PDF size: 836.54 Kb.
Melatonin (MEL) physiology has been related to the immune system regulation. The pharmacological inhibition of MEL synthesis decreased the primary antibody response produced by an antigenic stimulus. Moreover, the exogenous MEL administration antagonized the immunosupressive effects of corticosterone and acute stress in mice. Also, MEL induces the interleukin-12 release from monocytes and promotes the lymphocytes TH
1 differentiation. In these lymphocytes, MEL stimulates the interleukin-2 and interferon-γ release. The MEL receptors localized in the plasma membrane and in the nucleus, either from monocytes and lymphocytes, are the MEL effector signals in the cell. It has been suggested that the immunoenhancing effect produced by the hormone could be mediated by an opioid mechanism. Several lines of evidence have shown that lymphocytes, monocytes, and polymorphonuclear cells have the biochemical machinery to produce and release opioid peptides. In the central nervous system (CNS), the endogenous melatonin absence disrupted the enkephalin circadian rhythm and its tissue content was decreased as well. If the MEL absence in the CNS decreased the enkephalin tissue content, it is possible to consider that the opioid peptides content also decreased in the immune system. The present study was performed to evaluate the effect of endogenous MEL absence over opioid peptides concentration in the thymus and spleen of the rat.
Materials and methods
Sixty male Wistar rats, weighing each 220-240 g, were housed under a 12 h. light: 12 h. dark cycle in a temperature controlled room (21 ± 1°C); the illumination period started at 06:00. Water and food pellets were available ad libitum. This group was divided in six subgroups:
a) Naïve control group: Ten animals were housed under a 12 h. light: 12 h. dark cycle. The darkness period started at 18:00.
b) Control group + MEL: 10 animals kept with a 12 x 12 h. lightdarkness cycle were subcutaneously (s.c.) injected with melatonin (800 μg/kg) at 9:00. These rats were maintained four hours under light conditions before being sacrificed.
c) Control group + vehicle: Ten control animals were s.c. injected (at 9:00) with the same volume used to dissolve the hormone (ethanol: isotonic saline solution). These rats were maintained four hours under light conditions before being sacrificed.
d) Continuous light (CL): In order to reduce the melatonin plasma concentration, ten rats were kept in a room with continuous light during 15 days. Light intensity was ≤50 lux to avoid stress.
e) Continuous light + MEL: Ten CL animals were injected with melatonin (800 µg/kg s.c.) at 9:00. These rats were maintained four hours under light conditions before being sacrificed.
f) Continuous light + darkness: In order to enhance the melatonin plasma concentration, ten CL rats were kept in a dark room during four hours. The darkness period started at 9:00 and rats were sacrificed four hours later.
Animals were sacrificed by decapitation and thymuses and spleens were dissected and subjected to preparative processes before enkephalin determination. Opioid peptides IR-ME, IR-LE, IRHE, IR-OC content was measured using a radioimmunoassay technique. Statistical differences between groups were established by one-way ANOVA test (α=0.05), and then calculated using Tukey HSD and Tamhane as post hoc tests. A p‹0.05 level was accepted as significant. The concentration values were expressed as IR-peptide (pmol/mg protein).
In this work, three main features were found: 1. The endogenous melatonin absence produced by a chronic lighting exposure significatively reduced (50%) the opioid peptides content in both thymus and spleen. 2. Melatonin administration to CL rats produced an enkephalin tissue content increase (›100%), in both lymphoid organs. 3. No changes were found in control groups after melatonin or vehicle administration.
The immune system is susceptible to stress. Recent evidences in neuroimmunology have begun to define how mood alterations, stress, seasons, depression, and daily rhythms have profound effects on immune response through hormonal modulation. Several lines of evidence have suggested that immune system functions could be regulated by the melatonin-opioid peptides relation. In the present work, we found that endogenous melatonin absence significatively reduced the enkephalin content in both thymus and spleen of the rat. This reduction could be directly related to the cytokine and antibody production, since the primary response to an antigen could be mediated by opioids. Our results are related with those obtained for the CNS, where the melatonin absence significatively decreased the opioid peptide tissue content and disrupted the enkephalins circadian rhythm. It has been reported that MEL was able to induce the Proopiomelanocortin (POMC) synthesis in the immune system. These data suggest that MEL is related to the neuropeptides synthesis. However, the basic mechanisms underlying the melatonin effect over the opioid peptides synthesis remain unknown at present.
The signaling pathway may include the union to MT1 membrane receptors that had been located in the thymus and spleen, and the activation of phospholipase C. These events may cause an increase of intracellular calcium and the activation of the protein kinase C (PKC). PKC can phosphorylate and activate other kinases as the mitogen-activated protein kinases (MAPK), which include the ERK and JNK families. The ERK1/2 and the JNK activate the expression of transcription factors such as c-Fos and c-Jun, which form a heterodimer called AP-1. This protein can modulate the expression of the Proenkephalin A gene as described for the CNS. There is experimental evidence about the involvement of the MT1 receptor with the activation of EKR and JNK enzymes. Moreover, MEL had been described to stimulate the DNA binding activity of AP-1. Furthermore, the JNK phosphorylation had been associated to the regulation of Proenkephalin A gene expression, mediated by c-Fos and c-Jun. The induction of Proenkephalin A gene expression would produce an increase of the tissue content of the enkephalins.
ANTON-TAY F, RAMIREZ G, MARTINEZ I, BENITEZKING G: In vitro stimulation of protein kinase C by melatonin. Neurochem Res, 23(5):601-6, 1998.
ASAI M, VINDROLA O, ZUBIETA M, TALAVERA E, MASSARINI A: Diurnal variations of IR-Met-enkephalin in the brain of pentylenetetrazol-kindled rats. Brain Res, 442:81- 85, 1988.
ASAI M, ZUBIETA M, MATAMOROS-TREJO G, LINARES G, AGUSTIN P: Diurnal variations of opioid peptides and synenkephalin in vitro release in the amygdala of kindled rats. Neuropeptides, 32(3):293-299, 1998.
ASAI M: Efecto de la melatonina sobre el sistema endógeno opioide. XLII Congreso Nacional de Ciencias Fisiológicas, Cancún, QR. Septiembre 2000.
BARJAVEL MJ, MAMDOUH Z, RAGHBATE N, BAKOUCHE O: Differential expression of the melatonin receptor in human monocytes. J Immunol, 160(3):1191-1197, 1998.
BENITEZ-KING G: PKC activation by melatonin modulates vimentin intermediate filament organization in N1E-115 cells. J Pineal Research, 29(1):8-14, 2000.
BENITEZ-KING G, HERNANDEZ ME, TOVAR R, RAMIREZ G: Melatonin activates PKC–alpha but not PKCepsilon in N1E-115 cells. Neurochem Int, 39(2):95-102, 2001.
CHAN AS, LAI FP, LO RK, VOYNO-YASENETSKAYA TA, STANBRIDGE EJ, WONG YH: Melatonin MT1 and MT2 receptors stimulate c-Jun N-terminal kinase via pertussis toxinsensitive and –insensitive G proteins. Cell Signal, 14(3):249- 57, 2002.
CHEN Y, WU Q, SONG SY, SU WJ: Activation of JNK by TPA promotes apoptosis via PKC pathway in gastric cancer cells. World J Gastroenterol, 8(6):1014-8, 2002.
CHOE Y, LEE BJ, KIM M, Participation of protein kinase C alpha isoform and extracellular signal-regulared kinase in neurite outgrowth of GT1 hypothalamic neurons. J Neurochem, 83(6):1412-22, 2002.
FU W, SHAH SR, JIANG H, HILT DC, DAVE HP, JOSHI JB: Transactivation of proenkephalin gene by HTLV-1 tax1 protein in glial cells: involvement of Fos/Jun complex at an AP-1 element in the proenkephalin gene promoter. J Neurovirol, 3(1):16-27, 1997.
GARCIA-MAURINO S, POZO D, CARRILLO-VICO A, CALVO JR, GUERRERO JM: Melatonin activates Th1 lymphocytes by increasing IL-12 production. Life Sci, 65(20):2143-50, 1999.
GARCIA-MAURINO S, GONZALEZ-HABA MG, CALVO JR, RAFII-EL-IDRISSI M, SANCHEZ-MARGALET V, GOBERNA R, GUERRERO JM: Melatonin enhances IL-2, IL-6, and IFN-gamma production by human circulating CD4+ cells: a possible nuclear receptor-mediated mechanism involving T helper type 1 lymphocytes and monocytes. J Immunol, 159(2):574-81, 1997.
KAVELAARS A, HEIJNEN CJ: Expression of preproenkephalin mRNA and production and secretion of enkephalins by human thymocytes. Ann NY Acad Sci, 917:778-783, 2000.
KIM YH, CHOI SS, LEE JK, WON JS, CHOI MR, SUH HW: Possible roles of JNK pathway in the regulation of hippocampal proenkephalin and immediate early gene expression induced by kainic acid. Mol Cells, 11(2):144-50, 2001.
KUMAR MSA, CHEN CL, SHARP DC, LIU JM, KALRA PS, KALRA SP: Diurnal fluctuations in Methionineenkephalin levels in the hypothalamus and preoptic area of the male rat: effects of pinealectomy. Neuroendocrinology, 35:28-31, 1982.
LEVI NL, HANOCH T, BENARD O, ROZENBLAT M, HARRIS D, REISS N, NAOR Z, SEGER R: Stimulation of Jun N-terminal kinase (JNK) by gonadotropin releasing hormone in pituitary alpha T3-1 cell line is mediated by protein kinase C, c-Src, and CDC42. Mol Endocrinol, 12(6):815-24, 1998.
LOWRY OH, ROSEBROUGH NJ, FARR AL, RANDALL RJ: Protein measurement with the Folin phenol reagent. J Biol Chem, 193:265-275, 1951.
LUPOWITZ Z, RIMLER A, ZISAPEL N: Evaluation of signal transduction pathways mediating the nuclear exclusion of the androgen receptor by melatonin. Cell Mol Life Sci, 58(14): 2129-35, 2001.
MAESTRONI GJ, CONTI A, PIERPAOLI W: The pineal gland and the circadian, opiatergic, immunoregulatory role of melatonin. Ann NY Acad Sci, 496:67-77, 1987.
MAESTRONI GJ, CONTI A, PIERPAOLI W: Role of the pineal gland in immunity: III. Melatonin antagonizes the immunosuppressive effect of acute stress via an opiatergic mechanism. Immunology, 63(3):465-9, 1988.
MAESTRONI GJ: The photoperiod transducer melatonin and the immune-hematopoietic system. J Photochem Photobiol, 43(3):186-92, 1998.
MAESTRONI GJ, CONTI A: Immuno-derived opioids as mediators of the immuno-enhancing and anti-stress action of melatonin. Acta Neurol, 13(4):356-60, 1991.
MISHELL BB, SHIIGI SM: Selected methods in cellular immunology. WH. FREEMAN and Co. E.U.A. páginas 4,5. Nueva York, 1980.
MITSUTAKE N, NAMBA H, SHKLYAEV SS, TSUKAZAKI T, OHTSURU A, OHBA M, KUROKI T, AYABE H, YAMASHITA S: PKC delta mediates ionizing radiationinduced activation of c-Jun NH(2)-terminal kinase through MKK7 in human thyroid cells. Oncogene, 20(8):989-96, 2001.
MOCCHEGIANI E, BULIAN D, SANTARELLI L, TIBALDI A, MUZZIOLI M, LESNIKOV V, PIERPAOLI W, FABRIS N: The zinc pool is involved in the immune-reconstituting effect of melatonin in pinealectomized mice. J Pharmacol Exp Ther, 277(3):1200-8, 1996.
NADAL-WOLLBOLD F, PAWLOWSKI M, LEVY-TOLEDANO S, BERROU E, ROSA JP, BRYCKAERT M: Platelet ERK2 activation by thrombin is dependent on calcium and conventional protein kinases C but not Raf-1 or R-Raf. FEBS Lett, 531(3):475-82, 2002.
POZO D, DELGADO M, FERNANDEZ-SANTOS JM, CALVO JR, GOMARIZ RP, MARTIN-LACAVE I, ORTIZ GG, GUERRERO JM: Expression of the Mel1a-melatonin receptor mRNA in T and B subsets of lymphocytes from rat thymus and spleen. FASEB J, 11(6):466-73, 1997.
RAFII-EL-IDRISSI M, CALVO JR, GIORDANO M, GUERRERO JM: Specific binding of 2-[125I]iodomelatonin by rat spleen crude membranes: day-night variations and effect of pinealectomy and continuous light exposure. J Pineal Res, 20(1):33-8, 1996.
REITER RJ: The melatonin rhythm: both a clock and a calendar. Experientia, 49:654-664, 1993.
ROSEN H, BEHAR O, ABRAMSKY O, OVADIA H: Regulated expression of proenkephalin A in normal lymphocytes. J Immunol, 143:3703-3707, 1989.
ROY D, BELSHAM DD: Melatonin receptor activation regulates GnRH gene expression and secretion in GT1-7 GnRH neurons. Signal transduction mechanisms. J Biol Chem, 277(1):251-258, 2002.
URATA Y, HONMA S, GOTO S, TODOROKI S, IIDA T, CHO S, HONMA K, KOND T: Melatonin induces gammaglutamylcysteine synthetase mediated by activator protein-1 in human vascular endothelial cells. Free Radic Biol Med, 27(7-8):838-47, 1999.
VINDROLA O, PADROS MR, STERIN-PRYNC A, ASE A, FINKIELMAN S, NAHMOD V: Proenkephalin system in human polymorphonuclear cells. Production and release of a novel 1.0-kD peptide derived from synenkephalin. J Clin Invest, 86:531-537, 1990.
VINDROLA O, PADROS MR, BAUTISTA D: Opioides endógenos en la comunicación entre el sistema nervioso y el sistema inmune. Precongreso Actualización en Fisiología. Puebla, Pue. 1997.
VON GALL C, STEHLE JH, WEAVER DR: Mammalian melatonin receptors: molecular biology and signal transduction. Cell Tissue Res, 309(1):151-62, 2002.
WAJS E, KUTOH E, GUPTA D: Melatonin affects proopiomelanocortin gene expression in the immune organs of the rat. Eur J Endocrinol, 133:754-60, 1995.
WON JS, SONG DK, KIM YH, HUH SO, SUH HW: The stimulation of rat astrocytes with phorbol-12-myristate-13- acetate increases the proenkephalin mRNA: involvement of proto-oncogenes. Brain Res Mol Brain Res, 54(2):288-97, 1998.
WON JS, SONG DK, HUH SO, KIM YH, SUH HW: Effect of melatonin on the regulation of proenkephalin and prodynorphin mRNA levels induced by kainic acid in the rat hippocampus. Hippocampus, 10(3):236-43, 2000.
WON JS, KIM YH, SONG DK, HUH SO, LEE JK, SUH HW: Stimulation of astrocyte-enriched culture with arachidonic acid increases proenkephalin mRNA: involvement of proto-oncoprotein and mitogen activated protein kinases. Brain Res Mol Brain Res, 76(2):396-406, 2000.
ZIOLKOWSKA B, PRZEWLOCKA B, MIKA J, LABUZ D, PRZEWLOCKI R: Evidence for Fos involvement in the regulation of proenkephalin and prodynorphin gene expression in the rat hippocampus. Brain Res Mol Brain Res, 54(2):243- 51, 1998.