Serotonin provides an accessory signal to enhance T-cell activation by signaling through the 5-HT7 receptor

Abstract

Although typically considered a neurotransmitter, there is substantial evidence that serotonin (5-HT) plays an important role in the pathogenesis of inflammatory disorders. Despite these findings, the precise role of 5-HT in modulating immune function, particularly T-cell function, remains elusive. We report that naive T cells predominantly express the type 7 5-HT receptor (5-HTR), and expression of this protein is substantially enhanced on T-cell activation. In addition, T-cell activation leads to expression of the 5-HT(1B) and 5-HT(2A) receptors. Significantly, exogenous 5-HT induces rapid phosphorylation of extracellular signal-regulated kinase-1 and -2 (ERK1/2) and IkappaBalpha in naive T cells. 5-HT-induced activation of ERK1/2 and NFkappaB is inhibited by preincubation with a specific 5-HT(7) receptor antagonist. Thus, 5-HT signaling via the 5-HT(7) receptor may contribute to early T-cell activation. In turn, 5-HT synthesized by T cells may act as an autocrine factor. Consistent with this hypothesis, we found that inhibition of 5-HT synthesis with parachlorophenylalanine (PCPA) impairs T-cell activation and proliferation. Combined, these data demonstrate a fundamental role for 5-HT as an intrinsic cofactor in T-cell activation and function and suggest an alternative mechanism through which immune function may be regulated by indoleamine 2,3-dioxygenase-mediated catabolism of tryptophan.

Figures

Figure 1

Expression of 5-HTR by naive and activated T cells. T cells were negatively purified and activated with Con A or PMA plus ionomycin, and gene expression for 5-HTR subtype was examined by RT-PCR. (A) Primers for the type 1 and type 7 5-HTR families amplify a region with 1 exon; thus, both cDNA and RT-negative (RNA) samples are shown. (B) Gene expression of the 5-HT2-6 receptor subfamilies. Comparable quantities of cDNA were ensured by amplification of GAPDH. Data are representative of 3 independent experiments.

Figure 2

Naive T cells primarily express 5-HT7 receptors. Representative immunoblots showing relative expression of (A) 5-HT1B and (B) 5-HT7 receptors by T cells. Blots were stripped and reprobed for β-actin to ensure comparable quantities of protein were analyzed (lower blots). Data are 1 of 2 similar experiments.

Figure 3

Activated T cells are capable of 5-HT synthesis. (A) Expression of gene transcripts for SERT, TPH-1, and TPH-2 was determined by RT-PCR from naive T cells and from T cells activated with Con A or PMA plus ionomycin. (B) The relative expression of TPH-1 was quantified by q-PCR. Data are means ± 1 SD (n = 2, P < .001). (C) 5-HT was visualized by confocal microscopy in T cells activated with PMA plus ionomycin by labeling with 5-HT antisera (Alexa Fluor 546; red) (upper left). 5-HT antisera was preabsorbed with 5-HT as the negative control (lower left). Nuclei were counterstained with TO-PRO-3 (blue). Scale bar represents 10 μm. Data are representative of 3 independent experiments. (D) 5-HT concentrations in T-cell culture supernatants were determined by EIA. Data are mean from triplicate assays ± 1 SD (n = 2, *P < .001).

Figure 4

5-HT induces rapid phosphorylation of ERK1/2 that is inhibited by a 5-HT7 receptor–selective antagonist. Freshly isolated naive T cells were incubated with exogenous 5-HT (10 μM) at 37°C. Samples were lysed, immunoblotted, and probed for (A) phospho-ERK1/2 (top). Blots were stripped and reprobed for total ERK1/2 to confirm equal loading in each lane (bottom). Densitometric analysis was performed showing a maximal increase in phospho-ERK1/2 at 5 minutes following stimulation with exogenous 5-HT (relative to total ERK). (B) Freshly isolated naive T cells were incubated with SB 269970 (5-HT7 receptor antagonist) or SB 216641 (5-HT1B receptor antagonist) for 1 hour, 37°C. Samples were then pulsed with 5-HT (10 μM) for 5 minutes at 37°C) and analyzed for phospho-ERK1/2 (top) and total ERK1/2 (bottom). Densitometric analysis was performed, showing phospho-ERK p42 (relative to total ERK).

Figure 5

Exogenous serotonin induces rapid phosphorylation of IκBα that is inhibited by a 5-HT7 receptor–selective antagonist. Freshly isolated naive T cells were incubated with SB 269970 (5-HT7 receptor antagonist) or SB 216641 (5-HT1B receptor antagonist) for 1 hour, 37°C. Samples were then pulsed with exogenous 5-HT (10μM) for 5 minutes at 37°C and analyzed for phospho-IκBα (top). Blots were stripped and reprobed for β-actin to confirm equal loading in each lane (bottom). Densitometric analysis was performed showing phospho-IκBα (relative to β-actin).

Figure 6

Inhibition of endogenous 5-HT synthesis impairs T-cell proliferation ex vivo. Splenic CD3+ T cells were negatively enriched from C57BL/6 mice pretreated with PCPA or saline. (A) Basal 5-HT released from naive T cells isolated from saline- (□) or PCPA-treated mice (■) after overnight culture with IL-2 (10 IU/mL). Dashed line indicates background 5-HT resulting from complete media alone. Data are mean ± 1 SD from triplicate results (n = 2, *P < .01). (B) T cells from saline- or PCPA-treated mice were incubated for 48 hours with plate-bound anti-CD3 mAb (5 μg/mL). Data are the mean proliferation ± 1 SE from 6 independent assays, performed in triplicate (*P < .05). (C) T-cell proliferation in the presence of exogenous 5-HT (1 μM) or the 5-HT7 receptor agonist, AS19 (1 μM). Data are mean ± 1 SD from 1 representative experiment (n = 3) performed in triplicate (*P < .05).

Figure 7

Inhibition of endogenous 5-HT synthesis impairs T-cell expression of CD25. T cells from saline- or PCPA-treated mice were incubated for 48 hours with plate-bound anti-CD3 mAb (5 μg/mL). (A) Expression of CD25 and CD4 by T cells analyzed by flow cytometry. (B) Absolute number of CD25+ CD4+ T cells expressed as mean (percentage of control) ± 1 SE from triplicate assays (n = 4, *P < .001).