Patricia J. Lardone

A review of the multiple actions of melatonin on the immune system.

This review summarizes the numerous observations published in recent years which have shown that one of the most significant of melatonin’s pleiotropic effects is the regulation of the immune system. The overview summarizes the immune effects of pinealectomy and the association between rhythmic melatonin production and adjustments in the immune system as markers of melatonin’s immunomodulatory actions. The effects of both in vivo and in vitro melatonin administration on non-specific, humoral, and cellular immune responses as well as on cellular proliferation and immune mediator production are presented. One of the main features that distinguishes melatonin from the classical hormones is its synthesis by a number of nonendocrine extrapineal organs, including the immune system. Herein, we summarize the presence of immune system-synthesized melatonin, its direct immunomodulatory effects on cytokine production, and its masking effects on exogenous melatonin action. The mechanisms of action of melatonin in the immune system are also discussed, focusing attention on the presence of membrane and nuclear receptors and the characterization of several physiological roles mediated by some receptor analogs in immune cells. The review focuses on melatonin’s actions in several immune pathologies including infection, inflammation, and autoimmunity together with the relation between melatonin, immunity, and cancer.

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

Melatonin: Buffering the Immune System

Melatonin modulates a wide range of physiological functions with pleiotropic effects on the immune system. Despite the large number of reports implicating melatonin as an immunomodulatory compound, it still remains unclear how melatonin regulates immunity. While some authors argue that melatonin is an immunostimulant, many studies have also described anti-inflammatory properties. The data reviewed in this paper support the idea of melatonin as an immune buffer, acting as a stimulant under basal or immunosuppressive conditions or as an anti-inflammatory compound in the presence of exacerbated immune responses, such as acute inflammation. The clinical relevance of the multiple functions of melatonin under different immune conditions, such as infection, autoimmunity, vaccination and immunosenescence, is also reviewed.

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.

Abstract:  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‐α, IL‐12 and interferon‐γ 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.

Melatonin biosynthesis in the thymus of humans and rats

Abstract.Melatonin is an indoleamine widely distributed in the evolution that shows a great functional versatility, playing an important role as a transmitter of photoperiodic information and exhibiting antioxidant, oncostatic, anti-aging and immunomodulatory properties. In vertebrates, this molecule is produced by the pineal gland and other extrapineal sites. The present study was carried out to investigate the presence of melatonin in thymus and the possibility of an endogenous melatonin synthesis in this organ, in which T cells are matured. In this work, we demonstrate in humans and rats that thymus contains melatonin, expresses the mRNAs encoding N-acetyltransferase and hydroxyindol-O-methyltransferase, the two key enzymes of the melatonin synthesis, and has this biosynthetic machinery activated. In addition, rat thymocytes cultured for 24 h exhibited high levels of melatonin. The results presented here suggest that human and rat thymuses are able to synthesize melatonin, which could have intracrine, autocrine and paracrine functions.

Decreased MT1 and MT2 melatonin receptor expression in extrapineal tissues of the rat during physiological aging.

Abstract:  Aging is a complex process associated with a diminished ability to respond to stress, a progressive increase in free radical generation and a decline in immune function. Melatonin, a molecule with a great functional versatility exerts anti‐oxidant, oncostatic, immunomodulatory, and anti‐aging properties. Melatonin levels drop during aging and it has been speculated that the loss of melatonin may accelerate aging. This study was designed to elucidate whether aging involves responsiveness to reduced melatonin. Melatonin membrane receptor (MT1 and MT2) expression and MT1 protein expression were analyzed in extrapineal tissues (thymus, spleen, liver, kidney, and heart) of 3‐ and 12‐month‐old rats using real time polymerase chain reaction and western blotting analysis. Moreover, melatonin in tissues was measured by high performance liquid chromatography. We report for the first time, an age‐related reduction in mRNA MT1 and MT2 expression levels as well as MT1 protein expression in all tissues tested except the thymus, where surprisingly, both melatonin receptor levels were significantly higher in 12‐month‐old rats and MT1 protein expression maintained unchanged with age. Diminished melatonin concentrations were measured in spleen, liver, and heart during aging. As a conclusion, physiological aging seems to exert responsiveness to melatonin and consequently, the loss of this potent anti‐oxidant may contribute to onset of aging.

Melatonin synthesized by T lymphocytes as a ligand of the retinoic acid-related orphan receptor.

Abstract:  Melatonin modulates a wide array of physiological events with pleiotropic effects on the immune system. While the relevance of specific melatonin membrane receptors has been well established for several biological functions, retinoic acid‐related orphan receptor alpha (RORα) has been suggested as a mediator of nuclear melatonin signalling by results obtained from pharmacological approaches. However, a melatonin‐mediated downstream effect cannot be ruled out, and further evidence is needed to support a direct interaction between melatonin and RORα. Here, we show that RORα is mainly located in human Jurkat T‐cell nucleus, and it is co‐immunoprecipitated with melatonin. Moreover, immunocytochemistry studies confirmed the co‐localization of melatonin and RORα. Melatonin promoted a time‐dependent decrease in nuclear RORα levels, suggesting a role in the RORα transcriptional activity. Interestingly, RORα acts as a molecular switch implicated in the mutually exclusive generation of Th17 and Treg cells, both involved in the harm/protection balance of immune conditions such as autoimmunity or acute transplant rejection. Therefore, the identification of melatonin as a natural modulator of RORα gives it a tremendous therapeutic potential for a variety of clinical disorders.

A novel interplay between membrane and nuclear melatonin receptors in human lymphocytes : significance in IL-2 production

Abstract.Human lymphocyte melatonin, through membrane and nuclear receptors binding, acts as an activator in IL-2 production. Antagonism of membrane melatonin receptors using luzindole exacerbates the drop of the IL-2 production induced by PGE2 in peripheral blood mononuclear and Jurkat cells. This paper studies the melatonin membrane and nuclear receptors interplay in PGE2-diminished IL-2 production. The decrease in IL-2 production after PGE2 and/or luzindole administration correlated with downregulation in the nuclear receptor RORα. We also highlighted a role of cAMP in the pathway, because forskolin mimicked the effects of luzindole and/or PGE2 in the RORα expression. Finally, a significant RORα downregulation was observed in T cells permanently transfected with inducible MT1 antisense. In conclusion, we show a novel connection between melatonin membrane receptor signalling and RORα expression, opening a new way to understand melatonin regulation in lymphocyte physiology.

Evidence for melatonin synthesis in the rat brain during development

Abstract:  Melatonin production is not restricted to the pineal gland. Several extrapineal sources of this indole such as retina, Harderian gland, and immune system are well documented. Melatonin of pineal origin is not present in the rat at early stages of development. To assess the potential capacity of local melatonin synthesis by the immature brain and to gain insight into the relationship between melatonin production by the brain (without the pineal gland) and pineal gland during rat development, the melatonin content as well as the expression and activity of the melatonin‐synthesizing enzymes, N‐acetyltransferase (NAT) and hydroxyindole‐O‐methyltransferase (HIOMT), were studied at fetal and postnatal stages. Moreover, melatonin‐membrane receptor (MT1) expression was also analyzed. Both, the expression and activity of NAT and HIOMT were found in the brain with significant day/night differences in enzymes activities. Additionally, melatonin content was detected in all stages showing day/night differences depending on the stage of development. The brain nocturnal melatonin content was higher than diurnal content on postnatal day 16 and in adult rats which is in accordance with the pineal melatonin synthesis. To investigate the origin of this brain melatonin, pinealectomized rats were used and we found that the developing brain produced its own melatonin. Also, MT1 expression was detected in brain during development. These results demonstrate that, when the pineal is not yet producing melatonin, there is melatonin synthesis by the brain that could be used as protection from free radical damage and/or could exert some actions through MT1 receptors.

Melatonin synthesized by Jurkat human leukemic T cell line is implicated in IL‐2 production

Human lymphocytes have recently been described as an important physiological source of melatonin (N‐acetyl‐5‐methoxytryptamine), which could be involved in the regulation of the human immune system. On the other hand, stimulation of IL‐2 production by exogenous melatonin has been shown in the Jurkat human lymphocytic cell line. Furthermore, both melatonin membrane and nuclear receptors are present in these cells. In this study, we show that the necessary machinery to synthesize melatonin is present and active in resting and stimulated Jurkat cells. Accordingly, we have found that cells synthesize and release melatonin in both conditions. Therefore, we investigated whether endogenous melatonin produced by Jurkat cells was involved in the regulation of IL‐2 production. When melatonin membrane and nuclear receptors were blocked using specific antagonists, luzindole and CGP 55644, respectively, we found that IL‐2 production decreased, and this drop was reverted by exogenous melatonin. Additionally, PHA activation of Jurkat cells changed the profile of melatonin nuclear receptor mRNA expression. A previous study showed that exogenous melatonin is able to counteract the decrease in IL‐2 production caused by prostaglandin E2 (PGE2) in human lymphocytes via its membrane receptor. In our model, when we blocked the melatonin membrane receptor with luzindole, the inhibitory effect of PGE2 on IL‐2 production was higher. Therefore, we have demonstrated the physiological role of endogenous melatonin in this cell line. These findings indicate that endogenous melatonin synthesized by human T cells would contribute to regulation of its own IL‐2 production, acting as an intracrine, autocrine, and/or paracrine substance.