Sofía García-Mauriño

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

It has been historically assumed that the pineal gland is the major source of melatonin (N‐acetyl‐ 5‐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

Physiological levels of melatonin contribute to the antioxidant capacity of human serum

This work evaluates whether physiological concentrations of the pineal secretory product melatonin contribute to the total antioxidant status (TAS) of human serum. Day and nighttime serum samples were collected from healthy volunteers ranging from 2 to 89 years of age and used to measure melatonin and TAS. Results showed that both melatonin and TAS in human serum exhibited 24 hr variations with nocturnal peak values at 01:00 hr. Moreover, exposure of volunteers to light at night resulted in clear decreases of both TAS and melatonin. Furthermore, when melatonin was removed from sera collected at night, the TAS value of the sample was reduced to basal daytime values. In aging studies, it was found that nocturnal serum values of TAS and melatonin exhibited maximal values during the first four decades; thereafter, these values decreased as age advanced. In 60-year-old individuals, day/night differences in serum melatonin and TAS levels were clearly diminished, by more than 80%, with these differences being completely abolished in older individuals. Our results suggest that melatonin contributes to the total antioxidative capability of human serum. This antioxidant contribution of melatonin is reduced as age advances correlating with the age-related reduction of melatonin.

Correlation between nuclear melatonin receptor expression and enhanced cytokine production in human lymphocytic and monocytic cell lines

The report shows that melatonin enhances IL‐2 and IL‐6 production by two human lymphocytic (Jurkat) and monocytic (U937) cell lines via a nuclear receptor‐mediated mechanism. Jurkat cells express nuclear (RZRα, RORα1 and RORα2) and membrane (mt1) melatonin receptors, and melatonin binds to Jurkat nuclei and membranes with the same affinity described for human peripheral blood mononuclear cells (PBMCs). Melatonin enhances IL‐2 production by Jurkat cells activated by either phytohemagglutinin (PHA) or phorbol myristate acetate (PMA). PHA activation of Jurkat cells does not change the profile of melatonin receptor expression; on the contrary, PMA activation negatively regulates the mt1 receptor. In the absence of the membrane receptor, melatonin still activates IL‐2 production. U937 cells express only the mt1 receptor. Although melatonin binds to both U937 nuclei and membranes, CGP 52608, a ligand of the nuclear receptor for melatonin, does not inhibit melatonin binding to U937 nuclei, suggesting that a protein other than the RZR/RORα receptor was involved in the process. In U937 cells, melatonin did not modify basal production of IL‐6 or when activated by PMA plus LPS (lipopolysaccharide), a treatment that downregulates the expression of the mt1 receptor. However, in U937 cells activated with IFN‐Γ, which induces the expression of the RORα1 and RORα2 nuclear receptors and represses the expression of the mt1 receptor, melatonin can activate IL‐6 production. These results show that the expression of nuclear melatonin receptor is sufficient for melatonin to activate cytokine production in human lymphocytic and monocytic cell lines.

Melatonin activates Th1 lymphocytes by increasing IL-12 production

Melatonin could act on immune system by regulating cytokine production of immunocompetent cells. The hormone enhances IL-2, IFN-gamma and IL-6 production by cultured human mononuclear cells. As enhancement of IL-6 production is related to monocyte activation by melatonin, the hormone acts on human lymphoid cells causing a Th1-type response. This paper shows that melatonin seems to promote a Th1-response by increasing IL-12 production. The hormone enhances IL-12 production by cultured monocytes under suboptimal stimulation in a dose-dependent way. The effect of the hormone increases when PBMCs are incubated with melatonin before monocyte isolation. Enhanced IL-12 production by melatonin can also be shown in cultured human mononuclear cells.

High-affinity binding of melatonin by human circulating T lymphocytes (CD4+).

This paper shows the presence of high‐ affinity binding sites for melatonin in human circu‐lating T lymphocytes, but not in B lymphocytes. The binding of melatonin to T cells was dependent on time, stable, reversible, saturable, specific, and in‐versely correlated to the production of melatonin, expressed as the nocturnal 12 h production of its urinary metabolite 6‐sulfatoxymelatonin. The affin‐ity of these binding sites ( K d = 0.27 nM) suggests that they may recognize the physiological concentrations of melatonin in serum. Moreover, among the lymphocyte subpopulations studied, binding of melatonin was mostly found in CD4+ cells rather than in CD8+ cells. Results suggest that CD4+ cells may be the target of melatonin among the human circulating lymphocytes.—Gonzalez‐Haba, M. G., Garcia‐Mauriño, S., Calvo, J. R., Goberna, R., Guerrero, J. M. High‐affinity binding of melatonin by human circulating T lymphocytes (CD4+). FASEB J. 9, 1331‐1335(1995)

Involvement of nuclear binding sites for melatonin in the regulation of IL-2 and IL-6 production by human blood mononuclear cells

Many functional studies show that melatonin plays a fundamental role in neuroimmunomodulation. In this paper, we have extended our studies on the influence of melatonin on IL-2 and IL-6 production by human peripheral blood mononuclear cells (PBMCs) by comparing the effects of the specific membrane receptor agonist S 20098, the RZR/ROR(alpha) receptor agonist CGP 52608, and structurally related thiazolidinediones. Melatonin bound to membranes as well as to nuclei of human PBMCs with about the same affinity (IC50 values around 5 nM). S 20098 bound to PBMC membranes but not to PBMC nuclei, although the affinity was at least 100 times lower than that of melatonin; this compound did not stimulate cytokine production. In contrast, all four CGP compounds did not bind to PBMC membranes, while binding to nuclei exhibited IC50 values comparable to those of melatonin. The thiazolidinediones activating the RZR/ROR(alpha) receptor (CGP 52608, CGP 53079) also increased IL-2 and IL-6 production. CGP 55644 had no effect on cytokine production and antagonized the effects of CGP 52608 on IL-2 and IL-6 production; moreover, CGP 55644 decreased the enhanced IL-2 production caused by melatonin. Results obtained in monocyte cultures resembled closely those shown in PBMCs. The results reported in this paper confirm the involvement of a nuclear mechanism in the melatonin effects on cytokine production in human PBMCs. We have also shown a synergistic effect of S 20098 and CGP 52608, suggesting a possible link between nuclear and membrane melatonin receptors in PBMCs.

mRNA expression of nuclear receptor RZR/RORα, melatonin membrane receptor MT1, and hydroxindole‐O‐methyltransferase in different populations of human immune cells

Abstract:  We characterized the expression levels of the retinoid Z receptor α (RZR α), RORα mRNA isoforms (RORα1, RORα2, and RORα3), and both melatonin receptor MT1 and hydroxindole‐O‐methyltransferase (HIOMT) genes. For this purpose, the following human peripheral blood mononuclear cells populations were isolated: monocytes (CD14+ cells), B lymphocytes (CD19+ cells), T helper lymphocytes (CD14− CD4+), cytotoxic T lymphocytes (CD56− CD8+ cells), and natural killer (NK) lymphocytes (CD56+ cells). PBMCs subsets were obtained by Dynabeads M‐450 (Dynal) isolation procedure. We observed a strong gene expression signal for RZRα in all subpopulations studied, whereas both RORα1 and RORα2 transcripts were amplified only in CD8+ cells. Specific signal for RORα2 was obtained in all subpopulations studied, but we were not able to detect the RORα3 mRNA transcript in human immune cells studied. A weaker signal (especially in CD19+ cells) was also detected in all subsets of cells for the MT1 gene. With regard to HIOMT, a strong signal was achieved among all but one subpopulation of cells; the only exception was CD14+ cells. Thus, in addition to its classical function in the nervous and endocrine system, melatonin could act directly as a paracrine and/or autocrine agent in the human immune system.

New paradigms in chronic intestinal inflammation and colon cancer: role of melatonin.

Abstract:  In intestinal bowel disease (IBD), immune‐mediated conditions exert their effects through various cells and proinflammatory mediators. Recent data support a participation of the endoplasmic reticulum stress and mitochondrial dysfunctions in IBD. Moreover, it is evident that chronic degenerative pathologies, including IBD, share comparable disease mechanisms with alteration in the autophagy mechanisms. Chronic inflammation in IBD exposes these patients to a number of signals known to have tumorigenic effects. This circuitry of inflammation and cancer modifies apoptosis and autophagy, and promotes cellular cycle progression, invasion, and angiogenesis. Melatonin has been shown as a specific antioxidant reducing oxidative damage in both lipid and aqueous cell environments. However, several studies provide further insight into the molecular mechanisms of melatonin action in the colon. In this line, recent data suggest that melatonin modulates autophagy and sirtuin activity. An anti‐autophagic property of melatonin has been demonstrated, and it could contribute to its anti‐oncogenic activity. Nevertheless, there is no information about whether antitumoral effects of melatonin on colon cancer are dependent on autophagy. Sirtuins have pleiotropic effects on cancer development, being reported both as facilitator and as suppressor of colon cancer development. Sirtuins and melatonin are connected through the circadian clock machinery, and melatonin seems able to correct the alterations in sirtuin activity associated with several pathological conditions. Autophagy and sirtuin activities are linked through 5′AMP‐activated protein kinase (AMPK) activation, which switches on autophagy and increases sirtuin. The effect of melatonin on AMPK and the impact of this effect on IBD and colon cancer remain an open question.

Specific binding of melatonin by purified cell nuclei from spleen and thymus of the rat.

In the present paper, we show that pineal hormone melatonin interacts with purified cell nuclei from rat spleen and thymus. Binding of 2-[125I]iodomelatonin ([125I]melatonin) by cell nuclei fulfills all criteria for binding to a receptor site. Binding exhibited properties such as dependence on time and temperature as well as reversibility, saturability, high affinity, and specificity. Results suggested binding to single classes of binding sites. The dissociation constants (Kd) for the binding sites in the spleen and thymus nuclei were 68 and 102 pM, respectively. These data are in close agreement with data obtained from kinetic studies, in which the kinetically derived values of the dissociation constant in the spleen and thymus nuclei were 166 and 537 pM, respectively. The affinities for melatonin of these nuclear binding sites suggest that they may recognize the physiological concentrations of melatonin in the tissues. Finally, we have demonstrated that binding of [125I]melatonin by the nuclei is displaced by CGP 52608, a specific ligand of the putative nuclear melatonin receptor RZR/ROR. Results strongly suggest that in addition to membrane receptor-related mechanisms, nuclear receptors may be involved in the regulation of immune system by melatonin.

Involvement of Nuclear Receptors in the Enhanced IL-2 Production by Melatonin in Jurkat Cells

Abstract: This report shows that melatonin enhances IL‐2 production by Jurkat cells via a nuclear receptor‐mediated mechanism. Jurkat cells express nuclear (RZRα, RORα1, and RORα2) and membrane (mt1) melatonin receptors, and melatonin binds to Jurkat nuclei and membranes with the same affinity described for human peripheral blood mononuclear cells (PBMCs). Melatonin enhances IL‐2 production by Jurkat cells activated by either phytohemagglutinin (PHA) or phorbol myristate acetate (PMA). PHA activation of Jurkat cells does not change the profile of melatonin receptor expression; on the contrary, PMA activation negatively regulates the mt1 receptor. In the absence of the membrane receptor, melatonin still activates the IL‐2 production. These results show that the expression of the nuclear melatonin receptor is sufficient for melatonin to activate IL‐2 production by Jurkat cells.