5-HT1A-mediated promotion of mitogen-activated T and B cell survival and proliferation is associated with increased translocation of NF-κB to the nucleus

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Abstract

Mitogenic activation of T and B lymphocytes induces expression of the 5-HT1A receptor through an NF-κB-dependent signaling pathway. In the present study, it is shown that serotonin (5-HT), as well as the selective 5-HT1A receptor agonist R-DPAT, increase cell survival and S phase transition in mouse splenocytes stimulated by T or B cell mitogens. Further examination of the mechanisms underlying increased cell survival revealed that 5-HT and R-DPAT inhibited apoptotic cell death, assessed both by soluble DNA content, internucleosomal DNA cleavage, and hypodiploid DNA content. Additionally, 5-HT and R-DPAT treatment increased intranuclear levels of the p50 and p65 subunits of NF-κB. Potentiation by 5-HT and R-DPAT of mitogen-activated cell survival, S phase transition, and nuclear localization of NF-κB, as well as inhibition of apoptosis, were all reversed by the selective 5-HT1A receptor antagonist WAY-100635. These results indicate that 5-HT1A-mediated promotion of cell survival and proliferation of mitogen-activated T and B lymphocytes is associated with increased translocation of NF-κB in the nucleus.

Introduction

Serotonin (5-hydroxytryptamine; 5-HT) is a neurotransmitter of the central nervous system (CNS) that plays a key role in various cognitive functions such as feeding, sleep, pain, depression, learning, and anxiety (Blier et al., 1987; Charney et al., 1990; Saxena, 1995). Some of these processes are mediated by the 5-HT1A receptor, which has a widespread localization in several brain regions especially in the limbic system (Pompeiano et al., 1992; Törk, 1990). Agonists of the 5-HT1A receptor have some therapeutic effectiveness in anxiety and affective disorders (Olivier et al., 1999). Outside the CNS, 5-HT is synthesized and released in the circulation by enterochromaffin cells of the gut mucosa (Essman, 1978). It is taken up and released by other cell types, predominantly by platelets at sites of inflammation (Benedict et al., 1986; Endo et al., 1997; Essman, 1978; Timmons et al., 1986), and by rodent mast cells in response to antigens or neuropeptides (reviewed in Crivellato et al., 1991). Local tissue concentrations of 5-HT in the order of 10−4 M (100 μM) have been found in certain inflammatory conditions, such as thrombosis (Benedict et al., 1986). Also, 5-HT is stored by noradrenergic nerve terminals that are in close contact with immune cells in primary and secondary lymphoid organs (Ader et al., 1995; Bellinger et al., 1988), and the amine may be released upon stimulation of these sympathetic nerves as observed in other tertiary non-lymphoid tissues (Cohen, 1985; Junod, 1972; Verbeuren et al., 1983). Various immune cell types express specific 5-HT receptors that are the targets of the reported immunomodulatory effects of 5-HT (reviewed in Mössner and Lesch, 1998). Collectively, these findings provide evidence that 5-HT may play an important role in the immune system and in neuroimmune interactions. We recently showed that the 5-HT1A receptor is induced in mitogen-activated murine spleen T and B lymphocytes through the nuclear factor-κB (NF-κB)-dependent signaling pathways (Abdouh et al., 2001).

NF-κB plays an important role in regulation of the immune, inflammatory, and apoptotic responses (Baeuerle and Henkel, 1994; Barnes and Karin, 1997; Chen et al., 2001). NF-κB is mainly composed of p50 and p65 (RelA) subunits, which are normally retained in the cytosol of non-stimulated cells by inhibitory molecules of the IκB family. In response to diverse stimuli, including mitogens, inflammatory cytokines, several chemical toxicants, viral and bacterial products, IκBs are phosphorylated by the IκB kinase (IKK) complex, and rapidly degraded by the ubiquitin-dependent pathway. Degradation of IκBs allows translocation of free NF-κB dimers into the nucleus and subsequent activation of NF-κB elements (Baldwin, 1996; Karin, 1999). The target genes for NF-κB include those encoding cytokines, cell cycle regulators, and anti-apoptotic proteins (Barkett and Gilmore, 1999; Gerondakis et al., 1998; Ghosh et al., 1998; Grumont et al., 1998; Karin, 1998; Karin and Lin, 2002). As a consequence, agents that activate NF-κB are known to promote cell survival and/or cell proliferation, whereas agents that inhibit NF-κB are known to block lymphocyte activation and are immunosuppressive. One previous study has shown that the 5-HT1A receptor transfected in Chinese hamster ovary (CHO) cells is positively coupled with activation of NF-κB (Cowen et al., 1997), but it is still unknown whether such coupling exists for endogenous 5-HT1A receptors in primary lymphocyte cell cultures.

It has been shown that agonist stimulation of the 5-HT1A receptor potentiates production of the pro-inflammatory cytokines interleukin-2 (IL-2) and interferon-γ by T lymphocytes (Aune et al., 1994; Eugen-Olsen et al., 1997), as well as mitogen-activated proliferation of T lymphocytes (Abdouh et al., 2001; Aune et al., 1993), and B lymphocytes (Iken et al., 1995). Therefore, the aim of the present study was to investigate whether stimulation of the 5-HT1A receptor influences cell survival and/or nuclear translocation of NF-κB in mitogen-activated T and B lymphocytes under conditions where proliferation was increased. Experiments were designed to evaluate lymphocyte viability, and to assess apoptosis, cell cycle progression, and nuclear levels of NF-κB subunits. The results indicate that, through interaction with the 5-HT1A receptor, 5-HT promotes the survival and cell cycle progression of mitogen-activated T and B lymphocytes in association with increased nuclear translocation of NF-κB.

Section snippets

Animals

Female, BALB/c mice, 6–12 weeks of age, were purchased from Charles River (St. Constant, Canada) and maintained in our animal facility until use.

Reagents

All cell culture media were purchased from Life Technologies (Burlington, Canada), and fetal bovine serum (FBS) was supplied by Hyclone (Logan, UT). Escherichia coli lipopolysaccharide (LPS) serotype 0111:B4, phytohemagglutinin (PHA), concanavalin A (ConA), phorbol 12-myristate 13-acetate (PMA), 5-HT hydrochloride, and the diphenylamine reagent were

5-HT increases cell viability in mitogen-activated spleen lymphocytes

Spleen lymphocytes were cultured in the presence or absence of different mitogens in combination with 5-HT for various periods of time ranging from 24 to 72 h. Cell viability was assessed by trypan blue exclusion staining. As shown in Table 1, cell viability of mitogen-activated and control lymphocytes decreased with incubation time, although mitogen stimulation yielded better viability. In the presence of 5-HT (10−4 M), cell viability of mitogen-activated lymphocytes was further increased.

Discussion

Previously, we showed that 5-HT increases mitogen-induced T and B lymphocyte proliferation in a 5-HT1A receptor-dependent manner (Abdouh et al., 2001; Iken et al., 1995). Here, we demonstrate that 5-HT signals via the 5-HT1A receptor to increase S phase transition, and to inhibit apoptosis in activated T and B lymphocytes. The anti-apoptotic effect of 5-HT is confirmed by using three different markers, including the levels of soluble DNA content, internucleosomal DNA cleavage, and hypodiploid

Acknowledgements

This study was supported by Medical Research Council of Canada Grant MT-13259 (to E.K.) and by AstraZeneca/Fonds de la recherche en santé du Québec Grant 005086 (to E.K.).

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