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• W2054219549 abstract "Serotonin (5-HT) up-regulates B and T lymphocyte proliferation by activating mitogen-induced cell surface 5-HT1A receptors. The mechanism of 5-HT1Areceptor 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 (t 12 = 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-κB 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-κB 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. Serotonin (5-HT) up-regulates B and T lymphocyte proliferation by activating mitogen-induced cell surface 5-HT1A receptors. The mechanism of 5-HT1Areceptor 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 (t 12 = 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-κB 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-κB 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. 5-hydroxytryptamine or serotonin immunoglobulin antibody nuclear factor-κB lipopolysaccharide phytohemagglutinin phorbol 12-myristate 13-acetate phosphate-buffered saline concanavalin A phycoerythrin fluorescein isothiocyanate polymerase chain reaction reverse transcriptase base pair(s) pyrrolidinedithiocarbamate glyceraldehyde-3-phosphate dehydrogenase R(+)-8-OH-DPAT hydrobromide 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 (1Essman W.B. Serotonin in Health and Disease: Availability, Localization and Disposition. I. Spectrum Publications, NewYork1978: 15-179Google Scholar). 5-HT is also accumulated by and released from noradrenergic nerve terminals that are in close contact with lymphocytes in lymphoid organs (2Paiva M.Q. Caramona M. Osswald W. Naunyn Schmiedebergs Arch. Pharmacol. 1984; 325: 62-68Crossref PubMed Scopus (26) Google Scholar, 3Felten D.L. Felten S.Y. Carlson S.L. Olschowka J.A. Livnat S. J. Immunol. 1985; 135: 755s-765sPubMed Google Scholar, 4Fuchs B.A. Campbell K.S. Munson A.E. Cell Immunol. 1988; 117: 339-351Crossref PubMed Scopus (101) Google Scholar). 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 (5Crivellato E. Damiani D. Mallardi F. Travan L. Acta Anat. 1991; 141: 127-131Crossref PubMed Scopus (42) Google Scholar). 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 (6Hoyer D. Clarke D.E. Fozard J.R. Hartig P.R. Martin G.R. Mylecharane E.J. Saxena P.R. Humphrey P.A. Pharmacol. Rev. 1994; 46: 157-203PubMed Google Scholar, 7Mossner R. Lesch K.P. Brain Behav. Immun. 1998; 12: 249-271Crossref PubMed Scopus (379) Google Scholar). The 5-HT1A gene has been cloned previously in human (8Kobilka B.K. Frielle T. Collins S. Yang-Feng T. Kobilka T.S. Francke U. Lefkowitz R.J. Caron M.G. Nature. 1987; 329: 75-79Crossref PubMed Scopus (432) Google Scholar, 9Fargin A. Raymond J.R. Regan J.W. Cotecchia S. Lefkowitz R.J. Caron M.G. J. Biol. Chem. 1989; 264: 14848-14852Abstract Full Text PDF PubMed Google Scholar), rat (10Albert P.R. Zhou Q.Y. VanTol H.H.M. Bunzow J.R. Civelli O. J. Biol. Chem. 1990; 265: 5825-5832Abstract Full Text PDF PubMed Google Scholar), and mouse (11Charest A. Wainer B.H. Albert P.R. J. Neurosci. 1993; 13: 5164-5171Crossref PubMed Google Scholar), manifesting very high nucleotide and amino acid sequence homology in their respective putative transmembrane regions. 5-HT1AmRNA has been detected in various human tissues including lymph nodes, spleen, and thymus (8Kobilka B.K. Frielle T. Collins S. Yang-Feng T. Kobilka T.S. Francke U. Lefkowitz R.J. Caron M.G. Nature. 1987; 329: 75-79Crossref PubMed Scopus (432) Google Scholar), as well as in human peripheral blood mononuclear cells (12Marazziti D. Palego L. Canto B.D. Rotondo A. Pasqualetti M. Gino G. Lucacchini A. Ladinsky H. Nardi I. Cassano G.B. Life Sci. 1995; 57: 2197-2203Crossref PubMed Scopus (11) Google Scholar) and activated T lymphocytes (13Aune T.M. McGrath K.M. Sarr T Bombara M.P. Kelley K.A. J. Immunol. 1993; 151: 1175-1183PubMed Google Scholar). 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 (13Aune T.M. McGrath K.M. Sarr T Bombara M.P. Kelley K.A. J. Immunol. 1993; 151: 1175-1183PubMed Google Scholar, 14Hofmann B. Afzelius P. Iversen J. Kronborg G. Aabech P. Benfield T. Dybkjaer E. Nielsen J.O. AIDS. 1996; 10: 1339-1347Crossref PubMed Scopus (22) Google Scholar, 15Eugen-Olsen J. Afzelius P. Andresen L. Iversen J. Kronborg G. Aabech P. Nielsen J.O. Hofmann B. Clin. Immunol. Immunopathol. 1997; 84: 115-121Crossref PubMed Scopus (39) Google Scholar, 16Afzelius P. Nielsen S.D. Hofmann B. Nielsen J.O. Scand. J. Infect. Dis. 1997; 29: 117-120Crossref PubMed Scopus (13) Google Scholar), production of Th1 cytokines such as interleukin-2 and interferon-γ both in mice (17Aune T.M. Golden H.W. McGrath K.M. J. Immunol. 1994; 153: 489-498PubMed Google Scholar) and in human (15Eugen-Olsen J. Afzelius P. Andresen L. Iversen J. Kronborg G. Aabech P. Nielsen J.O. Hofmann B. Clin. Immunol. Immunopathol. 1997; 84: 115-121Crossref PubMed Scopus (39) Google Scholar, 16Afzelius P. Nielsen S.D. Hofmann B. Nielsen J.O. Scand. J. Infect. Dis. 1997; 29: 117-120Crossref PubMed Scopus (13) Google Scholar), and contact sensitivity reactions in mice (17Aune T.M. Golden H.W. McGrath K.M. J. Immunol. 1994; 153: 489-498PubMed Google Scholar). 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 (18Iken K. Chheng S. Fargin A. Goulet A.C. Kouassi E. Cell Immunol. 1995; 163: 1-9Crossref PubMed Scopus (126) Google Scholar). 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 (7Mossner R. Lesch K.P. Brain Behav. Immun. 1998; 12: 249-271Crossref PubMed Scopus (379) Google Scholar). 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 (18Iken K. Chheng S. Fargin A. Goulet A.C. Kouassi E. Cell Immunol. 1995; 163: 1-9Crossref PubMed Scopus (126) Google Scholar) and human T cells (13Aune T.M. McGrath K.M. Sarr T Bombara M.P. Kelley K.A. J. Immunol. 1993; 151: 1175-1183PubMed Google Scholar), but little is known about the molecular mechanisms underlying this effect. Nuclear factor-κB (NF-κB) 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 (19Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4599) Google Scholar, 20Barnes P.J. Karin M. N. Engl. J. Med. 1997; 336: 1066-1071Crossref PubMed Scopus (4284) Google Scholar, 21Sha W.C. J. Exp. Med. 1998; 187: 143-146Crossref PubMed Scopus (184) Google Scholar). NF-κB is mainly composed of p50 and p65 subunits, which are normally retained in the cytosol of nonstimulated cells by inhibitory molecules, IκB. In response to stimuli, IκB are rapidly phosphorylated and degraded, allowing translocation of NF-κB complexes into the nucleus and activation of NF-κB elements (22Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5579) Google Scholar). In this report, we used RNase protection assay to quantitate the expression of 5-HT1A receptor mRNA in unstimulatedversus mitogen-stimulated mouse splenocytes. In addition, we took advantage of the availability of pharmacological inhibitors of NF-κB (23Lin Y.Z. Yao S.Y. Veach R.A. Torgerson T.R. Hawiger J. J. Biol. Chem. 1995; 270: 14255-14258Abstract Full Text Full Text PDF PubMed Scopus (854) Google Scholar, 24Schreck R. Meier B. Mannel D.N. Droge W. Baueuerle P.A. J. Exp. Med. 1992; 175: 1181-1194Crossref PubMed Scopus (1448) Google Scholar, 25Pahl H.L. Kraub B. Schulze-Osthoff K. Decker T. Traenckner E.B. Vogt M. Myers C. Parks T. Warring P. Mühlbacher A. Czernilofsky A-P. Baeuerle P.A. J. Exp. Med. 1996; 183: 1829-1840Crossref PubMed Scopus (311) Google Scholar) 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 (26El Mestikawy S. Riad M. Laporte A.M. Vergé D. Duval G. Gozlan H. Hamon M. Neurosci. Lett. 1990; 118: 189-192Crossref PubMed Scopus (87) Google Scholar) 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-κB may be one of the important signaling mechanisms involved. 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 (26El Mestikawy S. Riad M. Laporte A.M. Vergé D. Duval G. Gozlan H. Hamon M. Neurosci. Lett. 1990; 118: 189-192Crossref PubMed Scopus (87) Google Scholar), and all other Ab were from PharMingen (San Diego, CA). 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-κB, splenocytes were incubated for 48 h with mitogens in the presence of 10–50 μg/ml SN50 (Calbiochem), 5–50 μmpyrrolidinedithiocarbamate (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 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 (27Grumont R.J. Rourke I.J. O'Reilly L.A. Strasser A. Miyake K. Sha W. Gerondakis S. J. Exp. Med. 1998; 187: 663-674Crossref PubMed Scopus (208) Google Scholar). 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%. Splenocytes were incubated for 30 min with or without 5 × 10−5mWAY100635 before stimulation for 72 h with mitogens in the presence or absence of 10−4m 5-HT or 5 × 10−5m 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. 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. 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 (11Charest A. Wainer B.H. Albert P.R. J. Neurosci. 1993; 13: 5164-5171Crossref PubMed Google Scholar) using the Pst I 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 Bss HII and transcribed with T3 RNA polymerase (Ambion) and 50 μCi of 800 Ci/mmol [α-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 [α-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-HT1Aand 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. 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 (26El Mestikawy S. Riad M. Laporte A.M. Vergé D. Duval G. Gozlan H. Hamon M. Neurosci. Lett. 1990; 118: 189-192Crossref PubMed Scopus (87) Google Scholar), 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. 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). 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 (18Iken K. Chheng S. Fargin A. Goulet A.C. Kouassi E. Cell Immunol. 1995; 163: 1-9Crossref PubMed Scopus (126) Google Scholar), 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 (28Gozlan H. Thibault S. Laporte A-M. Lima L. Hamon M. Eur. J. Pharmacol. 1995; 288: 173-186Crossref PubMed Scopus (168) Google Scholar). Previously, we demonstrated that 5-HT increases mitogen-stimulated murine B lymphocyte proliferation through a 5-HT1A receptor-mediated mechanism (18Iken K. Chheng S. Fargin A. Goulet A.C. Kouassi E. Cell Immunol. 1995; 163: 1-9Crossref PubMed Scopus (126) Google Scholar). 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 10−4m) and the selective 5-HT1A receptor agonist R-DPAT (10−11 to 10−4m) increased PHA-stimulated T lymphocyte proliferation in a dose-dependent manner with optimal concentrations of 10−4m and 5 × 10−5m, 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.1 A shows that 5 × 10−5m WAY100635 effectively abrogated 5-HT- and R-DPAT-mediated enhancement of activated T lymphocyte proliferation, thus implicating the 5-HT1Areceptor in the control of T cell proliferation. 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 (29Truneh A. Albert F. Golstein P. Schmitt-Verhulst A.M. Nature. 1985; 313: 318-320Crossref PubMed Scopus (610) Google Scholar, 30Klaus G.G. O'Garra A. Bijsterbosch M.K. Holman M. Eur. J. Immunol. 1986; 16: 92-97Crossref PubMed Scopus (99) Google Scholar, 31Li Y.S. Kouassi E. Revillard J.P. Eur. J. Immunol. 1989; 19: 1721-1725Crossref PubMed Scopus (23) Google Scholar). 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. 1 B 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. 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 (6Hoyer D. Clarke D.E. Fozard J.R. Hartig P.R. Martin G.R. Mylecharane E.J. Saxena P.R. Humphrey P.A. Pharmacol. Rev. 1994; 46: 157-203PubMed Google Scholar). 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. A quantitative analysis of 5-HT1Aup-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-HT1Aexpression 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).Table IBlast transformation and up-regulation of 5-HT1A mRNA expression in mitogen-stimulated mouse splenocytesMitogenNo. of replicated experimentsBlast transformation1-aResting and blast cells were distinguished by flow cytometry based on their forward scatter-side scatter profiles, and the values indicated represent the percentage of blast cells within the total cell population.Increase in 5-HT1A mRNA expression1-bRelative levels of 5-HT1AmRNA were determined by RNase protection assay using 18 S rRNA as an internal standard. -Fold increase in 5-HT1A expression induced by mitogens was calculated by using the relative 5-HT1Alevel in freshly isolated splenocytes as a reference.%-foldNo mitogen75 ± 20.98 ± 0.17LPS441 ± 63.43 ± 1.41PHA447 ± 133.03 ± 1.26ConA483 ± 163.76 ± 1.42PMA-ionomycin788 ± 114.90 ± 1.90Freshly 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 c" @default.
• W2054219549 created "2016-06-24" @default.
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• W2054219549 date "2001-02-01" @default.
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• W2054219549 title "Transcriptional Mechanisms for Induction of 5-HT1AReceptor mRNA and Protein in Activated B and T Lymphocytes" @default.
• W2054219549 cites W1495834546 @default.
• W2054219549 cites W1496381585 @default.
• W2054219549 cites W1942920640 @default.
• W2054219549 cites W1965583590 @default.
• W2054219549 cites W1969382469 @default.
• W2054219549 cites W1969572802 @default.
• W2054219549 cites W1979104877 @default.
• W2054219549 cites W1988111959 @default.
• W2054219549 cites W1989153933 @default.
• W2054219549 cites W1993516116 @default.
• W2054219549 cites W1994191168 @default.
• W2054219549 cites W2005533943 @default.
• W2054219549 cites W2012129851 @default.
• W2054219549 cites W2026108503 @default.
• W2054219549 cites W2028981503 @default.
• W2054219549 cites W2040970439 @default.
• W2054219549 cites W2041477655 @default.
• W2054219549 cites W2046209075 @default.
• W2054219549 cites W2049731820 @default.
• W2054219549 cites W2055080475 @default.
• W2054219549 cites W2067883239 @default.
• W2054219549 cites W2073793180 @default.
• W2054219549 cites W2080777409 @default.
• W2054219549 cites W2098767279 @default.
• W2054219549 cites W2100059138 @default.
• W2054219549 cites W2116937468 @default.
• W2054219549 cites W2119335473 @default.
• W2054219549 cites W2151892297 @default.
• W2054219549 cites W2159219386 @default.
• W2054219549 cites W2159567970 @default.
• W2054219549 cites W2159612818 @default.
• W2054219549 cites W2160858683 @default.
• W2054219549 cites W2166781237 @default.
• W2054219549 cites W2331176038 @default.
• W2054219549 cites W79466244 @default.
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