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Melatonin enhances cellular and humoral immune responses in the Japanese quail (Coturnix coturnix japonica) via an opiatergic mechanism
GENERAL AND COMPARATIVE
ENDOCRINOLOGY General and Comparative Endocrinology 131 (2003) 258–263 www.elsevier.com/locate/ygcen
Melatonin enhances cellular and humoral immune responses in the Japanese quail (Coturnix coturnix japonica) via an opiatergic mechanism C.B. Moore and T.D. Siopes* Department of Poultry Science, North Carolina State University, Raleigh, NC 27695-7608, USA Accepted 12 December 2002
Abstract It is known that melatonin has important immunomodulatory properties in the Japanese quail. However, the mechanism of melatonin action on the immune system is not clearly understood in avian species. In mammals, the immunostimulatory properties of melatonin are mediated by the release of opioid peptides from activated T-lymphocytes. The present study was performed to determine if these same melatonin-induced opioids (MIO) are involved with the immunoenhancing effects of melatonin in quail. Three treatment groups were given melatonin (50 lg/ml) in the drinking water ad libitum along with naltrexone, a known opioid receptor-blocking agent. Melatonin was administered throughout the 3 week study and each bird received a daily intramuscular injection of naltrexone at a dose of 0.1, 1.0, or 10.0 mg/kg. In addition, three control groups were established that received only melatonin, naltrexone, or diluent. Evaluation of the cellular and humoral immune responses was initiated after 2 weeks of treatments. A cutaneous basophil hypersensitivity reaction to phytohemagglutinin (PHA-P) was measured to evaluate the cellular immune response. To evaluate the humoral immune response, primary antibody titers were determined 7 days post-intravenous injection with a Chukar red blood cell (CRBC) suspension. Both the cellular and humoral immune responses were significantly increased by 22 and 34%, respectively, upon melatonin exposure as compared to quail receiving diluent only. Concomitant administration of naltrexone and melatonin significantly reduced the immunoenhancing effect of melatonin across all naltrexone doses. We conclude that melatonin enhances a cellular and humoral immune response in Japanese quail via an opiatergic mechanism. Ó 2003 Elsevier Science (USA). All rights reserved.
1. Introduction Knowledge about the physiological significance of the pineal gland has increased dramatically in recent years. This gland and its primary secretory product melatonin (N-acetyl-5-methoxytryptamine) has been linked to several physiological mechanisms including but not limited to gonadal, thyroid, adrenal and immune functions (Axelrod et al., 1981; Krieger, 1981; Reiter, 1984). It is thought that the pineal gland transduces environmental information through the circadian synthesis and secretion of melatonin. This allows the organism to respond appropriately to an environmental demand (Reiter, 1984). Consequently, the use of this
* Corresponding author. E-mail address: [email protected] (T.D. Siopes).
hormone in modulating several biological functions has been widely investigated in both mammalian and avian species. The existing literature in mammals and birds suggests that there is a close connection between melatonin and immune regulation. It has been observed in mammalian species that melatonin has important immunostimulatory properties, especially in immunocompromised states. Maestroni et al. (1986) demonstrated that inhibition of melatonin synthesis leads to depressed cellular and humoral immune responses in mice. In addition, exogenous melatonin administration antagonizes the immunodepressive effect of corticosterone and cyclophosphamide treatment in mice (Maestroni et al., 1986, 1987a). Furthermore, melatonin treatment ameliorates the survivability of mice immunodepressed by dexamethosone treatment or to exposure to Venezuelan equine encephalomyelitis virus (Bonilla et al., 2001). Therefore,
0016-6480/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0016-6480(03)00011-X
C.B. Moore, T.D. Siopes / General and Comparative Endocrinology 131 (2003) 258–263
the effectiveness of melatonin as a potent immunostimulatory agent is established in mammals. Similiar reports of immunoenhancement by melatonin exist in birds. Pinealectomized ring-doves have reduced immune responses as compared to sham-operated birds, suggesting an immunoenhancing effect for melatonin (Rodriguez and Lea, 1994). Also, pinealectomy reduced cellular and humoral immune responses in Japanese quail, which was improved to normal levels by melatonin replacement (Moore et al., 2002). Furthermore, birds kept in constant light, which suppresses endogenous melatonin levels, have reduced immune responses as compared to birds in light-dark cycles and melatonin can reconstitute this weakened immune system in a dose response manner (Moore and Siopes, 2000). Recently we have demonstrated that exogenous melatonin treatments can even boost immune responses in normal (immunologically unsuppressed) quail, suggesting potent immunoenhancing effects of melatonin in the quail (Moore and Siopes, 2002). In addition, it has been shown that exogenous melatonin enhances the activities of B and T lymphocytes in immature chickens (Brennan et al., 2002). Therefore, in both mammals and birds, melatonin is certainly an important immune regulator. However, the mechanism of melatonin action in boosting immune responses is not known in avian species. In mammals, it is known that melatonin receptors exist on circulating lymphocytes, suggesting direct communication between melatonin and immune cells (Liu and Pang, 1993; Poon et al., 1994; Calvo et al., 1995). Furthermore, it is known that the endogenous opioids have powerful immunomodulatory and analgesic properties (Sibinga and Goldstein, 1988). Melatonin has anticonvulsant and analgesic properties in mice, suggesting a possible relationship between melatonin and opioids (Lakin et al., 1981; Kumar et al., 1982). Based on these reports, Maestroni et al. (1987b, 1988a,b, 1989) investigated the opioid peptides as potential mediators of the observed immunoenhancing effects of melatonin in mice. It was found that the specific opioid antagonist naltrexone could completely abolish the immunoenhancing and anti-stress effects of melatonin. In addition, the immunoenhancing effects of melatonin were reproduced by administration of opioid peptides such as dynorphin 1–13 and b-endorphin (Maestroni and Conti, 1989). It was also shown that physiological concentrations of melatonin could stimulate secretion of opioid peptides from activated CD4þ T-helper lymphocytes in vitro (Maestroni and Conti, 1990). Therefore, it is thought that melatonin binds to specific receptors found on lymphocytes, which then secrete these melatonin-induced opioid (MIO) peptides to modulate immune function. It is through this pineal– immune–opioid network that melatonin appears to function as an immunostimulatory agent in mammals. Currently, there are no similar reports of opioid
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involvement for the observed melatonin immunoenhancement in avian species. Based on reports in mammals, we tested the hypothesis that melatonin immunoenhancement in the quail is mediated via an opiatergic mechanism. Analogous to experimentation in mice, the specific opioid antagonist naltrexone was utilized in an attempt to abolish the immunoenhancement by melatonin in the Japanese quail.
2. Materials and methods 2.1. Husbandry Adult female Japanese quail, greater than 10 weeks of age, were used in the study. All birds were randomly selected for each treatment group and placed in one of four light-controlled boxes, which were housed in a room maintained at 23 3 °C and constant darkness (DD). Each light-controlled box was maintained on LD16:8 (lights off at 1700). Two 40-W incandescent bulbs provided light with a mean intensity of approximately 700–800 lux at bird level and each box was mechanically ventilated. Each treatment group was provided ad libitum access to food and water. 2.2. Experimental Design This experiment was conducted to determine if melatonin enhanced a cellular and humoral immune response in the Japanese quail via an opiatergic mechanism. All birds were maintained on a long photoperiod (LD16:8) throughout the experiment. It is known that birds placed in long photoperiods have reduced melatonin secretion as compared to those in short photoperiods. Therefore, the photoperiod in the present study was used to allow for greater latitude in responsiveness to exogenous melatonin supplementation. One hundred and twenty female adult quail were selected at random and placed in one of six treatment groups (N ¼ 20 birds per treatment). The treatment groups were as follows: Control (diluent), melatonin only (MLT), naltrexone only (NAL 10 mg/kg), MLT + NAL (0.1 mg/kg), MLT + NAL (1.0 mg/kg), MLT + NAL (10.0 mg/kg). Melatonin was administered continuously to the appropriate treatment group via the drinking water. Doses of melatonin were prepared weekly by dissolving the appropriate amount in 1 ml of 95% ethanol and diluting to 1 L with distilled water. The melatonin dose (50.0 lg/ ml) used in the present experiment has been reported to produce immunoenhancement of Japanese quail in which endogenous melatonin levels were elevated under a short photoperiod (LD8:16) (Moore and Siopes, 2002). Therefore, it was reasonable to assume that this dose should produce immunoenhancement in quail
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maintained in longer photoperiods (LD16:8), in which endogenous melatonin secretion is reduced as compared to short photoperiods (LD8:16). A daily 0.2 ml injection of naltrexone was given to the appropriate treatment groups in the pectoral muscle just prior to lights-off time (1700). Naltrexone was prepared at three dose levels: 0.1, 1.0, and 10.0 mg/kg. This dose range was determined based on previous experimentation in mice, in which 1.0 mg/kg naltrexone was successful in completely abolishing the immunoenhancing effects of melatonin (Maestroni et al., 1988b). All naltrexone-only birds received the highest dose of naltrexone (10.0 mg/kg) in the same manner as all other naltrexone treatments. Birds in the control and naltrexone-only groups received a distilled water/ethanol solution (diluent) in the drinking water instead of melatonin. On day 15 of treatment, a cellular and humoral immune response was evaluated at the same time of day (1600) and within the same bird. In order to evaluate the cellular response, the cutaneous basophil hypersensitivity reaction to phytohemagglutinin (PHA-P), a lectin from Phaseolus vulgaris (Sigma Chemical, St. Louis, MO), was measured in the wing web of each bird (Stadecker et al., 1977). An intradermal injection of PHA-P was given in the wing web and the dermal swelling response was measured as the percent increase in wingweb thickness at the injection site 24 h post-PHA-P injection. The humoral immune response was evaluated by measurement of antibody titers following administration of a 0.5 ml intravenous injection of a 10% Chukar red blood cell (CRBC) suspension into the jugular vein. This injection was given at the same time as the PHA-P injection. The primary antibody response was measured 7 days following the CRBC injections using a microagglutination assay (Sever, 1962). All treatments were continued throughout the immune evaluations. The PHA-P and antibody data obtained for each treatment was averaged and expressed as a percentage increase or decrease from the averaged data obtained for the diluent-only treatments. 2.3. Statistical Analysis All data were analyzed by one-way analysis of variance (ANOVA) using the General Linear Model procedure of the SAS institute (SAS Institute, 1990). Significant differences among means were estimated using least-square means. Statements of statistical significance are based on P 6 0:05.
3. Results Fig. 1 illustrates the effect of concomitant melatonin and naltrexone supplementation on the cellular immune responses. It was clear that the cellular immune response
Fig. 1. Cellular immune response of adult Japanese quail following concomitant naltrexone and melatonin administration. All birds were in a long photoperiod (LD16:8) throughout the experiment. The treatment groups were as follows: control (diluent only), melatonin (no naltrexone), 0.1 mg/kg naltrexone + melatonin, 1.0 mg/kg naltrexone + melatonin, 10.0 mg/kg naltrexone + melatonin, naltrexone (10.0 mg/ kg) + diluent. Melatonin was provided continuously via the drinking water at a dose of 50 lg/ml. Naltrexone was administered daily via an intramuscular injection into the pectoral muscle just prior to the scotophase. On day 15 of treatment, the cellular immune response was determined in each treatment by subcutaneous injection of phytohemagglutinin (PHA-P) into the wing-web of each bird. The percent change in wing web thickness was calculated 24 h following injection and reported as the level of cellular immunity on the day of injection. Means with no common superscript differ significantly, P 6 0:05. N (range) 14–20 birds per treatment, root mean square error (RMSE) ¼ 34.5.
was significantly increased in birds receiving only melatonin (P ¼ 0:047) as compared to control birds (diluent only). This lymphoproliferative response to phytohemagglutinin (PHA-P) was increased by 22% in melatonin-treated birds as compared to untreated birds. In birds receiving melatonin and naltrexone, the 22% immunoenhancement of cellular immune responses by melatonin alone was significantly reduced. All naltrexone doses produced significant diminution of melatonin immunoenhancement in a dose response manner. In contrast to the effects observed following concomitant administration of naltrexone and melatonin, naltrexone administered alone produced significant immunoenhancement as compared to untreated controls. Fig. 2 illustrates the effect of naltrexone supplementation on the immunoenhancement of a humoral immune response by melatonin. The primary antibody response to Chukar red blood cells (CRBC) was significantly increased (P ¼ 0:0001) by melatonin treatment as compared to untreated birds. Melatonin supplementation increased antibody responses by 34% as compared to untreated birds. Consistent with the cellular immune data, all doses of naltrexone significantly decreased the melatonin-induced enhancement of antibody responses. However, in contrast to the cellular immune
C.B. Moore, T.D. Siopes / General and Comparative Endocrinology 131 (2003) 258–263
Fig. 2. Humoral immune response of adult Japanese quail following concomitant naltrexone and melatonin administration. All birds were in a long photoperiod (LD16:8) throughout the experiment. All treatments were as described in Fig. 1. On day 15 of treatment, the humoral immune response was determined in each treatment by intravenous injection of a Chukar red blood cell (CRBC) suspension into the jugular vein of each bird. The primary antibody titers were measured 7 days following injection with CRBC and reported as the level of humoral immunity on the day of immunization. Means with no common superscript differ significantly, P 6 0:05. N (range) 16–20 birds per treatment, RMSE ¼ 0.849.
data, naltrexone administered alone had no effect on antibody responses. No significant differences in egg production were observed between treatments therefore no significant difference in gonadal steroids should be expected.
4. Discussion From the results of the present study, it was clear that melatonin enhanced cellular and humoral immune responses in Japanese quail and that the response involved an opiatergic mechanism. This is consistent with our previous published results (Moore and Siopes, 2000). The opioid antagonist naltrexone abolished melatonin immunoenhancement of cellular and humoral immune responses across all dose levels and this is in agreement with published work by Maestroni on mice using 1.0 mg/ kg naltrexone (Maestroni et al., 1987b). Thus, in both birds and mammals the immunoenhancing effect of melatonin appears to be mediated by opioid peptides. Previous reports have established melatonin as an immune modulating hormone in mammals and birds. In mammals, several reports have detailed the immunoenhancing properties of melatonin and it has been suggested that melatonin may be part of a complex physiological system to coordinate reproductive, immunological, and other physiological processes to help cope with stresses associated with seasonal change and exposure to pathogen (Nelson and Drazen, 1999). It has also been suggested that this integrative system involves
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the opioid peptides. Maestroni (2001) reported that melatonin stimulated T-helper cells to secrete opioid peptides, which have an up-regulatory effect on a variety of immune cells. He indicated that melatonin functions as an immunoenhancing agent via this melatonin–immune–opioid network. Therefore, it appears that melatonin has a direct effect on immune cells, which is subsequently amplified within the immune system by opioids. The opioid peptides are known to have a wide range of effects on immunocompetent cells (Fischer and Falke, 1984). Analogous to the mammalian literature, recent reports in birds have suggested a close connection between melatonin and immune responses. It was found that exogenous melatonin could counteract the immunosuppressive effects of constant light and pinealectomy (Moore and Siopes, 2000; Moore et al., 2002). In addition, melatonin administration to immunologically unstressed quail produced significant immunoenhancement (Moore and Siopes, 2002). Therefore, as in mammals, melatonin is a regulator of immune function. It has been suggested that melatonin also acts as a mediator of environmental information in the bird, which can be transmitted quickly to the immune system for an appropriate response. As in mammals, this may be particularly useful for communication regarding changes in season and stress (Nelson and Demas, 1996). The current study is the first to implicate the opioid peptides as an important component of this melatonin–immune network in birds. Contrary to the effect of concomitant naltrexone and melatonin treatments on immune function, naltrexone administered alone had a stimulatory effect on cellular immune responses but not humoral responses. This was not completely surprising since the opioid peptides have been shown to exert diverse effects on the immune system (Fischer and Falke, 1984; Weber and Pert, 1984; Plotnikoff et al., 1986). In addition, naltrexone treatments have been shown to attenuate surgery-induced immunosuppression in the mouse, suggesting an immunosuppressive property of endogenous opioids (Nelson et al., 2000). The results in the present study suggest that endogenous opioid regulation of cell-mediated immune function can be different than that of humoral immune responses. In addition, this extends beyond the observed stimulatory effect in the presence of exogenous melatonin. It appears that in the absence of exogenous melatonin, endogenous opioids have an inhibitory role on the cellular immune response, whereas opioids do not seem to be affecting humoral immune responses in the absence of exogenous melatonin (Figs. 1 and 2). It was clear that naltrexone abolished the immunoenhancing effects of melatonin on the cellular immune response in a dose response manner. The highest dose (10.0 mg/kg) not only significantly reduced the immu-
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noenhancement by melatonin, but also produced significant reduction beyond that of controls. This was a surprising result considering the stimulatory response to naltrexone alone. At best, we expected the naltrexone treatment to abolish the immunoenhancement by melatonin and result in a response equivalent to controls. These birds were maintained on (LD16:8), therefore would have low levels of endogenous melatonin secretion relative to exposure to shorter photoperiods. However, this highest dose of naltrexone may have abolished the effect of the exogenous and endogenous melatonin on immune responses in this photoperiod. Another possibility is that exogenous melatonin has altered the overall action of opioids such that naltrexone reduced the cellular response below that of controls. A similar response occurred for the antibody response, but only at one dose level (1.0 mg/kg). This was 10-fold lower than the most effective dose for cellular immunity and suggests a different sensitivity to melatonin-induced opioids of the immunocompetent cells involved in each immune response. From the results in the present study, it was clear that melatonin immunoenhancement in the quail is mediated by the opioid peptides. These results further clarify the role of melatonin in modulating immune function in birds and confirm that the melatonin mechanisms of action are similar between mammals and birds.
References Axelrod, J., Fraschini, F., Velo, G.P., 1981. The pineal gland and its endocrine role. In: Proceedings of the NATO Advisory Study Institute, Erice, Italy. Plenum Press, New York. Bonilla, E., Rodon, C., Valero, N., Pons, H., Chacin-Bonilla, L., Garcia Tamayo, J., Rodriguez, Z., Medina-Leendertz, S., Anez, F., 2001. Melatonin prolongs survival of immunodepressed mice infected with the Venezuelan equine encephalomyelitis virus. Transactions of the Royal Society of Tropical Medicine and Hygiene 95 (2), 207–210. Brennan, C.P., Hendricks III, G.L., El-Sheikh, T.M., Mashaly, M.M., 2002. Melatonin and the enhancement of immune responses in immature male chickens. Poultry Science 81 (3), 371–375. Calvo, J.R., Rafii-el-Idrissi, M., Pozo, D., Guerrero, J.M., 1995. Immunomodulatory role of melatonin: specific binding sites in human and rodent lymphoid cells. Journal of Pineal Research 18 (3), 119–126. Fischer, E.G., Falke, N.E., 1984. Beta-endorphin modulates immune functions. Psychotherapy and Psychosomatics 42 (1–4), 195–204. Krieger, D.T., 1981. Endocrine Rhythms. Raven press, New York. Kumar, M.S., Chen, C.L., Sharp, D.C., Liu, J.M., Kalra, P.S., Kalra, S.P., 1982. Diurnal fluctuations in methionine-enkephalin levels in the hypothalamus and preoptic area of the male rat: effects of pinealectomy. Neuroendocrinology 35 (1), 28–34. Lakin, M.L., Miller, C.H., Stott, M.L., Winters, W.D., 1981. Involvement of the pineal gland and melatonin in murine analgesia. Life Science 19 (24), 2543–2547. Liu, Z.M., Pang, S.F., 1993. 125 I Iodomelatonin-binding sites in the bursa of Fabricius of birds: binding characteristics, subcellular
distribution, diurnal variations and age studies. Journal of Endocrinology 138 (1), 51–57. Maestroni, G.J., 2001. The immunotherapeutic potential of melatonin. Expert Opinon on Investigational Drugs 10 (3), 467– 476. Maestroni, G.J., Conti, A., Pierpaoli, W., 1986. Role of the pineal gland in immunity: circadian synthesis and release of melatonin modulates the antibody response and antagonizes the immunosuppressive effect of corticosterone. Journal of Neuroimmunology 13 (1), 19–30. Maestroni, G.J.M., Conti, A., 1989. b-Endorphin and dynorphin mimic the circadian immunoenhancing and anti-stress effects of melatonin. International Journal of Immunopharmacology 11 (4), 333–340. Maestroni, G.J.M., Conti, A., 1990. The pineal neurohormone melatonin stimulates activated CD4+, Thy-1+ cells to release opioid agonist(s) with immunoenhancing and anti-stress properties. Journal of Neuroimmunology 28 (2), 167–176. Maestroni, G.J.M., Conti, A., Pierpaoli, W., 1987b. Role of the pineal gland in immunity. II. Melatonin enhances the antibody response via an opiatergic mechanism. Clinical and Experimental Immunology 68 (2), 384–391. Maestroni, G.J.M., Conti, A., Pierpaoli, W., 1988a. Pineal melatonin, its fundamental immunoregulatory role in aging and cancer. Annals New York Academy of Science 521, 140–148. Maestroni, G.J.M., Conti, A., Pierpaoli, W., 1988b. Role of the pineal gland in immunity. III. Melatonin antagonizes the immunosuppressive effect of acute stress via an opiatergic mechanism. Immunology 63 (3), 465–469. Maestroni, G.J.M., Conti, A., Pierpaoli, W., 1989. Melatonin, stress, and the immune system. In: Reiter, R.J. (Ed.), Pineal Research Reviews, vol. 7. Alan R. Liss, New York, pp. 203–223. Maestroni, G.J.M., Conti, A., Pierpaoli, W., 1987a. The pineal gland and the circadian opiatergic immunoregulatory role of melatonin. Annals New York Academy of Science 496, 67– 77. Moore, C.B., Siopes, T.D., 2000. Effects of lighting conditions and melatonin supplementation on the cellular and humoral immune responses in Japanese quail Coturnix coturnix japonica. General and Comparative Endocrinology 119 (1), 95–104. Moore, C.B., Siopes, T.D., 2002. Melatonin can produce immunoenhancement in Japanese quail (Coturnix coturnix japonica) without prior immunosuppression. General and Comparative Endocrinology 129, 122–126. Moore, C.B., Siopes, T.D., Steele, C.T., Underwood, H., 2002. Pineal melatonin secretion, but not ocular melatonin secretion, is sufficient to maintain normal immune responses in Japanese quail (Coturnix coturnix japonica). General and Comparative Endocrinology 126 (3), 352–358. Nelson, C.J., Carrigan, K.A., Lysle, D.T., 2000. Naltrexone administration attenuates surgery-induced immune alterations in rats. Journal of Surgical Research 94 (2), 172–177. Nelson, R.J., Demas, G.E., 1996. Seasonal changes in immune function. Quarterly Review of Biology 71 (4), 511–548. Nelson, R.J., Drazen, D.L., 1999. Melatonin mediates seasonal adjustments in immune function. Reproduction, Nutrition, Development 39 (3), 383–398. Plotnikoff, N.P., Faith, R., Murgo, A.J., Good, R., 1986. Enkephalins and Endorphins: Stress and the Immune System. Plenum Press, New York. Poon, A.M., Liu, Z.M., Pang, C.S., Brown, G.M., Pang, S.F., 1994. Evidence for a direct action of melatonin on the immune system. Biological Signals 3 (2), 107–117. Reiter, R.J., 1984. The Pineal Gland. Raven Press, New York. Rodriguez, A.B., Lea, R.W., 1994. Effect of pinealectomy upon the nonspecific immune response of the ring-dove Streptopelia risoria. Journal of Pineal Research 16 (3), 159–166.
C.B. Moore, T.D. Siopes / General and Comparative Endocrinology 131 (2003) 258–263 SAS Institute, 1990. SAS/STATâ UserÕs Guide. Version 6, fourth ed., vol. 2, SAS Institute Inc., Cary, NC. Sever, J.L., 1962. Application of a microtechnique to viral serological investigations. Journal of Immunology 88 (3), 320–329. Sibinga, N.E., Goldstein, A., 1988. Opioid peptides and opioid receptors in cells of the immune system. Annual Review of Immunology 6, 219–249.
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Stadecker, M.J., Lukic, M., Dvorak, A., Leskowitz, S., 1977. The cutaneous basophil response to phytohemagglutinin in chickens. Journal of Immunology 118 (5), 1564–1568. Weber, R.S., Pert, C.B., 1984. Opiatergic modulation of the immune system. In: Miller, E.E., Genazzani, A.R. (Eds.), Central and Peripheral Endorphins: Basic and Clinical Aspects. Raven Press, New York, p. 35.
ENDOCRINOLOGY General and Comparative Endocrinology 131 (2003) 258–263 www.elsevier.com/locate/ygcen
Melatonin enhances cellular and humoral immune responses in the Japanese quail (Coturnix coturnix japonica) via an opiatergic mechanism C.B. Moore and T.D. Siopes* Department of Poultry Science, North Carolina State University, Raleigh, NC 27695-7608, USA Accepted 12 December 2002
Abstract It is known that melatonin has important immunomodulatory properties in the Japanese quail. However, the mechanism of melatonin action on the immune system is not clearly understood in avian species. In mammals, the immunostimulatory properties of melatonin are mediated by the release of opioid peptides from activated T-lymphocytes. The present study was performed to determine if these same melatonin-induced opioids (MIO) are involved with the immunoenhancing effects of melatonin in quail. Three treatment groups were given melatonin (50 lg/ml) in the drinking water ad libitum along with naltrexone, a known opioid receptor-blocking agent. Melatonin was administered throughout the 3 week study and each bird received a daily intramuscular injection of naltrexone at a dose of 0.1, 1.0, or 10.0 mg/kg. In addition, three control groups were established that received only melatonin, naltrexone, or diluent. Evaluation of the cellular and humoral immune responses was initiated after 2 weeks of treatments. A cutaneous basophil hypersensitivity reaction to phytohemagglutinin (PHA-P) was measured to evaluate the cellular immune response. To evaluate the humoral immune response, primary antibody titers were determined 7 days post-intravenous injection with a Chukar red blood cell (CRBC) suspension. Both the cellular and humoral immune responses were significantly increased by 22 and 34%, respectively, upon melatonin exposure as compared to quail receiving diluent only. Concomitant administration of naltrexone and melatonin significantly reduced the immunoenhancing effect of melatonin across all naltrexone doses. We conclude that melatonin enhances a cellular and humoral immune response in Japanese quail via an opiatergic mechanism. Ó 2003 Elsevier Science (USA). All rights reserved.
1. Introduction Knowledge about the physiological significance of the pineal gland has increased dramatically in recent years. This gland and its primary secretory product melatonin (N-acetyl-5-methoxytryptamine) has been linked to several physiological mechanisms including but not limited to gonadal, thyroid, adrenal and immune functions (Axelrod et al., 1981; Krieger, 1981; Reiter, 1984). It is thought that the pineal gland transduces environmental information through the circadian synthesis and secretion of melatonin. This allows the organism to respond appropriately to an environmental demand (Reiter, 1984). Consequently, the use of this
* Corresponding author. E-mail address: [email protected] (T.D. Siopes).
hormone in modulating several biological functions has been widely investigated in both mammalian and avian species. The existing literature in mammals and birds suggests that there is a close connection between melatonin and immune regulation. It has been observed in mammalian species that melatonin has important immunostimulatory properties, especially in immunocompromised states. Maestroni et al. (1986) demonstrated that inhibition of melatonin synthesis leads to depressed cellular and humoral immune responses in mice. In addition, exogenous melatonin administration antagonizes the immunodepressive effect of corticosterone and cyclophosphamide treatment in mice (Maestroni et al., 1986, 1987a). Furthermore, melatonin treatment ameliorates the survivability of mice immunodepressed by dexamethosone treatment or to exposure to Venezuelan equine encephalomyelitis virus (Bonilla et al., 2001). Therefore,
0016-6480/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0016-6480(03)00011-X
C.B. Moore, T.D. Siopes / General and Comparative Endocrinology 131 (2003) 258–263
the effectiveness of melatonin as a potent immunostimulatory agent is established in mammals. Similiar reports of immunoenhancement by melatonin exist in birds. Pinealectomized ring-doves have reduced immune responses as compared to sham-operated birds, suggesting an immunoenhancing effect for melatonin (Rodriguez and Lea, 1994). Also, pinealectomy reduced cellular and humoral immune responses in Japanese quail, which was improved to normal levels by melatonin replacement (Moore et al., 2002). Furthermore, birds kept in constant light, which suppresses endogenous melatonin levels, have reduced immune responses as compared to birds in light-dark cycles and melatonin can reconstitute this weakened immune system in a dose response manner (Moore and Siopes, 2000). Recently we have demonstrated that exogenous melatonin treatments can even boost immune responses in normal (immunologically unsuppressed) quail, suggesting potent immunoenhancing effects of melatonin in the quail (Moore and Siopes, 2002). In addition, it has been shown that exogenous melatonin enhances the activities of B and T lymphocytes in immature chickens (Brennan et al., 2002). Therefore, in both mammals and birds, melatonin is certainly an important immune regulator. However, the mechanism of melatonin action in boosting immune responses is not known in avian species. In mammals, it is known that melatonin receptors exist on circulating lymphocytes, suggesting direct communication between melatonin and immune cells (Liu and Pang, 1993; Poon et al., 1994; Calvo et al., 1995). Furthermore, it is known that the endogenous opioids have powerful immunomodulatory and analgesic properties (Sibinga and Goldstein, 1988). Melatonin has anticonvulsant and analgesic properties in mice, suggesting a possible relationship between melatonin and opioids (Lakin et al., 1981; Kumar et al., 1982). Based on these reports, Maestroni et al. (1987b, 1988a,b, 1989) investigated the opioid peptides as potential mediators of the observed immunoenhancing effects of melatonin in mice. It was found that the specific opioid antagonist naltrexone could completely abolish the immunoenhancing and anti-stress effects of melatonin. In addition, the immunoenhancing effects of melatonin were reproduced by administration of opioid peptides such as dynorphin 1–13 and b-endorphin (Maestroni and Conti, 1989). It was also shown that physiological concentrations of melatonin could stimulate secretion of opioid peptides from activated CD4þ T-helper lymphocytes in vitro (Maestroni and Conti, 1990). Therefore, it is thought that melatonin binds to specific receptors found on lymphocytes, which then secrete these melatonin-induced opioid (MIO) peptides to modulate immune function. It is through this pineal– immune–opioid network that melatonin appears to function as an immunostimulatory agent in mammals. Currently, there are no similar reports of opioid
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involvement for the observed melatonin immunoenhancement in avian species. Based on reports in mammals, we tested the hypothesis that melatonin immunoenhancement in the quail is mediated via an opiatergic mechanism. Analogous to experimentation in mice, the specific opioid antagonist naltrexone was utilized in an attempt to abolish the immunoenhancement by melatonin in the Japanese quail.
2. Materials and methods 2.1. Husbandry Adult female Japanese quail, greater than 10 weeks of age, were used in the study. All birds were randomly selected for each treatment group and placed in one of four light-controlled boxes, which were housed in a room maintained at 23 3 °C and constant darkness (DD). Each light-controlled box was maintained on LD16:8 (lights off at 1700). Two 40-W incandescent bulbs provided light with a mean intensity of approximately 700–800 lux at bird level and each box was mechanically ventilated. Each treatment group was provided ad libitum access to food and water. 2.2. Experimental Design This experiment was conducted to determine if melatonin enhanced a cellular and humoral immune response in the Japanese quail via an opiatergic mechanism. All birds were maintained on a long photoperiod (LD16:8) throughout the experiment. It is known that birds placed in long photoperiods have reduced melatonin secretion as compared to those in short photoperiods. Therefore, the photoperiod in the present study was used to allow for greater latitude in responsiveness to exogenous melatonin supplementation. One hundred and twenty female adult quail were selected at random and placed in one of six treatment groups (N ¼ 20 birds per treatment). The treatment groups were as follows: Control (diluent), melatonin only (MLT), naltrexone only (NAL 10 mg/kg), MLT + NAL (0.1 mg/kg), MLT + NAL (1.0 mg/kg), MLT + NAL (10.0 mg/kg). Melatonin was administered continuously to the appropriate treatment group via the drinking water. Doses of melatonin were prepared weekly by dissolving the appropriate amount in 1 ml of 95% ethanol and diluting to 1 L with distilled water. The melatonin dose (50.0 lg/ ml) used in the present experiment has been reported to produce immunoenhancement of Japanese quail in which endogenous melatonin levels were elevated under a short photoperiod (LD8:16) (Moore and Siopes, 2002). Therefore, it was reasonable to assume that this dose should produce immunoenhancement in quail
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maintained in longer photoperiods (LD16:8), in which endogenous melatonin secretion is reduced as compared to short photoperiods (LD8:16). A daily 0.2 ml injection of naltrexone was given to the appropriate treatment groups in the pectoral muscle just prior to lights-off time (1700). Naltrexone was prepared at three dose levels: 0.1, 1.0, and 10.0 mg/kg. This dose range was determined based on previous experimentation in mice, in which 1.0 mg/kg naltrexone was successful in completely abolishing the immunoenhancing effects of melatonin (Maestroni et al., 1988b). All naltrexone-only birds received the highest dose of naltrexone (10.0 mg/kg) in the same manner as all other naltrexone treatments. Birds in the control and naltrexone-only groups received a distilled water/ethanol solution (diluent) in the drinking water instead of melatonin. On day 15 of treatment, a cellular and humoral immune response was evaluated at the same time of day (1600) and within the same bird. In order to evaluate the cellular response, the cutaneous basophil hypersensitivity reaction to phytohemagglutinin (PHA-P), a lectin from Phaseolus vulgaris (Sigma Chemical, St. Louis, MO), was measured in the wing web of each bird (Stadecker et al., 1977). An intradermal injection of PHA-P was given in the wing web and the dermal swelling response was measured as the percent increase in wingweb thickness at the injection site 24 h post-PHA-P injection. The humoral immune response was evaluated by measurement of antibody titers following administration of a 0.5 ml intravenous injection of a 10% Chukar red blood cell (CRBC) suspension into the jugular vein. This injection was given at the same time as the PHA-P injection. The primary antibody response was measured 7 days following the CRBC injections using a microagglutination assay (Sever, 1962). All treatments were continued throughout the immune evaluations. The PHA-P and antibody data obtained for each treatment was averaged and expressed as a percentage increase or decrease from the averaged data obtained for the diluent-only treatments. 2.3. Statistical Analysis All data were analyzed by one-way analysis of variance (ANOVA) using the General Linear Model procedure of the SAS institute (SAS Institute, 1990). Significant differences among means were estimated using least-square means. Statements of statistical significance are based on P 6 0:05.
3. Results Fig. 1 illustrates the effect of concomitant melatonin and naltrexone supplementation on the cellular immune responses. It was clear that the cellular immune response
Fig. 1. Cellular immune response of adult Japanese quail following concomitant naltrexone and melatonin administration. All birds were in a long photoperiod (LD16:8) throughout the experiment. The treatment groups were as follows: control (diluent only), melatonin (no naltrexone), 0.1 mg/kg naltrexone + melatonin, 1.0 mg/kg naltrexone + melatonin, 10.0 mg/kg naltrexone + melatonin, naltrexone (10.0 mg/ kg) + diluent. Melatonin was provided continuously via the drinking water at a dose of 50 lg/ml. Naltrexone was administered daily via an intramuscular injection into the pectoral muscle just prior to the scotophase. On day 15 of treatment, the cellular immune response was determined in each treatment by subcutaneous injection of phytohemagglutinin (PHA-P) into the wing-web of each bird. The percent change in wing web thickness was calculated 24 h following injection and reported as the level of cellular immunity on the day of injection. Means with no common superscript differ significantly, P 6 0:05. N (range) 14–20 birds per treatment, root mean square error (RMSE) ¼ 34.5.
was significantly increased in birds receiving only melatonin (P ¼ 0:047) as compared to control birds (diluent only). This lymphoproliferative response to phytohemagglutinin (PHA-P) was increased by 22% in melatonin-treated birds as compared to untreated birds. In birds receiving melatonin and naltrexone, the 22% immunoenhancement of cellular immune responses by melatonin alone was significantly reduced. All naltrexone doses produced significant diminution of melatonin immunoenhancement in a dose response manner. In contrast to the effects observed following concomitant administration of naltrexone and melatonin, naltrexone administered alone produced significant immunoenhancement as compared to untreated controls. Fig. 2 illustrates the effect of naltrexone supplementation on the immunoenhancement of a humoral immune response by melatonin. The primary antibody response to Chukar red blood cells (CRBC) was significantly increased (P ¼ 0:0001) by melatonin treatment as compared to untreated birds. Melatonin supplementation increased antibody responses by 34% as compared to untreated birds. Consistent with the cellular immune data, all doses of naltrexone significantly decreased the melatonin-induced enhancement of antibody responses. However, in contrast to the cellular immune
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Fig. 2. Humoral immune response of adult Japanese quail following concomitant naltrexone and melatonin administration. All birds were in a long photoperiod (LD16:8) throughout the experiment. All treatments were as described in Fig. 1. On day 15 of treatment, the humoral immune response was determined in each treatment by intravenous injection of a Chukar red blood cell (CRBC) suspension into the jugular vein of each bird. The primary antibody titers were measured 7 days following injection with CRBC and reported as the level of humoral immunity on the day of immunization. Means with no common superscript differ significantly, P 6 0:05. N (range) 16–20 birds per treatment, RMSE ¼ 0.849.
data, naltrexone administered alone had no effect on antibody responses. No significant differences in egg production were observed between treatments therefore no significant difference in gonadal steroids should be expected.
4. Discussion From the results of the present study, it was clear that melatonin enhanced cellular and humoral immune responses in Japanese quail and that the response involved an opiatergic mechanism. This is consistent with our previous published results (Moore and Siopes, 2000). The opioid antagonist naltrexone abolished melatonin immunoenhancement of cellular and humoral immune responses across all dose levels and this is in agreement with published work by Maestroni on mice using 1.0 mg/ kg naltrexone (Maestroni et al., 1987b). Thus, in both birds and mammals the immunoenhancing effect of melatonin appears to be mediated by opioid peptides. Previous reports have established melatonin as an immune modulating hormone in mammals and birds. In mammals, several reports have detailed the immunoenhancing properties of melatonin and it has been suggested that melatonin may be part of a complex physiological system to coordinate reproductive, immunological, and other physiological processes to help cope with stresses associated with seasonal change and exposure to pathogen (Nelson and Drazen, 1999). It has also been suggested that this integrative system involves
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the opioid peptides. Maestroni (2001) reported that melatonin stimulated T-helper cells to secrete opioid peptides, which have an up-regulatory effect on a variety of immune cells. He indicated that melatonin functions as an immunoenhancing agent via this melatonin–immune–opioid network. Therefore, it appears that melatonin has a direct effect on immune cells, which is subsequently amplified within the immune system by opioids. The opioid peptides are known to have a wide range of effects on immunocompetent cells (Fischer and Falke, 1984). Analogous to the mammalian literature, recent reports in birds have suggested a close connection between melatonin and immune responses. It was found that exogenous melatonin could counteract the immunosuppressive effects of constant light and pinealectomy (Moore and Siopes, 2000; Moore et al., 2002). In addition, melatonin administration to immunologically unstressed quail produced significant immunoenhancement (Moore and Siopes, 2002). Therefore, as in mammals, melatonin is a regulator of immune function. It has been suggested that melatonin also acts as a mediator of environmental information in the bird, which can be transmitted quickly to the immune system for an appropriate response. As in mammals, this may be particularly useful for communication regarding changes in season and stress (Nelson and Demas, 1996). The current study is the first to implicate the opioid peptides as an important component of this melatonin–immune network in birds. Contrary to the effect of concomitant naltrexone and melatonin treatments on immune function, naltrexone administered alone had a stimulatory effect on cellular immune responses but not humoral responses. This was not completely surprising since the opioid peptides have been shown to exert diverse effects on the immune system (Fischer and Falke, 1984; Weber and Pert, 1984; Plotnikoff et al., 1986). In addition, naltrexone treatments have been shown to attenuate surgery-induced immunosuppression in the mouse, suggesting an immunosuppressive property of endogenous opioids (Nelson et al., 2000). The results in the present study suggest that endogenous opioid regulation of cell-mediated immune function can be different than that of humoral immune responses. In addition, this extends beyond the observed stimulatory effect in the presence of exogenous melatonin. It appears that in the absence of exogenous melatonin, endogenous opioids have an inhibitory role on the cellular immune response, whereas opioids do not seem to be affecting humoral immune responses in the absence of exogenous melatonin (Figs. 1 and 2). It was clear that naltrexone abolished the immunoenhancing effects of melatonin on the cellular immune response in a dose response manner. The highest dose (10.0 mg/kg) not only significantly reduced the immu-
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noenhancement by melatonin, but also produced significant reduction beyond that of controls. This was a surprising result considering the stimulatory response to naltrexone alone. At best, we expected the naltrexone treatment to abolish the immunoenhancement by melatonin and result in a response equivalent to controls. These birds were maintained on (LD16:8), therefore would have low levels of endogenous melatonin secretion relative to exposure to shorter photoperiods. However, this highest dose of naltrexone may have abolished the effect of the exogenous and endogenous melatonin on immune responses in this photoperiod. Another possibility is that exogenous melatonin has altered the overall action of opioids such that naltrexone reduced the cellular response below that of controls. A similar response occurred for the antibody response, but only at one dose level (1.0 mg/kg). This was 10-fold lower than the most effective dose for cellular immunity and suggests a different sensitivity to melatonin-induced opioids of the immunocompetent cells involved in each immune response. From the results in the present study, it was clear that melatonin immunoenhancement in the quail is mediated by the opioid peptides. These results further clarify the role of melatonin in modulating immune function in birds and confirm that the melatonin mechanisms of action are similar between mammals and birds.
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