Elsevier

Biochemical Pharmacology

Volume 70, Issue 3, 1 August 2005, Pages 369-380
Biochemical Pharmacology

The 5-HT3 receptor antagonist tropisetron inhibits T cell activation by targeting the calcineurin pathway

https://doi.org/10.1016/j.bcp.2005.04.031Get rights and content

Abstract

Tropisetron, an antagonist of serotonin type 3 receptor, has been investigated in chronic inflammatory joint process. Since T cells play a key role in the onset of several inflammatory diseases, we have evaluated the immunosuppressive activity of tropisetron in human T cells, discovering that this compound is a potent inhibitor of early and late events in TCR-mediated T cell activation. Moreover, we found that tropisetron specifically inhibited both IL-2 gene transcription and IL-2 synthesis in stimulated T cells. To further characterize the inhibitory mechanisms of tropisetron at the transcriptional level, we examined the DNA binding and transcriptional activities of NF-κB, NFAT and AP-1 transcription factors in Jurkat T cells. We found that tropisetron inhibited both the binding to DNA and the transcriptional activity of NFAT and AP-1. We also observed that tropisetron is a potent inhibitor of PMA plus ionomycin-induced NF-κB activation but in contrast TNFα-mediated NF-κB activation was not affected by this antagonist. Finally, overexpression of a constitutively active form of calcineurin indicated that this phosphatase may represent one of the main targets for the inhibitory activity of tropisetron. These findings provide new mechanistic insights into the anti-inflammatory activities of tropisetron, which are probably independent of serotonin receptor signalling and highlight their potential to design novel therapeutic strategies to manage inflammatory diseases.

Introduction

5-Hydroxytryptamine (5-HT1, serotonin) is a well-characterized neurotransmitter that plays a crucial role in the regulation of central processes, such as mode, appetite, sleep and other body rhythms. Moreover, 5-HT is found in the immune-inflammatory axis and has been shown to influence the immune response in mammals [1], [2]. The pleiotropic activity of 5-HT is due to the molecular complexity of 5-HT receptors (5-HTR) and their wide tissue expression [3]. Multiple serotoninergic receptors have been identified so far and among them the subtype 5-HT3 receptor (5-HT3R), which is an ionotropic receptor permeant to cations with high selectivity to Na+ inward movements, has been found to be expressed in cells of the immune system including T lymphocytes [4], [5], and evidence exists that 5-HT can modulate the T cells functionality through activation of 5-HT3R [4], [6]. Interestingly, highly selective 5-HT3R antagonists, such as tropisetron, have been investigated in chronic inflammatory joint process, although the antiphlogistic mechanisms of action are largely unknown [7], [8].

The signal transduction pathways triggered by the activation of the TCR–CD3 complex in T cells lead to the immediate activation of transcription factors that regulate a variety of activation-associated genes. Many of them are cytokines and surface receptors that play an important role in coordinating the immune response [9]. The signal transduction pathways involved in T cell activation are initiated by the clustering of lipids rafts at the cell surface, with formation of a supramolecular activation complex localized at the antigen-induced immunological synapse [10]. Several studies have demonstrated that the presence of specific signalling proteins such as Cot/Tpl-2, Vav-1, PKCθ and PLCγ1 within lipids rafts control lymphocyte signalling [11], [12]. Activated PLCγ1 cleaves phosphatidylinositol 4,5 bisphosphate yielding inositol (1,4,5) triphosphate (IP3) and diacylglycerol (DAG). While IP3 mobilizes Ca2+ from intracellular stores, DAG mediates activation of the protein kinase C (PKC) family members [13]. As a consequence of an increase in intracellular Ca2+ levels, several signalling pathways are activated in T cells [14]. In this sense, calcineurin, a Ca2+-calmodulin dependent protein phosphatase, is activated and subsequently dephosphorylates the nuclear factor of activated T cells (NFAT), allowing its nuclear shuttling [14]. This transcription factor was first described as an inducible regulatory complex critical for transcriptional induction of IL-2 in activated T cells, but was subsequently shown to regulate the transcription of many other genes, including cytokines, cell surface receptors and regulatory enzymes [14], [15]. In the nucleus, NFAT binds to the DNA either alone or in conjunction with AP-1 proteins [16]. Nevertheless, the coordinated induction and activation of the transcription factors NFAT, NF-κB and AP-1 is required to regulate cytokine gene expression [17].

Stimulation via TCR–CD3 complex alone is sufficient for NFAT activation, but it is insufficient for activation of NF-κB and AP-1. Thus, a second signal mediated by the CD28 co-stimulary receptor is required for the induction of NF-κB and AP-1 in antigen stimulated T cells [18]. The transcription factor NF-κB is one of the key gene regulators involved in the immune/inflammatory response as well as in survival from apoptosis [18]. NF-κB is an inducible transcription factor made up of homo- and heterodimers of p50, p65 (RelA), p52, RelB and c-rel subunits that interact with a family of inhibitory IκB proteins, of which IκBα is the best characterized [19], [20]. In most cell types, these proteins sequester NF-κB in the cytoplasm by masking its nuclear localization sequence, and in response to a variety of stimuli, including TCR and CD28 co-stimulation, IκBs are phosphorylated by the IκB kinase complex, followed by their ubiquitination and degradation in the proteasome. The release of IκBs unmasks the NLS and allows NF-κB to enter the nucleus [18], [21].

In this paper, we studied the effect of tropisetron, a 5-HT3R antagonist, on early and late T cell activation events and we have demonstrated that tropisetron inhibits antigen-induced proliferation and IL-2 production in human peripheral T cells. Moreover, we show here for the first time that tropisetron inhibits the signalling pathways that regulate the activation of the transcription factors NFAT, NF-κB and AP-1, which are known to play a critical role in the immune response.

Section snippets

Cell lines and reagents

The 5.1 clone (obtained from Dr. N. Isräel, Institut Pasteur, Paris, France) line is a Jurkat derived clone stably transfected with a plasmid containing the luciferase gene driven by the HIV-LTR promoter and was maintained in exponential growth in RPMI 1640 (Gibco BRL–Life technologies, Barcelona, Spain) supplemented with 10% heat inactivated foetal calf serum, 2 mM l-glutamine, 1 mM Hepes and antibiotics (completed medium) (Gibco BRL–Life technologies, Barcelona, Spain) and G418 (200 μg/ml).

Effects of tropisetron on T cell activation

Recent clinical data have shown that tropisetron, a 5-HT3R antagonist, is effective for the treatment of inflammatory and pain joint processes [7], [8]. Since 5-HT3R is expressed in human peripheral T cells we studied the effects of this 5-HT3R antagonist on several T cell activation events and we found that DNA synthesis measured by [3H]-TdR uptake in SEB and in PMA plus ionomycin-stimulated T cells was markedly inhibited by tropisetron in a concentration-dependent manner (Table 1). Due to

Discussion

5-HT is a neurotransmitter widely distributed in the central nervous system. Moreover, 5-HT is released in peripheral tissues by platelets, mast cells and noradrenergic nerve terminals that are in close contact with immune cells in lymphoid organs [30]. Compared to the immense amount of data elucidating the role of 5-HT and 5-HT receptors in the brain, the biological role of 5-HT in cells form the immune system is far to be understood and the pharmacological manipulation of the serotoninegic

Acknowledgements

We thank Dr. Nicole Israël (Institute Pasteur, Paris, France) for the 5.1 cells; Dr. Alain Israël (Institute Pasteur) for the anti-p65 antibody; Dr. R.T. Hay (CBMS, University of St. Andrews, Fife, Scotland) for the mAb 10B; and colleagues Dr. L. Schmitz (Bern, Switzerland), Dr. Manuel López-Cabrera (Hospital de la Princesa, Madrid, Spain) and Dra. Susana Alemany (IIB-CSIC, Madrid) for providing plasmids. Finally, we thank Carmen Cabrero-Doncel for assistance with the manuscript. This work was

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