Transcriptional Mechanisms for Induction of 5-HT1A Receptor mRNA and Protein in Activated B and T Lymphocytes*

Mohamed AbdouhDagger , John M. Storring§, Mustapha Riad, Yves Paquette||, Paul R. Albert**, Elliot Drobetsky||, and Edouard KouassiDagger Dagger §§

From the Dagger Dagger  Human Health Research Center, INRS-Institut Armand-Frappier, Pointe-Claire, Quebec H9R 1G6, the Departments of Dagger  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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-kappa 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-kappa 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-gamma 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-kappa B (NF-kappa 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-kappa B is mainly composed of p50 and p65 subunits, which are normally retained in the cytosol of nonstimulated cells by inhibitory molecules, Ikappa B. In response to stimuli, Ikappa B are rapidly phosphorylated and degraded, allowing translocation of NF-kappa B complexes into the nucleus and activation of NF-kappa 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-kappa 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-kappa B may be one of the important signaling mechanisms involved.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-kappa 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 [alpha -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 [alpha -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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

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.

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.

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.

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-kappa B in mitogen-stimulated 5-HT1A mRNA expression, splenocytes were pretreated with SN50, a cell-permeable peptide that specifically inhibits nuclear translocation of NF-kappa 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-kappa B activation. The effect of other NF-kappa B inhibitors acting through mechanisms different to SN50 were tested. These include PDTC that acts as both a radical scavenger and inhibitor of NF-kappa 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 Ikappa B-alpha (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-kappa 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-kappa 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-kappa 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-kappa 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-kappa B pathway, by stimulating the degradation of the inhibitory subunit, I-kappa B (32). Two consensus NF-kappa 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-kappa B are positive regulators of the rat 5-HT1A receptor promoter activity (36). Both a proximal NF-kappa B site (at -64) and a distal NF-kappa 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-kappa B in lymphocytes (19-21). Thus, we hypothesize that, like 5-HT1A agonists, immune stimulation may increase nuclear translocation of NF-kappa 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-kappa 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-kappa B, nuclear factor-kappa 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.


    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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