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May 21, 2004 - There is evidence from both human and animal research that 5-hydroxytryptamine (5-HT)3 receptor antagonists, particularly tropisetron, exert ...
Scand J Rheumatol 2004;33:1–5


Antiinflammatory effects of 5-HT3 receptor antagonists in Lipopolysaccharide-stimulated primary human monocytes BL Fiebich1,2, RS Akundi1, K Lieb1, E Candelario-Jalil1, D Gmeiner1, U Haus3, W Mu¨ller4, T Stratz4, and E Mun˜oz0 1

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

There is evidence from both human and animal research that 5-hydroxytryptamine (5-HT)3 receptor antagonists, particularly tropisetron, exert analgesic and antiinflammatory effects. However, the underlying mechanisms of these effects have not yet been investigated in detail. Therefore, the antiinflammatory effects of tropisetron and ondansetron were investigated in human monocytes. In human monocytes, both lipopolysaccharide (LPS)-stimulated tumour necrosis factor (TNF)-a and interleukin (IL)-1b secretion were dose-dependently inhibited by tropisetron starting at a concentration of 5 mg/mL and reaching maximal levels at 25 mg/mL (IC50: 32 mg/mL and 12 mg/mL, respectively). LPS-induced IL-6 and PGE2 release was only slightly inhibited at high doses, whereas LPS-induced release of IL-8 and matrix metalloprotease (MMP)-9 was not affected. In conclusion, our data show that the binding of tropisetron to 5-HT3 receptors results in antiinflammatory effects through inhibition of TNF-a/IL-1b, which might explain the antiphlogistic effects of 5HT3 antagonists.

Evidence for analgesic and antiphlogistic activity of 5-HT3 receptor antagonists, particularly tropisetron, has been found in clinical studies in recent years. The pain relieving effect of these substances was initially demonstrated in fibromyalgia, a syndrome characterized by generalized chronic pain of the musculoskeletal system, and confirmed in a double-blind study (1, 2). The results achieved by local application of tropisetron in patients with various inflammatory and non-inflammatory rheumatic diseases such as arthritis of different origin, periarthropathies, tendinopathies, and trigger points (3, 4, 5) indicate that 5-HT3 receptor antagonists seem to have an antiphlogistic effect in addition to their analgesic action. Experimental studies also revealed that 5-HT3 receptor antagonists possess both an analgesic and antiphlogistic effect. Experimentally-induced acute and chronic inflammatory pain (6, 7, 8) as well as chemically-induced pain (9, 10) responded well to 5HT3 receptor antagonists, probably because the latter inhibit nociceptor stimulation. Clinical observations concerning the analgesic and antiphlogistic effect of 5-HT3 receptor antagonists are of fundamental significance because they seem to open up new avenues for the treatment of rheumatic Bernd L. Fiebich, University of Freiburg Medical School, Dept. of Psychiatry and Psychotherapy, D – 79104 Freiburg, Germany. E-mail: [email protected] Received Accepted

diseases. Since clinical observations suggest antiinflammatory properties of the 5-HT3 receptor antagonist tropisetron (Navoban1), we were interested in identifying the molecular targets and mechanisms by which tropisetron exerts its antiinflammatory effect. As a cellular model, we used the well-established primary human monocyte model, which is commonly used to test antiinflammatory drugs.

Material and methods Cell lines and reagents Tropisetron (Navoban1) and ondansetron 1 (Zofran ) were purchased from a local pharmacy. Lipopolysaccharide (LPS) from Escherichia coli was obtained from Sigma (Deisenhofen, FRG). Isolation of human peripheral monocytes Monocytes from healthy human donors were prepared following a standardized protocol (Ficoll gradient preparation, Amersham-Biosciences) using completely endotoxin-free cultivation (11, 12). Using 50 ml tubes, 25 mL Ficoll was loaded with 25 mL blood of buffy coats from healthy blood donors. The gradient was established by centrifugation at 1800 revolutions-per-minute (rpm) and 20‡C for 40 min using slow acceleration and brakes. Peripheral blood mononuclear cells in the interphase were carefully removed and resuspended in 50 ml prewarmed phosphate buffered saline (PBS, from Invitrogen)

# 2004 Taylor & Francis on license from Scandinavian Rheumatology Research Foundation DOI:

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followed by centrifugation for 10 min at 1600 rpm and 20‡C. The supernatant was discarded and the pellet washed in 50 ml PBS and centrifuged as described above. The pellet was then resuspended in 50 ml RPMI-1640 low endotoxin-medium (from Invitrogen) supplemented with 10% human serum (PAA). After counting the number of cells in a particle counter (Euro Diagnostics, Krefeld), cells were seeded in 24-well plates and incubated at 37‡C and 5% CO2. The medium and non-adherent cells (lymphocytes) were removed and fresh RPMI-1640 medium containing 1% human serum was added. Then the monocytes were ready to be used for the experiments.

Determination of inflammatory parameters For the analysis of IL-6, TNF-a, IL-1b, MMP-9, IL8 and PGE2 release, cells were preincubated with the compounds for 30 min before stimulation with LPS (10 ng/mL) for an additional 24 h. After culture supernatants were harvested and centrifuged for 10 min at 10.000 centrifugation force (xg), levels of the cytokines, MMP-9, or PGE2 in the supernatant were measured by ELISA (IL-6, IL-8, TNF-a: Pelikine, distributed by HISS, Freiburg, Germany; MMP-9: Amersham-Biosciences, Freiburg, Germany) or EnzymeImmunoAssay (EIA) (AssayDesign, distributed by Biotrend, Cologne, Germany) according to the manufacturer’s instructions. All experiments were carried out with at least two buffy coats from different blood donors in triplicates.

Adenosine triphosphate (ATP) cell viability assay Cell viability was determined by measuring ATP metabolism. ATP levels were detected by using a sensitive luciferin-luciferase bioluminescence system from Promega (Madison, WI, USA) according the manufacturer’s instructions. Luminescence was recorded by means of a microplate luminometer (Berthold Detection Systems GmbH, Pforzheim, Germany).

Data analysis The statistical analysis was conducted by the Institute for Biochemical analysis and Method Developement GbR, Freiburg, Germany. The original data obtained were converted to percentage values relative to the LPS controls and their means and standard errors (Ses) were calculated. The IC50 values (mg/mL) were calculated using computerized non-linear regression analysis with 95% confidential intervals. In the figures, a regression curve was adjusted to the calculated mean values.

BL Fiebich et al

Results To investigate possible antiinflammatory effects of tropisetron, primary human monocytes were treated with LPS (10 ng/mL) as a proinflammatory stimulus in the absence or presence of tropisetron. By determining various inflammatory parameters in the supernatants of the monocyte cells cultures, we found PGE2 to be only slightly inhibited by tropisetron at doses of 25 and 50 mg/ml (Figure 1A), resulting in an IC50 of 168 mg/mL (11-2569 mg/mL). In contrast, tropisetron strongly inhibited both TNF-a and IL-1b release from monocytes induced by LPS. TNF-a secretion was dose-dependently inhibited by tropisetron starting at a 5 mg/ml concentration and reaching the plateau level at a dose 25 mg/ml (Figure 1B). The calculated IC50 was 32 mg/mL (18,9 – 55,1 mg/mL). LPS-induced IL-1b release was also significantly inhibited starting at a dose of 5 mg/mL, the maximal inhibition was obtained by using 25 mg/mL of tropisetron, and the IC50 was 12 mg/mL (8,7 – 15,9 mg/ml) (Figure 1C). LPS-induced IL-6 release was only slightly inhibited at a high dose of 50 mg/mL of tropisetron (Figure 1D), whereas LPS-induced release of IL-8 (Figure 1E) and MMP-9 (Figure 1F) was not affected. Tropisetron was non-toxic in the ATP cell viability assay and in cell cycle analyses at all doses tested (not shown). To exclude a tropisetron-specific effect independent of 5-HT3 receptors, another clinically used 5HT3 receptor antagonist, ondansetron (Zofran1), was used. As was shown for tropisetron, ondansetron also inhibited LPS-induced TNF-a release in a dose-dependent way. To exclude toxic effects of tropisetron being responsible for the antiinflammatory effects of tropisetron, an ATP viability assay was performed. It showed that tropisetron has no effect on cell viability.

Discussion We found the 5-HT3 receptor antagonist tropisetron to be a potent inhibitor of TNF-a and IL-1b in LPSstimulated human primary monocytes. PGE2 and IL-6 were slightly inhibited, whereas IL-8 and MMP9 were not affected. This indicates an IL-1b/TNF-aspecific effect of tropisetron, which suggests that the binding of tropisetron to monocytic 5-HT3 receptors somehow interferes with the signal transduction cascade of LPS-induced IL-1b/TNF-a release. The results showing that the two independent 5-HT3 antagonists (tropisetron and ondansetron) were equally potent in inhibiting TNF-a secretion in LPS-stimulated monocytes suggest that the molecular target for their inhibitory effect is at the 5-HT3 receptor itself. However, serotonin (5-HT) has been


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Antiinflammatory effects of 5-HT3 receptor antagonists


Figure 1. Effects of tropisetron on inflammatory parameters in LPS-treated primary human monocytes. Monocytes were either left untreated or treated for 24 h with LPS (10 ng/mL) alone or in combination with the indicated concentrations of tropisetron. PGE2 (A), TNF-a (B), IL-1b (C), IL-6 (D), IL-8 (E), and MMP-9 (F) immunoreactivity in cell supernatants was measured by ELISA or EIA according to the manufacturer’s protocol. Data are depicted as means¡standard deviations (n~9).

shown to play an important role in immune cells, and some reports have suggested, that this mediator can be also released by cells of the immune system (13). Thus, it is possible that LPS stimulation triggers a signal transduction pathway leading to the release of endogenous 5-HT, which acts in an autocrine manner and may activate the 5-HT3 receptor at the cell surface. However, although

exogenously added 5-HT may increase T-cell proliferation (14), it was found that 5-HT it or a 5-HT3 agonist (5-methylquizapine) failed to induce inflammatory cytokine synthesis (including TNF and IL-1) in monocytes (not shown). Nevertheless, 5-HT3 receptors might be necessary for the continuance of an existing inflammation via a so far unknown mechanism. An attractive and 5-HT-independent


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Figure 2. Effects of ondansetron on TNF-a release in LPStreated primary human monocytes Monocytes were either left untreated or treated for 24 h with LPS (10 ng/mL) alone or in combination with the indicated concentrations of ondansetron for 24 h. TNF-a immunoreactivity in cell supernatants was measured by ELISA according to the manufacturer’s protocol. Data are depicted as means¡standard deviations (n~4).

hypothesis to explain the immunosuppressive effects of tropisetron could be the agonizing antagonist activity hypothesis according to which tropisetron has an agonistic activity besides antagonising 5-HT at the 5-HT3 receptor. This kind of effect has not been described before for antagonists of 5-HT3 receptors. If this is the case, LPS stimulation in monocytes/macrophages or T-cell receptor stimulation in T-cells may induce post-transcriptional changes such as phosphorylation of the 5-HT3 receptor, which in turn may participate in the specific signalling events leading to both TNF-a and IL-1b secretion and the AP-1 activation in monocytes via a 5-HT-independent pathway. Thus, it is possible that tropisetron-binding to 5-HT3 receptor could inhibit its LPS-dependent phosphorylation and function. It has been shown recently that the 5-HT3 receptor can be phosphorylated either by the ubiquitous casein kinase II or by PKC (15, 16). Since the 5-HT3 receptor is a ionotropic receptor permeant to cations with a high selectivity to Naz inward movement, and since the role of Naz/Kz movement is implicated on several events of immune cells activation, we are currently investigating the role of tropisetron in the Naz mobilization in TCRstimulated T-cells and LPS-stimulated macrophage

BL Fiebich et al

cell lines. Moreover, the effects of specific 5-HT3 receptor antagonists on the phosphorylation status of the receptor warrant further research. In conclusion, we were able to demonstrate for the first time a specific IL-1/TNF inhibitory effect of tropisetron in primary human monocytes. However, the mechanism by which binding of the antagonists to the 5-HT3 receptor leads to antiinflammatory effects needs to be elucidated. The in vitro data shown here agree very well with in vivo data obtained by Mu¨ller and Stratz (5) who found tropisetron to be a potent inhibitor of inflammation in patients with inflammatory diseases such as rheumatic arthritis. The doses of the IC50 in vitro need to be compared to the doses used in vivo or the bioavailability in the inflamed tissues of interest. The benefit achieved in patients by a relative small dose of 10 mg tropisteron could be explained by the accumulation of basic tropisetron in the acidic inflamed tissue. The long-lasting effects of tropisetron on inflammation and immunological processes may suggest that this compound also acts on other cells of the immune system such as lymphocytes or T-cells, which needs to be substantiated by further studies. Overall, we have obtained evidence of a new and therapeutically promising benefit of the 5-HT3 receptor antagonist tropisetron.

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

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