Bidirectional communication between the pineal gland and the ...

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Bidirectional communication between the pineal gland and the immune system1,2 Krystyna Skwarlo-Sonta, Pawel Majewski, Magdalena Markowska, Ruslan Oblap, and Bozenna Olszanska

Abstract: The pineal gland is a vertebrate neuroendocrine organ converting environmental photoperiodic information into a biochemical message (melatonin) that subsequently regulates the activity of numerous target tissues after its release into the bloodstream. A phylogenetically conserved feature is increased melatonin synthesis during darkness, even though there are differences between mammals and birds in the regulation of rhythmic pinealocyte function. Membrane-bound melatonin receptors are found in many peripheral organs, including lymphoid glands and immune cells, from which melatonin receptor genes have been characterized and cloned. The expression of melatonin receptor genes within the immune system shows species and organ specificity. The pineal gland, via the rhythmical synthesis and release of melatonin, influences the development and function of the immune system, although the postreceptor signal transduction system is poorly understood. Circulating messages produced by activated immune cells are reciprocally perceived by the pineal gland and provide feedback for the regulation of pineal function. The pineal gland and the immune system are, therefore, reciprocally linked by bidirectional communication. Key words: pineal gland, melatonin, immunity, melatonin receptors, melatonin receptor transcripts. Résumé : Chez les vertébrés, la glande pinéale est un organe neuroendocrinien traduisant l’information photopériodique environnementale en un message biochimique (mélatonine) qui régule l’activité de nombreux tissus cibles après sa libération dans la circulation sanguine. Une caractéristique conservée phylogénétiquement est l’augmentation de la synthèse de la mélatonine durant l’obscurité, même s’il existe des différences entre les mammifères et les oiseaux au niveau de la régulation de la fonction rythmique des pinéalocytes. Des récepteurs membranaires de la mélatonine sont présents dans de nombreux organes périphériques, incluant les glandes lymphoïdes et les cellules immunitaires, d’où les gènes des récepteurs de la mélatonine ont été caractérisées et clonés. L’expression des gènes des récepteurs de la mélatonine dans le système immunitaire montre une spécificité d’espèce et d’organe. La glande pinéale, par l’intermédiaire de la synthèse et de la libération rythmiques de la mélatonine, influence le développement et la fonction du système immunitaire; toutefois, le mécanisme de traduction des signaux post-récepteurs n’est pas encore élucidé. Les messages produits par les cellules immunitaires activées sont perçus réciproquement par la glande pinéale et par un effet de rétroaction régulent la fonction pinéale. La glande pinéale et le système immunitaire sont, par conséquent, réciproquement liés par une communication bidirectionnelle. Mots clés : glande pinéale, mélatonine, immunité, récepteurs de la mélatonine, transcrits des récepteurs de la mélatonine. [Traduit par la Rédaction]

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Received 22 July 2002. Published on the NRC Research Press Web site at http://cjpp.nrc.ca on 2 April 2003. K. Skwarlo-Sonta,3 P. Majewski, and M. Markowska. Department of Vertebrate Physiology, Faculty of Biology, Warsaw University, Poland. R. Oblap4 and B. Olszanska. Institute of Genetics and Animal Breeding, Polish Academy of Sciences, Jastrzebiec, n/Warsaw, Poland. 1

This paper has undergone the journal’s usual peer review process. Presented at a Designated Meeting of the Physiological Society on Comparative Neuroscience and Comparative Physiology, University of Central Lancashire, Preston, U.K., May 2002. 3 Corresponding author (e-mail: [email protected]). 4 Present address: Institute of Agriecology and Biotechnology, Ukrainian Academy of Agricultural Sciences, 12 Metrologicheskaya str. Kyiv 03–143, Ukraine. 2

Can. J. Physiol. Pharmacol. 81: 342–349 (2003)

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doi: 10.1139/Y03-026

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Introduction In vertebrates, the pineal gland is a neuroendocrine organ that converts external (photoperiodic) information into an internal (hormonal) message for the regulation of peripheral target sites linked through the bloodstream (Reiter 1989). Melatonin is the hormone produced by the pineal gland and its synthesis is characteristically increased nocturnally or during darkness. The magnitude and duration of the nocturnal increase in melatonin synthesis is dependent upon the length of the dark phase of the photoperiodic cycle and it acts as a “clock” and “calendar” for the entrainment of other biological activities. It occurs in all vertebrate species, regardless of their diurnal or nocturnal locomotor activity, and it is thus a message of darkness and not of the rest or sleep period (Illnerova et al. 2000). In mammals, pineal melatonin synthesis is driven by a circadian oscillator in the hypothalamic suprachiasmatic nucleus (SCN), through a multisynaptic neural pathway that includes sympathetic nerve terminals that project from the superior cervical ganglia to the pineal gland. Nocturnal elevations in noradrenergic (NA) stimulation, via β- and α1adrenergic receptors on pinealocytes, increase the intracellular concentration of cyclic adenosine monophosphate (cAMP), which in turn activates arylalkylamine N-acetyltransferase (AA-NAT; EC 2.3.1.87) (Vacas et al. 1985), the penultimate and key enzyme in the synthesis of melatonin from tryptophan (Takahashi et al. 1989). Melatonin concentrations in the pineal and in blood therefore reflect the circadian rhythm in pineal AA-NAT activity (Fig. 1), which is also positively correlated with pinealocyte levels of cAMP (Falcon and Begay 1998). A similar circadian rhythm in melatonin production is also present in birds, although avian pinealocytes have direct photosensitivity (Collin et al. 1989; Cassone and Natesan 1997). Avian pinealocytes, moreover, have α 2 -adrenergic receptors rather than β- and α1-receptors and their activation occurs in the light phase, causing a decrease in pineal cAMP content, an inhibition of AA-NAT activity and a diminished release of melatonin (Zatz and Mullen 1988) (Fig. 2).

Melatonin: modulation of immune function It is now well established that amongst its numerous actions, melatonin is an immunomodulator, regulating the development, differentiation, and function of lymphoid tissues (Maestroni 1998; Lissoni 1999; Reiter et al. 2000; SkwarloSonta 2002). Indeed, diurnal and seasonal changes in immune function are thought to directly reflect changes in pineal melatonin production (Nelson and Drazen 1999; Molinero et al. 2000; Moore and Siopes 2000; Haldar et al. 2001). Such actions are thought to reflect a “strategic” role of circulating melatonin in the development and maintenance of immune function. The suppression of immunity and thymic atrophy that follow postnatal pinealectomy in rodents (Csaba and Barath 1975; Maestroni et al. 1986) supports this view, as does the reduced number of immune cells in the spleen and bursa of Fabricius of chickens following embryonic pinealectomy (Jankovic et al. 1994). Exogenous melatonin, in contrast, is known to increase the growth of immune tissues and their populations of immune cells (Haldar and

343 Fig. 1. Melatonin biosynthesis pathway. The graphs on the right show typical relations between the respective enzymes, light conditions, and melatonin levels in the pineal glands (according to Reiter 1993; Bernard et al. 1999).

Singh 2000), as well as the number of cells mediating nonspecific immunity (Currier et al. 2000). It has therefore been proposed as a putative anticancer therapy (for review see Lissoni 1999). Melatonin is also thought to have an “emergency” role in the immune system when it is activated by antigen stimulation (e.g., during infection). In this situation the actions of melatonin are, however, dependent on the species, experimental paradigm, and the immune function measured. For instance, it is generally accepted that cell-mediated immune function is increased in rodents by exogenous melatonin treatment (Maestroni et al. 1987; Demas and Nelson 1998) and is suppressed by pinealectomy (Haldar and Singh 2001). Contrary findings have, however, been presented by Provinciali et al. (1997). Melatonin treatment similarly elevates both cellular and humoral immune responses in quail (Haldar and Singh 2000; Moore and Siopes 2000), but blood levels of immune regulators are unaffected in young chickens by melatonin treatment and pinealectomy, although diurnal rhythms of these regulators are modified (Rosolowska-Huszcz et al. 1992, Skwarlo-Sonta et al. 1992). Furthermore, while melatonin has been reported to directly induce the proliferation of human peripheral blood mononuclear cells (PBMCs) in vitro (Kuhlwein and Irwin 2001), Rogers et al. (1997) found it inhibited PBMC proliferation. Similarly, melatonin has been reported to inhibit (Di Stefano et al. 1994), stimulate (Garcia-Maurino et al. 2000), or have no effect (Kuhlwein and Irwin 2001) on the synthesis of interferon-γ (IFN-γ) by human PBMCs in vitro. The relation between melatonin and the immune system is, therefore, complex. Activated immune cells in rodents (Maestroni et al. 1987, 1988) and chickens (Dziwinski et al. 1999) also synthesize opioid-like substances as part of the immune response following melatonin treatment. This mechanism is probably responsible for the antistress (Maestroni et al. 1988), antiviral © 2003 NRC Canada



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Fig. 2. Adrenergic regulation of melatonin synthesis in mammalian and avian pinealocytes. In mammals, noradrenaline (NA) release from sympathetic nerve terminals occurs in the dark. NA binding to β- and α 1 -adrenergic receptors on pinealocytes stimulates cAMP formation, which in turn activates AA-NAT, increasing melatonin levels (Vacas et al. 1985). In birds, NA release occurs in the light. NA binding to α 2 -adrenergic receptors on pinealocytes inhibits cAMP formation, blocking AA-NAT activity and decreasing melatonin levels (Collin et al. 1989; Zatz and Mullen 1988).

(Ben-Nathan et al. 1994), and hematopoietic actions of melatonin (Maestroni 1999). Melatonin is also immunoprotective in chickens, since it modifies the development of experimental peritonitis (induced by an injection of thioglycolate) by reducing the number of peritoneal leukocytes (Skwarlo-Sonta et al. 2002b). An antiinflammatory action of melatonin has also been demonstrated in rats (Cuzzocrea et al. 1998). Immune tissues are therefore target sites for melatonin, and these effects are likely to be receptor mediated.

and MT2, and are members of the G-protein-coupled receptor superfamily (Dubocovich 1995). In addition to membrane binding sites, melatonin binding to the RZR/ROR orphan nuclear receptor superfamily has also been demonstrated in the rat spleen and thymus (RafiiEl-Idrissi et al. 1998). These nuclear receptors are thought to mediate some of effects of melatonin on cytokine production, cell proliferation and oncostasis (Garcia-Maurino et al. 2000; Steinhilbert et al. 1995; Winczyk et al. 2001).

Melatonin: receptors in the immune system

Melatonin: receptor genes in the immune system

Melatonin is a small, lipophylic molecule and it can easily penetrate cell membranes. It is, moreover, also a free-radical scavenger and it therefore has this action within all tissues, including those of the immune system (reviewed by Reiter and Maestroni 1999). This function is not, however, tissue specific and seems to occur without the mediation of melatonin receptors. The presence of melatonin receptors in the immune system was first shown by the demonstration of 2-[125I]iodomelatonin binding sites in the spleen of hamsters (Niels 1989) and, thereafter, in other mammals and birds (Yu et al. 1991). Membrane-bound melatonin receptors have since been characterized in the thymus and spleen of many species and in the bursa of Fabricius in birds, as well as in peritoneal macrophages of mice and in human leukocytes (Table 1). The binding of 2-[125I]-iodomelatonin to these sites is specific, reversible, and time dependent, with binding parameters (Kd and Bmax) that indicate the presence of a single class of receptor. Melatonin binding sites in human leukocytes are, however, exceptional, since they have two classes of binding sites (of high and low affinity) (reviewed by Guerrero et al. 1994). In mammals, membrane-bound melatonin receptors are distinguished pharmacologically as subtypes mt1

The melatonin receptor gene was first cloned from an immortalized cell line of Xenopus laevis melanophores (Ebisawa et al. 1994), and since then numerous receptors and receptor fragments have been cloned (see Kokkola and Laitinen 1998 for review). In nonmammalian vertebrates, melatonin receptors can be divided into three subtypes, Mel1a, Mel1b, and Mel1c, based on their DNA and amino acid sequences. Within a subtype, these receptors have 65–96% homology, across species. Mel1a and Mel1b mRNA sequences, detectable by reverse-transcription PCR (RT–PCR) are present in all vertebrates, although Mel1c mRNA is only present in nonmammalian vertebrates (Kokkola and Laitinen 1998). According to the present terminology, in mammals the designation of mt1 and MT2 is accepted for the pharmacologically distinct products of Mel1a and Mel1b, respectively (Dubocovich et al. 1998). Within the immune system, Mel1a mRNA expression has been demonstrated in the rat thymus and spleen, as well as in isolated T and B cells (Pozo et al. 1997). In contrast, using the RT–PCR technique, we have demonstrated the presence of mRNA for Mel1b and Mel1c in the chicken spleen, thymus, bursa of Fabricius, and in isolated lymphocytes in © 2003 NRC Canada




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Table 1. Membrane-bound melatonin receptors within the immune system. (A) Organs Source Thymus Rat Duck Chicken Bursa of Fabricius Duck Chicken Spleen Hamster Mouse Rat Guinea pig Chicken

Pigeon

Duck Quail

Reference Lopez-Gonzales et al. 1993 Martin-Cacao et al. 1993 Poon et al. 1994 Skwarlo-Sonta et al. 1994 Liu and Pang 1992 Skwarlo-Sonta et al. 1994 Niels 1989 Yu et al. 1991 Yu et al. 1991 Yu et al. 1991 Poon and Pang 1992 Yu et al. 1991 Pang and Pang 1992 Skwarlo-Sonta et al. 1994 Yu et al. 1991 Wang et al. 1993 Poon et al. 1993 Yu et al. 1991 Wang et al. 1993

Fig. 3. Presence of melatonin receptor transcripts in chicken lymphoid glands. Hi-Line chickens of both sexes obtained from a local hatchery were kept from hatch under controlled conditions (12 h light:12 h dark). Thymus, bursa of Fabricius, and spleen were isolated from 1- and 2-week-old chickens; thymocytes and splenocytes were separated from the respective glands, as described previously (Markowska et al. 2001). Total RNA was isolated using the InViSorb RNA kit II (InViTec GmbH, Berlin, Germany) according to manufacturer’s protocol and used for RT–PCR. In brief, the material was lysed with the lysing solution, the DNA was removed with the sorbent, and the RNA was purified by phenol and chloroform extraction and precipitated with isopropanol. The RNA thus obtained was devoid of DNA contamination when tested by PCR. The primers for the melatonin receptor sequences were designed according to the published chick sequences: Mel1a (acc. No. U 31 821), Mel1b (acc. No. U 30 609), and Mel1c (acc. No. U 31 820). Primers were synthesized by InViTec GmbH; the sequence and RT–PCR conditions were as described in Oblap and Olszanska (2001). The identity of the RT–PCR products was attested by their size and by endonuclease restriction (Oblap and Olszanska 2001). NC, negative control without template DNA; M, molecular weight standard (100 bp DNA ladder, Promega); T, thymus; S, spleen; BF, bursa of Fabricius.

(B) Cells Source Lymphocytes Human Granulocytes Human Monocytes Human Peritoneal macrophages Mouse

Reference Lopez-Gonzalez et al. 1992 Guerrero et al. 1994 Barjavel et al. 1998 Garcia-Perganeda et al. 1999

Melatonin: signal transduction in immune cells the absence of Mel1a mRNA (Fig. 3; Table 2). Mel1a mRNA is, however, present in the chick central nervous system (CNS), indicating a tissue-specific pattern of melatonin receptor gene expression. This pattern would also appear to be age related, since Mel1c transcripts are present from the first week of life in the chick thymus but from the second week in the spleen. In the bursa of Fabricius, Mel1c mRNA has been found in one of five reactions (RT–PCR) performed for 1-week-old chicks and in six of eight reactions for 2-weekold chicks. The transcript for the Mel1b receptor is always seen in the bursa of Fabricius after 1 week of age, and it is present in the thymus by the second week. Its presence in the spleen is, however, equivocal in 1- and 2-week-old chicks (Table 2). As the immune system in the chick becomes functionally mature between 2 and 4 weeks post hatch (Chen et al. 1994), these changes in the expression of melatonin receptor mRNA may be related to developmental changes in the immune function.

The signal transduction pathways activated by melatonin are largely unknown, although a decrease in cAMP level, elevated by previous forskolin treatment, has been established in mammalian immune cells (Garcia-Perganeda et al. 1997, 1999). This effect is prevented by preincubation with pertussis toxin, indicating that it is mediated via a Gi protein. The inhibition of cAMP accumulation is also likely to be via α-subunit of the Gi protein and is accompanied by an increase in diacylglycerol production, most probably as a result of the activation of phospholipase C by βγ subunits (Garcia-Perganeda et al. 1997). At least some membrane-bound melatonin receptors in the immune system therefore appear to be coupled with Gi/o proteins. The increased concentration of cGMP in human monocytes following melatonin treatment does, however, suggest the involvement of other second messengers in the signal transduction of melatonin in mammalian immune cells (Sandler et al. 1975). In the avian immune system, melatonin inhibits mitogeninduced splenocyte proliferation in vitro, by an action reversed © 2003 NRC Canada



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Can. J. Physiol. Pharmacol. Vol. 81, 2003 Table 2. Expression of melatonin receptor transcripts in the chicken immune and central nervous system. One-week-old (female/male)

Two-week-old (female/male)

Material

Mel1a

Mel1b

Mel1c

Mel1a

Mel1b

Mel1c

Thymus Bursa of Fabricius Spleen Brain hemispheres Hypothalamus Thymocytes Splenocytes

–/– – – –/– – – –/– – – nd/++ nd/++ nd nd

–/– ++ +/+++ +/+– + nd/++ nd/++ nd nd

+/+++ –/– – – + –/– – + nd/++ nd/++ nd nd

– – – –/– – – – – – – –/– – – – – – – –/– – – – nd/++ nd/++ – – – –/– – – – – – – –/– – – –

++++/++++ ++++/++++ – – ++/– – ++ nd/++ nd/++ – + – –/– – ++ – +++/– +++

++++/++++ + – ++/+ – ++ ++++/++++ nd/++ nd/++ – ++ –/++++ +++–/– + – +

Note: The data represent RT–PCR results obtained for two female and two male birds, except for CNS tissues, where only two males were examined. Total RNA from the tissues of an individual bird was isolated and RT–PCR was performed with the individual RNA samples once (+ or –) or twice (++, – –, or – +) for each RNA sample. + and – represent a result of a single RT–PCR (positive or negative). nd, not determined.

by luzindole, an antagonist of membrane-bound melatonin receptors (Markowska et al. 2001, 2002). Melatonin similarly inhibits forskolin- and vasoactive intestinal peptide induced cAMP formation in chicken splenocytes (Markowska et al. 2002). It is therefore likely that melatonin acts through membrane-bound Mel1c and (or) Mel1b subtypes (see Fig. 3 and Table 2), coupled with G proteins, to modulate avian immune function.

Melatonin: modulation by immunological factors Communication between the pineal gland and the immune system is bidirectional, since circulating messages from activated immune cells (e.g., cytokines), inflammatory mediators (including histamine, prostaglandins activated complement), and hormones reciprocally act on the pineal gland (Fabris 1994). The feedback effect of these messages on the pineal gland is, however, poorly understood. Pineal gland function (measured as AA-NAT activity or melatonin concentration) has been shown to be modified by antigenic stimulation (Youbicier-Simo et al. 1996, Markowska et al. 2000), inflammation (Skwarlo-Sonta et al. 2002a), and treatment with cytokines (Withyachumnarnkul et al. 1991, Mucha et al. 1994), histamine (Zawilska et al. 1997), prostaglandins (Voisin et al. 1993), and opioids (Govitrapong et al. 1992). In addition, bursin (a unique tripeptide produced by the bursa of Fabricus) restores peak nocturnal melatonin levels in chicks in which the melatonin rhythm is suppressed by embryonic bursectomy (Youbicier-Simo et al. 1996). Although the pineal gland is part of the brain, circulating immune factors (e.g., cytokines) produced by activated immune cells have easy access to it, as the pineal is a circumventricular organ lacking the blood–brain barrier (Licino and Wong 1997). The expression of cytokine receptors on pinealocytes has, however, yet to be demonstrated, but their presence can be deduced from in vivo studies with cytokine receptor ligands (Mucha et al. 1994). Constitutive expression of interleukin-1β (IL-1β) in the rat pineal gland has also been shown to correlate with the diurnal melatonin rhythm, since IL-1β mRNA is higher during the day than during darkness (Tsai and McNulty 1999). The number of IL-1βpositive cells in the pineal is also increased by interferon (IFN) or lipopolysaccharide (LPS) stimulation, suggesting

an important role for IL-1β in the immune system – pineal axis. Moreover, systemic inflammation induced by an i.p. injection of LPS induces IL-1β gene expression in the pineal gland, whereas expression of the cytokines that counterregulate IL-1β activity (e.g., IL-1 receptor agonist, IL-10, and IL-13) (Wong et al. 1997) is limited. In addition to direct actions on pinealocytes, cytokines and other immune factors may regulate pineal function by indirect actions on pineal glial cells. The presence of cytokine receptors in glial cells of the CNS (Brzezinski 1997) supports this view. Microglia in the pineal have, moreover, been shown to affect the morphology and function of rat pinealocytes. Furthermore, in recent studies, Tsai et al. (Tsai and McNulty 1997, 1999; Tsai et al. 2001) have found that the actions of some cytokines (IFN-γ and IL-1β) on pineal function in vitro are mediated by microglia, whereas other inflammatory mediators (e.g., tumor necrosis factor-α and transforming growth factor β exert their effects directly on pinealocytes. The mechanisms mediating the actions of immunoregulators within and between these cells are, however, unknown.

Concluding remarks The pineal gland and the immune system appear to be linked by bidirectional communication. Melatonin functions as an immunoregulatory factor in the development and maturation of the immune system and in the progression of the immune response. The immune system, in turn, appears to reciprocally regulate pineal gland function, mainly via cytokines produced by activated immune cells, although the precise mechanism(s) involved are, as yet, largely unknown.

Acknowledgements This work was supported by grants from the State Committee for Scientific Research (KBN) 6 P04C 04320 and 6 P04C 08921, agreed to the Department of Vertebrate Physiology, Warsaw University. The work of B.O. and R.O. was performed within the Statutory Project No. S.1.2. of the Institute of Genetics and Animal Breeding of Polish Academy of Sciences. R.O. was recipient of a NATO fellowship (Poland). © 2003 NRC Canada




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