The FASEB Journal express article 10.1096/fj.03-0694fje. Published online January 8, 2004.
Evidence of melatonin synthesis by human lymphocytes and its physiological significance: possible role as intracrine, autocrine, and/or paracrine substance Antonio Carrillo-Vico,* Juan R. Calvo,* Pedro Abreu,† Patricia J. Lardone,* Sofía García-Mauriño,* Russel J. Reiter,‡ and Juan M. Guerrero* *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, TX, USA Corresponding author: Juan M. Guerrero, Department of Medical Biochemistry and Molecular Biology, The University of Seville School of Medicine, Avda. Sánchez Pizjuan 4, 41009 Seville, Spain. E-mail:
[email protected] ABSTRACT It has been historically assumed that the pineal gland is the major source of melatonin (N-acetyl5-methoxytryptamine) in vertebrates. Melatonin plays a central role in fine-tuning circadian rhythms in vertebrate physiology. In addition, melatonin shows a remarkable functional versatility exhibiting antioxidant, oncostatic, antiaging, and immunomodulatory properties. Melatonin has been identified in a wide range of organisms from bacteria to human beings. Its biosynthesis from tryptophan involves four well-defined intracellular steps catalyzed by tryptophan hydroxylase, aromatic amino acid decarboxylase, serotonin-N-acetyltransferase, and hydroxyndole-O-methyltransferase. Here, for the first time, we document that both resting and phytohemagglutinin-stimulated human lymphocytes synthesize and release large amounts of melatonin, with the melatonin concentration in the medium increasing up to five times the nocturnal physiological levels in human serum. Moreover, we show that the necessary machinery to synthesize melatonin is present in human lymphocytes. Furthermore, melatonin released to the culture medium is synthesized in the cells, because blocking the enzymes required for its biosynthesis or inhibiting protein synthesis in general produced a significant reduction in melatonin release. Moreover, this inhibition caused a decrease in IL-2 production, which was restored by adding exogenous melatonin. These findings indicate that in addition to pineal gland, human lymphoid cells are an important physiological source of melatonin and that this melatonin could be involved in the regulation of the human immune system, possibly by acting as an intracrine, autocrine, and/or paracrine substance Key words: neuroimmunology • NAT • HIOMT • IL-2
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elatonin, the major secretory product of the pineal gland, is involved in the regulation of circadian rhythms and seasonal changes in vertebrate physiology via the daily nocturnal increase and release from the pineal. Because melatonin production is
highest at night, this mediator has been described as the chemical expression of darkness (1). In addition, melatonin shows a remarkable functional versatility exhibiting antioxidant (2), oncostatic (3), antiaging (4), and immunomodulatory (5) properties. Its biosynthesis from tryptophan involves four well-defined intracellular steps (6) catalyzed by tryptophan hydroxylase (TPH), aromatic amino acid decarboxylase (AADC), serotonin-N-acetyltransferase (NAT), and hydroxyndole-O-methyltransferase (HIOMT). Its remarkable functional versatility is reflected in its wide distribution within phylogenetically distant organisms from bacteria (7) to human beings (1). In recent years, melatonin has been shown to play a fundamental role in neuroimmunomodulation (8). In vivo studies show that melatonin exerts immunoenhancing properties. Thus, pinealectomy on newborn rats causes disorganization of thymic structure and suppression of pineal function, whereas constant light diminishes antibody responses to T-celldependent antigens (9, 10). Moreover, pinealectomy inhibits IL-2 production and NK cell activity (11), whereas melatonin treatment enhances antibody-dependent cellular cytotoxicity (12) and IFN-γ production by murine splenocytes (13). Furthermore, in vitro studies show that melatonin acts on immune cells by regulating cytokine production. Melatonin activates T helper cells by increasing IL-2 production (5) as well as activating monocytes by increasing the production of IL-1, IL-6, tumor necrosis factor (TNF), reactive oxygen species (ROS), and NO (5, 14, 15). Melatonin also enhances IL-12 production by monocytes driving T-cell differentiation toward the Th1 phenotype and causing an increase of IFN-γ production (16). In this sense, cytokine production could be considered as one of the main mechanisms to modulate the immune system by melatonin. Moreover, the existence of specific melatonin binding sites in lymphoid cells provides evidence for a direct effect on the regulation of the immune system. Thus, using the melatonin agonist 2[125I]melatonin, high-affinity binding sites and a signal transduction pathway for melatonin have been characterized in human lymphocytes (17, 18). In addition, a recent study described a physiological role of the MT1 membrane melatonin receptor in the human system in which melatonin counteracts the inhibitory effect of prostaglandin E2 on IL-2 production in human lymphocytes via its MT1 membrane receptor (19). Heretofore, these melatonin-mediated effects were presumed to be driven by pineal-derived melatonin, not only because of the previously mentioned pinealectomy and constant light exposure experiments, but also because the reported circadian and seasonal variations in melatonin production are associated with changes in immune cell activity (20). However, we believe that melatonin synthesized by lymphoid cells may also play a physiological role in lymphocyte regulation. Here, we describe high melatonin synthesis in cultured human lymphocytes, with the concentrations of melatonin in these cells reaching values five times higher than nocturnal physiological levels of melatonin in human serum. Moreover, we observe that this melatonin is involved in the regulation of the human immune system, at least, through modulation of IL-2 production.
MATERIALS AND METHODS Human peripheral blood mononuclear cells (PBMC) culture Human peripheral venous blood was obtained from healthy volunteers (aged 25–55 years). PBMCs were then obtained by centrifugation over 1.077 g/ml Ficoll-Hypaque gradient (Seromed Biochrom KG, Berlin, Germany) (21). Cells were washed in saline and finally resuspended in RPMI 1640 medium and cultured (0.25×106 cells/ml) in 24-well flat-bottom culture plates in medium supplemented with 25 mM HEPES, 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 U/ml penicilin, and 100 µg/ml streptomycin (Sigma-Aldrich, Dorset, UK). After incubation at 37°C in 5% CO2 humidified atmosphere, cell free culture supernatants were collected, filtered, and stored at –20°C for melatonin and IL-2 determinations. NAT and HIOMT expression mRNA was isolated by Chomcynski’s method (22). Single-stranded cDNA was then synthesized from cells using the following method. RNA (2 µg) was preincubated with 1 µg of oligo(T)15 in 20 µL Rnase-free H2O at 85°C for 10 min, and then rapidly chilled on ice. To this reaction were added 1 µl RNase inhibitor (RNasin) (40 U/µl), 8 µl reverse transcriptase 5× reaction buffer, 8 µl dithiothhreitol (100 mM), and 2 µl deoxyribonucleotides (dNTP; 10 mM of each), and the mixture was incubated at 42°C for 3 min. Finally, 1 µl of Mo-MuLVRT (200 U/µl) were added to give a final volume of 40 µl, and the reaction was incubated at 42°C for 60 min, then terminated by placing it on ice after deactivation at 95°C for 5 min (all reagents from Promega, Madison, WI). After each extraction, one sample was run by RT-PCR without adding reverse transcriptase enzyme in order to check for possible DNA contamination. The following oligonucleotides (5′ to 3′) (Roche, Mannheim, Germany) were used as primers for RT-PCR: human NAT, CCA GTG AGT TTC GCT GCC TCA C (exon 2) and CAG AGC GAG CCG ATG ATG AAG G (exon 3), which amplified a single 459 bp band; and human HIOMT, CAT GAC TGG GCA GAC GGA AA (exon 8) and GTT AGT TCC AGG TCA CAA GAA ACA GTT (exon 9) (23), which amplified a single 300 bp band. For the NAT PCR reactions (1× PCR reaction buffer, 1.5 mM MgCl2, 400 µM dNTPs), 5 µl of RT product was amplified after “hot start” procedure in a final volume of 25 µl using 2.5 U TaqDNA-polymerase (Promega). Thirty PCR cycles were performed (94°C, 1 min; 60°C, 1 min; 72°C, 1 min), followed by a final 10 min extension at 72°C (PTC-150 Minicycler, MJ Research, Watertown, MA). For the HIOMT PCR reactions (1× PCR reaction buffer, 1.5 mM MgCl2, 400 µM dNTPs), 5 µl of reverse transcription product was amplified after “hot start” procedure in a final volume of 25 µl using 2.5 U Taq-DNA-polymerase (Promega). Thirty-five PCR cycles were performed (94°C, 1 min; 55°C, 2 min; 72°C, 3.5 min), followed by a final 10 min extension at 72°C (PTC-150 Minicycler, MJ Research). Specific primers for human β-actin were used to test the efficiency of reverse transcription. The β-actin forward (exon 3) and reverse (exon 6) primers were (5′ to 3′) TTG TAA CCA ACT GGG ACG ATA TGG and GAT CTT GAT CTT CAT GGT GCT AGG (746-bp fragment), respectively.
Southern blotting After amplification, 5 µl PCR reaction was electrophoresed in 2% agarose gel in 1× TAE buffer and visualized by staining with ethidium bromide and UV illumination using DNA molecular size marker. The cDNA was transferred to a hy+-nylon membrane (Amersham-Pharmacia Biotech, Uppsala, Sweden) using a vacuum blotting system (Hoeffer, San Francisco, CA), with 10× SSC as transfer solution and cross-linked to the nylon membrane using a calibrated UV light source. Blots were prehybridized at 68°C for 1 h in prehybridization buffer (5× SSC, 0.1% Nlaurylsarcosyl, 0.02% SDS, 1% blocking reagent). The hybridization was performed at 60°C overnight in the same prehybridization buffer containing 25 ng/ml of labeled probe with oligonucleotide tailing kit (Roche). Thereafter, blots were washed twice for 5 min in 2× SSC/0.1% SDS at room temperature and twice for 5 min in 0.1× SSC/0.1% SDS at 60°C. To detect the hybridization signal, we incubated the blots for 30 min in 0.1 M maleic acid containing 0.15 M NaCl and 1% blocking reagent and for 30 min with anti-DIG-AP (anti-digoxigenin conjugated to alkaline phosphatase). Finally, they were washed and incubated in CSPD. Blots were then exposed to Kodak (Rochester, NY) X-OMAT AR film at room temperature. The probes used in this study were NAT probe, GCT CGA TCT CAA AGG CGC TGA CAG CGT CCT, and HIOMT probe, GGT CAC ACA TAA GTG GGA ACA CTG ACA GGT, both of which were directed against fragment amplified by PCR. NAT and HIOMT activity PBMC were resuspended in 20 mM Tris/1 mM EDTA buffer, pH 7.4, containing 1 mM DTT and disrupted at 4°C using a cell sonicator (Sonics and Materials, Danbury CT). The cell lysate was centrifuged for 3 min at 16,000g, and the supernatant was retained for assay of NAT and HIOMT activity as previously described (24). Protein concentration was determined by the Bradford method (25). Melatonin determination Melatonin content in the culture medium was assayed by HPLC and fluorimetric detection (26). Aliquots of 500 µl were mixed with 1 ml of chloroform, shaken for 20 min, and centrifuged at 9000g for 10 min. After washing the organic phase twice with 0.05 M carbonate buffer, pH 10.25, this was dried. The residue was redissolved in 100 µl of HPLC mobile phase, and 70 µl were injected into chromatograhic system. The samples were injected onto a µBondapack-C18 ODS reversed-phase column (10 µm particle size, Waters S.A., Barcelona, Spain). The mobile phase consisted of 0.1 M sodium phosphate, 50 mg/l of EDTA, and 30% acetonitrile, pH 5.1. The system was run at a flow rate of 0.9 ml/min (Waters 600E pump, Waters S.A.). The fluorescence detector (LS 40, Perkin Elmer, Buckinghamshire, UK) was set at excitation/emission wavelength of 285/345 nm. The identification of peaks by retention time and their quantification by peak height was done using a HP 3396 integrator (Hewlett-Packard, Palo Alto, CA). The detection limit was 10 pg/injection, and the inter- and intra-assay variation coefficients were 600 pg/ml; this concentration is approximately five times higher than nocturnal physiological levels of melatonin in human serum; indeed, they are in the same concentration range as the dissociation constant of melatonin binding sites described in human lymphocytes (18). 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 merely released after accumulating before lymphocytes were isolated. To answer this question, we cultured cells in the presence of a mixture of antisense oligonucleotides that 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 probably explained by the fact that HIOMT is the final enzyme involved in melatonin synthesis. These results strongly support the conclusion that, at least, a part of melatonin found in the culture medium was synthesized by the lymphocytes.
Effect of cycloheximide (CHX) on melatonin levels To confirm the endogenous origin of melatonin, PBMCs were incubated for 24 h in the presence of a protein synthesis inhibitor CHX (10 µg/ml). Incubation with CHX resulted in a significant reduction of melatonin levels in the culture medium (Table 2). The results indicate that translational blockade induced by CHX leads to a loss in melatonin production by PBMCs. Consequently, these data together with those already expounded strongly support that melatonin found in culture medium is synthesized by cells and subsequently released to the medium. Effect of PCPA on IL-2 production To define a possible physiological role for lymphocytes melatonin, we incubated cells in the presence of PCPA and/or melatonin and studied IL-2 production. The results show that PCPA significantly reduced IL-2 production by lymphocytes and that by adding melatonin to the medium, the inhibitory effect of PCPA was reverted (Fig. 2D). However, we have also shown melatonin production in unstimulated lymphocytes, which apparently is not related to IL-2 release. Therefore, we can speculate that endogenous melatonin produced by lymphocytes is effective in terms of stimulating IL-2 release only when cells are stimulated, acting as a modulator, which has been extensively suggested by several authors (29). We propose that melatonin synthesized by the lymphocyte could somehow contribute to regulation of its own IL2 production, possibly by acting as an intracrine, autocrine, and/or paracrine substance (Fig. 3). DISCUSSION In vivo models to test the immunomodulatory role of melatonin have been widely used. As summarized in reference 29, most authors agree that pinealectomy and in vivo models of melatonin administration clearly show the immunoenhancing properties of the melatonin. Many immune function tests by numerous investigators leave little doubt that, among many other functions, melatonin should be considered as a physiological immunomodulatory compound. However, when melatonin is used in vitro, that is, adding melatonin to different immune cells in culture, the results seem contradictory. Although many authors reveal a direct effect of melatonin on T and B lymphocytes in vitro (5), many others have claimed no effect of melatonin on resting lymphocytes or lymphocytes activated with PHA, Con A, or PMA. Thus, melatonin at low or high concentrations failed to activate the proliferation of human (30–33), rat (34), or chicken (35) lymphoid cells. In some cases, an inhibitory effect of melatonin on lymphocyte proliferation has been described as being coupled to NK activity inhibition (36) or IFN-γ and TNF-α production (37). These results disagree with those obtained in in vivo models in which melatonin behaved as a positive modulator of lymphocyte proliferation and cytokine production. The reasons for the apparent contradictions are not clear, but several possibilities exist. First, the effect of melatonin on immune cells is mediated via other tissues, cells, hormones, and/or cytokines that are not present in the culture medium. However, the presence of melatonin receptors in immune cells supports the hypothesis of a direct effect of melatonin on these cells (38). Second, melatonin efficiency in culture has been tested primarily in cells fully activated with PHA, Con A, or PMA. Under these conditions, immunomodulators, including melatonin, frequently fail to achieve further activation of immune cells (5). Third, the presence of melatonin in the culture medium released by immune cells may mask the effect of exogenous melatonin. In
this sense, the presence of high concentrations of melatonin in rat and human bone marrow (39, 40) has been described, as well as the presence of TPH, NAT, and HIOMT activities in human PBMCs, tumor-derived human cell lines, and rodent bone marrow, respectively (39–42). Thus, endogenously generated melatonin may interfere with exogenously added melatonin in in vitro experiments. Some in vivo studies only prove the presence of melatonin in human or rodent bone marrow together with the enzymatic machinery involved in its synthesis. These studies do not demonstrate that melatonin is, in fact, synthesized in these cells. For example, the melatonin that accumulated in these tissues before the cells were isolated may have been released after cells were placed in culture. On the other hand, in vitro studies by Finocchiaro et al. (41, 43) showed that melatonin was synthesized by cultured human PBMCs only when stimulated with serotonin or IFN-γ but not in unstimulated cells. Moreover, Conti et al. (40) also showed that different resting human cell lines (Nalm-6, Jurkat, and U-937) are able to synthesize melatonin. 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 (Fig. 1). Thus, when PBMCs were stimulated, no changes were observed in NAT expression, whereas a light decrease in HIOMT mRNA levels was observed in comparison to unstimulated cells. With regard to enzymatic activity, we observed an increase in NAT activity in stimulated cells, whereas HIOMT activity was inhibited significantly in comparison to unstimulated cells. Furthermore 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 (Fig. 2A–C and Table 2). The PHA-induced rise in melatonin synthesis could be due to an increase in NAT activity. Although HIOMT activity is inhibited by PHA, there is apparently enough enzyme activity remaining to convert the presumably higher amounts of N-acetylserotonin into melatonin. These results are in accordance with previous studies performed in Y79 human retinoblastoma cells (44). 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 (Fig. 2D). 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. Therefore, these results clearly demonstrate that the human immune system is a source of melatonin. The stimulatory effect of exogenous melatonin on IL-2 production by human lymphocytes is not a new finding (5). However, here, we show for the first time that melatonin synthesized by lymphocytes is involved in IL-2 production. Because numerous papers support the effect of melatonin on the production of several cytokines, through their membrane and nuclear receptors (5, 19, 29), we postulate that endogenous melatonin would act on IL-2 production in the same cells from which it was derived or on cells in its immediate vicinity, thereby behaving as an intracrine, autocrine, and/or paracrine substance (Fig. 3), as Simonneaux et al. have recently proposed in other structures (45). In addition, because melatonin production is also found in resting lymphocytes, although there is no IL-2 production, some factors other than melatonin may be involved in the production of the cytokine.
These results raise two questions. First, in view of the apparent high melatonin production by human PBMCs, what is its contribution to the serum where melatonin is known to be derived from the pineal gland? We propose that high concentrations of melatonin would only be found in close proximity to PBMCs in which it has been synthesized; this melatonin, we theorize, would act on the same cells from which it was derived or on cells in its immediate vicinity. Therefore, any excess melatonin would have only a minor effect on serum concentrations of the indole. This idea is consistent with previous studies showing that pinealectomy does not completely abolish serum melatonin levels (46). In the same way, we are led to believe that part of melatonin found in PBMCs come from the pineal gland, because PCPA, HIOMT-directed antisense treatments, and CHX treatments were not able to completely inhibit melatonin synthesis. However, it is well-known that using antisense treatment, researchers have rarely obtained inhibitions of >50% (47). Moreover, studies using PCPA never showed a complete inhibition of serotonin production (48). It has also been described that CHX is not able to completely inhibit the NAT protein levels in PBMCs (42). Second, some reports clearly show that the pinealectomy itself influences the immune system in mammals. How, then, does pineal-derived melatonin participate (along with locally produced melatonin) in the regulation of the immune system? Although additional experiments are necessary to answer this question, we hypothesize that melatonin derived from the pineal gland, especially at night, acts on the immune system by either supplementing the effect of synthesized melatonin by the immune cells (producing further increases in local melatonin concentrations) or acting via indirect means, that is, modulating steroid hormone production, which in turn influences the immune system as proposed by Nelson et al. (20). In summary, we propose that lymphoid cells are an important source of melatonin for the immune system, and this melatonin is somehow involved in the regulation of IL-2 production and, probably, other immune functions driven by IL-2. It is already known that IL-2 plays an essential role in promoting T-cell division, release of mediators such as IFN-γ, potentiation Bcell growth, and monocytes and NK cell activation, possibly by acting as an intracrine, autocrine, and/or paracrine substance (Fig. 3). Therefore, in future studies, during in vitro experiments with lymphocytes, an inhibitor of the melatonin synthesis 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. ACKNOWLEDGMENTS This work was supported by grants of the Spanish government (DGI, SAS 2002-00939; DGES, PM98-0156; PETRI, 95-04510P, BFI 2002-03544). A.C.V. was supported by a fellowship of the Asociación Sanitaria Virgen Macarena. REFERENCES 1.
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Table 1 Phosphorothioate oligonucleotides used in the study Oligonucleotide
Sequence (5' to 3')
Position
Antisense 1
CCG GAG CCT CTG GAG CGC TTG
5'UTR (–46 to –26)
Antisense 2
TGA GGA TCC CAT CTT GTC TCC
5'UTR-Exon 1 (–9 to +12)
Antisense 3
CTT GCA TGA ACT GTA GCC GC
Exon 5 (456 to 475)
Antisense 4
TGG CAT CAT AAA TGG CTC CTG
Exon 9 (1085 to 1105)
Sense 1
GTT CGC GAG GTC TCC GAG GCC
Sense 2
CCT CTG TTC TAC CCT AGG AGT
Sense 3
CGC CGA TGT CAA GTA CGT TC
Sense 4
GTC CTC GGT AAA TAC TAC GGT
Random 1
GCT GAT GAG GCC TCG GCT GCC
Random 2
CTT TAC GGC AAT GTG CCT CCT
Random 3
CAG GAT TAC CGC TAG GTC TC
Random 4
TAG CAG CTT AGC TAA GGC TCT
Table 2 Effect of cycloheximide (CHX) on melatonin levelsa Melatonin levels (percentage of maximum) Without CHX With CHX
100 13.7 ± 5.85b
PHA-stimulated cells were cultured for 24 h in the presence or absence of CHX (10 µg/ml). Data are expressed as mean ± SE of six experiments performed in triplicate. b Significant differences between cells treated with or without CHX were observed (P
