Transcriptional Mechanisms for Induction of 5-HT1A
Receptor mRNA and Protein in Activated B and T
Lymphocytes*
Mohamed
Abdouh
,
John M.
Storring§,
Mustapha
Riad¶,
Yves
Paquette
,
Paul R.
Albert**,
Elliot
Drobetsky
, and
Edouard
Kouassi
§§
From the 
Human Health Research Center,
INRS-Institut Armand-Frappier, Pointe-Claire, Quebec H9R 1G6, the
Departments of
Pharmacology and ¶ Pathology and
Cellular Biology, University of Montreal, Montreal, Quebec H3C 3J7,
the § Department of Pharmacology and Therapeutics, McGill
University, Montreal, Quebec H3G 1Y6, and the
Guy-Bernier
Research Center, Maisonneuve-Rosemont Hospital, and the ** Neuroscience
Research Institute, University of Ottawa, Ottawa,
Ontario K1H 8M5, Canada
Received for publication, May 26, 2000, and in revised form, October 13, 2000
 |
ABSTRACT |
Serotonin (5-HT) up-regulates B and T lymphocyte
proliferation by activating mitogen-induced cell surface
5-HT1A receptors. The mechanism of 5-HT1A
receptor induction by B and T cell mitogens at the mRNA and protein
levels in mouse splenocytes was addressed. Quantitation by RNase
protection assay showed maximal increases of 3.4-, 3.0-, 3.8-, and
4.9-fold in relative 5-HT1A mRNA levels after 48 h
of stimulation of splenocytes with lipopolysaccharide, phytohemagglutinin, concanavalin A, or phorbol 12-myristate 13-acetate plus ionomycin, respectively, as compared with unstimulated cells. Mitogens did not alter 5-HT1A mRNA stability
(t1/2 = 26 h), but induction of
5-HT1A mRNA was blocked by the transcriptional inhibitor actinomycin D (10 µg/ml) and by inhibition of nuclear factor-
B signaling. Additionally, mitogenic stimulation of
transcription was paralleled by increased cell surface
5-HT1A receptor immunoreactivity in splenocytes. Thus,
mitogen-induced 5-HT1A receptor expression appears to
involve transcriptional regulation by the nuclear factor-
B signaling
cascade. Increased expression of the 5-HT1A receptor in
activated B and T lymphocytes may enhance the immune response and
provide therapeutic target for tissue inflammation and immune stimulation.
 |
INTRODUCTION |
Serotonin (5-HT)1 is a
neuroimmunomodulator that is widely distributed in brain and peripheral
tissues, and which is released by activated platelets during the course
of tissue inflammation (1). 5-HT is also accumulated by and released
from noradrenergic nerve terminals that are in close contact with
lymphocytes in lymphoid organs (2-4). Rodent mast cells are another
important source which release their stored 5-HT following exposure to
antigen and IgE-sensitizing Ab, or to neuropeptides such as
somatostatin, substance P, calcitonin gene-related peptide, and
vasoactive intestinal peptide, the latter being released from
peripheral nerves (5).
Among the numerous 5-HT receptors, 5-HT1A belongs to
G-protein-coupled receptor superfamily and is also widely distributed in brain and immune tissues (6, 7). The 5-HT1A gene has been cloned previously in human (8, 9), rat (10), and mouse (11),
manifesting very high nucleotide and amino acid sequence homology in
their respective putative transmembrane regions. 5-HT1A
mRNA has been detected in various human tissues including lymph
nodes, spleen, and thymus (8), as well as in human peripheral blood
mononuclear cells (12) and activated T lymphocytes (13). In functional
studies using selective agonists and antagonists, it has been shown
that the 5-HT1A receptor is implicated in the regulation of
T cell responses including human T-cell proliferation (13-16),
production of Th1 cytokines such as interleukin-2 and interferon-
both in mice (17) and in human (15, 16), and contact sensitivity
reactions in mice (17). We have shown previously that
mitogen-stimulated B lymphocyte proliferation in rodents is
up-regulated by 5-HT via specific interaction with the
5-HT1A receptor (18). Thus, immune and inflammatory
responses may be regulated in part through 5-HT1A receptor
expression in B and T lymphocytes.
A recent review of the role of 5-HT in the immune system and in
neuroimmune interactions has underscored the necessity of characterizing the distribution of the various 5-HT receptors in
different immune cell populations, preferably by using molecular biological methods (7). The previous studies cited above using essentially functional and radioligand binding criteria suggest that
5-HT1A receptor expression is increased following mitogenic stimulation of both murine B cells (18) and human T cells (13), but
little is known about the molecular mechanisms underlying this effect.
Nuclear factor-
B (NF-
B) is a ubiquitous and inducible transcription factor involved in many immune and inflammatory responses, including activation and proliferation of B and T
lymphocytes stimulated by mitogens such as LPS, PHA, and PMA (19-21).
NF-
B is mainly composed of p50 and p65 subunits, which are normally retained in the cytosol of nonstimulated cells by inhibitory molecules, I
B. In response to stimuli, I
B are rapidly phosphorylated and degraded, allowing translocation of NF-
B complexes into the nucleus and activation of NF-
B elements (22).
In this report, we used RNase protection assay to quantitate the
expression of 5-HT1A receptor mRNA in unstimulated
versus mitogen-stimulated mouse splenocytes. In addition, we
took advantage of the availability of pharmacological inhibitors of
NF-
B (23-25) to explore its role in regulation of
5-HT1A receptor mRNA expression following mitogenic
stimulation. Additionally, we used an affinity-purified anti-5-HT1A antiserum (26) to evaluate the expression of
the 5-HT1A receptor protein in the splenocytes. Our data
demonstrate that 5-HT1A receptor mRNA and protein are
markedly increased following mitogenic stimulation of B and T
lymphocytes with similar quantitative variation in these lymphocyte
populations. Furthermore, our data indicate that up-regulation of
mitogen-stimulated B and T lymphocyte 5-HT1A receptor
occurs at the transcriptional level, and that mitogen-induced nuclear
translocation of NF-
B may be one of the important signaling
mechanisms involved.
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EXPERIMENTAL PROCEDURES |
Mice and Reagents--
Female BALB/c mice, 6-12 weeks of age,
were purchased from Charles River (St-Constant, Canada) and maintained
in our animal facilities until use. All culture media were purchased
from Life Technologies, Inc. (Burlington, Canada). Fetal bovine serum
was purchased from HyClone (Logan, UT), and dialyzed against PBS to remove molecules of molecular weight <12-14 kDa.
Escherichia coli LPS (serotype 0111:B4), PHA,
ConA, PMA, and 5-HT hydrochloride were from Sigma,
R(+)-8-OH-DPAT hydrobromide (R-DPAT) and WAY100635 maleate
from RBI (Natick, MA), and ionomycin from Calbiochem (La Jolla, CA).
[3H]Thymidine (specific activity 2 Ci/mmol) was obtained
from PerkinElmer Life Sciences (Mississauga, Canada), and
[3H]WAY100635 (specific activity 81 Ci/mmol) from
Amersham Pharmacia Biotech (Little Chalfont, United Kingdom).
Anti-5-HT1A antiserum was produced as described previously
(26), and all other Ab were from PharMingen (San Diego, CA).
Isolation and Stimulation of Splenocytes--
BALB/c mice were
killed by cervical dislocation. Spleens were then aseptically harvested
and gently teased into a single-cell suspension in Hanks' balanced
salt solution. Red blood cells were removed by osmotic shock with
NH4Cl, and splenocytes were resuspended in a culture medium
consisting of RPMI 1640 medium supplemented with penicillin (100 units/ml), streptomycin (100 µg/ml), L-glutamine (2 mM), and 10% decomplemented fetal bovine serum. Cells were cultured in flat-bottomed 96-well culture plates (Life Technologies, Inc.) in a humidified atmosphere containing 5% CO2 at
37 °C at a density of 4 × 105 cells/well in a
total volume of 200 µl. Cells were stimulated by incubation for
different periods of time in the presence or absence of LPS (10 µg/ml), PHA (20 µg/ml), ConA (5 µg/ml), or a combination of PMA
(1 ng/ml) and ionomycin (500 ng/ml). In some experiments, splenocytes
were incubated with 10 µg/ml actinomycin D (ICN, Saint-Laurent,
Canada), to distinguish between existing and newly transcribed
mRNA. To prevent the activation of the transcription factor
NF-
B, splenocytes were incubated for 48 h with mitogens in the
presence of 10-50 µg/ml SN50 (Calbiochem), 5-50 µM
pyrrolidinedithiocarbamate (PDTC, Sigma), or 0.01-10 µg/ml gliotoxin
(Sigma). As controls for SN50 and gliotoxin specificity, their
respective inactive analogues SN50M (50 µg/ml) and
methylthiogliotoxin (1-10 µg/ml) were also used. Cell counting and
viability were assessed by trypan blue exclusion, and all chemicals
were used at noncytotoxic concentrations.
Purification of Resting and Activated B and T
Lymphocytes--
Purification of B and T lymphocytes was achieved by
negative selection of splenocytes using flow cytometry sorting with Ab directed against granulocytes and macrophages (anti-CD11b-PE), NK cells
(anti-Ly49C, 5E6-PE), and T lymphocytes (anti-Thy-1.2-PE), or B
lymphocytes (anti-CD19-FITC), as described previously (27). Dead cells
were stained with the vital dye propidium iodide (1 µg/ml; Molecular
Probes, Eugene, OR). Resting and activated lymphocytes were gated
appropriately and separated in two different regions using forward
scatter and side scatter profiles. Cells that were negative for the
indicated cell surface markers and for propidium iodide staining were
sorted on a FACStar-Plus cell sorter (Becton Dickinson, San Jose, CA).
The purity of the resulting B or T cells was assessed by flow
cytometry with anti-Thy-1.2-PE and anti-CD19-FITC, and it ranged
between 93% and 97%.
Proliferation Assay--
Splenocytes were incubated for 30 min
with or without 5 × 10
5 M
WAY100635 before stimulation for 72 h with mitogens in the presence or absence of 10
4 M 5-HT
or 5 × 10
5 M R-DPAT, and
cultures were pulsed with 1 µCi of [3H]thymidine for
the last 6 h of incubation. Cell nuclei were harvested, and
radioactivity was counted with a Wallac System 1409 scintillation counter (Wallac Oy, Turku, Finland). Determinations of
[3H]thymidine uptake were made in triplicate wells, and
results were expressed as arithmetic means of counts per minute
(cpm) ± S.E.
RNA Preparation and RT-PCR--
Total cellular RNA was isolated
from cell suspensions by Trizol reagent (Life Technologies, Inc.)
according to the manufacturer instruction. For RT-PCR, 1 µg of total
RNA was treated for 15 min at 37 °C with 2 units of amplification
grade DNase I (Life Technologies, Inc.) to remove genomic DNA. After
denaturation for 10 min at 75 °C, cDNA was synthesized for
1 h at 42 °C by adding Superscript II reverse transcriptase
(Life Technologies, Inc.) and 1 µM random hexamer primers
(Roche Molecular Biochemicals, Laval, Canada). A 1/8 volume of the
resulting first strand cDNA was used as template during the
subsequent PCR amplification in a PCR machine (GeneAmp PCR System 9600, PerkinElmer Life Sciences) using 1.25 units of Taq DNA
polymerase (Roche Molecular Biochemicals) in the buffer provided with
10 mM Tris (pH 8.3), 50 mM KCl, and 1.5 mM MgCl2, in the presence of 200 µM dNTPs, and 250 nM primers (synthesized by
Life Technologies, Inc.) in a total volume of 25 µl. The thermocycle
conditions were 22 cycles of 94 °C, 60 s, 62 °C, 60 s,
72 °C, 60 s. There was also an initial denaturation step at
94 °C for 5 min and a terminal extension step at 72 °C for 10 min. The sense primer for 5-HT1A was
5'-ACCCCGACGCGTGCACCATCAG-3', and the antisense primer was
5'-GCAGGCGGGGRCATAGGAG-3' derived, respectively, from the second
extracellular loop and the third intracytoplasmic loop of the rat and
mouse 5-HT1A genes, which gave a 413-bp PCR product. This
set of primers allowed detection of 5-HT1A mRNA in
several positive controls including the cell lines LZD-7 and LM1A,
which are derived from the mouse fibroblasts Ltk- cells transfected
with the rat and mouse 5-HT1A cDNA, respectively, and
in RNA extracts from rat and mouse brain. The sense primer for GAPDH
was 5'-CAACGACCCCTTCATTGACCTC-3', and the antisense primer was
5'-GGAAGGCCATGCCAGTGAGC-3', which gave a 602-bp PCR product. The PCR
products were separated on a 1.5% agarose gel, stained with ethidium
bromide, and visualized with UV light.
RNase Protection Assay--
Detection and quantitation of
5-HT1A mRNA expression was carried out using an RNase
protection assay (Direct Protect Lysate Ribonuclease Protection Assay
Kit from Ambion) with 18 S ribosomal RNA as an internal standard. To
prepare the template for 5-HT1A riboprobes, the first 860 bp of the mouse 5-HT1A cDNA were cut from the M1A-KS+
vector (11) using the PstI enzyme. This cDNA fragment
was subsequently inserted in the antisense orientation with respect to
the T3 RNA polymerase promoter found in the pBluescript II KS+ plasmid
(Promega). To synthesize radiolabeled 5-HT1A antisense cRNA, the plasmid was linearized with the enzyme BssHII and
transcribed with T3 RNA polymerase (Ambion) and 50 µCi of 800 Ci/mmol
[
-32P]UTP (Mandel, Guelph, Canada) using the
MAXIscript in vitro transcription kit (Ambion) at 37 °C
for 1 h. The resulting transcripts were then treated with 2 units
of RNase-free DNase I at 37 °C for 15 min. The 18 S ribosomal RNA
antisense probe was synthesized using a 18 S cDNA template
(Ambion), which was transcribed with T3 RNA polymerase in the presence
of 30 µCi of [
-32P]UTP. Total RNA was extracted from
samples of 106 cells in 50 µl of Lysis/Denaturing
solution (Ambion) and coprecipitated with the freshly radiolabeled
5-HT1A (0.25 µCi) and 18 S (0.015 µCi) riboprobes, and
incubated overnight at 37 °C. A volume of 500 µl of a RNase mix
containing 5 units of RNase A and 200 units of RNase T1 (Ambion) was
then added to the samples and incubated at 37 °C for 1 h to
digest the unprotected riboprobes and RNA. The reaction was stopped by
adding proteinase K and sodium sarkosyl, and by re-incubating at
37 °C for 30 min. The protected fragments were precipitated with 500 µl of isopropanol, resuspended in a gel loading buffer, and resolved
on a 8 M urea, 5% acrylamide gel. The sizes of the
expected protected fragments were 124 and 80 bp for 5-HT1A
and 18 S, respectively. Radiolabeled RNA transcripts from Century
Marker Template set (Ambion) were used as size markers. The results
were quantitated on a PhosphorImager (GS-525 Molecular Imager System,
Bio-Rad). Relative 5-HT1A levels were calculated by
normalizing the 5-HT1A mRNA band to that of the 18 S
ribosomal RNA.
Immunocytofluorometry Analysis of 5-HT1A Receptor
Protein--
A rabbit polyclonal anti-rat 5-HT1A receptor
antiserum was used for this study. It is directed against a synthetic
antigenic polypeptide that is derived from the third intracytoplasmic
loop of the rat 5-HT1A receptor, with 92% homology with
the corresponding region of mouse 5-HT1A protein. Extensive
characterization of this antiserum has been reported elsewhere (26),
and it cross-reacts with mouse 5-HT1A receptor. Samples of
106 cells were permeabilized with absolute ethanol (95%)
at 4 °C for 30 min, and fixed with 2% (w/v) paraformaldehyde in PBS
for 30 min at 4 °C. Cells were then incubated overnight with
anti-rat 5-HT1A receptor antiserum (1:1000) in Ab buffer
consisting of PBS containing 1% (v/v) normal goat serum (Cederlane,
Hornby, Canada). After several washings in PBS (three times for 10 min each time), cells were incubated in PE-labeled goat anti-rabbit Ig
(1:250) for 1 h, and washed again in PBS (three times for 10 min
each time). Cells were analyzed on a FACScan flow cytometer (Becton
Dickinson, San Jose, CA) using the LYSIS program provided by the
manufacturer. For double staining of B or T lymphocytes, cells were
stained first with FITC-conjugated anti-CD19 Ab or FITC-conjugated
anti-Thy-1.2 Ab, and then with the anti-5-HT1A receptor
antiserum followed by goat anti-rabbit-Ig-PE as described above.
Immunocytochemistry Analysis of 5-HT1A Receptor
Protein--
Cells (106) were layered 1 h at room
temperature on microscope slides pretreated with 50 µg/ml
poly-D-lysine. Slides were rinsed with PBS (50 mM, pH 7.4), fixed for 1 h at room temperature with 2% paraformaldehyde in PBS, and washed in PBS. Cells were then preincubated for 1 h in a blocking solution of PBS containing 5%
normal goat serum, 0.2% Triton X-100, and 0.5% gelatin to saturate nonspecific sites, and incubated for 2 h with a 1/1000 dilution of
rabbit anti-5-HT1A antiserum. After washes in PBS (three
times for 10 min each time), the slides were incubated for 1 h
with biotinylated goat anti-rabbit IgGs diluted 1/1000 in blocking solution, rinsed in PBS (three times for 10 min each time), and incubated for 1 h with a 1/1000 dilution of horseradish
peroxidase-conjugated streptavidin. This was followed by successive
washes in PBS (two times for 10 min each time) and in Tris-HCl buffer
(0.05 M, pH 7.4; two 10-min washes), and then incubated in
hydrogen peroxide (0.01%) in the presence of 3,3'-diaminobenzidine
(0.05%) in Tris-HCl buffer. The reaction was stopped by several washes
in the same buffer. The slides were then dehydrated in a graded series
of ethanol, followed by toluene, and coverslipped with DPX mountant (Fluka, Oakville, Canada). Immunocytochemical control consisted of
processing slides as above, except for replacement of the
anti-5-HT1A antiserum by preimmune rabbit serum at the same
dilutions. Staining was examined by light microscopy (final
magnification, ×400).
Radioligand Binding Assay--
Binding studies of
[3H]WAY100635 were performed on unstimulated and
mitogen-stimulated lymphocytes, following the procedures described
previously by us for [3H]8-OH-DPAT (18), except that
[3H]WAY100635 was used at 0.5-15 nM, and
that the Whatman GF/B filters through which cell suspensions were
filtered were presoaked in a 0.5% aqueous solution of polyethylenimine
for 30 min to limit nonspecific binding of the radioligand (28).
 |
RESULTS |
5-HT1A Receptor-mediated Up-regulation of
Mitogen-stimulated B and T Lymphocyte Proliferation--
Previously,
we demonstrated that 5-HT increases mitogen-stimulated murine B
lymphocyte proliferation through a 5-HT1A receptor-mediated mechanism (18). Here, we used mouse splenocytes stimulated by the T
cell mitogen PHA to determine whether T lymphocyte proliferation is
influenced by 5-HT1A receptor ligands. Preliminary dose
reponse studies indicated that 5-HT (10
11 to
10
4 M) and the selective
5-HT1A receptor agonist R-DPAT
(10
11 to 10
4
M) increased PHA-stimulated T lymphocyte proliferation in a
dose-dependent manner with optimal concentrations of
10
4 M and 5 × 10
5 M, respectively. Those
maximally effective concentrations were used in combination with the
relatively selective 5-HT1A receptor antagonist WAY100635
to evaluate receptor specificity of 5-HT and R-DPAT action. Fig.
1A shows that 5 × 10
5 M WAY100635 effectively
abrogated 5-HT- and R-DPAT-mediated enhancement of activated T
lymphocyte proliferation, thus implicating the 5-HT1A
receptor in the control of T cell proliferation.

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Fig. 1.
5-HT1A-mediated up-regulation of
mitogen-stimulated mouse T and B lymphocyte proliferation. Mouse
splenocytes were pre-incubated for 30 min at 37 °C in the presence
or absence of the 5-HT1A receptor antagonist WAY100635
(WAY; 5 × 10 5
M), and then cells were stimulated with 20 µg/ml PHA
(A) or 1 ng/ml PMA plus 500 ng/ml ionomycin (B)
in the presence or absence of 10 4
M 5-HT or 5 × 10 5
M R-DPAT as indicated. Cells were incubated for 72 h,
and proliferation was measured by [3H]thymidine uptake
during the last 6 h of culture. Student's t test was
performed. For mitogen-stimulated splenocytes versus
mitogen-stimulated splenocytes in the presence of WAY100635,
p value was not statistically significant; for
mitogen-stimulated splenocytes versus mitogen-stimulated
splenocytes in the presence of 5-HT or R-DPAT (§), p < 0.05; for mitogen-stimulated splenocytes + 5-HT or R-DPAT
versus mitogen-stimulated splenocytes + WAY100635 + 5-HT or
R-DPAT (*), p < 0.05.
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The combination of PMA plus ionomycin is known to bypass antigen
receptor signaling in both B and T lymphocytes, engendering a potent
activation and proliferation of these cells (29-31). To test whether
5-HT1A ligands can influence B and T cell proliferation in
this model, splenocytes were stimulated with a mitogenic combination of
PMA (1 ng/ml) and ionomycin (500 ng/ml), in the presence of 5-HT or
R-DPAT, with or without WAY100635. Fig. 1B shows that 5-HT
and R-DPAT increased splenocyte proliferation induced by PMA plus
ionomycin, and that WAY100635 reversed agonist-induced mitogenic
potentiation, further indicating a role for 5-HT1A receptor activation. Thus, we chose the model of mouse splenocytes incubated in
the presence or absence of PMA plus ionomycin for most of the following
experiments to further characterize the 5-HT1A receptor mRNA and protein which are expressed in B and T lymphocytes.
5-HT1A Receptor mRNA Expression in
Mitogen-stimulated Splenocytes--
The 5-HT1A receptor
belongs to the family of G protein-coupled receptors. These receptors
are characterized by the presence of seven putative transmembrane
domains showing a high degree of similarity between members of this
family, whereas most sequence differences are seen in the extracellular
and intracellular loops (6). We used primers derived from the second
extracellular loop, and from the third cytoplasmic loop, to carry out
PCR assays for 5-HT1A receptor on cDNA generated by RT
of total RNA isolated from splenocytes before and after mitogenic
stimulation with PMA (1 ng/ml) plus ionomycin (500 ng/ml). RNA samples
from the mouse Ltk
and LM1A cell lines were used as
negative and positive controls, respectively. Fig.
2 shows the presence of a
5-HT1A transcript in mitogen-stimulated splenocytes that
was identical in size to the signal obtained in LM1A cells. Among a
total of six experiments, 5-HT1A mRNA was expressed in
all splenocyte samples stimulated by PMA plus ionomycin. In marked
contrast, 5-HT1A mRNA was not detectable
(n = 4) or only barely detectable (n = 2), in samples of unstimulated splenocytes, and in the latter case only
if the amount of cDNA introduced in the PCR reaction was increased
by a factor of at least 4-fold. Each of the RNA samples were also subjected to PCR assays without RT, and no DNA fragment was obtained, indicating that the product observed represented amplification of
5-HT1A cDNA, and did not result from amplification of
contaminating genomic DNA.

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Fig. 2.
Mitogen-stimulated mouse splenocytes express
5-HT1A receptor mRNA as determined by RT-PCR. RNA
samples were reverse transcribed (+RT) or not
( RT) and subjected to PCR for 5-HT1A and
GAPDH. No PCR products were present if these RNA samples were not
reverse transcribed ( RT). Total RNA was extracted from
freshly isolated splenocytes (lane 1), splenocytes cultured
during 48 h in the presence of culture media (lane 2)
or in the presence of 1 ng/ml PMA plus 500 ng/ml ionomycin (lane
3), Ltk mouse fibroblast cells (lane 4),
and LM1A cells (lane 5). Ltk and LM1A cells
were used as negative and positive controls, respectively, for
5-HT1A mRNA expression. Lane 6 corresponds
to a PCR with neither cDNA nor RNA, which were replaced by
H2O, to ensure the specificity of the PCR reactions.
Lane M was loaded with the Life Technologies, Inc. DNA size
marker, with sizes as indicated.
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5-HT1A Receptor mRNA Is Up-regulated in Activated B
and T Lymphocytes--
A quantitative analysis of 5-HT1A
up-regulation following treatment with various B and T cell mitogens
was performed using the RNase protection assay. Splenocytes were
incubated for different periods of time in the presence of culture
medium (unstimulated control), LPS, PHA, ConA, or a combination of PMA
plus ionomycin, and 5-HT1A mRNA levels were determined
and normalized to 18 S ribosomal RNA expression. Fig.
3 shows that 5-HT1A mRNA
was expressed in unstimulated splenocytes and was increased by all four
mitogens in a time-dependent manner. The level of
5-HT1A receptor mRNA was significantly enhanced after
24 h of incubation, reached a maximum at 48 h, and declined
toward the level in unstimulated cells after 72 h of culture. As
shown in Table I, relative to 5-HT1A mRNA level in freshly isolated splenocytes, the
level of increase in 5-HT1A mRNA in splenocytes treated
for 48 h with mitogens was 3.4-, 3.0-, 3.8-, and 4.9-fold with
LPS, PHA, ConA, or PMA plus ionomycin, respectively. There was no
increase in 5-HT1A expression in cells incubated for
48 h in the absence of mitogen. The level of 5-HT1A
expression correlated positively with the frequency of mitogen-induced
blast transformation which averaged 41%, 47%, 83%, and 88% in
splenocytes stimulated for 48 h with LPS, PHA, ConA, and PMA plus
ionomycin, respectively (Table I).

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Fig. 3.
Time-dependent up-regulation of
5-HT1A mRNA expression in mitogen-activated B and T
lymphocytes. Total RNA from splenocytes incubated for different
periods of time in the presence or absence of mitogens was hybridized
with radiolabeled 5-HT1A and 18 S riboprobes. The protected
RNA fragments were separated on a 5% polyacrylamide-urea gel and
quantitated by PhosphorImager analysis. Mitogens used: medium control
(lanes 4, 5, 10, and 15),
LPS (lanes 6, 11, and 16), PHA
(lanes 7, 12, and 17), ConA
(lanes 8, 13, and 18), and PMA plus
ionomycin (lanes 9, 14, and 19). The
incubation times were: 0 h (lane 4), 24 h
(lanes 5-9), 48 h (lanes 10-14), and
72 h (lanes 15-19), as indicated. Lane 1 was loaded with the RNA size marker, lane 2 with the
undigested 5-HT1A and 18 S antisense probes, which migrate
at 180 and 99 bp, respectively, and lane 3 with
RNase-digested 5-HT1A and 18 S antisense probes. Relative
5-HT1A levels were calculated by normalizing the
5-HT1A mRNA band to that of the 18 S rRNA, and -fold
increases in 5-HT1A expression induced by mitogens were
calculated by using the relative 5-HT1A level in freshly
isolated splenocytes (incubation time, 0 h) as a reference.
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Table I
Blast transformation and up-regulation of 5-HT1A mRNA
expression in mitogen-stimulated mouse splenocytes
Freshly isolated mouse spleen cells were incubated for 48 h in the
presence of culture medium or mitogen: LPS (10 µg/ml), PHA (20 µg/ml), ConA (5 µg/ml), or a combination of PMA (1 ng/ml) and
ionomycin (500 ng/ml). All values represent the mean ± S.D. of at
least four separate experiments.
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Since mitogen-stimulated splenocytes contain mixtures of different cell
types in different activation states, a more rigorous approach was
required to distinguish between B and T lymphocytes, and between
resting and activated lymphocytes. To this end, resting and activated
cells were separated by flow cytometry on the basis of their light
scatter properties, while CD19-positive B cells and Thy-1.2-positive T
cells were sorted by negative selection to 93-97% purity.
5-HT1A mRNA and 18 S rRNA expressions were measured in
unsorted as well as in sorted B and T lymphocyte populations by the
RNase protection assay. As shown in Fig.
4, 5-HT1A mRNA was
detected in both resting B and T cells purified from freshly isolated
splenocytes, and its level was increased in both activated B and
activated T cells purified from PMA plus ionomycin-stimulated lymphocyte populations. Quantitation by PhosphorImager analysis or
densitometry indicated that the increase in the relative level of
5-HT1A mRNA after stimulation with PMA plus ionomycin
was similar in RNA samples from unsorted lymphocytes, purified B
lymphocytes, or purified T lymphocytes (Fig. 4), suggesting similar
regulation of 5-HT1A mRNA expression in the two cell
types.

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Fig. 4.
5-HT1A mRNA expression in
purified B and T lymphocytes. Freshly isolated splenocytes were
used for sorting of resting B and T lymphocytes, while cells treated
with PMA plus ionomycin during 36-48 h were used for sorting of
activated B and T lymphocytes. Resting and activated cells were gated
on the basis of their forward scatter-side scatter profiles in flow
cytometry, and B and T lymphocytes were sorted in the desired region by
negative selection. The purity of the sorting was verified by
immunophenotyping, and it was 93-97%. Total RNA isolated from resting
lymphocytes (lanes 1-3) and activated lymphocytes
(lanes 4-6) was analyzed by RNase protection assay.
Lanes 1 and 4 represent unsorted lymphocytes,
lanes 2 and 5 are purified B lymphocytes, and
lanes 3 and 6 are purified T lymphocytes.
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Transcriptional Mechanisms of Mitogen-induced 5-HT1A
Receptor mRNA Expression--
Since 5-HT1A receptor
mRNA accumulation in activated lymphocytes could be attributed to
enhanced stabilization of existing mRNA and/or to enhanced
transcription of new mRNA, studies were performed to distinguish
between these two possibilities. Splenocytes were incubated or not with
a combination of PMA and ionomycin for 36 h prior to inhibition of
de novo mRNA transcription by addition of 10 µg/ml
actinomycin D. Total RNA was then extracted at fixed time intervals for
quantitation by RNase protection assay. As shown in Fig.
5 (A-C), the profiles of
mRNA degradation were superimposable in PMA-ionomycin-treated and
untreated cells with a similar half-life of 26 h, indicating an
absence of stabilization of 5-HT1A transcripts upon
mitogenic stimulation. Additional experiments using splenocytes
pretreated for 15 min with actinomycin D (10 µg/ml) and subsequently
stimulated with PMA-ionomycin for 36-48 h, showed that
5-HT1A mRNA expression did not increase over the level
in unstimulated cells (data not shown), indicating that induction of
5-HT1A mRNA is dependent on enhanced RNA transcription in mitogen-stimulated cells.

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Fig. 5.
Time course of 5-HT1A mRNA
degradation in splenocytes. Freshly isolated splenocytes were let
unstimulated (A) or were stimulated with PMA-ionomycin
(PMA-Iono) for 36 h (B). Actinomycin D
(Act. D, 10 µg/ml) was then added to stop all de
novo RNA transcription. Cells were harvested at the indicated
times after actinomycin D treatment and analyzed for 5-HT1A
mRNA and 18 S rRNA by an RNase protection assay. Plots in
C show the linear regression of the percentage of remaining
5-HT1A mRNA relative to time 0 and after normalization
to the 18 S rRNA. The coefficient of regression
(r2) is shown for unstimulated cells and for
PMA-ionomycin-stimulated cells; the calculated half-life was the same
(26 h).
|
|
To determine the potential role of the transcription factor NF-
B in
mitogen-stimulated 5-HT1A mRNA expression, splenocytes were pretreated with SN50, a cell-permeable peptide that specifically inhibits nuclear translocation of NF-
B (23). Fig.
6 shows that SN50
dose-dependently blocked the increase in 5-HT1A
mRNA expression induced by PMA plus ionomycin. In contrast, SN50M
(50 µg/ml), an inactive analogue of SN50, was devoid of any effect on
5-HT1A mRNA expression (Fig. 6), indicating the
specificity of the inhibitory action of SN50 on NF-
B activation. The
effect of other NF-
B inhibitors acting through mechanisms different
to SN50 were tested. These include PDTC that acts as both a radical
scavenger and inhibitor of NF-
B activation (24). Results showed that
PDTC (5-50 µM) caused a dose-dependent
inhibition of mitogen-induced up-regulation of lymphocyte
5-HT1A mRNA (Fig. 6). The immunosuppressive fungal metabolite gliotoxin (0.01-10 µg/ml), which appears to prevent degradation of I
B-
(25), also caused a significant
dose-dependent inhibition of mitogen-induced
5-HT1A up-regulation, while its inactive derivative
methylthiogliotoxin (1-10 µg/ml) had no significant effect (data not
shown).

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Fig. 6.
Blockage of mitogen-induced
5-HT1A mRNA up-regulation by
NF- B inhibitors. Total RNA was extracted
from freshly isolated splenocytes or from splenocytes stimulated with
PMA plus ionomycin for 48 h and analyzed by RNase protection assay
for 5-HT1A mRNA and 18 S rRNA expression. PMA plus
ionomycin stimulation was performed in the presence or absence of the
indicated NF- B inhibitors used at the indicated concentrations.
Shown are the bands corresponding to the protected 5-HT1A
and 18 S fragments, and the values of -fold increase in the relative
amount of 5-HT1A expression in PMA-ionomycin-treated cells
compared with freshly isolated cells.
|
|
5-HT1A Receptor Protein Expression in Splenocytes and
Up-regulation by B and T Cell Mitogens--
To evaluate the expression
of 5-HT1A receptor protein in unstimulated and
mitogen-stimulated lymphocytes, cells were permeabilized, fixed, and
subsequently analyzed by indirect immunofluorescence and flow cytometry
using a specific anti-peptide antiserum directed against the third
intracellular loop of the 5-HT1A receptor (26). Unstimulated splenocytes constitutively expressed the
5-HT1A protein, since greater than 90% of the cells were
positive (Fig. 7A). After stimulation with PMA plus ionomycin, the mean fluorescence intensity of
5-HT1A immunoreactivity was 4 times greater (Fig.
7B) as compared with unstimulated cells, indicating an
increased expression of 5-HT1A receptor protein. Cell
incubation with buffer or with preimmune serum yielded a much lower,
nonspecific fluorescence signal compared with the
anti-5HT1A antiserum, without any variation between
unstimulated (Fig. 7A) and mitogen-stimulated cells (Fig.
7B). Moreover, binding of the antiserum to an intracellular
epitope was revealed by the absence of any consistent signal above
background, unless the cells were permeabilized (Fig. 7, C
and D). Double staining with anti-CD19 or anti-Thy1.2 and
the anti-5HT1A receptor antiserum showed similar levels of
5-HT1A receptor protein expression in activated B and T
cells (data not shown), consistent with the similar level of induction
of 5-HT1A receptor RNA in the cells.

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Fig. 7.
5-HT1A protein expression in
splenocytes as detected by immunocytofluorometry and
immunocytochemistry. A-D, immunocytofluorometry
analysis. Freshly isolated splenocytes (A and C)
and splenocytes stimulated during 48 h with PMA plus ionomycin
(B and D) were incubated with a rabbit
anti-5-HT1A antiserum followed by a second step PE-labeled
goat anti-rabbit Ig Ab. Cells were permeabilized and fixed before
incubation with the antiserum (A and B). As a
control for intracellular labeling with anti-5-HT1A
antiserum, cells were stained with the antiserum without prior
permeabilization and fixation (C and D).
Histograms of fluorescence of cells incubated with the
anti-5-HT1A antiserum (bold line), or with
buffer (dashed line), or preimmune serum (thin
line) are shown, as well as the values of the mean fluorescence
intensity corresponding to cells positive for anti-5-HT1A
antiserum. E and F, immunocytochemistry analysis.
Unstimulated lymphocytes that were in the resting state of cell
activation and exhibited a small size (E), and lymphocytes
treated with PMA plus ionomycin for 48 h that underwent blast
transformation and exhibited higher cell size (F) were
subsequently permeabilized and incubated with the
anti-5-HT1A receptor antiserum. Staining was revealed by
the horseradish peroxidase system and visualized under photonic
microscope. The intensity of the staining was low (open
arrowheads) and high (filled arrowheads) in
unstimulated and mitogen-stimulated lymphocytes, respectively, and it
was localized at the plasma membrane in both cell types. Results are
representative of three separate experiments.
|
|
To visualize the localization of the 5-HT1A receptor
immunoreactivity, unstimulated and PMA plus ionomycin-stimulated cells were permeabilized and incubated with the anti-5-HT1A
antiserum whose binding was revealed by immunocytochemistry using the
horseradish peroxidase system. Labeling with the
anti-5-HT1A receptor antiserum yielded a little staining in
the unstimulated cells (Fig. 7E), while labeling of
mitogen-stimulated cells showed a marked and uniform staining of the
cell membrane, without any consistent staining of the cytoplasm (Fig.
7F). Labeling with the preimmune serum manifested no
detectable signal in unstimulated and mitogen-stimulated lymphocytes
(data not shown).
 |
DISCUSSION |
We showed previously that rat and mouse B lymphocyte in
vitro proliferation in response to mitogens is up-regulated by
5-HT and 5-HT1A agonists, and that selective
5-HT1A antagonists reverse the effect (18). Others have
shown that exposure to 5-HT1A agonists potentiates
mitogenic responses in human T cells, both in vivo (14) and
in vitro (13, 15, 16). Conversely, exposure to inhibitors of
5-HT synthesis or to 5-HT1A antagonists, leads to inhibition of mouse T cell responses in vivo and human T
cell responses in vitro (17). Additionally, previous
radioligand binding studies using [3H]8-OH-DPAT, a
relatively selective 5-HT1A agonist, have shown an increased level of
specific binding sites on murine B lymphocytes (18), and human T
lymphocytes (13) after mitogenic stimulation. To further characterize
the mechanisms of 5-HT1A receptor regulation in
lymphocytes, we used a quantitative RNase protection assay to assess
mRNA expression in mouse splenocytes. Our results demonstrate that
unstimulated B and T lymphocytes express low levels of
5-HT1A receptor mRNA that is markedly increased after
mitogenic stimulation in vitro, in accord with the previous
operational studies cited above. The results also show that purified B
and T lymphocytes behave similarly in their basal and mitogen-induced
5-HT1A mRNA expression. The increased expression of
5-HT1A in mitogen-stimulated B and T cells is detectable at
24 h, and reaches a maximum after 48 h. This delayed
induction of 5-HT1A mRNA correlates with the delayed
augmentation of mitogen-induced B and T lymphocyte proliferation, which
peaks at 72 h of cell incubation in the presence of
5-HT1A agonists. The late induction of 5-HT1A
mRNA by mitogens also suggests an indirect action including,
e.g., mitogen-induced cytokine synthesis that may in turn
regulate expression of the mRNA for 5-HT1A in target B
and T cells.
Our studies further elucidate the possible mechanism of
mitogen-stimulated increase in 5-HT1A mRNA. In
particular, 5-HT1A mRNA stability was not altered by
mitogen treatment, indicating that increased RNA stabilization plays no
detectable role in the induction. In contrast, the RNA synthesis
inhibitor actinomycin D completely blocked the mitogen-induced
overexpression of lymphocyte 5-HT1A mRNA, indicating
that induction is due to transcriptional stimulation, as opposed to
post-transcriptional mRNA stabilization. Moreover, we show that
exposure to several NF-
B inhibitors, including SN50, PDTC, and
gliotoxin, prevents any increase in 5-HT1A mRNA expression in mitogen-treated cells, suggesting a role for nuclear translocation of NF-
B in the up-regulation of lymphocyte
5-HT1A mRNA. Treatment of transfected Chinese hamster
ovary cells with 5-HT1A agonists has been shown previously
to increase 5-HT1A receptor density via activation of the
NF-
B pathway, by stimulating the degradation of the inhibitory
subunit, I-
B (32). Two consensus NF-
B binding sites (at
64 and
365 bp upstream from the initiation ATG) are located in a region with
strong enhancer activity that is highly conserved in rat and mouse
(33-35). In addition, recent studies have shown that the p50/p65
subunits of NF-
B are positive regulators of the rat
5-HT1A receptor promoter activity (36). Both a proximal
NF-
B site (at
64) and a distal NF-
B site (at
365) contribute
to this activity, whereas corticosteroids can repress it via their
glucocorticoid receptor. A variety of immune and inflammatory stimuli
are well known activators of nuclear translocation of NF-
B in
lymphocytes (19-21). Thus, we hypothesize that, like
5-HT1A agonists, immune stimulation may increase nuclear translocation of NF-
B to enhance transcription of the
5-HT1A receptor gene in B and T lymphocytes. Conversely,
part of the immunosuppressive and anti-inflammatory action of drugs
such as glucocorticoids may be explained by repression of
NF-
B-mediated induction of 5-HT1A receptor gene
transcription in immune cells.
Immunostaining with the anti-5-HT1A antiserum followed by
flow cytometry or by immunocytochemistry analysis demonstrates that the
expression of the receptor is low in unstimulated lymphocytes, while it
increased markedly upon mitogenic stimulation. This is consistent with
previous binding studies with radiolabeled agonists as performed by us
on murine B lymphocytes (18), and by others on human T cells (13).
Additional binding studies with the 5-HT1A antagonist
[3H]WAY100635 also indicate the existence of few specific
binding sites on unstimulated murine splenocytes, and greater binding on PMA plus ionomycin-stimulated cells (data not shown). Moreover, the
immunocytochemical studies show clearly that the receptor is localized
to the plasma membrane both in unstimulated and in mitogen-treated
cells, and not in intracellular compartment. Similar plasma membrane
localization of 5-HT1A receptor was demonstrated in
neuronal cell bodies and dendrites in adult rat brain (37), using
immunocytochemistry with the same anti-5-HT1A antiserum as
in this study. Together, the findings suggest that mitogenic stimulation of transcription is paralleled by increased cell surface 5-HT1A receptor immunoreactivity in lymphocytes.
The role of the 5-HT1A receptor in the immune response
suggests that pharmacological manipulations which alter levels of 5-HT (e.g. reuptake blockers, or depletion) or directly modulate
the 5-HT1A receptor (e.g. agonists, antagonists)
may constitute important strategies for immunomodulation. It is likely
that, in the course of tissue inflammation or immune response, the
activation of B and T cells may trigger a recurrent enhancement of
proliferation that is supported, in part, by induction and signaling of
the 5-HT1A receptor. Blockage of this enhancement in
5-HT1A receptor transcription or signaling may provide a
useful clinical approach to modulate immune and inflammatory responses.
On the other hand, enhancement of 5-HT1A induction or
signaling may augment the immune response under conditions (such as
immunodeficiency diseases) where an enhanced immune response is
desirable. This hypothesis is consistent with previous reports showing
that in vivo administration of the partial
5-HT1A agonist and anxiolytic/antidepressant drug buspirone
increases CD4 T-cell counts and in vitro T-cell
proliferation in human immunodeficiency virus-seropositive patients
(14).
 |
ACKNOWLEDGEMENTS |
We thank Sylvie Arbour for technical
assistance with the RNase protection assay and the immunocytochemical
studies, Sophie Ouellet for sorting of B and T lymphocytes by flow
cytometry, and Louis Senécal and Francine Leclerc for computer work.
 |
FOOTNOTES |
*
This work was supported by Medical Research Council of
Canada Grant MT-13259 (to E. K.) and by AstraZeneca/Fonds de la
recherche en santé du Québec Grant 981120 (to
E. K.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§§
To whom all correspondence should be addressed. Tel.:
514-630-8851; Fax: 514-630-8850; E-mail:
edouard.kouassi@inrs-iaf.uquebec.ca.
Published, JBC Papers in Press, November 15, 2000, DOI 10.1074/jbc.M004559200
 |
ABBREVIATIONS |
The abbreviations used are:
5-HT, 5-hydroxytryptamine or serotonin;
Ig, immunoglobulin;
Ab, antibody;
NF-
B, nuclear factor-
B;
LPS, lipopolysaccharide;
PHA, phytohemagglutinin;
PMA, phorbol 12-myristate 13-acetate;
PBS, phosphate-buffered saline;
ConA, concanavalin A;
PE, phycoerythrin;
FITC, fluorescein isothiocyanate;
PCR, polymerase chain reaction;
RT, reverse transcriptase;
bp, base pair(s);
PDTC, pyrrolidinedithiocarbamate;
GAPDH, glyceraldehyde-3-phosphate
dehydrogenase;
R-DPAT, R(+)-8-OH-DPAT hydrobromide.
 |
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