Scand J Rheumatol 2004;33:1–3

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Expression of 5-HT3A receptors in cells of the immune system BL Fiebich1,2, RS Akundi1, M Seidel3, V Geyer1, U Haus4, W Mu¨ller5, T Stratz5, E Candelario-Jalil1 1

Department of Psychiatry and Psychotherapy, University of Freiburg Medical School, 2VivaCell Biotechnology GmbH, Freiburg, 3University Outpatient Clinic of Internal Diseases, Rheumatology, Bonn, 4Novartis Pharma GmbH, Nu¨rnberg, and 5Hochrhein Institute of Rehabilitation Research, Bad Sa¨ckingen, Germany

There is evidence from both human and animal research that 5-hydroxytryptamine3 (5-HT3) receptor antagonists, particularly tropisetron, exert analgesic and antiinflammatory effects. However, the underlying mechanisms of these effects including the expression of 5-HT3 receptors in cells of the immune system have not yet been investigated in detail. Therefore, we investigated the expression of the 5-HT3A receptor in primary human monocytes, chondrocytes, T cells, dendritic cells, and synovial tissue. We found that 5-HT3A receptors are expressed in monocytes, chondrocytes, T-cells, and synovial tissue but not in dendritic cells. Our data show that 5-HT3A receptors are widely expressed in cells of the immune system and that they might play an important role in inflammatory events and in the observed antiphlogistic effects of 5-HT3 receptor antagonists.

5-HT3 receptors occur mainly in the peripheral nervous system, particularly on nociceptive sensory neurons autonomic neurons and enteric neurons, on which 5-HT exerts a strong excitatory effect (1). The central 5-HT3 receptors are concentrated in regions that are involved among other things in the integration of the vomiting reflex and pain processing (2). The physiological role of 5-HT3 receptors is far from being well understood, but it has been postulated that excitation of vascular sensory nerve terminals by 5-HT released from platelets may be involved in the pathogenesis of migraine. Unlike other 5-HT receptors that are coupled to G-proteins, 5-HT3 receptors are exceptional in being directly linked to membrane ion channels and not involving any second messengers in their transduction mechanism. The 5-HT3 receptor is a ligandgated cation channel belonging to the nicotine/ gamma-aminobutyrate(GABA) receptor superfamily (3, 4). The receptor contains a large extracellular domain containing a conserved cysteine loop, four hydrophobic transmembrane segments, a large intracellular loop between the third and fourth transmembrane region and an extracellular Cterminus (5). The ion channel itself is an oligomeric complex composed of five subunits (6). Two forms of 5-HT3 receptor subunits, 5-HT3A and 5-HT3B, have been identified in the nineties, and recently three other 5-HT3 receptor-like genes, HT3C, Bernd L Fiebich, University of Freiburg Medical School, Dept. of Psychiatry and Psychotherapy, D-79104 Freiburg, Germany. E-mail: [email protected] Received Accepted

HT3D, and HT3E, have been cloned (7) which show homology to the other subunits in the four transmembrane regions and the huge intracellular loop, both known to affect channel function. It has been suggested that the five 5-HT3 receptor subtypes are differentially expressed in tissues, and there is evidence that the 5HT3B subtype is not expressed in the CNS, which helps to account for the complexity of 5-HT3 receptors observed (7, 8). Activation of 5-HT3 receptors followed by rapid depolarisation of the peripheral or central neuron causes a rapid increase in free cytosolic Ca2z levels by inducing Ca2z influx and mobilisation of intracellular Ca2z stores, which modulates the release of various neurotransmitters and neuropeptides such as dopamine, cholecystokinin, acetylcholine, GABA, substance P or 5-HT itself (9). 5-HT3 receptor antagonists are novel potent antiemetics (10). Experimental studies have also revealed that 5-HT3 receptor antagonists possess both an analgesic and antiphlogistic effect, the latter suggesting the presence of 5-HT3 receptors in various cell types involved in inflammation. But so far the expression of 5-HT3 receptors has not been investigated in most cells of the immune system. Therefore, we studied the expression of 5-HT3A receptors [long and short splice variant (3)] in a variety of cells involved in inflammation.

Material and methods Human peripheral monocytes, T-cells, synovial tissue, and dendritic cells were isolated. Monocytes, T cells, and dendritic cells from healthy human donors were prepared as described by Norgauer et al (11). Synovial tissue of osteoarthritic patients was

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prepared as described by Seidel et al (unpublished observations). Chondrocytes were obtained from PromoCell (Heidelberg, Germany). Cells were seeded in 6-well plates for ribonucleic acid (RNA) analysis and incubated at 37‡C and 5% CO2.

BL Fiebich et al

RNA extraction and polymerase chain reaction (PCR) analysis Total RNA was extracted using the guanidine isothiocyanate method (12). For RT-PCR, 1 mg total RNA was reverse transcribed using MuMLV-reverse

Figure 1. Expression of 5-HT3A receptors in human cells and tissues. RNA was isolated from monocytes (A), T cells (B), synovial tissue of six patients (C), and primary chondrocytes (D) as described in Material and methods, and then RT-PCR was carried out for the two splice variants of the 5-HT3A receptor (short and long). MW: molecular weight marker (100 bp ladder). H2O: water control.

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5-HT3A expression in immune cells

transcriptase (Gibco, Eggenstein, Germany) and random hexamers. Primers for human 5-HT3 receptors were designed using PrimerSelect Software from DNA Star Inc. (Madison, WI, USA). PCR was then carried out by adding Taq polymerase (Promega) and a pair of primers for human 5-HT3 (sense: 5’-CCGGCGGCCCCTCTTCTAT-3’, anti-sense: 5’GCAAAGTAGCCAGGCGATTCTCT-3’, 64‡C, 40 cycles), or human S12 (sense: 5’-GGAGGTGTAATGGACGTTA-3’, anti-sense: 5’-CTGAGACTCCTTGCCATAG-3’, 54‡C, 30 cycles). PCR products were then separated electrophoretically on a 2% agarose gel. Results Since clinical observations suggested antiinflammatory properties of the 5-HT3 receptor antagonist tropisetron (Navoban1), we were interested in investigating the expression of 5-HT3A receptors in cells involved in immune function. By using the reverse transcriptase (RT)-PCR method, we assessed the expression of 5-HT3A receptors in monocytes, T-cells, dendritic cells, and chondrocytes. As shown in Figure 1A, B, C, and D, monocytes, T-cells, synovial tissue, and primary chondrocytes strongly expressed both splice variants of 5-HT3A receptors, whereas dendritic cells showed no signal in the RTPCR analysis (not shown). Discussion We recently found the 5-HT3 receptor antagonist tropisetron to be a potent inhibitor of TNF-a and IL-1b in LPS-stimulated human primary monocytes (Fiebich et al, J Pharmacol Exp Ther 2004). After concluding that those antiinflammatory effects are mediated by 5-HT3 receptors, we investigated the expression of 5-HT3A receptors in various cells of the immune system. Here we demonstrated that most cells of the immune system tested expressed both splice variants of the 5-HT3A receptors, which suggests that this receptor plays a crucial role in inflammation. This is supported by data showing that serotonin can also be released by cells of the immune system [reviewed by Gordon J, Barnes NM (13)] and that exogenously added serotonin increases T-cell proliferation (14). The 5-HT3 receptor is an ionotropic receptor with a high selectivity to Naz inward movement. It has been shown that Naz/Kz movement has an impact on several events of immune cell activation. However, little is known about the role of 5-HT3 receptors in inflammatory

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processes and the molecular mechanisms by which this receptor might mediate antiinflammatory effects. Additional studies are needed to assess the role of 5-HT3 receptors in immune cells. In conclusion, the presence of 5-HT3 receptors on immune cells suggests a promising new role of the 5-HT3 receptor antagonists such as tropisetron in therapeutics.

Acknowledgement The technical assistance of Sonja Orlikowsky and Brigitte Guenter is gratefully acknowledged.

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