Georges J.M. Maestroni

MELATONIN: NATURE'S MOST VERSATILE BIOLOGICAL SIGNAL

Melatonin is a ubiquitous molecule and widely distributed in nature, with functional activity occurring in unicellular organisms, plants, fungi and animals. In most vertebrates, including humans, melatonin is synthesized primarily in the pineal gland and is regulated by the environmental light/dark cycle via the suprachiasmatic nucleus. Pinealocytes function as ‘neuroendocrine transducers’ to secrete melatonin during the dark phase of the light/dark cycle and, consequently, melatonin is often called the ‘hormone of darkness’. Melatonin is principally secreted at night and is centrally involved in sleep regulation, as well as in a number of other cyclical bodily activities. Melatonin is exclusively involved in signaling the ‘time of day’ and ‘time of year’ (hence considered to help both clock and calendar functions) to all tissues and is thus considered to be the bodys chronological pacemaker or ‘Zeitgeber’. Synthesis of melatonin also occurs in other areas of the body, including the retina, the gastrointestinal tract, skin, bone marrow and in lymphocytes, from which it may influence other physiological functions through paracrine signaling. Melatonin has also been extracted from the seeds and leaves of a number of plants and its concentration in some of this material is several orders of magnitude higher than its night‐time plasma value in humans. Melatonin participates in diverse physiological functions. In addition to its timekeeping functions, melatonin is an effective antioxidant which scavenges free radicals and up‐regulates several antioxidant enzymes. It also has a strong antiapoptotic signaling function, an effect which it exerts even during ischemia. Melatonins cytoprotective properties have practical implications in the treatment of neurodegenerative diseases. Melatonin also has immune‐enhancing and oncostatic properties. Its ‘chronobiotic’ properties have been shown to have value in treating various circadian rhythm sleep disorders, such as jet lag or shift‐work sleep disorder. Melatonin acting as an ‘internal sleep facilitator’ promotes sleep, and melatonins sleep‐facilitating properties have been found to be useful for treating insomnia symptoms in elderly and depressive patients. A recently introduced melatonin analog, agomelatine, is also efficient for the treatment of major depressive disorder and bipolar affective disorder. Melatonins role as a ‘photoperiodic molecule’ in seasonal reproduction has been established in photoperiodic species, although its regulatory influence in humans remains under investigation. Taken together, this evidence implicates melatonin in a broad range of effects with a significant regulatory influence over many of the bodys physiological functions.

Physiological effects of melatonin: Role of melatonin receptors and signal transduction pathways

Melatonin, an endogenous signal of darkness, is an important component of the bodys internal time-keeping system. As such it regulates major physiological processes including the sleep wake cycle, pubertal development and seasonal adaptation. In addition to its relevant antioxidant activity, melatonin exerts many of its physiological actions by interacting with membrane MT1 and MT2 receptors and intracellular proteins such as quinone reductase 2, calmodulin, calreticulin and tubulin. Here we review the current knowledge about the properties and signaling of melatonin receptors as well as their potential role in health and some diseases. Melatonin MT1 and MT2 receptors are G protein coupled receptors which are expressed in various parts of the CNS (suprachiasmatic nuclei, hippocampus, cerebellar cortex, prefrontal cortex, basal ganglia, substantia nigra, ventral tegmental area, nucleus accumbens and retinal horizontal, amacrine and ganglion cells) and in peripheral organs (blood vessels, mammary gland, gastrointestinal tract, liver, kidney and bladder, ovary, testis, prostate, skin and the immune system). Melatonin receptors mediate a plethora of intracellular effects depending on the cellular milieu. These effects comprise changes in intracellular cyclic nucleotides (cAMP, cGMP) and calcium levels, activation of certain protein kinase C subtypes, intracellular localization of steroid hormone receptors and regulation of G protein signaling proteins. There are circadian variations in melatonin receptors and responses. Alterations in melatonin receptor expression as well as changes in endogenous melatonin production have been shown in circadian rhythm sleep disorders, Alzheimers and Parkinsons diseases, glaucoma, depressive disorder, breast and prostate cancer, hepatoma and melanoma. This paper reviews the evidence concerning melatonin receptors and signal transduction pathways in various organs. It further considers their relevance to circadian physiology and pathogenesis of certain human diseases, with a focus on the brain, the cardiovascular and immune systems, and cancer.

Role of the pineal gland in immunity. Circadian synthesis and release of melatonin modulates the antibody response and antagonizes the immunosuppressive effect of corticosterone.

Inhibition of synthesis of the pineal neurohormone melatonin (MEL) in mice, by administration of propranolol (PRO) in the evening, and daily injections of p-chlorophenylalanine (PCPA), resulted in a significant depression of the primary antibody response to sheep red blood cells (SRBC). Spleen cells from these mice showed a reduced reactivity against antigens in the autologous mixed lymphocyte reaction (AMLR). In contrast, alloreactivity remained normal. Reconstitution of the night-time peak of plasma MEL by evening injections to the mice completely reversed the suppression of the humoral response and the AMLR. MEL administration was able to antagonize the depression of antibody production induced by corticosterone in vivo. These results suggest that the pineal gland has important immunomodulatory functions through its cyclic, circadian release of MEL.

Evidence for melatonin synthesis in mouse and human bone marrow cells

Recently, it was demonstrated that inbred strains of mice have a clearcut circadian rhythm of pineal and serum melatonin. Moreover, it is known that melatonin is involved in many immunoregulatory functions. Among them, hematopoiesis is influenced by the action of melatonin via melatonin‐induced opioids on kappa‐opioid receptors, which are present on stromal bone marrow cells. Therefore, the present study was carried out to investigate the presence of melatonin in the bone marrow in which immunocompetent cells are generated. Specifically, we aimed at answering the following question: are bone marrow cells involved in melatonin synthesis? In the present study, we demonstrate that (1) bone marrow cells contain high concentrations of melatonin; (2) bone marrow cells have a N‐acetyltransferase activity and they express the mRNA encoding hydroxy‐O‐methyltransferase and (3) bone marrow cells cultured for a prolonged period exhibited high levels of melatonin. Results presented here suggest that mouse and human bone marrow and bone marrow cells are capable of de novo synthesis of melatonin, which may have intracellular and/or paracrine functions.

Role of melatonin in neurodegenerative diseases

The pineal product melatonin has remarkable antioxidant properties. It scavenges hydroxyl, carbonate and various organic radicals, peroxynitrite and other reactive nitrogen species. Melatonyl radicals formed by scavenging combine with and, thereby, detoxify superoxide anions in processes terminating the radical reaction chains. Melatonin also enhances the antioxidant potential of the cell by stimulating the synthesis of antioxidant enzymes like superoxide dismutase, glutathione peroxidase and glutathione reductase, and by augmenting glutathione levels. The decline in melatonin production in aged individuals has been suggested as one of the primary contributing factors for the development of age-associated neurodegenerative diseases,e.g., Alzheimer’s disease. Melatonin has been shown to be effective in arresting neurodegenerative phenomena seen in experimental models of Alzheimer’s disease, Parkinsonism and ischemic stroke. Melatonin preserves mitochondrial homeostasis, reduces free radical generation, e.g., by enhancing mitochondrial glutathione levels, and safeguards proton potential and ATP synthesis by stimulating complex I and IV activities. Therapeutic trials with melatonin have been effective in slowing the progression of Alzheimer’s disease but not of Parkinson’s disease. Melatonin’s efficacy in combating free radical damage in the brain suggests that it may be a valuable therapeutic agent in the treatment of cerebral edema after traumatic brain injury.

T‐Helper‐2 lymphocytes as a peripheral target of melatonin*

Abstract: In the past several years we demonstrated that the pineal neurohormone melatonin has immunoenhancing properties and can counteract the immunodepres‐sion that may follow acute stress, drug treatment, and viral diseases or aging. Sev‐eral laboratories have subsequently confirmed and extended our findings. It soon appeared evident that T‐derived cytokines constitute the main mediators of the im‐munological effect of melatonin. We have recently found a high affinity (Kd: 346 |Mp 24 pM) binding site for 125I‐melatonin on T‐helper‐type 2 lymphocytes in the bone marrow. Activation of this putative melatonin receptor, with both physiologi‐cal and pharmacological concentrations of melatonin, resulted in an enhanced pro‐duction of interleukin‐4 (IL4), which in turn acted on bone marrow stromal cells and induced the release of hematopoietic growth factors. This melatonin‐cytokine cascade showed the remarkable capacity of rescuing hematopoietic functions in mice treated with cancer chemotherapeutic compounds without interfering with the anticancer action of these agents. The very low concentration (0.1 nM) at which melatonin is active may well reflect a physiological function of endogenous melatonin. The pineal gland has been, in fact, reported to signal the blood forming system. The evidence of IL4 involvement is relevant to our understanding of many melatonin effects and may be part of a pineal‐immune axis involving also Th 1 cy‐tokines. The ability of rescuing hematopoiesis against the toxic action of cancer chemotherapeutic compounds and the presence of high‐affinity IL4 receptors on human tumors provide a further promising rationale for the clinical use of melatonin

Pineal Melatonin, Its Fundamental Immunoregulatory Role in Aging and Cancer

A closely interwoven network of immune and neuroendocrine mechanisms protects our organism against a variety of environmental threats. Derangements of neuroendocrine functions such as those associated with or caused by failure to cope with stressful events or “distress” may lead in turn to an impairment of immunologic functions and possibly to an increased incidence of infections, autoimmune diseases and cancer.’.’ As a matter of fact, functional connections and feedback loops between the immune and the neuroendocrine systems are being increasingly Thus, primary defects in the neuroendocrine system or psychologic disturbances may adversely affect the immunologic machinery. The opposite is also true. In the case of aging, for example, the associated decline of both psychic and immune performance might, therefore, be triggered either by primary psycho-neuroendocrinologic or by immune alterations. The pineal gland is a fundamental modulator of the entire neuroendocrine system. The pineal gland functions as a true “biologic clock” secreting in a circadian fashion its main neurohormone melatonin or N-acetyl-5-methoxytryptamine. Melatonin synthesis and release is regulated mainly by the light-dark cycle with a peak during the night, darkness hours.’.* However, other environmental variables such as temperature, humidity and, perhaps, pheromones and magnetism may influence its rhythm.’.’ Also various physiopathologic states can affect melatonin rhythms. For example, in man alterations of melatonin production have been associated, amongst other things, with aging and cancer. In particular, low or impaired melatonin production has been described in aging’ and various modifications of the melatonin rhythm have been found in cancer patients.” We have recently found that the circadian synthesis and release of melatonin exerts an important immunomodulatory Melatonin appears to be a physiologic “upregulator” of the immune system and to operate via the endogenous opioid system (EOS) on antigen-activated cells.12*” Here we report further on other immunologic

The pineal neurohormone melatonin stimulates activated CD4+, Thy-1+ cells to release opioid agonist(s) with immunoenhancing and anti-stress properties

In previous studies we showed that in mice the pineal gland modulates the immune response via the circadian synthesis and release of melatonin. Exogenous melatonin proved also to exert immunoenhancing effects and to counteract completely the immunologic effect of acute stress. Melatonin was active only in vivo, in mice primed with T-dependent antigens and its effects on the primary antibody response and thymus weight were abolished by the specific opioid antagonist naltrexone. Here we demonstrate that physiologic concentrations of melatonin stimulate, in vitro, activated L3T4+ (CD4+) cells to release opioid agonist(s) that can reproduce in vivo the immunoenhancing and anti-stress effects on thymus cellularity and antibody production of melatonin and compete with specific binding of [3H]naloxone to mouse brain membranes. Similar results were obtained when mitogen-activated human immunocompetent cells were incubated with melatonin. In the human model the results were, however, less consistent than those obtained with murine cells, in that only four out of ten blood donors provided cells that were responsive to melatonin. This finding elucidates the mechanism of a novel immuno-neuroendocrine connection with relevant implications for our understanding of the neuroendocrine factors that may influence the immune response in vivo in normal and stressful situations. In addition, it opens new perspectives in a wide range of research fields.

The role of melatonin in immuno-enhancement : potential application in cancer

Melatonin, a neurohormone produced mainly by the pineal gland, is a modulator of haemopoiesis and of immune cell production and function, both in vivo and in vitro. Physiologically, melatonin is associated with T‐helper 1 (Th1) cytokines, and its administration favours Th1 priming. In both normal and leukaemic mice, melatonin administration results in quantitative and functional enhancement of natural killer (NK) cells, whose role is to mediate defenses against virus‐infected and cancer cells. Melatonin appears to regulate cell dynamics, including the proliferative and maturational stages of virtually all haemopoietic and immune cells lineages involved in host defense – not only NK cells but also T and B lymphocytes, granulocytes and monocytes – in both bone marrow and tissues. In particular, melatonin is a powerful antiapoptotic signal promoting the survival of normal granulocytes and B lymphocytes. In mice bearing mid‐stage leukaemia, daily administration of melatonin results in a survival index of 30–40% vs. 0% in untreated mice. Thus, melatonin seems to have a fundamental role as a system regulator in haemopoiesis and immuno‐enhancement, appears to be closely involved in several fundamental aspects of host defense and has the potential to be useful as an adjuvant tumour immunotherapeutic agent.

The Pineal Gland and the Circadian, Opiatergic, Immunoregulatory Role of Melatonin

Earlier work in our laboratory led to the proposition that lymphocyte products act as messengers to the central nervous system.’ Those findings, which have been recently confirmed and extended,’,’ originated from the observation that antigens evoke specific and rapid hormonal responses early after injection into mice. We could demonstrate that the interference with those endocrine changes by a combination of psychotropic drugs specifically depressed humoral and cellular immune responseseh In the course of those studies, however, the idea progressively emerged that another basic modulator of neuroendocrine mechanisms, namely the pineal gland, plays a fundamental role in neuroimmunomodulation besides the better known hypothalamicpituitary system.’ The pineal gland functions as a neuroendocrine “transducer” by translating the most basic environmental information (photic, thermic, magnetic) into signals which modulate most neuroendocrine mechanisms.’.* This remarkable task seems to be accomplished via the circadian synthesis and release of melatonin (N-acetyl-5-methoxytryptamine), the most studied and best known pineal neurohormone. Melatonin is synthesized and released upon activation of pineal beta-adrenergic receptors in man and most vertebrates during the nocturnal, dark hours and maintains a consistently regular circadian rhythm? In humans, alterations of this rhythm have been associated with sleep disturbances, anxiety states, affective disorders, and psychosomatic diseases.I0.I1 Scant experimental findings exist on a possible connection between the pineal gland and the lymphohemopoietic system.” It has been reported that immune reactivity and circulating lymphocytes fluctuate according to a circadian r h ~ t h m , ’ ~ ’ ~ although the significance of these variations is not clear. Evidence for an inhibitory role of the pineal gland on carcinogenesis and tumor growth has also been reported (reviewed in Ref. 16). Recently, our early pharmacologic studies on the pineal-immune system connections’ have been replicated by other authors and our results have been confirmed.”