The role of melatonin in the cells of the innate immunity: a review.

Melatonin is the major secretory product synthesized and secreted by the pineal gland and shows both a wide distribution within phylogenetically distant organisms from bacteria to humans and a great functional versatility. In recent years, a considerable amount of experimental evidence has accumulated showing a relationship between the nervous, endocrine, and immune systems. The molecular basis of the communication between these systems is the use of a common chemical language. In this framework, currently melatonin is considered one of the members of the neuroendocrine-immunological network. A number of in vivo and in vitro studies have documented that melatonin plays a fundamental role in neuroimmunomodulation. Based on the information published, it is clear that the majority of the present data in the literature relate to lymphocytes; thus, they have been rather thoroughly investigated, and several reviews have been published related to the mechanisms of action and the effects of melatonin on lymphocytes. However, few studies concerning the effects of melatonin on cells belonging to the innate immunity have been reported. Innate immunity provides the early line of defense against microbes and consists of both cellular and biochemical mechanisms. In this review, we have focused on the role of melatonin in the innate immunity. More specifically, we summarize the effects and action mechanisms of melatonin in the different cells that belong to or participate in the innate immunity, such as monocytes-macrophages, dendritic cells, neutrophils, eosinophils, basophils, mast cells, and natural killer cells.

Melatonin present in beer contributes to increase the levels of melatonin and antioxidant capacity of the human serum.

BACKGROUND & AIM: Melatonin is a molecule with antioxidative properties including direct free radical scavenging and indirect stimulatory actions on a variety of antioxidative enzymes which further promote its ability to reduce the toxicity of radicals and their associated reactants. Beer is an integral element of the diet of numerous people and is rich in antioxidants. We analyzed if melatonin is present in beer and if so, at what concentration. It further determines whether the moderate consumption of beer has an effect on the total antioxidant status (TAS) of human serum. METHODS: We analyzed 18 brands of beer with different percentage of alcohol content in order to determine the concentration of melatonin. Serum samples were collected from 7 healthy volunteers. These samples were used to measure melatonin and TAS on basal conditions and after drinking beer. RESULTS: Showed that all the beer analyzed did indeed contain melatonin and the more they have got, the greater was its degree of alcohol. Both melatonin and TAS in human serum increased after drinking beer. CONCLUSIONS: Melatonin present in the beer does contribute to the total antioxidative capability of human serum and moderate beer consumption can protect organism from overall oxidative stress.

Beneficial pleiotropic actions of melatonin in an experimental model of septic shock in mice: regulation of pro-/anti-inflammatory cytokine network, protection against oxidative damage and anti-apoptotic effects.

Septic shock, the most severe problem of sepsis, is a lethal condition caused by the interaction of a pathogen-induced long chain of sequential intracellular events in immune cells, epithelium, endothelium, and the neuroendocrine system. The lethal effects of septic shock are associated with the production and release of numerous pro-inflammatory biochemical mediators including cytokines, nitric oxide and toxic oxygen and nitrogen radicals, together with development of massive apoptosis. As melatonin has remarkable properties as a cytokine modulator, antioxidant and anti-apoptotic agent, the present study was designed to evaluate the possible protective effect of melatonin against LPS-induced septic shock in Swiss mice. We observed that intraperitoneally (i.p.) administered-melatonin (10 mg/kg) 30 min prior, and 1 hr after i.p. LPS injection (0.75 mg/animal) markedly protected mice from the LPS lethal effects with 90% survival rates for melatonin and 20% for LPS-injected mice after 72 hr. The melatonin effect was mediated by modulating the release of pro-/anti-inflammatory cytokine levels, protection from oxidative damage and counteracting apoptotic cell death. Melatonin was able to partially counteract the increase in LPS-induced pro-inflammatory cytokine levels such as tumor necrosis factor-alpha, IL-12 and interferon-gamma at the local site of injection, while it increased the production of the anti-inflammatory cytokine IL-10 both locally and systemically. Furthermore, melatonin inhibited the LPS-induced nitrite/nitrate and lipid peroxidation levels in brain and liver and counteracted the sepsis-associated apoptotic process in spleen. In conclusion, we have demonstrated that melatonin improves the survival of mice with septic shock via its pleiotropic functions as an immunomodulator, antioxidant and anti-apoptotic mediator.

Human lymphocyte-synthesized melatonin is involved in the regulation of the interleukin-2/interleukin-2 receptor system.

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 E(2)-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.

mRNA expression of nuclear receptor RZR/RORalpha, melatonin membrane receptor MT, and hydroxindole-O-methyltransferase in different populations of human immune cells.

We characterized the expression levels of the retinoid Z receptor alpha (RZR alpha), RORalpha mRNA isoforms (RORalpha1, RORalpha2, and RORalpha3), 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 RZRalpha in all subpopulations studied, whereas both RORalpha1 and RORalpha2 transcripts were amplified only in CD8+ cells. Specific signal for RORalpha2 was obtained in all subpopulations studied, but we were not able to detect the RORalpha3 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.

Expression of membrane and nuclear melatonin receptors in mouse peripheral organs.

Previous studies have shown that melatonin acts through specific receptors, including MT(1) and MT(2) membrane receptors as well as a nuclear receptor belonging to the orphan nuclear receptor family. Therefore, the goal of this study was to determine whether melatonin receptors mRNA is expressed in mouse peripheral tissues. To study the different receptors subtype expression, we have used a reverse-transcription polymerase chain reaction (RT-PCR) procedure followed by Southern hybridization with specific digoxigenin-labeled probes. RT-PCR studies revealed the presence of both MT(1) membrane receptors and ROR(alpha)1 nuclear receptors in all the peripheral tissues examined (brain, heart, lung, liver and kidney). Moreover, the expression of ROR(alpha)1 nuclear receptors was also demonstrated by Western-blot. In contrast, expression of MT(2) membrane receptors was only observed in brain and lung. These results suggest that melatonin, acting through its different subtypes receptors, plays a role in the neuroendocrine regulation of peripheral tissues function.

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 hydroxyindole-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.

Melatonin inhibits telomerase activity in the MCF-7 tumor cell line both in vivo and in vitro.

In this study for the first time the relationship between melatonin and telomerase activity was investigated. Melatonin exhibits oncostatic properties, but the actual mechanism of action by which the indole reduces tumor cell activity is not clear. Telomerase is an enzyme responsible of telomere elongation and is activated in most human cancers. In the current in vivo study, eight nude mice received a MCF-7 xenograft and thereafter they were treated for 5 weeks with 0.1 mg/mL of melatonin in the drinking water. Melatonin treatment caused a significant reduction in the weight of tumors and reduced metastases when compared with the control group. As indicated by the Telomerase Repeats Amplification Protocol (TRAP) assay, a significant decrease in telomerase activity was observed in the group treated with melatonin. In related in vitro studies, cultured MCF-7 cells were treated with three different concentrations of melatonin and a control without indole treatment. A significant dose-dependent decrease in Telomerase reverse transcriptase (TERT), the catalytic subunit of telomerase, mRNA expression was observed in the melatonin-treated cells. We also observed a significant reduction in TR, the RNA telomerase subunit, mRNA expression at physiological concentrations of melatonin (1 nm). Significant differences in TEP1, an associated telomerase protein, mRNA expression were also observed. In conclusion, melatonin influences telomerase both in vivo and in vitro, decreasing its activity in the tumors of nude mice and the mRNA expression of the TERT and TR subunits, essential factors for the proper function of the telomerase enzyme.

Melatonin counteracts the inhibitory effect of PGE2 on IL-2 production in human lymphocytes via its mt1 membrane receptor.

It is well known that melatonin plays a fundamental role in human neuro-immunomodulation. Thus, melatonin regulates the production of a large number of cytokines, including interleukin-2 (IL-2) in the human system. Both membrane and nuclear receptors for melatonin are present in lymphoid cells. However, most of these effects have been shown to be mediated by the putative nuclear receptor for the neurohormone. In this paper, we show that prostaglandin E2 (PGE2), a potent inflammatory mediator, inhibits IL-2 production in human lymphocytes by a cyclic AMP (cAMP)-dependent mechanism. In this model, melatonin counteracts the effects of PGE2 on IL-2 and cAMP production. We propose that the effect of melatonin is mediated by a membrane receptor, since similar results were obtained when cells were cultured in the presence of S 20098, a specific melatonin membrane receptor agonist. No effect was observed by using CGP 52608, a nuclear receptor agonist. Moreover, when cells were stimulated with phorbol myristate acetate (PMA), which has been shown to inhibit mt1 melatonin membrane receptor expression, the neurohormone failed to counteract the effect of PGE2. Therefore, we postulate, for the first time, a physiological role of the mt1 melatonin membrane receptor in the human immune system.

Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide inhibit LPS-stimulated MIP-1alpha production and mRNA expression.

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are neuropeptides with immunomodulatory properties, including the regulation of several proinflammatory mediators. Such mediators, for example chemokines, influence trafficking of inflammatory cells and contribute to shaping the immune response. In the present work, we studied the effect of VIP and PACAP on the CC chemokine macrophage inflammatory protein-1 alpha (MIP-1alpha) production in LPS-stimulated RAW 264.7 macrophage cell line. VIP and PACAP inhibited the production of MIP-1alpha in a dose-dependent manner and over a broad spectrum of LPS concentrations. The use of selective agonists and antagonists of VIP/PACAP receptors showed that type 1 VIP receptor (VPAC1) is the major receptor involved, but the type 2 VIP receptor (VPAC2) may be also implicated. By using selective PKA and PKC inhibitors and cAMP mimicked agents, we demonstrated a cAMP-dependent signalling pathway for the inhibitory effect of VIP/PACAP on MIP-1alpha production, although a minor non-mediated cAMP pathway was also involved. mRNA expression studies showed a down-regulation of MIP-1alpha gene expression by VIP and PACAP. Taken together, the present work strongly supports an anti-inflammatory role of VIP and PACAP by a new mechanism associated with impairment of a key component of the chemokine network.

Inhibitory effect of melatonin on homocysteine-induced lipid peroxidation in rat brain homogenates.

Oxidative damage is implicated in several pathologies including cardiovascular disease. As a model system to study the response of cells to oxidative insults, homocysteine toxicity was examined since it is an independent risk factor for atherosclerotic disease. The levels of malondialdehyde and 4-hydroxyalkenals were assayed as an index of oxidatively damaged lipid. In in vitro experiments, the increase of lipid peroxidation products induced by homocysteine were concentration- and time-dependent. To study the protective effect of melatonin on homocystine induced lipid peroxidation, brain homogenates were treated with different concentrations of melatonin. The accumulation of malondialdehyde and 4-hydroxyalkenals induced by homocysteine was significantly reduced by melatonin in a concentration-dependent manner. Additionally, a melatonin concentration of 1.5 mM reduced the levels of oxidatively damaged lipid products below those measured in control homogenates (no homocysteine, no melatonin). These data suggest that melatonin, an endogenous antioxidant may have a role in protecting cells from oxidative damage due to homocysteine and they support the idea that pharmacological concentrations could be used as a therapeutic agent in reducing cardiovascular disease where homocysteine may be a causative or contributing agent.