Experimental Pathology, Karl-Franzens Universität, Heinrichstrasse 31, A-8010 Graz, Austria. Serotonin (5-hydroxytryptamine, 5-HT) has been shown to play a ...
Brain, Behavior, and Immunity 14, 219–224 (2000) doi:10.1006/brbi.1999.0579, available online at http://www.idealibrary.com on
BRIEF COMMUNICATION mRNA Expression of Serotonin Receptors in Cells of the Immune Tissues of the Rat Jasminka Stefulj,*,† Branimir Jernej,* Lipa Cicin-Sain,* Ingo Rinner,† and Konrad Schauenstein† *Department of Molecular Genetics, Rudjer Boskovic Institute, Bijenicka 54, and Croatian Institute for Brain Research, Salata 12, HR-10000 Zagreb, Croatia; and †Department of General and Experimental Pathology, Karl-Franzens Universita¨t, Heinrichstrasse 31, A-8010 Graz, Austria Serotonin (5-hydroxytryptamine, 5-HT) has been shown to play a role in immunoregulation; however, little is known about specific subtypes of 5-HT receptors involved in peripheral immunomodulation. In the present study we used RT-PCR methods to examine the mRNA expression of 5-HT receptors in the cells of lymphoid tissues of the rat. All 13 rat 5-HT receptor genes cloned so far were examined in ex vivo isolated spleen, thymus, and peripheral blood lymphocytes, as well as in mitogen-stimulated spleen cells. Positive signals were obtained for 5-HT1B, 5-HT1F, 5-HT2A, 5-HT2B, 5-HT6, and 5-HT7 receptor mRNAs in all three compartments. Mitogen (ConA and PWM) stimulated cells additionally expressed mRNA corresponding to the 5HT-3 receptor subtype. In contrast, 5-HT1A, 5HT1D, 5-HT2C, 5-HT4, 5-HT5A, and 5-HT5B mRNAs were not detected in any of the examined cell populations. These results may be useful as a starting point for future functional studies on immunomodulatory effects of 5-HT and may help to understand conflicting serotonergic effects on immune functions as found in the literature. 2000 Academic Press
Key Words: serotonin; 5-HT receptor mRNA; immune tissues; lymphocytes; mitogen stimulation.
1. INTRODUCTION
Serotonin (5-hydroxytryptamine, 5-HT) has been shown to affect various functions of immune cells (for review see Roszman & Brooks, 1985; Mo¨ssner & Lesch, 1998). Being released early during immune responses, 5-HT is essential for the optimal activation of T cells and natural killer cells, initiation of delayed-type hypersensitivity responses, natural immunity delivered by macrophages, production of a variety of chemotactic factors, etc. (Mo¨ssner & Lesch, 1998). Membrane receptors for this amine represent a highly polymorphic group of proteins, and are, at present, classified into 7 families (Hoyer & Martin, 1997). In the rat, 13 distinct 5-HT receptor subtypes have been identified so far by means of molecular biological techniques. With the exception of one cation channel (5-HT3 receptor), all subtypes are G-protein-coupled receptors and are linked to adenylylcyclase (5-HT1, 5-HT4, 5-HT6, 5-HT7) or phospholipase C (5-HT2), while the second messenger system of the 5-HT5 receptors still remains to be identified (Olivier, van Wijngaarden, & Soudijn, 1997). 5-HT receptors in the immune system have mostly been studied by pharmacological methods, and only a few have been demonstrated on the mRNA level (for review 219 0889-1591/00 $35.00
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see Mo¨ssner, 1998). In the present study using the reverse transcription–polymerase chain reaction (RT–PCR) we examined the presence of mRNA for each of the 13 cloned rat 5-HT receptor subtypes in ex vivo isolated cells of spleen, thymus, and peripheral blood lymphocytes (PBL), as well as in mitogen-stimulated spleen cells. 2. MATERIALS AND METHODS
Oligonucleotide Primers Primers used in PCR amplification were synthesized by Life Technologies (Vienna, Austria) according to sequences previously published by Ullmer, Schmuck, Kalkman, and Lu¨bbert (1995) for the rat 5-HT4 receptor, and by Pierce, Xie, Levine, and Peroutka (1996) for all other 5-HT receptor subtypes. Primers specific for βactin mRNA were used to monitor the quality of RNA and the efficiency of reverse transcription. Tissues, RNA Preparation, and RT-PCR Male Wistar or Sprague Dawley rats of 200–300 g were used. Spleen and thymus cell suspensions were prepared by passing tissue fragments through a nylon sieve and PBL were obtained by isolation on a Percoll gradient (Sigma). Tissues known to express particular 5-HT receptors (positive control tissue), i.e., brain cortex, corpus striatum, and stomach fundus, were taken from few animals and frozen immediately in liquid nitrogen. RNA was isolated from either 1 ⫻ 10 7 cells or 20–30 mg of tissue according to Chomczynski and Sacchi (1987). All RNA preparations were treated with DNase I and analyzed by agarose gel electrophoresis. One microgram of each RNA was reverse transcribed in a final volume of 20 µl with 15 U of AMV reverse transcriptase (Promega) and 0.5 µg oligo (dT)15 primer (Promega), for 30 min at 42°C, followed by 5 min at 99°C. A control reaction lacking reverse transcriptase was also prepared from each RNA sample (negative tissue control). PCR amplification was performed with 1.5 µl aliquots of cDNA. The optimal concentrations of MgCl2 and of the primers were determined for each receptor, and PCR amplification (94°C for 45 s, 59°C for 1 min, 72°C for 50 s) was performed in a final volume of 25 µl with 0.6 U of AmpliTaq Gold DNA-polymerase (Perkin–Elmer) and 0.2 mM dNTP (Promega). Each PCR assay included a positive control tissue, a water control, and negative tissue control for each sample to exclude genomic DNA contamination. The length of PCR products separated on 1.6% agarose gels was determined using computer program (ONE-Dscan, Version 1.0). Cell Cultures Mitogen activation of spleen cells was performed for 48 h in U-bottomed microtiter plates (Greiner) using 106 cells per well in 200 µl medium (RPMI 1640, supplemented with 10⫺5M 2-mercaptoethanol and antibiotics) containing 2.5 µg/ml Concanavalin A (ConA, Sigma) or 20 µg/ml Pokeweed mitogen (PWM, Sigma). To monitor mitogen stimulation the cellular uptake of 3H-thymidine was determined as previously described (Felsner, Hofer, Rinner, Mangge, Gruber, Korsatko, & Schauenstein, 1992). Experimental Design Two independent groups of experiments were carried out with ex vivo isolated lymphocytes. In the first group, total RNA was isolated from pools of PBL, spleen,
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and thymus cells, each derived from five animals, while in the second group spleens and thymuses from individual animals (N ⫽ 10 and 6, respectively) were analyzed. Mitogen activation of spleen lymphocytes was performed in two independent experiments with individual organs of three animals each. RT–PCR results were confirmed in at least two independent reactions throughout. 3. RESULTS
Oligonucleotide Primers All primers were pretested by RT–PCRs with RNA from positive control tissues (see Table 1). A single band of the expected size was obtained with each primer pair. Serotonin Receptor mRNAs Expression in Immune Tissues mRNAs for six out of 13 receptor subtypes, i.e., 5-HT1B, 1F, 2A, 2B, 6, and 7, were found in resting cells of three different immune compartments (Fig. 1A, Table 1). The same six mRNAs were present in mitogen—both ConA and PWM—activated spleen cells, but, in addition, 5-HT3 mRNA was also positive (Fig. 1B). Preliminary experiments showed the same with activated PBL (not shown). In contrast, 5HT1A, 1D, 2C, 4, 5A, and 5B messages were not detected in any of the cell populations. No differences in 5HT receptor mRNAs expression were noted either between individual animals or between animals of the two strains used (not shown). Although internal standards were not used to compare the relative abundance of particular mRNAs, differences in the number of PCR cycles needed to obtain clearly positive RT–PCR signals for different receptors in immune tissues (see note to Table 1) indicated different levels of mRNA expression, i.e., high for 5HT6 and 5-HT7, moderate for 5HT-2A, 5-HT2B, and 5-HT3, and low for 5HT-1B and 5-HT1F receptor subtypes. TABLE 1 Serotonin Receptor mRNAs in Rat Immune Tissues Receptor subtype 5-HT 1A 1B 1D 1F 2A 2B 2C 3 4 5A 5B 6 7
PCR product length (bp)
Positive control
635 643 630 618 611 341 612 415 397 637 620 374 253
brain cortex brain cortex brain cortex brain cortex brain cortex stomach fundus brain cortex brain cortex striatum brain cortex brain cortex brain cortex brain cortex
Thymus
Peripheral blood lymphocytes
Spleen
Mitogen activated spleen cells
⫺ ⫹ ⫺ ⫹ ⫹ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫹
⫺ ⫹ ⫺ ⫹ ⫹ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫹
⫺ ⫹ ⫺ ⫹ ⫹ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫹
⫺ ⫹ ⫺ ⫹ ⫹ ⫹ ⫺ ⫹ ⫺ ⫺ ⫺ ⫹ ⫹
Note. ⫹/⫺ indicate positive/negative RT-PCR signals. All negative results were confirmed at 45 PCR cycles, while clear positive signals were obtained at 35 (5-HT6, 5-HT7), 40 (5-HT2A, 5-HT2B, 5-HT3), or 45 (5-HT1B, 5-HT1F) cycles. With the respective positive control tissues all primers yielded signals already after 30 PCR cycles.
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FIG. 1. Agarose gel electrophoresis of serotonin receptor RT-PCR products obtained from cells of immune tissues of the rat. As control for the absence of genomic DNA contamination, all RT–PCRs were performed with (⫹) or without (⫺) reverse transcriptase added to the cDNA synthesis reaction. Rat tissues known to express particular 5-HT receptor subtypes, i.e., brain cortex (b.c), stomach fundus (s.f ), and striatum, were used as positive controls. (A) RNA of peripheral blood lymphocytes (PBL), spleen, and thymus was isolated from cell pools of the respective tissues from five animals. RT–PCR products derived at 45 PCR cycles are presented. (B) RT–PCR products corresponding to 5-HT3 receptor, derived at 40 PCR cycles from resting (1), and Concanavalin A (2) or Pokeweed mitogen (3) activated spleen cells.
4. DISCUSSION
As an extension of our previous research on the effects of 5-HT on the immune response in rats (Gabrilovac, Cicin-Sain, Osmak, & Jernej, 1992; Poljak-Blazi, Jernej, Cicin-Sain, & Boranic, 1990), we performed a systematic screening for the presence of mRNA for all known rat 5-HT receptor subtypes mRNAs in central and peripheral immune compartments of the rat. We showed that mRNA for six 5-HT receptors, i.e., 5-HT1B, 1F, 2A, 2B, 6, and 7, is constitutively present in rat immune tissues, while 5-HT3 mRNA was found after mitogenic activation only. The identical set of six 5-HT receptor mRNAs, consistently found in resting cells of three different immune compartments, as well as in mitogen activated (i.e., cultured) spleen cells and PBL, strongly suggests that the detected molecules were derived from immune cells and not from other tissue components of lymphoid organs. Furthermore, the induction of transcription of the 5-HT3 receptor gene by mitogenic stimulation is indicative
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for a role of this receptor in lymphocyte activation and points out that even low levels of mRNA (detectable not before 40 PCR cycles) might be of functional significance. Future studies will focus on the differential expression of those mRNAs in various types of purified leukocyte subsets. mRNA encoding the 5-HT1A receptor has previously been reported in resting lymphocytes of humans (Marazziti, Palego, Dal Canto, Rotondo, Pasqualetti, Gino, Lucacchini, Ladinsky, Nardi, & Cassano, 1995; Eugen-Olsen, Afzelius, Andresen, Iversen, Kronborg, Aabech, Nielsen, & Hofmann, 1997) and mice (Fillion, Fillion, Cloez-Tayarani, Sarhan, Haour, & Bolanos, 1993). The discrepancy between these results and the absence of 5-HT1A mRNA in rat immune tissue may reflect species differences. It cannot be explained by findings reported by Aune, McGrath, Sarr, Bombara, and Kelly (1993) that the expression depends on the level of cell activation, since we could not detect 5-HT1A mRNA in mitogen activated rat lymphocytes either. Pharmacological studies with 8-OH-DPAT, which was considered to be a selective 5-HT1A receptor agonist, suggested the presence of 5-HT1A receptor on immune cells too (Hellstrand & Hermodsson, 1993; Aune, Golden, & McGrath, 1994; Iken, Chheng, Fargin, Goulet, & Kouassi, 1995). However, this compound has recently been shown to bind also to the 5-HT7 receptor (Shen, Monsma, Metcalf, Jose, Hamblin, & Sibley, 1993), whose mRNA was demonstrated in human (Heidmann, Metcalf, Kohen, & Hamblin, 1997) and rat (Shen et al., 1993; our results) immune tissues. Thus, some of the described effects may be mediated through 5-HT7 receptors rather than 5-HT1A. Much less was reported on mRNA expression of the other 5-HT receptors in immune tissues. 5-HT2C and 5-HT1D mRNAs were demonstrated in human spleen (Bonhaus, Bach, Desouza, Salazar, Matsouka, Zuppan, Chan, & Eglen, 1995) and mouse thymus (Fillion et al., 1993), respectively, being different from our findings in rats, and again most likely due to species differences. On the other hand, 5-HT1B, 2A, 2B, and 7 mRNAs have been demonstrated in human and mouse (Fillion et al. 1993; Bonhaus et al. 1995; Heidmann et al., 1997), as well as in rat (this paper) lymphoid tissues. In conclusion, the present study provides the basic data on the presence/absence of 5-HT receptor subtype mRNAs in cells of rat immune tissues, and it should give guidelines for pharmacological research on immunoregulatory effects of 5-HT. Experiments are presently underway to determine to what extent 5-HT receptor messages are successfully translated into functionally active proteins on the surface of immune cells. ACKNOWLEDGMENT Supported by the Jubila¨umsfonds der Oesterreichischen Nationalbank (Project 5692).
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