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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2004-1429
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 2 992-1000
Copyright © 2005 by The Endocrine Society

Human Lymphocyte-Synthesized Melatonin Is Involved in the Regulation of the Interleukin-2/Interleukin-2 Receptor System

Antonio Carrillo-Vico, Patricia J. Lardone, José M. Fernández-Santos, Inés Martín-Lacave, Juan R. Calvo, Michal Karasek and Juan M. Guerrero

Departments of Medical Biochemistry and Molecular Biology (A.C.-V., P.J.L., J.R.C., J.M.G.) and Normal and Pathological Cytology and Histology (J.M.F.-S., I.M.-L.), The University of Seville School of Medicine and Virgen Macarena Hospital, 41009 Seville, Spain; and Laboratory of Electron Microscopy (M.K.), Department of Pathology, Medical University of Lodz, 92-216 Lodz, Poland

Address all correspondence and requests for reprints to: Juan M. Guerrero, Department of Medical Biochemistry and Molecular Biology, The University of Seville School of Medicine, Avda. Sánchez Pizjuán 4, 41009 Seville, Spain. E-mail: guerrero{at}us.es.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Since melatonin was first isolated in 1958 up to the last few years, this substance was considered a hormone exclusive to the pineal gland. Although melatonin has lately been identified in a large number of extrapineal sites, its potential biological actions have not yet been studied. This paper shows that human lymphocyte-synthesized melatonin plays a crucial role modulating IL-2/IL-2 receptor system because when blocking melatonin biosynthesis by the tryptophan hydroxylase inhibitor, parachlorophenylalanine, both IL-2 and IL-2 receptor levels fell, restoring them by adding exogenous melatonin. Moreover, we demonstrated that this endogenous melatonin interfered with the exogenous melatonin effect on IL-2 production. Melatonin exerted these effects by a receptor-mediated action mechanism because both IL-2 and IL-2 receptor expressions significantly decreased when lymphocytes were incubated in the presence of the specific membrane and/or nuclear melatonin receptor antagonists, luzindole, and/or CGP 55644, respectively. Finally, we made the real significance of the membrane melatonin receptors in this process clear, so prostaglandin E2-induced inhibition on IL-2 production increased when we blocked the membrane receptors using luzindole. In conclusion, these data show that endogenous melatonin is an essential part for an accurate response of human lymphocytes through the modulation of IL-2/IL-2 receptor system.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE INDOLAMINE MELATONIN was first isolated from the bovine pineal gland by Lerner et al. (1). Melatonin, synthesized and released by the pineal gland, plays a central role in fine-tuning circadian rhythms and seasonal changes through its daily nocturnal increase in blood levels (2). Additionally, melatonin shows an outstanding functional versatility by exhibiting antioxidant (3), oncostatic (4), antiaging (5), and immunomodulatory (6) properties.

Regarding the effects of melatonin on immune system, in vivo studies have revealed that pinealectomy promotes thymic disorganization (7) as well as inhibition of IL-2 production and natural killer cell activity in rodents (8), whereas melatonin treatment or pineal grafting prevents thymic involution in very old mice (9). Besides, melatonin promotes immunostimulatory effects on many immune parameters such as antibody-dependent cellular cytotoxicity (10), antigen presentation by splenic macrophages to T cells (11), levels of gene expression of TGFß, macrophage-colony stimulating factor, TNF{alpha}, and stem cell factor in peritoneal exudated cells and IL-1ß, macrophage-colony stimulating factor, TNF{alpha}, interferon (IFN)-{gamma} and stem cell factor in splenocytes (12). Moreover, melatonin enhances the production of thymic peptides such as thymosin-{alpha}1 and thymulin through a nocturnal increase in prothymosin-{alpha} gene expression (13). Furthermore, administration of melatonin triggers Crohn’s disease symptoms (14), an IL-2-mediated autoimmune disease. On the other hand, in vitro studies show that melatonin acts mostly on the immune system by regulating cytokine production. Thus, melatonin activates the production of IL-2, IL-6, and IFN{gamma} by T helper cells and monocytes (15) as well as counteracting the inhibitory effect of prostaglandin (PG)E2 on IL-2 production in human lymphocytes (16). Melatonin also increases IL-12 production by monocytes driving T-cell differentiation toward the Th1 phenotype and causing an increase of IFN{gamma} production (17). Melatonin is also involved in the activation of human monocytes by the generation of cytotoxicity and secretion of IL-1, IL-6, TNF, and reactive oxygen intermediates (18, 19). Several of these effects occur via a direct action of melatonin on its receptors because both membrane and nuclear melatonin receptors have been identified in a large number of tissues including the immune system. To date, three mammalian melatonin membrane receptors have been either cloned (MT1 and MT2), or affinity-purified [MT3 (20)]. With regard to melatonin nuclear receptors, they belong to the RZR/ROR orphan receptor subfamily (21).

Since melatonin was first isolated five decades ago (1) until it was identified in extrapineal tissues such as retina, Harderian gland (22), and enterochromaffin cells (23) 20 yr later, melatonin was considered as a hormone exclusive to the pineal gland. Over the last few years, the presence of melatonin in a large number of extrapineal sites has been confirmed (24). In relation to the immune system, melatonin has been localized in thymus and cells such as mast, natural killer, eosinophilic leukocytes, platelets, and endothelial cells (24). In addition, high concentrations of the neurohormone together with the enzymatic machinery involved in its synthesis have been described in human, mouse, and rat bone marrow (25, 26). Recently we described that cultured human lymphocytes synthesize and release large amounts of melatonin that could act, in addition to its endocrine effect, as an intracrine, autocrine, and/or paracrine substance for the local coordination of the immune response (27). Although the presence of melatonin in extrapineal sites is becoming more and more evident, the potential physiological actions of this endogenous melatonin have not yet been studied. In this paper, we show that both IL-2 and IL-2 receptor (IL-2R) levels fall when blocking melatonin biosynthesis using parachlorophenylalanine (PCPA), an irreversible tryptophan hydroxylase (TPH) inhibitor, which were restored by adding exogenous melatonin. Moreover, we show that the endogenous melatonin is biologically active because it exerts these effects by means of action mechanisms in which both membrane and nuclear receptors are implicated. Finally, we demonstrated that melatonin membrane receptors have a real significance in this process.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

Melatonin, phytohemagglutinin (PHA), PCPA, and PGE2 were purchased from Sigma-Aldrich (Dorset, UK). Luzindole was from Tocris (Northpoint, Bristol, UK), and CGP 55644 was kindly provided by M.K.

Peripheral blood mononuclear cell (PBMC) cultures

Human peripheral venous blood was obtained from healthy volunteers (aged 25–50 yr). Volunteers were excluded if they displayed any immunological affection or were taking medication that could influence the immune function. Blood samples were obtained by venipuncture of the brachial vein and blood was collected in sterile EDTA K3-containing tubes.

PBMCs were then obtained by centrifugation over 1.077 g/ml Ficoll-Hypaque gradient (Seromed Biochrom KG, Berlin, Germany). Cells were washed in saline and finally resuspended in RPMI 1640 medium and cultured (0.25 x 106 cells/ml) in 96-well flat-bottom culture plates in medium supplemented with 25 mM HEPES, 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Sigma-Aldrich). After incubation at 37 C in 5% CO2 humidified atmosphere, cell-free culture supernatants were collected, filtered, and stored at –20 C for both IL-2 and melatonin determinations.

IL-2 assay

IL-2 concentrations were measured using a specific BD OptEIA set ELISA (PharMingen, San Diego, CA). Mouse antihuman IL-2 in 0.2 M sodium phosphate (pH 9.0) was coated overnight to high-binding microtiter plates. The plates were washed twice with PBS/0.05% Tween 20, incubated with 1% BSA in PBS/Tween 20 for 1 h as a blocking step, and washed again. Samples and standards (recombinant human IL-2) were diluted in 1% BSA PBS/Tween 20 and incubated overnight. After being washed five times, biotinylated mouse antihuman IL-2 monoclonal antibody was added, and bound IL-2 was detected using streptavidin-horseradish peroxidase conjugate, acting 3,3',5,5'tetramethylbenzidine and hydrogen peroxide as substrates of the enzyme.

Melatonin determination

Melatonin content in the culture medium from 72-h cultured PBMCs was assayed by HPLC with fluorometric detection (28). Aliquots of 500 µl were mixed with 1 ml chloroform, shaken for 20 min, and centrifuged at 9000 x g for 10 min. After washing the organic phases twice with 0.05 M carbonate buffer (pH 10.25), these were dried. The residue was redissolved in 100 µl HPLC mobile phase, and 80 µl were injected into chromatograhic system. The samples were injected onto a µBondapack-C18 ODS reversed-phase column (10 µm of particle size, Waters S.A., Barcelona, Spain). The mobile phase consisted of 0.1 M sodium phosphate, 50 mg/liter of EDTA, and 30% acetonitrile (pH 5.1). The system was run at a flow rate of 0.9 ml/min (soft-start 1350 pump, Bio-Rad Laboratories S.A., Barcelona, Spain). The fluorescence detector (Jasco 821-FP, Japan Spectroscopic Co. Ltd., Hachioji City, Japan) 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 an 1100 integrator (Hewlett Packard Co., Palo Alto, CA). The detection limit was 50 pg/injection and the inter- and intraassay variation coefficients were smaller than 7 and 4%, respectively

Flow cytometric analysis of CD25 expression

Cells stimulated with PHA (8 µg/ml) were incubated 72 h at 37 C without or with melatonin (10–8 M) and PCPA (10–4 M). Thereafter, cells were incubated 20 min in the presence of APC-conjugated anti-CD3 antibodies and phycoerythrin-conjugated anti-CD25 (PharMingen) and then analyzed on a FACScalibur flow cytometer (Becton Dickinson, San Jose, CA) using CELLQuest software (Becton Dickinson Immunocytometry Systems). Ten thousand cells were routinely acquired for analysis.

Proliferation assay

The effect of the antagonists on PBMCs viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) proliferation assay (Roche, Mannheim, Germany). Cells were seeded onto a 96-well flat-bottom plate at a concentration of 5 x 104 cells/well in 200 µl RPMI 1640. After an incubation period of 72 h, MTT reagent was added at a concentration of 0.5 mg/ml, and the plates were incubated for 4 h at 37 C. Afterward, 100 µl of solubilization buffer (10% sodium dodecyl sulfate in 0.01 M HCl) was added, and the plates were incubated overnight at 37 C. After being thoroughly mixed to dissolve the dark blue crystals, the plates were read on a microplate reader (Dynex Technologies, Chantilly, VA) at 595 nm to quantify absorbance.

Immunofluorescence visualization of CD25

PBMCs were cultured in 8-well slide chambers (Lab-Tek II chamber slide, Nunc, Naperville, IL). After activation with PHA for 72 h in either the presence or absence of both membrane and nuclear antagonists, cells were fixed by immersion in methanol at –20 C for 6 min. Then samples were allowed to rehydrate in PBS twice for 5 min. All the subsequent incubations were performed in a humidity chamber. Background labeling was prevented by incubation with normal horse serum for 30 min. Afterward samples were incubated with mouse antihuman CD25-PE-conjugated (PharMingen) for 1 h at 37 C, followed by three 10-min washes in PBS. Slides were finally mounted in glycerol/PBS containing 2% n-propyl-gallate (Sigma, St. Louis, MO) as antifading agent and observed under confocal laser scanning microscopy (TCS SP2, Leica Microsystems, Heidelberg GmbH, Germany), using the Leica confocal software (version 2.0). AOFT was set up to excite R-PE using the 488-nm line of a krypton-argon ion laser, and fluorescent emission of antigen-antibody complexes was monitored between 535 and 642 nm. Samples were observed using medium- and high-power magnification lens (1:40, NA = 1.25; 1:63, NA = 1.63; 1:100 NA = 1.40); confocal pinhole size was always set at Airy 1 position to minimize the thickness of the optical slice. Optical sections of cell aggregates were generated by the same microscope and software parameter settings, and finally, image outputs were compared for intensity.

Statistical analysis

Data are expressed as mean ± SEM of the indicated number of experiments. A t test was used to determine differences between pairs of treatments, as indicated in Results and Figs. 1Go and 2Go (also see Fig. 7Go). One-way ANOVA followed by a Student-Newman-Keuls post hoc test was used to determine differences between the mean values of the different treatment groups, as indicated in Results and Figs. 3–5GoGoGo. P < 0.05 was considered significant. The program used for the analyses of these data was SYSTAT 10 (Systat Software Inc., Richmond, CA).



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  		FIG. 1. Effect of PCPA on IL-2 production induced by increasing concentrations of melatonin (MEL). Cells were incubated for 72 h with the indicated concentrations of PHA and melatonin in the presence or absence of PCPA (10–4 M), and IL-2 levels were determined by ELISA in culture supernatants. Data are expressed as percentage of mean values ± SE of 20 experiments performed in triplicate. Control values of IL-2 production: 0.5 µg/ml PHA, 1521 ± 333 pg/106 cells (– PCPA) and 1306 ± 270 pg/106 cells (+ PCPA); 2 µg/ml PHA, 2384 ± 446 pg/106 cells (–PCPA) and 1982 ± 453 pg/106 cells (+ PCPA); 8 µg/ml PHA, 4875 ± 1704 pg/106 cells (–PCPA) and 3329 ± 1139 pg/106 cells (+ PCPA). Statistically significant differences were determined (*, P < 0.05; **, P < 0.01; ***, P < 0.001), compared with cells cultured in the absence of melatonin.

 


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  		FIG. 2. Inhibitory effect of PCPA on melatonin production. A, Cells stimulated with PHA (8 µg/ml) were incubated in the presence or absence of PCPA (10–4 M) for 72 h, and melatonin content was determined by HPLC in culture supernatants. Data are expressed as mean values ± SE of 12 experiments performed in triplicate. *, Significant differences were observed between cells treated with PCPA and those untreated (P < 0.05). B, Representative chromatograms of melatonin formed in the medium with or without PCPA and authentic standard of melatonin, 1000 pg/ml.

 


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  		FIG. 7. Effect of luzindole (LUZ) on PGE2-inhibited IL-2 production. Cells were incubated for 72 h with PHA (8 µg/ml), the indicated concentrations of PGE2, and when indicated 10–6 M LUZ, and IL-2 content was determined by ELISA in culture supernatants. Data are expressed as mean values ± SE of six experiments performed in triplicate. Control values of IL-2 production: 8000 ± 621 pg/106 cells (–LUZ) and 6095 ± 880 (+ LUZ). Significant differences were observed between cells treated with LUZ and group without LUZ (***, P < 0.001; **, P < 0.01; *, P < 0.05).

 


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  		FIG. 3. Effect of PCPA on IL-2 production. Cells were incubated for 72 h with indicated concentrations of PHA, melatonin (Mel, 10–8 M), and/or or PCPA (10–4 M), and IL-2 levels were determined by ELISA in culture supernatants. Data are expressed as percentage of mean values ± SE of 32 experiments performed in triplicate. Control values of IL-2 production: 0.5 µg/ml PHA, 1037 ± 201 pg/106 cells; 2 µg/ml PHA, 3019 ± 409 pg/106 cells; 8 µg/ml PHA, 8710 ± 1773 pg/106 cells. Significant differences between cells treated with PCPA and other groups were observed (*, P < 0.05 vs. control; §, P < 0.05 and §§§, P < 0.001 vs. PCPA/Mel).

 


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  		FIG. 4. Effect of PCPA on CD25 expression. Cells were incubated for 72 h with PHA (8 µg/ml) and, when indicated, 10–8 M melatonin (Mel) and/or 10–4 M PCPA, and CD25 expression was determined by flow cytometry. Data are expressed as percentage of mean values ± SE of 12 experiments performed in triplicate. Control values: 17.9 ± 1.24 (percent) of cells that coexpress CD3 and CD25. Significant differences were observed between cells treated with PCPA and control group (***, P < 0.001) and between cells treated with PCPA/Mel and PCPA (§§, P < 0.01).

 


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  		FIG. 5. A, Effect of luzindole (LUZ) and CGP 55644 (CGP) on IL-2 production. Cells stimulated with PHA (8 µg/ml) were incubated with luzindole and/or CGP 55644 (10–6 M) for 72 h, and IL-2 content was determined by ELISA in culture supernatants. Data are expressed as percentage of mean values ± SE of 12 experiments performed in triplicate. Control values of IL-2 production: 3298 ± 798 pg/106 cells. B, Effect of antagonists on cell viability. Cells were cultured in the same conditions as A and were subjected to MTT proliferation assay. Data are expressed as mean values ± SE of 12 experiments performed in triplicate Significant differences were observed between cells treated with LUZ, CGP, and LUZ/CGP and control group (***, P < 0.001) and between cells treated with CGP, LUZ/CGP, and LUZ (§, P < 0.05).

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PCPA effect on exogenous melatonin-induced IL-2 production

To determine whether human lymphocyte-synthesized endogenous melatonin could be interfering in the production of IL-2 induced by exogenous melatonin, we cultured PBMCs with optimal (8 µg/ml) and suboptimal concentrations of PHA (15, 29, 30), which promoted the IL-2 production in cells. Under these conditions we studied the effect of increasing concentrations of exogenous melatonin on IL-2 production when cells were cultured in the presence or absence of PCPA, a TPH inhibitor and therefore inhibitor of endogenously synthesized melatonin (Fig. 1Go). We observed that melatonin increased IL-2 production when PBMCs were cultured only in the presence of 0.5 µg/ml PHA (Fig. 1AGo). This effect was dose dependent, reaching significant values when cells were cultured in the presence of physiological levels of melatonin (10–8 M, P = 0.01; 10–7 M, and 10–6 M, P < 0.001, t test), i.e. the physiological concentrations known to exist in the blood of humans, whereas no effect was observed in the presence of higher concentrations of PHA (Fig. 1Go, B and C). On the other hand, when cells were incubated with PCPA, we observed a dose-dependent effect of endogenous melatonin under all conditions of stimulation tested (0.5 µg/ml PHA: 10–7 M, P = 0.034, and 10–6 M, P < 0.001; 2 µg/ml PHA: 10–8 M, P = 0.005; 10–7 M, P = 0.01, and 10–6 M, P < 0.001; 8 µg/ml PHA: 10–8 M, 10–7 M, and 10–6 M, P < 0.001, t test).

PCPA inhibits melatonin production by PBMCs

To confirm the crucial role of endogenous melatonin in the regulation of IL-2 production by PBMCs, we studied the presence of melatonin synthesis in these cells by HPLC (Fig. 2Go). We detected the presence of high concentrations of melatonin in the culture medium when cells were incubated for 72 h. When the cells were incubated in the presence of PCPA, melatonin levels dropped significantly (P = 0.04, t test). No melatonin was detected in culture medium alone and was insignificant in those containing FCS (data not shown). To exclude that PCPA effect was mediated through a decrease in cellular viability, we performed a proliferation assay in which we did not detect any effect of PCPA on the cellular viability (data not shown).

PCPA inhibits IL-2 production by a decrease in endogenous melatonin

When we cultured human PBMCs in the presence of exogenous melatonin together with PCPA, we observed a clear effect of exogenous melatonin on IL-2 production under any of the conditions of stimulation (Fig. 1Go). Because this PCPA effect appears to be mediated by a drop in endogenous melatonin synthesized by lymphocytes, we expected that the incubation of PHA-stimulated cells only with PCPA would promote a fall in the IL-2 levels, which could be restored by adding of exogenous melatonin. Thus, we cultured PBMCs in the presence of suboptimal and optimal doses of PHA. When these PHA-stimulated cells were incubated with PCPA, we observed a significant fall in the levels of IL-2 (2 µg/ml PHA, P = 0.012; 8 µg/ml PHA, P = 0.029, ANOVA) (Fig. 3Go, B and C) except when cells were stimulated with 0.5 µg/ml PHA (P = 0.386) (Fig. 3AGo). As indicated in Fig. 3Go, these falls in IL-2 levels were counteracted by adding exogenous melatonin (0.5 µg/ml PHA: P = 0.019; 2 µg/ml PHA: P = 0.021; 8 µg/ml PHA: P < 0.001, ANOVA).

PCPA inhibits CD25 expression

Although results shown so far strongly support that the effect of PCPA on IL-2 production is mediated through a decrease of endogenous melatonin synthesized by human PBMCs, we investigated a possible non-melatonin-mediated effect of PCPA in the expression of the {alpha}-chain (CD25) of the high-affinity IL-2R by flow cytometry. Thus, when we studied the expression CD25 in PBMCs treated with PCPA, we detected a significant fall in CD25 levels (P < 0.001). Moreover, when PCPA-treated cells were incubated in the presence of melatonin, the effect of PCPA was counteracted (P = 0.005), reaching CD25 levels close to control values (Fig. 4Go).

Endogenous melatonin acts on IL-2 production via its membrane and nuclear receptors

Because we have shown that lymphocyte-synthesized melatonin is involved in the regulation of IL-2 production, we studied through which mechanism of action endogenous melatonin was acting. Accordingly, PHA-stimulated cells were incubated with the specific membrane and nuclear receptor antagonists, luzindole and CGP 55644, respectively, and IL-2 levels were studied (Fig. 5Go). We observed a significant decrease in IL-2 production when cells were incubated with either luzindole or CGP 55644 or both together, although the fall was higher when CGP 55644 was used (P < 0.001) than with luzindole (P < 0.001). In the presence of both antagonists together, the drop was even higher (P < 0.001) (Fig. 5AGo). To rule out that the antagonist effect would be due to a decrease in cellular viability, we performed a proliferation assay (Fig. 5BGo) in which we did not detect any effect of antagonists on the cellular viability (P = 0.8).

Luzindole and CGP 55644 inhibit the CD25 expression

To confirm the endogenous melatonin action on IL-2/IL-2R system, we also studied the effects of both antagonists in CD25 expression (Fig. 6Go). The use of confocal laser-scanning microscopy allowed us to analyze the expression of CD25 in individualized cells, although PBMCs are usually aggregated after PHA treatment. PBMC samples showed a pattern of immunoreactivity for IL-2R that, as expected, was restricted to the plasma membrane area and, only occasionally, appeared as a diffuse nuclear and cytoplasmic signal probably due to tangential optical sections. A decrease in immunofluorescence for CD25 was displayed when cells were incubated in the presence of luzindole together with CGP 55644. Furthermore, the micrographs revealed that the antagonist-induced decrease in CD25 expression was homogeneously observed in most of the cells. These results are consistent with those observed in IL-2 production (Fig. 5AGo).



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  		FIG. 6. Effect of luzindole and CGP 55644 on CD25 expression. Cells stimulated with PHA (8 µg/ml) were incubated with luzindole and CGP 55644 (10–6 M) for 72 h, and immunofluorescence detection of CD25 was detected by using confocal laser-scanning microscopy. Micrographs show the comparison of CD25 expression on optical sections of PHA-induced cell aggregates in either the absence (A) or presence (B) of both antagonists.

 
Luzindole enhances the PGE2-induced inhibition of IL-2 production

We have recently shown that exogenous melatonin counteracts the inhibitory effect of PGE2 on IL-2 production via its membrane receptor MT1 (16). Using this model as a base, we studied whether lymphocyte-synthesized melatonin could itself be involved in PGE2-inhibited IL-2 production through a mechanism in which only the membrane receptors were implicated. Thus, we observed that PGE2 decreased IL-2 production and this was a dose-dependent effect. When we incubated these PGE2-treated cells with luzindole, we saw that the inhibitory effect of PGE2 was significantly higher than that in the absence of luzindole (Fig. 7Go), almost reaching complete inhibition at micromolar concentration of PGE2 (10–8 M, P = 0.003; 10–7 M, P < 0.001; 10–6 M, P = 0.038, t test).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Over the last two decades, an increasing number of reports have documented the existence of a relationship between pineal melatonin and the immune system. In this way, in vivo data that confirm this connection have been provided. Thus, a correlation between melatonin production and circadian and seasonal variations in the immune system (31) as well as the effects of surgical or functional pinealectomy on the immune system (13) has been described. This connection is also supported by the presence of melatonin receptors in many different immune tissues and cells from many species (29, 32, 33, 34). Melatonin in vivo administration usually promotes stimulation of the immune system (6). However, when melatonin is used in vitro, the results seem contradictory. Thus, many authors have demonstrated the direct effects of melatonin on T and B lymphocytes (15, 35), whereas others have claimed no effect of melatonin on fully activated lymphocytes (36). In some cases, an inhibitory effect of melatonin on lymphocyte proliferation has been described as being coupled to IFN{gamma} and TNF{alpha} production (37). These results disagree with those obtained in vivo, in which melatonin behaved as a positive modulator of lymphocyte proliferation and cytokine production (10, 11, 13, 38, 39, 40). The reasons for the apparent contradictions are not clear, but we believe that the presence of endogenous melatonin released by immune cells may mask the effect of exogenous melatonin. In this sense, the presence of high concentrations of melatonin and its biosynthetic machinery have been described in mouse, rat, and human immune system (25, 26, 41, 42, 43).

Numerous in vitro studies have shown that cytokine production could be considered as one of the main mechanisms by which melatonin modulates the immune system. In this way, our group has described that administration of melatonin to human PBMC cultures enhances IL-2 production only under PHA suboptimal stimulation (15, 44). Additionally, a recent paper (27) has shown that human PBMCs stimulated with optimal doses of PHA are able to synthesize and release large amounts of melatonin into the medium culture. Actually, this could be the reason we find exogenous melatonin effect only under suboptimal conditions of stimulation. In other words, endogenous melatonin would be masking the effect of exogenous melatonin on IL-2 production. Consequently, we studied the effect of exogenous melatonin on IL-2 production by PBMCs using a model in which melatonin synthesis was blocked by PCPA. We showed that exogenous melatonin significantly increased IL-2 production when PBMCs were cultured only in the presence of suboptimal conditions of stimulation (0.5 µg/ml PHA), whereas no effect was observed in the presence of higher concentrations of PHA. On the other hand, when cells were incubated with PCPA, we observed a dose-dependent effect of exogenous melatonin under all conditions of stimulation tested. These data strongly suggest that production of PHA-induced IL-2 is, at least partly, mediated through endogenous melatonin. Therefore, when cells are stimulated with high doses of PHA, endogenous melatonin production is either really high or lymphocytes are prepared to respond to this endogenous melatonin, resulting in an inability of exogenous melatonin to activate IL-2 production.

Moreover, we detected the presence of high concentrations of melatonin in the culture medium when cells were incubated for 72 h. Although the comparison of the amount of melatonin derived from cultured cells to physiological levels in serum is not so meaningful, melatonin concentration rose to values higher than 600 pg/ml, which are 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 previously described in human lymphocytes (45). When the cells were incubated in the presence of PCPA, melatonin levels significantly dropped. These results not only show the presence of melatonin in these cells but also demonstrate that the PCPA effect on IL-2 production is actually mediated by a fall in endogenous melatonin levels. The reason PCPA did not exert a higher effect on melatonin levels could be due to serotonin external contribution coming from FCS used in the culture of cells because this inhibitor acts on the TPH, the first enzyme involved in melatonin synthesis.

Bearing in mind these data we expected that the incubation of cells with only PCPA would lead to a decrease in IL-2 levels. When these PHA-stimulated cells were incubated with PCPA, we observed a significant fall in the levels of IL-2, which was not significant when cells were stimulated with 0.5 µg/ml PHA. These falls in IL-2 levels were counteracted by adding exogenous melatonin. These data reveal that the decrease in PCPA-induced IL-2 production is mediated by the inhibition of melatonin synthesis in the cell because the IL-2 levels returned to control values when we added exogenous melatonin, indicating, once again, a clear implication of endogenous melatonin in this process. The presence of this endogenous melatonin could be the reason many authors are able to find in vitro effects of exogenous melatonin only when cells are not activated or are just slightly activated with PHA (15, 29).

Although results strongly support that the effect of PCPA on IL-2 production is mediated through a decrease in PBMC-synthesized endogenous melatonin, some papers (46) revealed that PCPA reduced the capacity of splenic T cells to express IL-2R in response to concanavalin A. Consequently, we investigated a possible non-melatonin-mediated effect of PCPA in the expression of the {alpha}-chain (CD25) of the high-affinity IL-2R. Thus, when we cultured PBMCs in the presence of PCPA, we detected a significant fall in CD25 expression levels that were counteracted by using exogenous melatonin, which indicated that PCPA action on IL-2R expression is mediated by endogenous melatonin.

Because we demonstrated that endogenous melatonin is responsible, at least in part, for the IL-2 production by lymphocytes, we investigated through which mechanism of action endogenous melatonin was acting. It has been described that melatonin can act by mechanisms either mediated or not mediated by receptors. In this way, melatonin membrane and nuclear receptors have been identified in the human immune system (16, 29, 44). Furthermore, over the last years, several specific melatonin membrane and nuclear receptor agonists and antagonists have been described (20, 47), which have been extensively used in the study of melatonin effects in several systems, including the immune system (15, 32, 44). Basing our investigation on these works, we studied the effects of both luzindole and CGP 55644 as antagonists of membrane and nuclear receptors, respectively, on IL-2/IL-2R system expression. We observed that IL-2 levels significantly decrease when lymphocytes were incubated with luzindole or CGP 55644 or both together, although the fall was higher when CGP 55644 was used than with luzindole. In the presence of both antagonists together, the drop was even higher, exhibiting a synergistic effect that has previously been described using melatonin membrane and nuclear receptor agonists (44). These results could indicate a possible link between nuclear and membrane melatonin receptors in these cells. On the other hand, we also detected that the incubation of lymphocytes with both antagonists caused a decrease in IL-2R expression. Therefore, human lymphocytes not only synthesize melatonin, but this endogenous melatonin is also biologically active because it is involved in the regulation of the IL-2/IL-2R expression through a mechanism of action in which both membrane and nuclear receptors are involved (summarized in Fig. 8Go). The action of a substance through both membrane and nuclear receptor pools in the same cell is not a new finding (48), so we hypothesize that endogenous melatonin might act through either one receptor or both receptors at once, depending on the physiological state of the cell.



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  		FIG. 8. Schematic diagram of how melatonin (Mel) could act on the IL-2/IL-2R system as an autocrine, intracrine, and/or paracrine substance in human PBMCs. AADC, Aromatic amino acid decarboxylase; HIOMT, hydroxyndole-O-methyltransferase; 5-H-Trp, 5-hydroxytryptophan; NAT, serotonin-N-acetyltransferase; Ser, serotonin; Trp, tryptophan.

 
Finally, with the aim of determining the actual importance of membrane receptors in endogenous melatonin action, we studied a model in which IL-2 production was modified only through a membrane mechanism. PGE2 is known to inhibit human T-cell activation by inhibiting IL-2/IL-2R levels through a cAMP-coupled membrane receptor (49), and recently our group described that exogenous melatonin is able to counteract this action via its membrane receptor MT1 (16). Using this model as a base, we raised the question of whether endogenous melatonin itself, by binding to the membrane receptors, would also be able to counteract the PGE2 effect on IL-2 production. We observed that the inhibitory effect of PGE2 on IL-2 production was significantly higher when we blocked these membrane receptors by using luzindole. Consequently, when endogenous melatonin is bound to its membrane receptors, it causes an increase in IL-2 levels by an action mechanism in which cAMP probably is involved. These data support that endogenous melatonin could be acting in the immune system through either membrane and/or nuclear receptor pools, depending on the physiological state of the cell.

Taken together, the results show for the first time that human lymphocyte-synthesized melatonin plays a fundamental intra-, auto-, and/or paracrine role in cell activation through regulation of the IL-2/IL-2R system (summarized in Fig. 8Go) and, probably, other cytokine and immunological mediators. In conclusion, this work is potentially important for understanding hormonal control of immune activation.


   Acknowledgments
 
The authors thank Mr. John Brown for correcting the English language.


   Footnotes
 
This work was supported by Spanish Government grants (DGI, SAS 2002-00939; DGES, PM98-0156; PETRI, 95-04510P, BFI 2002-03544, MCYT P2003/1339, PAI CTS0160, PAI CTS439). A.C.-V. and P.J.L. were supported by fellowships from the Asociación Sanitaria Virgen Macarena and Andalusian Regional Government, respectively.

First Published Online November 23, 2004

Abbreviations: FCS, Fetal calf serum; IFN, interferon; IL-2R, IL-2 receptor; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBMC, peripheral blood mononuclear cell; PCPA, parachlorophenylalanine; PG, prostaglandin; PHA, phytohemagglutinin; TPH, tryptophan hydroxylase.

Received July 22, 2004.

Accepted November 16, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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