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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 19, 2003 as doi:10.1096/fj.02-0501fje. |
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Department of Medical Biochemistry and Molecular Biology, The University of Seville School of Medicine and Virgen Macarena Hospital, 41009 Seville, Spain
2Correspondence: Department of Medical Biochemistry and Molecular Biology, The University of Seville School of Medicine, Avda. Sánchez Pizjuan 4, 41009 Seville, Spain. E-mail: guerrero{at}us.es
SPECIFIC AIMS
It is well known that melatonin plays a fundamental role in human neuroimmunomodulation. Thus, melatonin regulates the production of a large number of cytokines including interleukin-2 (IL-2), and membrane and nuclear receptors for melatonin are present in lymphoid cells. However, the putative nuclear receptor for the neurohormone has mediated most of these melatonin effects. In this paper, we describe that melatonin, through its mt1 membrane receptor and via a cyclic adenosine monophosphate (cAMP)-dependent signal transduction pathway, is able to counteract the inhibitory effect of prostaglandin E2 (PGE2) on IL-2 production in human lymphocytes. Therefore, we postulate, for the first time, a physiological role of the mt1 melatonin membrane receptor in the human immune system.
PRINCIPAL FINDINGS
1. Effect of melatonin and PGE2 on IL-2 production by human lymphocytes
We observed that melatonin and PGE2 exert opposed effects on IL-2 production in human peripheral blood mononuclear cells (PBMCs). Thus, we detected that melatonin increases IL-2 production, whereas PGE2 decreased this production. Melatonin and PGE2 effects were dose-dependent, reaching significant values when cells were cultured in the presence of physiological concentrations of PGE2 or melatonin (data not shown).
2. Effect of melatonin, S 20098, and CGP 52608 on PGE2-inhibited IL-2 and PGE2-stimulated cAMP production
As we observed that PGE2-inhibited IL-2 production was overcome when PGE2-treated cells were cultured in the presence of melatonin, we studied the mechanism involved in this effect. Thus, we cultured PGE2-treated cells with the melatonin membrane receptor agonist S 20098 or the nuclear receptor agonist CGP 52608, and we observed that the melatonin effect was reproduced by S 20098, and no effect was seen in the presence of CGP 52608 (Fig. 1
A). Conversely, it had been shown that PGE2 inhibits PBMCs IL-2 production by elevating cAMP levels and melatonin inhibits cAMP production in PBMCs stimulated by forskolin via its membrane receptor coupled to a G protein. Therefore, we studied the possibility that a cAMP-dependent signal transduction pathway could be implicated in the effect of melatonin. Thus, we cultured PBMCs in the presence of PGE2, observing a significant increase of cAMP levels. When cells treated with PGE2 were cultured in the presence of melatonin, cAMP production significantly decreased. The effect of melatonin was reproduced, even overcome, when cells were treated with S 20098, whereas in cells cultured with CGP 52608, no changes in cAMP levels were observed (Fig. 1B
).
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3. The effect of melatonin via its membrane receptor is prevented by phorbol 12-myristate 13-acetate (PMA)
When cells were stimulated with PMA in addition to PHA, we found that neither melatonin nor S 20098 was able to counteract the inhibitory effect of PGE2 on IL-2 production (Fig. 2
A). Previous studies had shown that PMA, a protein kinase C (PKC) activator that also stimulates the production of IL-2 production, causes a partial or total reduction in the levels of mt1 mRNA. Trying to find a correlation between the effect of melatonin and expression of its mt1 receptor, we studied the ability of PMA-stimulated PBMCs to express the mt1 mRNA using the method of RT-PCR with specific mt1 primers. A single amplified band of 285 bp was obtained when cells were stimulated only with PHA, whereas in cells stimulated with PHA plus PMA, no band was detected (Fig. 2B
).
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CONCLUSIONS AND SIGNIFICANCE
Currently, two melatonin membrane receptors, mt1 and MT2, have been widely studied. In addition to membrane receptors, the presence of melatonin nuclear receptors corresponding to orphan members of the nuclear receptor superfamily, RZR/ROR, has been described. Although in the human immune system, melatonin membrane and nuclear receptors have been described, no biological effect mediated by the mt1 receptor had been shown. However, it has been observed that melatonin, via nuclear receptors, is involved in several immune processes, including the production of cytokines. Conversely, the effect of PGs as potent, local regulators of the immune response is well known. In this way, the capacity of PGE2 to inhibit human T cell responses through an IL-2 decrease is well established.
As we observed that PGE2 decreased IL-2 production, whereas melatonin exerts an opposed effect, increasing IL-2 production in human PBMCs, we studied a possible interaction between melatonin and PGE2 on IL-2 production. Thus, we observed that physiological concentrations of melatonin were able to counteract the inhibitory effect of PGE2 on IL-2 production. To study whether the effect of melatonin was a receptor-mediated mechanism, we compared the effects of the specific membrane receptor agonist S 20098 and the nuclear receptor agonist CGP 52608 on the inhibitory effect of PGE2 on IL-2 production. We observed that S 20098 was able to overcome the inhibitory effect of PGE2, whereas no effect was observed when cells were cultured in the presence of CGP 52608 (Fig. 1A
).
The capacity of PGE2 to inhibit IL-2 production through an increase in cAMP levels has been well established. Moreover, it has been observed that the melatonin membrane receptor in human lymphocytes is involved in the inhibition of adenylyl cyclase. Therefore, we studied whether a cAMP-dependent mechanism was involved in the effect of melatonin. Thus, we observed that melatonin and S 20098 counteracted the PGE2-induced increase in cAMP levels, whereas no effect was observed in the presence of CGP 52608 (Fig. 1B
).
When PHA-treated cells were stimulated with PMA, a PKC activator that also stimulates the production of IL-2 production, we observed that melatonin was not able to counteract the effect of PGE2 (Fig. 2A
). Some studies have revealed that PMA reduced, even eliminated, the expression of the mt1 receptor in lymphoid cells. Thus, we studied the effect of PMA stimulation on mt1 expression. We found that the stimulation by PMA caused the disappearance of mt1 mRNA expression (Fig. 2B
).
These results, together with previous studies performed by our group regarding the involvement of melatonin nuclear receptor in the regulation of cytokine production, allow us to hypothesize a schematic diagram of the melatonin effect on IL-2 production (Fig. 3
). Thus, conversely, melatonin would prevent the inhibitory effect on IL-2 production induced by some regulators of lymphocytes, such as PGE2. However, previous studies have revealed that melatonin directly activates IL-2 production via binding nuclear receptors. Although this schematic diagram seemingly shows a jointly effect of melatonin on IL-2 production, via membrane and nuclear receptor, this scheme only summarized results from two different studies.
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Taken together, these data show that melatonin counteracts the inhibitory effect of PGE2 on IL-2 production via its mt1 membrane receptor through a cAMP-dependent mechanism in human lymphocytes. Therefore, the results reported in this paper describe, for the first time, a physiological effect of melatonin via its mt1 membrane receptor on the human immune system.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0501fje; to cite this article, use FASEB J. (February 19, 2003) 10.1096/fj.02-0501fje 
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