**2. Mechanisms of Melatonin**

Endocrine circadian rhythms are regulated by the endogenous hormone melatonin (*N*-acetyl-5-methoxytryptamine), which activates specific high-affinity melatonin receptors expressed on several different types of cells, including immunocompetent cells [8]. The pineal gland is the primary source of melatonin, which is also synthesized by tissues and organs of multicellular organisms including the retina, gastrointestinal tract, skin, and leukoctyes (in peripheral blood and in bone marrow) [9]. In the skin, melatonin can regulate cutaneous pigmentation and perform photoprotective and anticancer activities [10,11]. *N*-acetylserotonin, a precursor to melatonin, enters the systemic circulation from different peripheral organs for transformation to melatonin [12]. Furthermore, the effects of melatonin can be mediated by its metabolites because of the rapid metabolism of melatonin at the peripheral sites [13,14]. As the production of melatonin by the non-endocrine organs responds to signals other than circadian cycles, the endocrine, autocrine, and paracrine effects of melatonin mean that this substance is extensively involved in the regulation of the human immune system [9].

The biological effects of melatonin involve three pathways: (i) G-protein-coupled membrane receptor signaling; (ii) nuclear signaling; and (iii) receptor-independent signaling that accounts for radical scavenging activities of melatonin [7,15]. The binding of melatonin to the G protein-coupled melatonin receptors (MT1 and MT2) on the plasma membrane of the target cells enables melatonin to stimulate signaling pathways and reduce cell proliferation [16]. MT1 receptors are expressed throughout the body but mainly in the central nervous system, in the thymus and the spleen, B cells, CD4, and CD8 cells [7]. Neuroanatomical mapping of melatonin receptors has revealed marked differences in distribution patterns of MT1 and MT2 proteins in the adult rat brain, with for instance MT2 receptors identified in the reticular thalamic nucleus, mediating neuronal firing and burst activity related to nonrapid eye movement (NREM) sleep, whereas no MT1 receptors are found in this area, confirming the highly specific functioning of melatonin receptors in sleep neurophysiology [17]. MT1 and MT2 affect gene transcription activities through extracellular signal-regulated kinase (ERK) pathways and CREP phosphorylation [7]. Melatonin receptors have also been identified in RA synovial macrophages [18]. A positive correlation has been observed between the polymorphism of the melatonin receptor type 1B (*MTNR1B*) and levels of rheumatoid factor in Korean patients with RA [19]. Other researchers have reported significantly lower levels of MT1 expression in RA synovial tissue compared with normal healthy tissue, and the finding that siRNAs against MT1 reverse melatonin-mediated inhibition of TNF-α and IL-1β production, confirming that melatonin suppresses TNF-α and IL-1β via the MT1 receptor [20]. Thus, the activity of membrane melatonin receptors and their specific agonists is implicated in circadian rhythmicity [7]. Retinoic acid-related orphan receptor alpha (RORα) is an important member of the ROR subfamily of nuclear receptors, which mediate several physiological functions, such as metabolic, immunologic, and circadian actions [21]. The identified endogenous ligands for RORα consist of sterols and their derivatives [22]. It appears that RORα mediates the indirect effects of melatonin in the periphery, such as immunomodulation, cellular growth,

and bone differentiation [23]. Moreover, while RORα is not a receptor for melatonin or its metabolites, the constitutive activity of RORα may be modulated by membrane melatonin receptors [21,23].

Melatonin also displays antioxidant and anti-inflammatory activity, depending on the cellular state [24,25]. Evidence suggests that melatonin serves as a link between circadian rhythms and joint diseases, including RA and osteoarthritis (OA) [26,27]. For instance, oxidative stress induced by RA is reduced by melatonin and/or its metabolites, which not only neutralize the reactive oxygen (ROS) and reactive nitrogen species (RNS), but also upregulate levels of glutathione and antioxidant enzyme expression and activity [28,29]. In RA and OA, melatonin and its metabolites modulate several molecular signaling pathways including those governing inflammation, proliferation, and apoptosis [28,29].

#### **3. A Role for Melatonin in Rheumatoid Arthritis Therapy?**

In investigations involving melatonin in animal models of inflammatory autoimmune diseases (multiple sclerosis, systemic lupus erythematosus, inflammatory bowel disease, and type 1 diabetes), melatonin has demonstrated beneficial effects in these diseases, including prophylactic and therapeutic effects in rats with adjuvant-induced arthritis (AA), in which melatonin dose-dependently repressed the inflammatory response and enhanced proliferation of thymocytes and secretion of IL-2 [30]. In addition, melatonin decreased the elevated level of cyclic 3 ,5 -AMP (cAMP) induced by forskolin. The drop in thymocyte proliferation induced by injection of Freund's complete adjuvant was highly correlated with a decrease in the levels of Met-enkephalin (Met-Enk) in the thymocytes, which were strikingly augmented by melatonin; this effect was blocked by the Ca2<sup>+</sup> channel antagonist, nifedipine. The anti-inflammatory and immunoregulatory actions of melatonin involved a G protein-adenyl cyclase-cAMP transmembrane signal and Met-Enk release by thymocytes [30].

#### *3.1. Modulation of the Circadian Clock by Melatonin in RA*

Importantly, circadian rhythms exist in almost all cells of the body, and are regulated by circadian clock gene expression [7,15]. Any disruption in these circadian clocks is associated with the onset of inflammatory-related disease states and joint diseases, including RA [7,15]. Patients with RA exhibit abnormal clock gene expression, with disturbances in the hypothalamic-pituitary-adrenal axis influencing changes in circadian rhythms of circulating serum levels of melatonin, IL-6, cortisol and in chronic fatigue [15]. Melatonin exerts its effects in RA by modulating clock gene expression, including the *Cry1* gene [7,15]. By attenuating the expression of the *Cry1* gene, melatonin upregulates levels of cAMP production and increases activation of protein kinase A (PKA) and nuclear factor kappa B (NF-κB), which increases CIA severity in rats [31,32]. As detailed earlier, the diurnal secretion of melatonin is also closely related to the production of IL-12 and NO among RA synovial macrophages and human monocytic myeloid THP-1 cells [33].

A positive correlation has been reported between elevated early morning serum melatonin concentrations and disease activity scores as well as erythrocyte sedimentation rate (ESR) levels in patients with juvenile rheumatoid arthritis, although higher melatonin concentrations did not correlate with disease severity [34], echoing the findings of Forrest and colleagues reported earlier, who noted that elevated ESR and neopterin concentrations following melatonin treatment did not worsen the severity of RA disease [35]. El-Awady and colleagues suggested that melatonin may promote the activity of RA disease, rather than its severity [34]. However, as reported above, Akfhamizadeh and colleagues found no link between elevated morning serum melatonin concentrations and RA disease activity or other disease characteristics, despite also observing significantly higher melatonin values in newly diagnosed RA patients compared with those who had established RA disease [36].

There also appears to be a relationship between melatonin and the *Bmal1* and *ROR* clock genes [15]. It is speculated that high melatonin concentrations in RA patients may modulate *ROR* activation [15]. *ROR* acts as a negative regulator of inflammation via the NF-κB signaling pathway and is essential in the activity of both melatonin and the clock gene *Bmal1*, which helps to maintain 24-h rhythms and regulate immune responses [15,37]. Moreover, ROR proteins bind into the promoter region and

drive *Bmal1* gene expression [38]. This activity at the binding site is inhibited by reverse-eritroblastosis viruses (REV-ERBs), which may contribute to *Bmal1* suppression and exacerbation of RA [15].

### *3.2. Adverse E*ff*ects of Melatonin in RA*

Evidence suggests that melatonin is not beneficial in RA. For instance, the development of collagen-induced arthritis (CIA) in DBA/1 mice is exacerbated by constant darkness [39] and by daily exogenous administration of melatonin 1 mg/kg [40]. Hansson and colleagues then investigated the effects of surgical pinealectomy in DBA/1 and NFR/N mice with collagen-induced arthritis (CIA) [41]. Serum melatonin levels were reduced in the pinealectomized mice to around 30% of levels in normal or sham-operated controls [41]. In both mouse strains, pinealectomy was associated with a delay in onset of arthritic disease, less severe arthritis (lower clinical scores), and lower serum anti-CII levels compared with sham-operated animals [41]. The researchers interpreted these findings as showing that high physiological levels of melatonin stimulate the immune system and worsen CIA, while inhibiting the release of melatonin is beneficial [41]. Their speculation was supported by observations from mice subjected to 30 days of Bacillus Calmette-Guérin (BCG) inoculations into the left hind paw, inducing chronic granulomatous inflammation [42]. Higher vascular permeability was seen around the granulomatous lesions at midnight than at midday; this rhythmic variation was eliminated by pinealectomy and restored by nocturnal replacement of melatonin [42].

This ability of melatonin to modulate immune response was further illustrated by experiments in which the production of IL-12 and nitric oxide (NO) was significantly increased in the media of melatonin-stimulated RA synovial macrophages and cultured THP-1 cells compared with RPMI-treated synovial macrophage controls [33]. Unexpectedly, the opposite effects in IL-12 and NO levels were seen when RA synovial macrophages were pretreated with lipopolysaccharide (LPS) prior to melatonin, as compared with synovial macrophages treated with LPS alone [33]. This study explained the possible mechanism of joint morning stiffness in relation to diurnal rhythmicity of neuroendocrine pathways [33].

These conclusions are supported by later evidence from in vitro and in vivo studies, as well as clinical investigations, showing how melatonin stimulates the production of NO, T helper type 1 (Th1)-type and other inflammatory cytokines besides IL-12 (IL-1, IL-2, IL-4, IL-5, IL-6, TNF-α, granulocyte-macrophage colony-stimulating factor [GM-CSF], and transforming growth factor [TGF]-β, interferon [IFN]-γ), and enhances both cell-mediated and humoral responses [43–45]. In the early morning, patients with RA exhibit high serum levels of proinflammatory cytokines, especially TNF-α and IL-6, when melatonin serum concentrations are also higher [6,43]. The effects of these circadian rhythms are thought to promote the joint pain and morning stiffness that characterizes RA [6]. Animal studies have shown that melatonin treatment (10 mg/kg) dysregulates circadian clock genes, which may promote the progression of RA [31]. Intriguingly, a dual effect of melatonin as a proinflammatory agent and antioxidant has been observed in CIA rats [32]. In that study, a lower dosage of melatonin (30 μg/kg) increased anti-collagen antibodies, IL-1β, and IL-6 levels in the serum and joints of arthritic rats, worsening the severity of joint damage, while simultaneously lowering oxidative markers nitrite/nitrate and lipid peroxidation in serum, but not in joints [32].
