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Evidence of melatonin synthesis by human lymphocytes and its physiological significance: possible role as intracrine, autocrine, and/or paracrine substance¹

ANTONIO CARRILLO-VICO^*, JUAN R. CALVO^*, PEDRO ABREU, PATRICIA J. LARDONE^*, SOFÍA GARCÍA-MAURIÑO^*, RUSSEL J. REITER and JUAN M. GUERRERO^*,2

^* Department of Medical Biochemistry and Molecular Biology, The University of Seville School of Medicine and Virgen Macarena Hospital, Seville, Spain;
Department of Physiology, School of Medicine, University of La Laguna, Tenerife, Spain; and
Department of Cellular and Structural Biology, The University of Texas, Health Science Center at San Antonio, San Antonio, Texas, USA

²Correspondence: Department of Medical Biochemistry and Molecular Biology, The University of Seville School of Medicine, Avda. Sánchez Pizjuan 4, 41009 Seville, Spain. E-mail: guerrero{at}us.es

SPECIFIC AIMS

It has been historically assumed that the pineal gland is the major source of melatonin in vertebrates. Melatonin plays a central role in fine tuning circadian rhythms in vertebrate physiology. Additionally, melatonin shows a remarkable functional versatility exhibiting antioxidant, oncostatic, anti-aging, and immunomodulatory properties. Its biosynthesis from tryptophan involves four well-defined intracellular steps catalyzed by tryptophan hydroxylase (TPH), aromatic amino acid decarboxylase (AADC), serotonin-N-acetyltransferase (NAT), and hydroxyndole-O-methyltransferase (HIOMT). This paper shows that both resting and stimulated human lymphocytes have the necessary machinery to synthesize melatonin as well as synthesize and release large amounts of melatonin. Moreover, melatonin released to the culture medium is synthesized in the cells since blocking the enzymes required for its biosynthesis produced a significant reduction in melatonin release. This inhibition caused decrease in IL-2 production, which was restored by adding exogenous melatonin. These findings indicate that human lymphoid cells are an important physiological source of melatonin which could be involved in the regulation of the human immune system.

PRINCIPAL FINDINGS

1. Presence of two key enzymes involved in melatonin synthesis (NAT and HIOMT) in human peripheral blood mononuclear cells (PBMCs)
The synthesis of melatonin requires the presence of the enzymes involved in its metabolic pathway. We investigated the presence of the two key enzymes involved in melatonin biosynthesis in PBMCs. NAT is the rate-limiting enzyme in melatonin synthesis, while HIOMT is the final enzyme of the biosynthetic pathway. To determine the expression of NAT and HIOMT mRNA in lymphoid cells, mRNA from stimulated and unstimulated cells was subjected to RT-PCR analysis using specific primers to both genes. The RT-PCR amplification pattern obtained using the NAT and HIOMT primers revealed the presence of the expected bands in stimulated and unstimulated cells (Fig. 1 A). Southern blot analysis performed with DIG-labeled NAT and HIOMT probes confirmed the identity of PCR products (Fig. 1B ). To study whether the presence of NAT and HIOMT mRNA was related with a functional response of both enzymes, we measured NAT and HIOMT activity in these cells. This study revealed a strong NAT and HIOMT activity in both groups of cells (Fig. 1C ).

View larger version (53K): [in this window] [in a new window]   Figure 1. Presence of two key enzymes involved in melatonin synthesis (NAT and HIOMT) in PBMCs. A) RT-PCR analysis of NAT (left) and HIOMT (right) mRNA expression. B) Southern blot hybridization of the PCR products shown in panel A with the DIG-labeled specific probes. = PCR and Southern molecular size; C = PCR reaction without cDNA substrate used as PCR control. C) NAT and HIOMT activity. Cells were cultured for 24 h in the presence or absence of PHA (8 µg/mL). The pineal glands from animals injected with isoproterenol were used as positive control of NAT activity, whereas not injected animals were used as positive control of HIOMT activity. Data are expressed as mean values ± SE of 6 experiments performed in triplicate. *, Significant differences between stimulated and unstimulated cells were observed (P<0.001).

2. Melatonin production by PBMCs
We observed the presence of high concentrations of melatonin in the PBMC culture supernatants using HPLC assay (Fig. 2 A). Melatonin concentration was dependent on time and the presence of the mitogen PHA. After 72 h incubation (Fig. 2A, B : B4) melatonin concentration was significantly higher than at 24 h (Fig. 2A, B : B3), while in PHA stimulated-cells, melatonin concentration was also significantly higher than in resting cells. No melatonin was detected in medium alone (Fig. 2A, B : B1) and insignificant containing FCS (Fig. 2A, B : B2). Similar results were obtained when an ELISA assay was used to measure melatonin (data not shown).

View larger version (27K): [in this window] [in a new window]   Figure 2. Melatonin produced by PBMCs can be inhibited by PCPA or HIOMT-directed antisense oligonucleotides and has biological effects on IL-2 production. A) Melatonin production assayed by HPLC. Melatonin was determined in RPMI 1640 medium with and without FCS and PBMC culture supernatants. B) Representative chromatograms of melatonin. (B1) RPMI medium. B(2) RPMI with FCS. (B3) and (B4), Melatonin formed in the medium after 24 or 72 h of incubation. (B5) Authentic standard of melatonin. Data are expressed as mean values ± SE of 20 experiments performed in triplicate. *, Significant differences in melatonin levels between 24 and 72 h (P<0.001) and between cells stimulated and unstimulated (P<0.05). C) HIOMT-directed antisense oligonucleotides and PCPA inhibited melatonin synthesis by PBMCs. (C1) Inhibition of melatonin synthesis using antisense mixture to HIOMT. Cells were incubated for 24 h with DOTAP, and a mixture of sense, random, and antisense oligonucleotides. Data are expressed as mean values ± SE of 12 experiments performed in triplicate. *, Significant differences between cells treated with antisense and other groups were observed (P<0.001). (C2) Inhibitory effect of PCPA on melatonin production. Cells were incubated in the presence or absence of PHA (8 µg/mL) and PCPA (10-4 M) at indicated times. Data are expressed as mean values ± SE of 12 experiments performed in triplicate. *, Significant differences between cells treated with PCPA and untreated were observed (P<0.05). D) Effect of PCPA on IL-2 production. Cells were incubated for 72 h with PHA (8 µg/mL) and, where indicated, 10-8 M melatonin or 10-4 M PCPA. Data are expressed as mean values ± SE of 20 experiments performed in triplicate. *, Significant differences between cells treated with PCPA and other groups were observed (P<0.001).

3. HIOMT-directed antisense oligonucleotides and para-cholorophenylalanine (PCPA) inhibited melatonin synthesis by human PBMCs
Previous studies raised the question of whether melatonin in the culture medium is synthesized by the cells or whether it was merely released after being accumulated before lymphocytes were isolated. To answer this question, cells were cultured in the presence of a mixture of antisense oligonucleotides which was directed to mRNA encoding HIOMT. Blocking HIOMT expression resulted in a significant reduction in melatonin concentration in the culture medium (Fig. 2C : C1). Another method to inhibit the melatonin synthetic pathway was the addition of PCPA, a reversible TPH inhibitor. Significant decreases in melatonin concentration in both stimulated and unstimulated cells at 24 and 72 h were observed (Fig. 2C : C2). A decrease in melatonin content was more obvious after HIOMT blockade than after PCPA treatment. This is likely explained by the fact that HIOMT is the final enzyme involved in melatonin synthesis. Moreover, when cells were incubated with the protein synthesis inhibitor cycloheximide, the melatonin levels fell significantly (data not shown).

4. Effect of PCPA on IL-2 production
Finally, to define a possible physiological role for lymphocytes melatonin, cells were incubated in the presence of PCPA and/or melatonin, and IL-2 production was studied. The results show that PCPA significantly reduced IL-2 production by lymphocytes, and adding melatonin to the medium the inhibitory effect of PCPA was reverted (Fig. 2D ). Therefore, we propose that melatonin synthesized by the lymphocyte could somehow contribute to regulation of its own IL-2 production.

CONCLUSIONS AND SIGNIFICANCE

In vivo models to test the immunomodulatory role of melatonin have been widely used. Most of authors agree that pinealectomy and in vivo models of melatonin administration clearly show the immunoenhancing properties of the melatonin. However, when melatonin is used in vitro, the results seem contradictory. The reasons for the apparent contradictions are not clear but several possibilities exist. 1) The effect of melatonin on immune cells is mediated via other tissues, cells, hormones and/or cytokines that are not present in in vitro studies. 2) melatonin efficiency in culture has been tested primarily in cells fully activated. Under these conditions, immunomodulators, including melatonin, frequently fail to achieve further activation of immune cells. 3) presence of endogenously generated melatonin may interfere or mask with exogenously added melatonin in in vitro experiments.

In this report, we document that both in vitro cultured resting and stimulated human lymphocytes show a strong NAT and HIOMT mRNA expression as well as a clear activity of both enzymes. Moreover, these cells release large amounts of melatonin, and melatonin is actually synthesized by the cells because the HIOMT mRNA blockage, inhibition of TPH activity, or protein synthesis reduced melatonin release. Taken together, these data show a new experimental approach to demonstrate that at least, a portion of the melatonin found in the cells is actually synthesized by the human immune system. These results clearly demonstrate that human immune system is a source of melatonin. Furthermore, a physiological role for melatonin produced by human lymphocytes has been described. Thus, the inhibition of melatonin synthesis by PCPA caused a reduction in IL-2 production and the PCPA inhibitory effect was counteracted by exogenous melatonin.

This study shows for the first time that melatonin synthesized by lymphocytes is involved in the regulation of IL-2 production, and probably, other immune functions driven by IL-2, possibly by acting as an intracrine, autocrine and/or paracrine substance (Fig. 3 ). Therefore, in future studies, during in vitro experiments with lymphocytes, a melatonin synthesis inhibitor should be added to the culture to prevent the endogenous synthesis of melatonin by immune cells. The development of specific inhibitors of melatonin synthesis would be very useful in future studies of melatonin effects on the human immune system.

View larger version (53K): [in this window] [in a new window]   Figure 3. Schematic model of how melatonin could act as intracrine, autocrine or paracrine substance in immune system.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0694fje;

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