Pineal melatonin is normally synthesized and secreted during the dark phase of the day in all species studied to date. The primary physiological function of melatonin is to convey information about daily cycles of light and darkness to body physiology. By its pattern of secretion during darkness, melatonin indicates the length of the scotophase, thus representing the chemical code of the night. This information is used for the organization of functions, which respond to changes in photoperiod such as circadian and seasonal rhythms (Arendt, Middleton, Stone, and Skene 1999; Cardinali and Pevet 1998).
Melatonin is secreted by the pineal gland by simple diffusion and its lipophilicity contributes to an easy passive diffusion across cell membranes as well as through cell layers. This assures a rapid distribution of melatonin throughout the body and effects on virtually every cell type of the organism. Radioactive melatonin administered intravenously rapidly disappears from the blood with a half-life of about 30 min; about 60 to 70% of melatonin in plasma is bound to albumin (Cardinali, Lynch, and Wurtman 1972).
The first experiments on brain melatonin receptor sites were carried out in the late seventies by employing 3H-melatonin as a ligand indicating the existence of melatonin acceptor sites in bovine brain (Cardinali 1981). By using a 2-125I-iodomelatonin analog, melatonin binding was then detected in several brain areas, the choroid plexus and in some brain arteries as well as in peripheral organs, like primary and secondary lymphoid organs, the Harderian glands, the adrenals, heart and lungs, the gastrointestinal tract, the mammary glands, the kidney and male reproductive organs. Indeed, to have an organ devoid of melatonin binding site may constitute an exception rather than the rule (Brydon et al. 1999).
A first classification of putative melatonin receptor sites into the ML1 and ML2 was based on kinetic and pharmacological differences of 2-125I-iodomelatonin binding and a major achievement in the field was the cloning of the ML1 melatonin receptors (Brydon et al. 1999; Reppert, Weaver, and Ebisawa 1994). A nomenclature of melatonin receptors has been proposed by the International Union of Pharmacology (IUPHAR) (Dubocovich et al. 2000). Besides membrane acceptor sites, evidence has accumulated on nuclear binding of melatonin as well (Acuna-Castroviejo, Reiter, Menendez-Pelaez, Pablos, and Burgos 1994). Melatonin appears to interact with the orphan nuclear hormone receptor superfamily RZR/ROR (Missbach, Jagher, Sigg, Nayeri, Carlberg, and Wiesenberg 1996). Among presumptive second messengers for melatonin action, the cAMP generating system has received paramount attention. The main signal transduction pathway of high affinity MT1 receptors in both neuronal and nonneuronal tissues is the inhibition of cAMP formation through a pertussis toxin sensitive inhibitory Gi protein.
Coupling of the high affinity melatonin receptors to other signaling pathways has also been reported. Melatonin modifies cGMP levels, decreases Ca2+ influx and inhibits arachidonate acid conversion and prostaglandin synthesis and the NO synthase/NO system. Direct effects of melatonin on calmodulin and other intracellular proteins, nuclear receptor activity for melatonin, and the free radical scavenging properties of melatonin should be also considered (for ref. see Cardinali, Golombek, Rosenstein, Cutrera, and Esquifino 1997b; Reiter, Tan, and Burkhardt 2002).
Pineal ablation, or any other experimental procedure that inhibits melatonin synthesis and secretion, induces a state of immunodepression which is partly counteracted by melatonin in several species (Beskonakli, Palaoglu, Aksaray, Alanoglu, Turhan, and Taskin 2001; Fraschini, Demartini, Esposti, and Scaglione 1998; Liebmann, Wolfler, Felsner, Hofer, and Schauenstein 1997; Maestroni 2001; Martins et al. 2001; Skwarlo-Sonta 2002). Melatonin treatment increases T-cell proliferation (Konakchieva, Kyurkchiev, Kehayov, Taushanova, and Kanchev 1995; Pioli, Caroleo, Nistico, and Doria 1993), enhances antigen presentation by macrophages to T cells by increasing the expression of MHC class II molecules (Pioli et al. 1993), activates splenic, lymph node, and bone marrow cells (Drazen and Nelson 2001; Wajs, Kutoh, and Gupta 1995), stimulates antibody-dependent cellular cytotoxicity (Giordano and Palermo 1991) and augments natural and acquired immunity (Bonilla et al. 2001; Negrette et al. 2001; Poon, Liu, Pang, Brown, and Pang 1994). Melatonin also stimulates NK cell activity (Currier, Sun, and Miller 2000; del Gobbo, Libri, Villani, Calio, and Nistico 1989), activates monocytes (Morrey, McLachlan, Serkin, and Bakouche 1994), increases the number of Th-2 lymphocytes (Lissoni et al. 1995), augments CD4+ lymphocytes and decreased CD8+ lymphocytes in submaxillary lymph nodes (Castrillon et al. 2001), restores impaired Th cell activity in immuno-depressed mice (Maestroni, Conti, and Pierpaoli 1988) and augments antibody responses in vivo (Akbulut, Gonul, and Akbulut 2001; Fraschini et al. 1998; Maestroni 1995; Pioli et al. 1993; Poon et al. 1994). Concerning B cells, there is information on melatonin inhibition of apoptosis during early B-cell development in mouse bone marrow (Yu, Miller, and Osmond 2000).
Besides the release of proinflammatory Th-1 cytokines, such as IFN-y and IL-2, administration of melatonin to antigen-primed mice increased the production of IL-10, indicating that melatonin also activates anti-inflammatory Th-2-like immune responses (Raghavendra, Singh, Kulkarni, and Agrewala 2001a; Raghavendra, Singh, Shaji, Vohra, Kulkarni, and Agrewala 2001b). It is not yet clear whether melatonin acts only on Th-1 cells or also on Th-2 cells. This is an important subject as the Th-1/Th-2 balance is significant for the immune response (Maestroni 2001). Relevant to this, melatonin treatment suppressed the subsequent in vitro stimulation by the mitogenic agents LPS (that stimulates B cells) and Con A (that stimulates T cells) in submaxillary lymph nodes (Castrillon et al. 2001). In addition, an inhibitory influence of melatonin on parameters of the immune function has also been demonstrated, i.e., in human lymphocytes NK cell activity, DNA, IFN-y, and TNF-a synthesis, as well as the proliferation of T lymphocytes and lymphoblastoid cell lines were depressed by melatonin (Arzt et al. 1988; Lewinski, Zelazowski, Sewerynek, Zerek-Melen, Szkudlinski, and Zelazowska 1989; Persengiev and Kyurkchiev 1993). In our own studies, melatonin treatment exerted an inhibitory influence on submaxillary lymph node cytolytic, CD8+ cells (Esquifino et al. 2001).
Melatonin possesses anti-inflammatory activity (Cuzzocrea and Reiter 2002). It reduces tissue destruction during inflammatory reactions by a number of means, among them scavenging of free radicals (Reiter et al. 2002). Additionally, melatonin prevents the translocation of nuclear factor-kappa B to the nucleus and its binding to DNA, thereby reducing the up-regulation of a variety of proinflammatory cytokines (for ref. see Guerrero and Reiter 2002). Finally, there is evidence that melatonin inhibits the production of adhesion molecules that promote the sticking of leukocytes to endothelial cells, attenuating transendothelial cell migration and edema (Sasaki et al. 2002).
The subcellular mechanisms involved in the immunomodulatory activity of melatonin remain to be elucidated. There is evidence of MTt receptors for melatonin, mainly in human circulating CD4+ T helper lymphocytes with few in CD8+ T cytolytic cells and none in B lymphocytes (Garcia-Maurino et al. 1997). Such a predominant effect on CD4+ cells is supported by the observations on melatonin efficacy to augment CD4+ cells in submaxillary lymph nodes (Castrillon et al. 2001). However, expression of Mel1a-melatonin receptor was found in rat thymus and spleen, melatonin receptor mRNA being expressed in all the thymic lymphocyte subpopulations (CD4+,CD8+, doubled positive, doubled negative, and B cells), indicating possible effects of melatonin on all these cells (Pozo et al. 1997; Yu et al. 2000). Nuclear melatonin receptors may also mediate immunomodulation, since drugs that bind to RZR/ROR receptors are active in experimental models of autoimmune diseases (Missbach et al. 1996). Melatonin is also a potent antioxidant, acting by itself rather than through specific binding sites (Reiter 1998; Reiter et al. 1999). In addition, melatonin could affect centrally the release of hormone in the hypothalamic-hypophyseal unit (Cardinali et al. 1997b) as well as the activity of autonomic pathways to the lymphoid organs (Brusco, Garcia Bonacho, Esquifino, and Cardinali 1998).
Our studies on melatonin role in arthritis have been mainly addressed to examine the participation of melatonin in regulation of circadian rhythmicity of immune parameters in rats (Castrillon et al. 2001). Pretreatment for 11 days with a pharmacological dose of melatonin (100 ^g) affected some aspects of the early phase of the immune response elicited by FCA injection, at the preclinical phase of disease. Cell proliferation in rat submaxillary lymph nodes and spleen during the immune reaction (as assessed by ODC activity) exhibited a pineal-dependent diurnal rhythmicity, as it was reduced by pinealectomy or pineal sympathetic denervation (Cardinali, Cutrera, Castrillon, and Esquifino 1996b; Cardinali, Cutrera, Garcia Bonacho, and Esquifino 1997a). This effect was counteracted by a pharmacological melatonin dose (100 ^g/day). Exogenous melatonin also restored the reduced amplitude in diurnal rhythms of lymph node or splenic tyrosine hydroxylase (TH) activity and lymph node acetylcholine synthesis (Cardinali et al. 1996b, 1997a).
Further examination of melatonin activity on circadian rhythmicity of cell proliferation in submaxillary lymph nodes and spleen at the clinical phase of arthritis was conducted in young and old Sprague-Dawley rats (Cardinali et al. 1998b). Pineal melatonin content was measured, as well as the efficacy of melatonin treatment to recover modified circadian rhythmicity of submaxillary lymph node and splenic ODC and TH activities and of lymph node 3H-acetylcholine synthesis. After 17 daily injections of 10 or 100 of melatonin at the evening, the treatment restored the inflammatory response in old rats (assessed plethysmographically in hind paws) to the level found in young animals. In young rats, an inflammation-promoting effect of 100 melatonin could be demonstrated. As a consequence of the immune reaction, submaxillary lymph node and splenic lymph cell proliferation augmented significantly, with acrophases of 24-h rhythms at the afternoon for lymph nodes or in the morning for spleen. Mesor and amplitude of ODC rhythm were lowest in old rats, while melatonin injection generally augmented its amplitude. Lymph node and splenic TH activity attained maximal values at early night while maxima in lymph node. 3H-acetylcholine synthesis occurred at the afternoon. Amplitude and mesor of these rhythms were lowest in old rats, an effect generally counteracted by melatonin treatment. The results were compatible with an age-dependent, significant depression of pineal melatonin synthesis during adjuvant-induced arthritis and with decreased amplitude of circadian rhythms in immune cell proliferation and autonomic activity in lymph nodes and spleen at the clinical phase of the disease. This picture was generally counteracted by melatonin injection, mainly in old rats (Cardinali et al. 1998b).
A number of studies were carried out to examine the participation of melatonin in altered 24-h rhythms of serum ACTH, GH, prolactin, LH and insulin in rats at the preclinical phase of Freund's adjuvant arthritis (Esquifino, Castrillon, Garcia Bonacho, Vara, and Cardinali 1999a). The data indicated that several early changes in levels and 24-h rhythms of circulating ACTH, PRL and LH in FCA-injected rats were sensitive to treatment with melatonin (100 ^g). An effect of melatonin treatment on 24-h variations in hypothalamic 5-HT and DA turnover during the preclinical phase of Freund's adjuvant arthritis was also apparent (Pazo et al. 2000). FCA injection suppressed circadian rhythmicity of 5-HT turnover in the anterior hypothalamus, an effect prevented by the previous injection of melatonin. Melatonin decreased 5-HT turnover rate in the anterior hypothalamus. Melatonin also prevented the changes in 5-HT turnover of medial hypothalamus evoked by Freund's adjuvant. As far as hypothalamic DA turnover, the preventing effect of melatonin was less clear, sometimes synergizing with the mycobacterial adjuvant to modify the normal 24-h pattern detected in hypothalamic regions (Pazo et al. 2000).
Physiological circulating levels of melatonin at midnight in rats are about 90 pg/ml in rats (Chan, Pang, Tang, and Brown 1984) while the melatonin levels achieved within 15 min after the systemic administration of a 100 ^-dose are about 30 or 200 ng/ml plasma (Raynaud, Mauviard, Geoffriau, Claustrat, and Pevet 1993), with a half-life of about 20 min (Chan et al. 1984). We addressed this subject by examining whether the administration of melatonin to pinealectomized rats in a way that reproduced the plasma values and daily rhythm of endogenous melatonin could affect immune responses during arthritis development (Cardinali, Garcia, Cano, and Esquifino 2004). Pinealectomized rats exhibited a significantly less pronounced inflammatory response, which was restored to normal by physiological melatonin administration. The physiological doses of melatonin employed were effective to counteract the impaired response of lymph node ODC seen in pinealectomized rats.
It must be thus noted that the pharmacological effect of melatonin on the immune response may not always be beneficial, particularly in young subjects. In autoimmune arthritis developed in mice with type II rat collagen melatonin administration (1 mg/kg) induced a more severe arthritis. Accordingly, pinealectomy in two strains of mice immunized with rat type II collagen reduced severity of the arthritis as shown by a slower onset of the disease, a less severe course of the disease (reduced clinical scores) and reduced serum levels of anti- collagen II antibodies (Hansson, Holmdahl, and Mattsson 1992, 1993). Using a 100-^g dose of melatonin an inflammation-promoting effect could be demonstrated in young rats injected with FCA. In contrast, melatonin administration (10 or 100 p,g) to old rats restored the inflammatory response in hind paws of FCA-injected rats to levels found in young rats (Cardinali et al. 1998b). Therefore, high levels of melatonin in young animals may stimulate the immune system and cause exacerbation of both autoimmune collagen II and mycobacterial arthritis. Indeed, recent data indicate that rheumatoid arthritis patients have increased nocturnal plasma levels of melatonin and their synovial macrophages respond to melatonin with an increased production of IL-12 and NO (Maestroni, Sulli, Pizzorni, Villaggio, and Cutolo 2002; Sulli et al. 2002). In these patients, inhibition the antagonism of melatonin synthesis or effect could be therapeutically desirable.
Acknowledgments. Work in authors' laboratories was supported in part by DGES, Spain, Agencia Nacional de Promoción Científica y Tecnológica, Argentina, and the University of Buenos Aires and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina.
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