes of rat and rhesus monkeys, resting or
activated, and human activated peripheral blood mono-
nuclear cells [19].
The effects of 5-HT on monocyte apoptosis seems to
be mediated through 5-HT1 or 5-HT7 receptors, which
produce phosphorylation of extracellular signal-regulat-
ed kinase 1/2, ERK1/2 [20]. Complex signaling has been
shown in T lymphocytes, including ERK1/2 and κBα, me-
diated by endogenous 5-HT as an intrinsic cofactor in the
activation of these cells [21]. However, the main route of
second messenger production by 5-HT7 receptors is me-
diated through Gs proteins with a corresponding increase
in cAMP [22]. Thus, based on quoted reports and since
5-HT7 receptors are expressed in lymphocytes and mod-
ulated by glucocorticoids, the aims of this study were to:
(i) evaluate the presence of 5-HT7 receptors in T lympho-
cytes, (ii) study the effect of activation with Con A, phys-
ical restraint and treatment with reserpine, and (iii) en-
hance the role of 5-HT as an autocrine modulator by
showing the presence of the synthetic enzyme tryptophan
hydroxylase.
Materials and Methods
Chemicals and Equipment
The following chemicals and equipment were used in this
study: LymphoprepTM (from Nycomed); sodium heparin (from
Biogalenic); TRIzol reagent, deoxyribonucleotide triphosphate
mix (DNTP) and platinum Taq DNA polymerase (all from Invit-
Mitogen, Stress and Lymphocyte 5-HT7
Receptors
rogen); Chlorophorm GR (from EM Science); reverse transcrip-
tion system and primers for β-actin (from Promega); primers for
5-HT7 receptors (from Eurogentech); agarose low electroendos-
mosis (from Scietrade); diethyl-piro-carbamato (DEPC; from Cal-
biochem); refrigerated centrifuge Evolution RC (from Sorvall);
table centrifuge with basculant rotor (from Damon/IEC Division);
incubator 4200 (from Napco); a fluorescence microscope with dif-
ferential interference contrast Eclipse 600 (from Nikon); Minicy-
clerTM (from MJ Research), and GelDoc 2000TM and electrophore-
sis camera SubCell-GT (both from BioRad). Histopaque 1077, eth-
ylendiaminotetracetic acid (EDTA), reserpine, Con A IV grade,
Trizma base, Roswell Park Medical Institute (RPMI) 1640 medi-
um, gentamicin, L-glutamine, bovine serum albumin (BSA), fetal
calf serum (FCS; with levels of 5-HT in a very low nanomolar
range, less than 0.2 ng per well), [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide] (MTT) were all supplied by Sigma.
Diethyl ether, isopropyl alcohol, sodium chloride and other salts
were obtained from Merck. All antibodies were purchased from
Santa Cruz Biotechnology Inc. Other general chemicals were ac-
quired from Sigma.
Animals and Treatments
Male Sprague-Dawley rats (250 ± 40 g) were obtained from the
Animal House of our institute (IVIC). The rats were housed, 1 per
cage, in a room controlled for temperature, humidity and lighting
(12 h/12 h) for at least 72 h prior to the experiments. Food and
water were available ad libitum. The treatments involved admin-
istration of a T cell mitogen as an inflammatory stress, as well as
two other protocols of stress, one physical and one pharmacologi-
cal. Controls, according to each protocol, either received vehicle or
were not restrained. Con A (0.5 mg/ml), was prepared in a phos-
phate-buffered saline (PBS; all measurements in mM: 137.9 of
NaCl, 0.9 of KCl, 1.15 of KH2PO4, 8.06 of Na2HPO4; pH 7.4) and
i.p. injections were done on 2 consecutive days, 500 μg/kg (200–
300 μl), between 08.00 and 09.30 h [3]. The rats were physically
restricted in a different room in individual ventilated Plexiglass
boxes, 16 × 7 × 6 cm, adjustable for length to 9, 11 or 14 cm, for 5 h
per day between 09.00 and 14.00 h for 5 days [4, 5]. Reserpine was
injected i.p., 2.5 mg/kg daily for 3 consecutive days in 200–300 μl
of 0.5% methylcellulose [23]. The rats were anesthetized with ether
and blood samples were taken by intracardial puncture 24 h after
the last intervention.
Isolation of Lymphocytes from Rat Blood
Blood was placed in tubes with heparin, 20 IU/ml, or 10% w/v
EDTA. After centrifugation at 1,000 rpm at room temperature for
10 min, plasma was separated and white layers plus some red
blood cells were diluted up to 12 ml with PBS in another tube.
Then, 6 ml of cell preparation were carefully placed in 3 ml of
LymphoprepTM followed by centrifugation at 145 g for 30 min.
Mononuclear blood cells were collected, washed twice with PBS
and centrifuged at 580 g for 10 min. In order to eliminate adher-
ent cells (monocytes/macrophages), cell suspensions were placed
into culture flasks with 5 ml of RPMI with gentamicin (100 μg/
ml), L-glutamine (2 mM) and 10% FCS. Incubation was done at
room temperature for 1 h. Nonadherent cells (∼90%) were taken,
washed with PBS and separated by centrifuge at 145 g for 10 min
and suspended in culture medium. Cells were used for culture,
immunocytochemistry and extraction of RNA. Membrane integ-
rity membrane was evaluated by exclusion of Trypan blue (>96%).
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Neuroimmunomodulation 2014;21:240–249 241
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According to previous results from the laboratory more blood
was obtained by cardiac puncture and each measurement corre-
sponded to 1 rat [3].
Culture and Evaluation of Proliferation in the Absence and in
the Presence of Con A
In an incubator, lymphocytes were cultured in 96-well, con-
cave-bottomed dishes, in RPMI medium with 10% FCS, 0.1% gen-
tamicin and 0.2% sodium bicarbonate, pH 7.4, for 72 h at 37 ° C     20 μl of the sample, and absorbance was measured at 260 and
with 5% CO2. Con A (2.5 μg/ml) [3, 24] was added to some of the
wells. Aliquots of cells were separated before plating for immuno-
chemical labeling. Proliferation was measured with MTT [25].
MTT was prepared in PBS (5 mg/ml), 20 μl were added to each well
and incubation was done for 3 h at 37 ° C. Next, 100 μl of solution
   
and 100 μl of 0.04 N HCl in isopropanol were mixed and read in a
GENios lector (Tecan) at 570 nm using the Megallan program.
Controls were done for each type of experiment.
Immunochemical Localization of 5-HT7 Receptors in
Subpopulations of T Lymphocytes and the Presence of
Tryptophan Hydroxylase
Cells (∼500,000), fixed with 2% paraformaldehyde in PBS for
1 h, washed with PBS and centrifuged, were plated on poly-L-ly-
sine-coated slides. After drying they were covered with 0.3% Tri-
ton X-100 in PBS for 20 min at room temperature, followed by
incubation with 5% BSA for 30 min. Three washes were performed.
In order to enhance the signal, incubation was done with two poly-
clonal goat antibodies, one directed to the amino terminal and the
other to the carboxyl terminal of the receptor, from a preparation
of the IgGs of 200 μg/ml at a dilution of 1:25. For labeling, CD4+
or CD8+ lymphocyte mice monoclonal antibodies were used, 100
μg/ml at a dilution of 1:20. In both case incubation was done for
1 h at room temperature. Controls were incubated with 1% BSA in
PBS. Anti-goat IgG was conjugated with fluorescein isothicianate
(FITC; 1:25) for 5HT7 receptors, and with anti-mouse IgG conju-
gated to rhodamine (1: 40), either for CD4+ or for CD8+ cells.
Slides were washed with PBS, dried and covered with Immuno-
MountTM. The observation was done using a Nikon fluorescence
microscope, 490 nm for FITC and 546 nm for rhodamine. The cells
(n = 300–600) were counted and expressed as a percentage of the
total lymphocytes for 5-HT7 receptors, CD4+ or CD8+. The per-
centage of 5-HT7 receptor-positive cells was determined in CD4+
and CD8+ lymphocytes. Two goat antibodies (1:100) against tryp-
tophan hydroxylase were used, one directed to internal and the
other to external domains of the enzyme. After washing with PBS,
incubation took place at 4 ° C overnight, and anti-goat IgG conju-
   
gated with FITC was used as a second antibody. All were basically
done according to Fazzino et al. [26].
Extraction of RNA from Lymphocytes of Rat Blood
RNA extraction was modified from the method used by Chom-
czynski and Sacchi [27]. The pellet of cells was homogenized in new
sterilized plastic tubes of 1.5 ml (Eppendorf) with TRIzol, 1 ml per
107 cells, and kept at –80 ° C. Samples were placed at room tem-
   
perature for 15 min. Then 0.2 ml of chloroform were added to each
ml of TRIzol, followed by vigorous agitation for 30 s and standing
for 10 min prior to centrifugation at 12,000 g at 4 ° C for 20 min.
   
From the three phases formed, the aqueous phase was transferred
to a tube, 0.5 ml of isopropyl alcohol was added per milliliter of
TRIzol, the tubes were kept on the bench for 15 min and centrifu-
242 Neuroimmunomodulation 2014;21:240–249
DOI: 10.1159/000357148
gation was performed at 12,000 g at 4 ° C for 20 min. The pellet of    
RNA was suspended in 1 ml of 75% ethanol in 0.1% DEPC deion-
ized water. After mild mixing in a vortex for 15–30 s, centrifugation
was performed at 7,500 g at 4 ° C for 5 min. This procedure was re-
peated, the tubes were dried for 10 min and the RNA was suspend-
ed in 15–20 μl of DEPC water using a pipette. Samples were incu-
bated at 55 ° C for 10 min and rapidly centrifuged. For quantifying
RNA, 330 μl of DEPC water was added to each well of a dish plus
280 nm. The amount of RNA was calculated according to Beer-
Lambert law, and the ratio of A260/A280 >1.8 indicated the purity.
The integrity of RNA was verified by electrophoresis in 1.2% aga-
rose gel in Tris/borate/EDTA buffer, pH 8, giving two bands of
ribosomal RNA: 28S (4.8 kb) and 18S (1.87 kb). Genomic DNA
bands of high molecular weight were not observed. Linearity of
β-actin was obtained with increasing concentrations, and also be-
tween relative units, in 3% agarose gels (R = 0.99) and 5-HT7 recep-
tors (R = 0.97) up to 35 cycles (data not shown).
Treatment with DNase I
Total RNA, 2 μg, were diluted in 16 μl of DEPC water, plus 4 μl
of DNase RQ1 free of RNases in buffer for DNase (10×). Incuba-
tion was done at 37 ° C for 30 min and the reaction was stopped
with the addition of 2 μl of 20 mM EGTA, followed by incubation
at 65 ° C for 10 min for inactivation of the enzyme. This treatment
eliminated all residual genomic DNA.
Synthesis of Complementary DNA
A reverse transcriptase system was employed to obtain cDNA.
Total RNA (1 μg) was placed in the following mix: 2 μl of reverse
transcriptase buffer (10×), 2 μl of 10 mM dNTPs equimolar solution,
0.5 ml of RNasin, 15 U of AMV reverse transcriptase and 0.5 μg of
oligo(dT)15. A final volume of 20 μl was reached with DEPC water.
Incubation was carried out at 42 ° C for 1 h, followed by heating at
99 ° C for 5 min for inactivation of the reverse transcriptase.
Polymerase Chain Reaction
Primers for rat 5-HT7 receptors (GenBank No. L19654)
were: sense 5′-AGGATTTTGGCTACACGATC-3′, and antisense
5′-GAGGAAAAACGGCAGCCAGCA-3′. Negative controls were
done with total RNA to ensure avoidance of genomic contamina-
tion. Positive controls were done with total RNA from the prefron-
tal cortex of rats. β-Actin constitutive gene was used as an internal
standard with the primers: sense 5′-TCATGAAGTGTGACGTT-
GACATCCGT-3′, and antisense 5′-CTTAGAAGCATTTGCG-
GTGCACGATG-3′. The coamplification of 5-HT7 receptors and
β-actin performed with various numbers of cycles was exponen-
tial. For all experimental groups RT-PCR was done in a final vol-
ume of 25 μl: 1.8 μl of cDNA, buffer for PCR (1.2×), 2.36 mM of
MgCl2, 200 μM of DNTPs, 500 nM of primers for 5-HT7 receptors
and 100 nM of primers for β-actin. The incubation was as follows:
94 ° C for 2 min; 33 cycles at 94 ° C for 30 s, at 55 ° C for 30 s and at
72 ° C for 1 min; 72 ° C for 5 min. Fragments were visualized in 3%
agarose gels in buffer TBE (1×) and 0.0001% ethidium bromide.
The product of RT-PCR, 12 μl plus 2.2 μl of charge buffer (6×) were
placed in the gel. The run was done at 120 V (9 V/cm) for 1 h. Im-
ages were taken for densitometry analysis with Quantity One soft-
ware, v.4.2.0. The relative levels of mRNA are expressed as densi-
tometry intensities of the corresponding bands for the receptors
divided by those of β-actin.
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 60
Control
Con A in vitro
50
Fluorescent cells (%)
40 a
30 a ment significantly elevated the number of 5-HT7-positive
20 a, c
10 b b
0 cantly elevated to 43% by Con A treatment (fig. 3a). There
CD4 CD8 5HT7 CD4-5HT7 CD8-5HT7
Fig. 1. Percentage of CD4+, CD8+ and 5HT7 receptor-positive lym-
phocytes, the coexistence of these markers and the effect of Con A
(2.5 μg/ml) in vitro. Cultures were performed for 72 h at 37 ° C with
5% CO2. Total cells were counted as >300 and specific labeling was
detected. For CD4+ or CD8+ cells, the total number of cells corre-
sponded with total CD4+ or CD8+, respectively. n = 6; a p < 0.05 vs.
control; b p < 0.05 vs. CD4 or CD8; c p < 0.05 vs. 5HT7.
Statistical Analysis
Results are expressed as the mean ± standard error of the mean
(SEM). The percentage of labeled cells was calculated by counting
the total cells or specific populations, with more than 300 for each
condition. One-way analysis of variance, ANOVA, was performed
and p values were corrected by Tukey’s post hoc test. Statistical
significance was considered with p < 0.05. The program used was
GraphPad InStat 3.
Results
CD4, CD8, 5-HT7 Receptors and Tryptophan
Hydroxylase-Expressing Cells in the Absence and in
the Presence of Con A in vitro
The presence of Con A significantly increased prolif-
eration (by about 60%) in all cultures. CD4+ cells corre-
sponded to 39 and 36% in the absence and in the presence
of Con A, respectively. For CD8+ lymphocytes, there was
a significant elevation from 22% in the absence and 34%
in the presence of Con A. 5-HT7 receptors were present
in 9% of all cells, and in 26% of those cultured in the pres-
ence of Con A. Six percent of CD4+ lymphocytes ex-
pressed 5HT7 receptors, and in the presence of Con A
there was a significant increase to 14%. CD8+ lympho-
cytes expressed 5-HT7 receptors in 4%, and Con A sig-
nificantly augmented the percentage of 5-HT7-positive
cells to 15% (fig.  1). Tryptophan hydroxylase-positive
cells rose from 12 to 18% in the absence of Con A, and
from 52 to 68% in the presence of Con A (fig. 2).
Mitogen, Stress and Lymphocyte 5-HT7
Receptors
Effect of in vivo Treatment with Con A on CD4, CD8
and 5-HT7 Receptor-Expressing Cells
The percentages of CD4+ lymphocytes were 53 and
48% in control and Con A-treated rats, respectively,
which corresponded to a significant difference. The treat-
cells from 14 to 23%. Of the total cells, 13% were positive
a, c
for CD4 and 5-HT7 receptors, and Con A administration
significantly increased this to 20%. Of all CD4+ lympho-
cytes, 27% expressed 5-HT7 receptors, which was signifi-
was a significant increase of CD8+ lymphocytes following
Con A, rising from 32 to 42%, and also of cells expressing
5-HT7 receptors, increasing from 12 to 17% (fig. 3b).
   
CD4, CD8 and 5-HT7 Receptor-Expressing Cells after
Physical Restriction
There was a significant reduction in the CD4+ lym-
phocytes of physically restrained rats from 48 to 41%, and
an increase of 5-HT7 receptor-expressing cells from 10 to
20%. The presence of 5-HT7 receptors calculated from the
total of CD4+ cells was higher after physical restriction,
increasing from 17 to 35% (fig. 4a). CD8+ lymphocytes
and 5HT7 receptor cells increased from 24 to 34%, and
CD4+ lymphocytes increased from 11 to 28%. A higher
number of cells with 5-HT7 receptors and CD8+ were ob-
served after physical restraint when considering both to-
tal cells (6–12%) and CD8+ (24–35%; fig. 4b).
CD4, CD8 and 5HT7 Receptor-Expressing Cells after
Reserpine Administration
Reserpine treatment did not significantly affect the
percentage of CD4+ lymphocytes but did produce a sig-
nificant elevation of 5-HT7 receptor cells from 11 to 17%.
When the percentage of both cells was determined over
the total cells, an increase from 9 to 13% was observed for
5-HT7 receptor cells, and from 20 to 32% for CD4+ lym-
phocytes (fig. 5a). CD8+ cells were increased by the treat-
ment from 27 to 34%, and 5-HT7 receptor cells rose from
9 to 19%. There were also increases in the coexistence of
CD8 and 5-HT7 receptors from total cells, from 6 to 13%,
and CD8+, from 20 to 37% (fig. 5b).
mRNA of 5-HT7 Receptors in Lymphocytes after
Con A Administration, or Physical Restriction and
Reserpine Treatment to Rats
The number of cycles was standardized to 33 for 5HT7
receptors, β-actin and the rest of the experiments, as in-
dicated in Materials and Methods. The administration of
Con A and physical restriction produced an elevation of
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Neuroimmunomodulation 2014;21:240–249 243
DOI: 10.1159/000357148
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 10 μm

10 μm 10 μm

a With Con A Without Con A

b With Con A Without Con A

Fig. 2. a Corresponding light microscopy

images of each immunocytochemistry re- 70
sult for detecting tryptophan hydroxylase- 60
positive lymphocytes in enriched prepara- 50
tions (>90% CD4+ and CD8+). b Images of 40
fluorescence marking with primary and 30
secondary antibody conjugated with FITC 20
in negative controls, in the absence and 10
presence of Con A, as indicated in Materi- 0
als and Methods. c Percentage of positive Without With
cells for tryptophan hydroxylase without c Con A Con A
and with Con A. p < 0.05.
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244 Neuroimmunomodulation 2014;21:240–249 Urbina/Arroyo/Lima

DOI: 10.1159/000357148
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 60 60
Control Control
a Con A in vivo Con A in vivo
50 a, c 50
a
Fluorescent cells (%)

Fluorescent cells (%)

40 40

30 c 30 a, c
a
a, b c
20 20 a
b
a, b
10 10 b

0 0
a CD4 5HT7 CD4-5HT7 CD4-5HT7/CD4 b CD8 5HT7 CD8-5HT7 CD8-5HT7/CD8

Fig. 3. Percentage of CD4+ and 5HT7 receptor-positive lympho-
cytes and the coexistence of these markers (a), and CD8+ and
5HT7 receptor-positive lymphocytes, and coexistence of these
markers (b). Effect of Con A in vivo (500 μg/kg i.p. for 2 consecu-
tive days, with blood obtained on the third day). Total cells were
counted as >300 and specific labeling was detected. For CD4+ or
CD8+ cells, the total number of cells corresponded with total
CD4+ or CD8+, respectively. n = 6; a p < 0.05 vs. control; b p < 0.05
vs. CD4 or CD8; c p < 0.05 vs. CD4-5HT7 or CD8-5-HT7.

60 60
Control Control
Physical restriction Physical restriction
50 50
a
Fluorescent cells (%)

Fluorescent cells (%)

40 a, c 40 c
a

30 30 a
c
a c
20 a, b 20
a, b
b
10 10 b

0 0
a CD4 5HT7 CD4-5HT7 CD4-5HT7/CD4 b CD8 5HT7 CD8-5HT7 CD8-5HT7/CD8

Fig. 4. Effect of physical restriction (5 h daily for 5 days, with blood
obtained on the 6th day) on the percentage of CD4+ and 5HT7
receptor-positive lymphocytes, and the coexistence of these mark-
ers (a), and CD8+ and 5HT7 receptor-positive lymphocytes, and
the coexistence of these markers (b). Total cells were counted as
>300 and specific labeling was detected. For CD4+ or CD8+ cells,
the total number of cells corresponded with total CD4+ or CD8+,
respectively. n = 6; a p < 0.05 vs. control; b p < 0.05 vs. CD4 or CD8;
c p < 0.05 vs. CD4-5HT or CD8-5-HT .
7 7

the relative mRNA concentration of 5-HT7 receptors in
lymphocytes, which was not observed in reserpine-treat-
ed rats (fig. 6, 7).
Discussion
5-HT7 receptors have previously been studied in the
central nervous system using genetic, behavioral and
pharmacological strategies and have been involved in
sleep, mood, anxiety, thermoregulation, electricity of the
brain, pain, memory and impulsivity testing, among oth-
ers [12–14, 17, 21]. However, little has been done in the
immune system.
The concentration of Con A used in the cultures sig-
nificantly increased proliferation as previously described
[28, 29]. Con A-mediated proliferation of rat blood lym-
phocytes is inhibited by antagonism of 5-HT1A receptors,
but it is not further elevated by agonists of this receptor
[29], and Con A activation of spleen CD4+ and CD8+ T
cells after 5-HT depletion is increased by 5-HT2A receptor
agonists [30]. The culture of lymphocytes in the presence
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Mitogen, Stress and Lymphocyte 5-HT7 Neuroimmunomodulation 2014;21:240–249 245

Receptors DOI: 10.1159/000357148
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 60 60
Control Control
Reserpine Reserpine
50 50
Fluorescent cells (%)

Fluorescent cells (%)

a, c
40 40 a
a, c

30 30
c c
a
20 a 20
a, b a, b
b
10 10 b

0 0
a CD4 5HT7 CD4-5HT7 CD4-5HT7/CD4 b CD8 5HT7 CD8-5HT7 CD8-5HT7/CD8

Fig. 5. Effect of treatment with reserpine (2.5 mg/kg for 3 days,
with blood obtained on the 4th day) on the percentage of CD4+
and 5HT7 receptor-positive lymphocytes, and the coexistence of
these markers (a), and CD8+ and 5HT7 receptor-positive lympho-
cytes, and the coexistence of these markers (b). Total cells were
counted as >300 and specific labeling was detected. For CD4+ or
CD8+ cells, the total number of cells corresponded with total
CD4+ or CD8+, respectively. n = 6; a p < 0.05 vs. control; b p < 0.05
vs. CD4 or CD8; c p < 0.05 vs. CD4-5HT7 or CD8-5-HT7.

Control Con A 1.2 D

1.0

5HT7/DŽDFWLQ
0.8
0.6
Fig. 6. Agarose gel (3%) of representative
5HT7 0.4
samples of DNA in control and Con A-
treated rats expressing 5-HT7 receptors in DŽDFWLQ 0.2
lymphocytes and β-actin (a) as constitutive 0
standard for semi-quantities of mRNA for a b &RQWURO &RQ$
the receptor (b). a p < 0.05 vs. control.

of Con A increased the number of cells presenting 5-HT7
receptors or tryptophan hydroxylase, indicating enhanced
5-HT transmission produced by lymphocyte activation.
5-HT7 expression was augmented in CD4+ and CD8+
lymphocytes. The in vivo administration of lipopolysac-
charide or Con A augment the number of 5-HT1A recep-
tors labelled with [3H]8-hydroxy-2-(di-n-propylamino)
teralin in lymphocyte membranes [3], and several mito-
gens, including lipopolysaccharide and Con A, have been
shown to induce 5-HT1A receptor mRNA [31]. As shown
in figure 2, in vivo administration of Con A also increased
the number of CD4+ and CD8+ lymphocytes expressing
5-HT7 receptors. mRNAs of 5-HT1B, 5-HT1F, 5-HT2A,
5-HT2B, 5-HT6 and 5-HT7 present in isolated spleen cells
are elevated by Con A and pokeweed mitogen, and addi-
tionally express 5-HT3 receptors [18]. No previous report
has been published concerning the differential presence of
5-HT7 receptors in a subpopulation of lymphocytes.
Immobilization stress, 1 h daily for 7 days, significant-
ly increases the capacity of 5-HT1A receptors in lympho-
cytes [3] and physical restriction, 5 h daily for 5 days, el-
evates the sensitivity of 5-HT1A [4]. Restraint-produced
impairment of the extra-dimensional set-shifting (a cog-
nitive task presenting more than one stimuli in which the
subject must choose one according to relevant rules) abil-
ity is reversed by the antagonist of the 5-HT7 receptor SB-
269970, which improves extra-dimensional performance
in unstressed rats [11]. Interestingly, corticosterone up-
regulates 5-HT7 receptor mRNA in primary hippocampal
cultures [31]. The increase in the number of lymphocytes
expressing 5-HT7 receptors by physical restriction could
be the result of elevation of corticosterone due to stress.
On the other hand, dexamethasone produces a significant
reduction in 5-HT7 receptor mRNA in frontocortical as-
trocytes [32]. Modifications in the percentages of CD4+
and CD8+ were also observed, indicating modification of
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246 Neuroimmunomodulation 2014;21:240–249 Urbina/Arroyo/Lima

DOI: 10.1159/000357148
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 0.5
Control Physical restriction D
0.4

5HT7/DŽDFWLQ
0.3

0.2
5HT7
0.1
DŽ-actin
0
&RQWURO 3K\VLFDO
a b UHVWULFWLRQ

Control Reserpine
0.5

0.4

5HT7/DŽDFWLQ
0.3

5HT7 0.2
DŽDFWLQ 0.1

0
c d &RQWURO 5HVHUSLQH

Fig. 7. Agarose gel (3%) of representative samples of DNA in control and physical restricted rats (a) or reserpine-
treated rats (c) expressing 5-HT7 receptors in lymphocytes and β-actin as constitutive standard for semi-quanti-
ties of mRNA for the receptor (b, d). a p < 0.05 vs. control.

immune function by physical restriction. Dysregulation
of T cell pathways and interplay could be significant in the
pathophysiology of stress and consequent alterations
leading to autoimmunity.
Autoradiography with of [3H]carboxamidotriptamine
and in situ hybridization, as well as expression, revealed
concordance for 5-HT7 receptors in rat and guinea pig
brains [33, 34], as well as in other tissues [35–37]. Beside
neurons and astrocytes, microglial cells present two splice
variants of 5-HT7 (a/b) visualized by RT-PCR [38]. In a
study of rhesus macaques, peripheral blood mononuclear
cells expressed mRNA of several 5-HT receptors, includ-
ing 5-HT7, but the authors indicated that it did not occur
in all samples [39]. Positive signals of mRNA detected by
RT-PCR have been obtained from isolated spleen, thy-
mus and peripheral blood lymphocytes [19]. Naïve T cells
also express protein of 5-HT7 receptors, which is en-
hanced by T cell activation [20]. To our knowledge, there
is no other evidence concerning 5-HT7 receptor expres-
sion in T cells, although, in agreement with the elevation
of 5-HT7 receptors in activated lymphocytes, the in vivo
administration of Con A produced a considerable in-
crease of mRNA levels in lymphocytes. The observed el-
evation of 5-HT7 receptors could contribute to the bal-
ance with 5-HT1A receptors, which augment proliferation
by decreasing cAMP, while 5-HT7 receptors decrease
cAMP, and could then be a regulatory process in order to
maintain the function of lymphocytes.
The elevation of mRNA for these receptors after phys-
ical restraint is in accordance to the elevation in the num-
ber of cells expressing it, and the lack of significant eleva-
tion in pharmacological stress produced by reserpine
constitutes evidence of the differential mechanisms in-
volved in each protocol. For instance, physical restraint
activates the neuroendocrine axis, and reserpine mainly
acts through the sympathetic system.
Conclusions
In vitro, Con A increased lymphocyte proliferation,
the number of CD8+ and 5-HT7 receptor-positive cells,
and lymphocytes expressing tryptophan hydroxylase.
Treatment with Con A increased CD4+, CD8+ and
5-HT7 receptor-positive cells, as well as the correspond-
ing mRNA for the receptors. Physical restraint de-
141.20.60.108 - 4/24/2014 7:36:08 PM
Humbold-Universität zu Berlin

Mitogen, Stress and Lymphocyte 5-HT7 Neuroimmunomodulation 2014;21:240–249 247

Receptors DOI: 10.1159/000357148
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creased CD4+ lymphocytes, increased CD8+, 5-HT7 re-
ceptor-positive cells, and the mRNA of these receptors.
Treatment with reserpine increased CD4+, CD8+ and
5-HT7 receptor-positive cells. The modifications pro-
duced by mitogen activation and two protocols of stress,
either physical or pharmacological, could result in rel-
evant changes in immune system functions. The differ-
ential presence of 5-HT7 receptors in lymphocytes,
which was not previously documented, indicates the
potential physiopathological role of this subtype of
5-HT receptors.
Acknowledgements
This work was supported by grant G-1387 from Fondo de
Ciencia, Tecnología e Innovación (FONACIT), Venezuela.
Rubén Arroyo, MSc, was a graduate student of the Centro de
Estudios Avanzados at Laboratorio de Neuroquímica of Instituto
Venezolano de Investigaciones Científicas.
Disclosure Statement
There are no conflicts of interest.

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