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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, 95–104 (2000) doi:10.1006/gcen.2000.7496, available online at http://www.idealibrary.com on
Effects of Lighting Conditions and Melatonin Supplementation on the Cellular and Humoral Immune Responses in Japanese Quail Coturnix coturnix japonica C. B. Moore and T. D. Siopes 1 Department of Poultry Science, Box 7608, North Carolina State University, Raleigh, North Carolina 27695-7608 Accepted April 3, 2000
The humoral immune response increased to a peak at the 5.0 g/ml dose while the cellular immune response increased across all dose levels. From the present study it was clear that quail placed in daily light-dark cycles (LD), possessing a diurnal rhythm of melatonin, had significantly elevated immune responses as compared to those birds in LL. Furthermore, melatonin supplemented to birds exposed to LL was immuno-enhancing. This suggests that melatonin may be a mediator of the differences seen between LD and LL lighting conditions and may have important immune modulating properties. © 2000
Two experiments were conducted to determine the effects of lighting conditions and melatonin supplementation on the cellular and humoral immune responses in Japanese quail. The first experiment was designed to evaluate differing light regimes as immune modulators in both adult and juvenile quail. The cellular and humoral immune responses were determined for three lighting conditions; short days (8:16LD), long days (16:8LD), and constant light (LL). In the second experiment, melatonin was administered in varying doses to adult quail placed in LL. The doses used in this experiment were 0.0, 0.5, 5.0, and 50.0 g/ml melatonin given in the drinking water for 16 h per day for 2 weeks. The cellular and humoral immune responses were evaluated after 1 week of melatonin treatment. In both experiments, 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 calculated 7 days postintravenous injection with a Chukar red blood cell suspension. In the adult birds of experiment 1, both the 8:16LD and 16:8LD treatments produced similar cellular and humoral immune responses but these responses were significantly greater than those observed in LL. The juvenile birds held under 8:16LD also had significantly greater cellular and humoral immune responses as compared to juvenile birds held in LL. In experiment 2, there was a clear melatonin dose response on immune function in LL. 1
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Key Words: melatonin; photoperiod; immune response; Japanese quail. It is well established that melatonin biosynthesis and secretion in essentially all vertebrates are affected by environmental light. The enzymatic processes leading to melatonin biosynthesis are suppressed by light and enhanced by darkness (Reiter, 1988). Thus, animals respond to increased daylengths (decreased dark period) by reducing the duration of nocturnal melatonin secretion (Kumar and Follett, 1993; Underwood and Siopes, 1985) while circulating melatonin levels may be completely suppressed to basal levels in constant light (LL) (Ralph et al., 1975; Siopes, unpublished). In recent years, researchers have found an association between the duration of environmental light and some immune parameters of mammals. Most of this
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research in mammals has indicated that in short photoperiods both the cellular and the humoral components of the immune system were enhanced as compared to mammals kept in long photoperiods (Champney and McMurray, 1991; Mahmoud et al., 1994; Blom et al., 1994; Nelson and Blom, 1994; Demas et al., 1996; Demas and Nelson, 1996). Furthermore, the pineal hormone melatonin may be the mediator of these photoperiod-immune effects due to its direct and indirect effects on immune function (Maestroni et al., 1986; Caroleo et al., 1992; Maestroni, 1993; Nelson et al., 1995; Liebmann et al., 1997). There are few reports about melatonin as an immune modulator for avian species and there is no literature establishing a short-day immuno-enhancing effect. It has been suggested that melatonin injections do not have an immunostimulatory effect on immune indices in the chicken (Skwarlo-Sonta, 1991; SkwarloSonta, 1992). However, pinealectomized ring doves have reduced immune responses as compared to sham-operated birds, suggesting an immuno-enhancing effect for melatonin (Rodriguez et al., 1994). Furthermore, a link between melatonin and certain nonspecific immune responses has been established in the ring dove (Rodriguez et al., 1997, 1999). Melatonin has also been shown to have some useful antioxidant properties in this species (Rodriguez et al., 1998, 1999). Also, exogenous melatonin boosted cellular immune function in photostimulated male starlings (Bentley et al., 1998). However, melatonin has been shown to reduce primary and secondary immune responses in the chicken (Giannessi et al., 1992). Therefore, unlike the extensive mammalian literature, the avian literature is sparse and inconsistent about a role for lighting conditions or melatonin in immune function. Most of the literature for photoperiod effects on the avian immune system is indirect, reflected by growth, performance, and reproductive changes (Lewis and Morris, 1995). However, Kirby and Froman (1991) reported a suppressed cellular immunity and secondary antibody response in immature cockerels reared under LL as compared to 12:12LD (light-dark) daily cycles. Therefore the objective of our research was to determine if changes in lighting conditions could alter immune function in the Japanese quail and, if so, to determine if melatonin is involved. This was accomplished by establishing a LD vs LL treatment difference in immune function and then determining if
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Moore and Siopes
melatonin was the mediator of this effect, by use of a replacement therapy technique. We hypothesized an immuno-inhibition in LL, due to suppressed levels of melatonin, and immuno-enhancement as melatonin was increased artificially (replacement) or naturally (light-dark cycle).
MATERIALS AND METHODS Husbandry Adult and juvenile Japanese quail were used in the study. All adult birds were females and greater than 5 weeks of age whereas the mixed-sex juvenile quail were 21 days old at the start of the experiments. Japanese quail are considered mature and produce sperm or ovulate by 5 to 6 weeks of age on long daylength. Males mature in advance of females and testicular weights and testosterone remain very low until 21 days of age (Ottinger and Brinkley, 1979). Birds were randomly selected for each treatment group and placed in one of three light-controlled boxes which were housed in a room maintained at 23 ⫾ 3°C and constant darkness (DD). The lighting conditions used in the study were 8:16LD (light:dark), 16:8LD (light:dark), and constant light (LL). The daily lights-on time was 0800 for both LD cycle treatment groups. Four 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.
Experiment 1 This experiment was designed to evaluate lighting condition as an immune modulator in adult and juvenile birds. Three different lighting conditions were used, short days (8:16LD), long days (16:8LD), and constant light (LL), to provide long, short, and no daily dark period, respectively. It was expected that LL continually suppressed circulating melatonin to basal levels whereas the dark periods in the LD cycles provided increased daily melatonin levels in proportion to the length of the dark period.
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Fifteen female adult birds were selected for each lighting condition and remained in this treatment for 3 weeks prior to immune response evaluation. As a consequence, the birds on short days had regressed ovaries whereas those on long days had fully developed ovaries and were ovulating. Likewise, 15 unsexed juvenile birds, 14 days posthatch, received 1 week of exposure to 8:16LD and LL treatments. At 21 days of age the gonads remained undeveloped and immune challenges were started. All birds remained on their respective lighting protocols during the period of immune response evaluation. The cutaneous basophil hypersensitivity reaction to phytohemagglutinin (PHA-P), a lectin from Phaseolus Vulgaris (Sigma Chemical Co., St. Louis, MO), was measured in the wing web of each bird to evaluate the cellular immune response (Stadecker et al., 1977). An intradermal injection of PHA-P was given in the wing web of each bird and at the same time of day (1400) in all treatment groups. The dermal swelling response was measured as the percentage increase in wing-web thickness at the injection site 24 h post-PHA-P injections. 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. The primary antibody response was measured 7 days following the CRBC injections using a microagglutination assay (Sever, 1961). Both the CRBC injections and the primary antibody titers were performed at the same time of day as the cellular immune response (1400). In addition, the total and differential white blood cell (WBC) counts of the adult quail were counted using a method provided by Natt and Herrick (1952). Differential counts of 100 leukocytes per blood smear were made following staining of the smear with a Diff-Quik stain set (Baxter Scientific, McGaw Park, IL) which is a modified WrightGiemsa stain. Total WBC and differentials were measured at four time intervals: 0, 2, 4, and 7 days postCRBC. Blood was obtained from the brachial vein to perform the WBC counts during the middle of the dark phase, in the LD groups, with use of a dim, site-localized light and for a maximum exposure of 3 min. Blood was collected from birds in the LL group at the same time of day. The juvenile birds were euthanized by carbon monoxide asphyxiation 7 days
postimmunization (28 days of age) and gonadal weights were obtained.
Experiment 2 This experiment was conducted to determine if exogenous melatonin administered to simulate daily plasma melatonin patterns typical in short photoperiods (8:16LD) could enhance the cellular and humoral immune responses of birds maintained in LL. Forty adult female birds were placed in two light-controlled boxes. These birds were allowed to adjust to LL for 2 weeks before the treatments were started. Four treatment groups (n ⫽ 10) were formed with 0.0, 0.5, 5.0, and 50 g/ml of melatonin given in the drinking water while the birds remained in LL. It has been reported that 100 g/ml melatonin given in the drinking water to birds placed in DD produced approximately 4 times the physiological levels of melatonin as compared to the normal peak nighttime levels seen in birds entrained to LD 12:12 (Underwood and Edmonds, 1995; Underwood et al., 1984). Since quail have a robust melatonin rhythm in DD, this must be due to additive effects of the exogenous and endogenous melatonin. Therefore, although plasma melatonin concentrations were not measured in the present study, the melatonin dose range used should result in plasma melatonin levels approximating physiological levels. In order to simulate melatonin plasma profiles typically observed on an 8:16LD photoperiod, the melatonin doses were administered for 16 h (1700 to 0900) and substituted with tap water for the remaining 8 h. Doses of melatonin were prepared daily by dissolving the appropriate amount in 1 ml of 95% ethanol and diluting to 1 L with distilled water. A control group (0.0 g/ml melatonin) was treated similarly but received a distilled water/ethanol solution (diluent) instead of melatonin at the same time of day as the treatment groups. After 1 week of melatonin supplementation, the cellular and humoral immune responses were evaluated by PHA-P and primary antibody responses, respectively, as described in experiment 1. The total and differential WBC counts were determined at three time intervals; 0, 3, and 7 days post-CRBC injection in the control group and 5.0 g/ml melatonin treatment group only. As in experiment 1, all injections and measurements were made at the same time of day in
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TABLE 1 Effect of Daily Lighting Conditions on Immune Responses in Adult and Juvenile Quail Cellular response 1
Humoral response 2
Photoperiod
Adult
Juvenile
Adult
Juvenile
8:16LD 16:8LD LL RMSE 3 N (Range) P⬎F
137.8 a 112.6 a 102.8 b 28.6 11–13 0.0001
230.7 a — 196.1 b 76.3 14–16 0.048
2.81 a 2.93 a 2.08 b 0.36 11–13 0.0001
2.65 a — 1.85 b 0.33 12–14 0.0001
a,b
Means within a column and with no common superscript differ significantly (P ⱕ 0.05). 1 Cellular data are expressed as a percentage change in wing web thickness, 24 h postphytohemagglutinin (PHA-P) injection. 2 Humoral data are expressed as a log 2 of the primary antibody titer. 3 Root-mean-square error.
each treatment group (1400). Melatonin or distilled water/ethanol administration was continued throughout all immune tests.
Statistical Analyses All data were analyzed by one-way analysis of variance (ANOVA) using the General Linear Model procedure of the SAS institute (SAS Institute, 1990). Repeated-measures ANOVA was used to assess time effects on white blood cell counts. Significant differences among means were estimated using least-square means. Statements of statistical significance are based on P ⱕ 0.05.
humoral immune response compared to LL. In all juvenile birds the mean gonadal weights at the end of the test (28 days of age) were 57 and 23 mg for ovaries and 125 and 8 mg for testes in LL and 8:16LD, respectively. Furthermore, there was no cloacal gland foam in the birds placed in LL, which is dependent on testosterone. The total WBC counts of adult quail under each lighting condition tested are expressed in Table 2. Prior to immunization with antigen, there was a significant elevation in total WBCs within the 8:16LD treatment group compared to the 16:8LD or LL groups, which were similar. At 2 and 4 days postimmunization, the two LD treatment groups had similar numbers of white blood cells and both were elevated compared to the LL group. There was a significant time effect as well as treatment main effect ⫻ time interaction. The data in Table 3 are the differential WBC counts before antigen stimulation and represent lighting effects on the differential counts. No significant differences occurred among treatments after inoculation compared to preinoculation; accordingly, only the results prior to inoculation (Day 0 of Table 2) are presented. The lymphocyte populations were significantly greater and heterophils significantly less for birds in the 8:16LD or 16:8LD treatments, compared to those in LL. Therefore, the heterophil/lymphocyte ratio was significantly increased within those birds kept
TABLE 2 Effect of Daily Lighting Conditions on Total White Blood Cell Counts in Adult Quail Following Immunization Total white blood cell (cells/mm 3 ⫻ 10 4) 1 Days
RESULTS Experiment 1 The data in Table 1 represent the cellular and humoral immune responses of adult and juvenile birds in each light treatment. Within the adult bird data, it was clear that both the 8:16LD and the 16:8LD groups had similar responses to PHA-P and CRBC and both were greater compared to the LL group. The juvenile bird responses were consistent with the adult data. Therefore LD treatments have a greater cellular and
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Photoperiod
0
2
4
7
8:16LD 16:8LD LL RMSE 2 N (Range) P⬎F
5.19 a 4.18 b 4.17 b 0.486 11–13 0.0039
6.34 a 6.46 a 4.66 b 0.401 11–13 0.0001
6.41 a 6.15 a 4.14 b 0.250 11–13 0.0001
3.88 a 3.93 a 3.06 a 0.197 10–12 0.162
a,b
Means within a column with no common superscript differ significantly, (P ⱕ 0.05). 1 Counts were taken at four time intervals: 0, 2, 4, and 7 days postantigenic challenge. Repeated-measures ANOVA for Days 0 to 7: Probability (P ⱖ F) for treatment ⫽ 0.0001; time ⫽ 0.0001; time ⫻ treatment ⫽ 0.0001. Pooled SEM ⫽ 0.34. 2 Root-mean-square error.
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TABLE 3 Effect of Daily Lighting Conditions on the Mean Differential White Blood Cell Counts in Adult Quail prior to Immunization
on the red blood cell count, therefore these data were not presented.
Differential cell count (%) 1 Photoperiod
Lymphocyte
Heterophil
H/L ratio
8:16LD 16:8LD LL RMSE 2 Pr ⬎ F
79 a 70 a 60 b 5.362 0.0001
13 a 19 a 35 b 5.585 0.0001
0.165 a 0.271 a 0.583 b 0.129 0.0001
a,b Means within a column with no common superscript differ significantly (P ⱕ 0.05). 1 Differential counts of 100 leukocytes per blood smear; N ⫽ 15. 2 Root-mean-square error.
in LL compared to the LD treatments. The white blood cell populations other than lymphocytes and heterophils were not different among the treatment groups. Also, there was no effect of lighting condition
Experiment 2 Figure 1 illustrates the cellular and humoral immune responses of quail placed in LL and given different oral doses of melatonin. All melatonin doses resulted in elevated cellular and humoral immune responses compared to the control group. The cellular immune response generally increased with the dose of melatonin and no clearly defined peak response occurred over the dose range used. The humoral immune response to oral melatonin doses significantly increased across all melatonin treatments compared to the control. A significant elevation occurred between each treatment except the 5.0 and 50.0 g/ml treatment groups.
FIG. 1. Cellular immune response (top) and primary antibody response (bottom) of adult Japanese quail given different doses of melatonin. All birds were in constant light (LL) throughout the experiment. The PHA-P-induced response (top) was measured 1 week following initiation of melatonin treatments. Immunizations were given at this time and titers (bottom) were determined 7 days postimmunization. (Top) N ⫽ 15 per treatment, RMSE ⫽ 45.2, P ⫽ 0.012. (Bottom) N ⫽ 6 – 8 per treatment, RMSE ⫽ 0.38, P ⫽ 0.0014. Means with no common superscript differ significantly (P ⱕ 0.05). Control group ⫽ 0.0 g/ml melatonin.
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DISCUSSION
TABLE 4 Mean Total and Differential White Blood Cell Count in Adult Quail Held in Constant Light and Following 7 Days Melatonin Treatment Total WBC (cells/mm ⫻ 10 ) Days postimmunization 3
4 1
Treatment
0
3
7
0.0 g/ml 5.0 g/ml RMSE 3 P⬎F
2.68 a 4.41 b 0.372 0.0001
3.59 a 8.02 b 0.486 0.0001
4.78 a 5.41 a 0.291 0.078
Differential WBC (%) 2 Cell type Treatment
Lymphocyte
Heterophil
H/L ratio
0.0 g/ml 5.0 g/ml RMSE 3 P⬎F
59 a 71 b 3.692 0.0001
28 a 17 b 3.861 0.0001
0.475 a 0.239 b 0.073 0.0001
a,b
Means within a column and with no common superscript differ significantly (P ⱕ 0.05). 1 Counts were taken at three time intervals: 0, 3, and 7 days postantigenic challenge. N (range) ⫽ 8 –9. 2 Differential counts of 100 leukocytes per blood smear, prior to immunization: N (range) ⫽ 8 –9. 3 Root-mean-square error.
Before antigen stimulation, and after 7 days of melatonin treatment (Day 0 postimmunization), the total WBC counts were elevated within the melatonin treatment group compared to the untreated control group (Table 4). On Day 3 postantigenic challenge, the melatonin group was still significantly elevated from controls. By the seventh day postimmunization, the WBC counts were not significantly different between treatment groups. The differential WBC in the two treatment groups was not significantly different following CRBC antigen stimulation as compared to preimmunization. Therefore, only the preimmunization results (Day 0) are given (Table 4). The melatonin treatment significantly enhanced the lymphocyte percentage while the heterophil percentage was significantly reduced. Accordingly, the heterophil/lymphocyte ratio was significantly elevated in the control group compared to the melatonin treatment. Also, melatonin treatment had no effect on red blood cell numbers therefore these data were not presented.
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From the results in experiment 1, it was clear that differing lighting conditions had similar qualitative effects on immune responses in both adult and juvenile Japanese quail. Both the cellular and humoral immune responses were greater when birds were placed in daily LD treatments as compared to LL. Clearly, the responses were not age dependent and were not dependent on state of sexual development because gonadal weights of the juvenile birds were similar among treatments and all were clearly immature in development. In addition, the adult immune responses in 16:8LD and LL were different yet both had similar ovarian status, that is, both were laying eggs. Our results are consistent with the report by Kirby and Froman (1991) in which 12:12LD photoperiods enhanced the phytohemagglutinin and secondary antibody responses of immature cockerels compared to LL treatment. We are not aware of any similar reports using adult birds. However, Bentley et al. (1998) reported that immune function was suppressed in adult starlings photostimulated with long days (18:6LD). In the mammalian literature, Blom et al. (1994) and Demas and Nelson (1996) reported an enhanced immune response in adult deer mice placed in short photoperiods. It was interesting that the boost in cellular immunity was greater in the juvenile birds as compared to the adult birds. This likely is related to the presence of immune tissue such as the thymus that subsequently regresses in the adult. Mahmoud et al. (1994) reported that photoperiod influences thymus morphology in rats and suggested an involvement by melatonin. The immuno-enhancing effect of LD vs LL light treatments in the present study was strong enough to be observed in the total number of white blood cells, with and without antigen challenge (Table 2). Interestingly, not only did LL treatment suppress WBC as compared to the LD treatments before antigen stimulation, but also birds in LL did not significantly increase WBC following an antigenic challenge, as did those birds in the light-dark cycles. This of course could mean decreased ability to resist certain antigenic challenges and result in adverse physiological consequences. The preimmunization WBC increase, in LD versus LL-treated birds, was associated with a preim-
Melatonin and Immunity in Quail
munization rise in the number of lymphocytes and a fall in the number of heterophils for birds placed in LD cycles. Therefore, the heterophil/lymphocyte ratio was lower in the LD treatments than for those birds placed in LL. An elevated H/L ratio is well established as a general indicator of stress, suggesting that LL was more stressful than long or short daily photoperiods. This is consistent with the adverse effects of LL on immune responses. Experiment 1 was designed to roughly mimic the range of natural lighting conditions experienced by birds during changes in temperate zone seasons. That is, the 8:16LD (short day) group was indicative of a “winter-like” condition while the 16:8LD (long day) group was more “summer-like.” It has been reported that seasonal changes in photoperiod may alter the immune responses of birds and ultimately the health of an overall population. Immunity is enhanced in the wintertime (short days) when the animal is subjected to many environmental stresses such as hypothermia and malnutrition (Nelson and Demas, 1996). The fact that there was no significant difference seen between fixed short and long photoperiods in the present study indicated that other factors might be involved in the reported seasonal variations of immune responses or that certain quantitative and/or qualitative characteristics of the light exposure may be important. Interestingly, Bentley et al. (1998) have reported that changes in reproductive state, and not simply a change in photoperiod, had a significant effect on the immune response of adult starlings. Although the results from experiment 1 demonstrated that LD cycles increase immune responses as compared to LL, a possible mechanism for this effect was not addressed. Certainly, the presence or absence of gonadal steroids does not seem to be an essential factor because the immuno-enhancing effect of LD cycles was seen in both adult and juvenile birds. This is in agreement with Blom et al. (1994) using adult deer mice. Likewise immuno-enhancement in 16:8LD as compared to LL occurred in the presence of fully functional ovaries. From research with mammals it has been suggested that differing photoperiods influence immune responses by altering the amount of circulating melatonin (Nelson et al., 1995). Also, melatonin supplementation can have immuno-enhancing effects in immunocompromised mammals (Maestroni, 1993; Piolo et al., 1993).
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In Japanese quail, we may assume that melatonin is completely suppressed to basal levels in LL and increases with an increased duration of the dark phase (Kumar and Follett, 1991). Because there can be substantial circulating melatonin present in LD treatment but only minimal (basal) levels present in LL, it would be consistent with melatonin as a possible mediator of the LD treatment-induced immuno-enhancement exhibited in experiment 1. Furthermore, although there was no significant difference seen between the 8:16LD and the 16:8LD treatments, the longer photoperiod was intermediate in every immune parameter measured. There certainly seems to be a trend toward increased photoperiods (decreased scotophase) causing a decreased immune response. Although this lends support to melatonin being a mediator of these effects, there is little evidence of this within the avian literature, which is sparse and contradictory. Experiment 2 was done to evaluate melatonin as a mediator of the LD effects on immune responses. The results indicated that exogenous melatonin given as a simulation of the plasma profile of melatonin typical in 8:16LD, to quail placed in LL, has an immunoreconstituting effect. This is consistent with the mammalian literature in that immuno-enhancement in mammals due to exogenous administration of melatonin is almost always seen as a reconstituting effect in immunocompromised or stressed animals (Maestroni et al., 1988; Maestroni and Conti, 1991; Caroleo et al., 1992; Mocchegiani et al., 1994). The reduced immune responses and increased heterophil/lymphocyte ratios (stress) occurring in our birds in LL effectively “immunocompromised” them, which could then be alleviated by melatonin supplements. These results provide evidence for melatonin functioning as a stimulator of both the cellular and the humoral immune response in quail and we are suggesting that melatonin plays a role in mediating the effects of LD cycles on immune responses. Since melatonin levels are directly proportional to the duration of the dark period, stepwise increases in the dark period would result in stepwise increases in plasma melatonin. Therefore all of our treatment groups had different degrees of melatonin secretion with 8:16LD and LL being the highest and lowest, respectively. There was certainly an immune response difference between the LD and LL treatments and a consistent, but not statistically significant, difference was ob-
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served between 8:16LD and 16:8LD. Thus, both increases in the daily dark period or increasing amounts of melatonin should result in increasing immune function, and this was generally observed in our study. It is known that a circadian rhythm in immune response occurs in mammals and birds (Haus and Smolensky, 1999; Rodriguez et al., 1999). Certainly birds placed in different LD cycles can have out-ofphase immune function rhythms. Furthermore, the LL treatment may abolish the melatonin rhythm along with perhaps the immune rhythm. Therefore a potential problem may arise if measurements of certain acute responses are made at a single time point during the day and compared between treatment groups. However, the PHA-P and primary antibody responses are prolonged responses measured after a 24-h and 7-day period, respectively. Circadian variations in immune responses should not be significant under these conditions. Also, it has been suggested that the T cell (PHA-P)- and B cell (CRBC)-mediated immune responses are not significantly effected by varying the injection time during a 24-h period (Stinson et al., 1980). Furthermore, there is evidence that the pineal gland via its hormone melatonin controls the diurnal rhythm of certain immune parameters in mammals and birds (Rosolowska et al., 1991; Pevet, 1998; Skwarlo-Sonta, 1996). If melatonin indeed has important immune properties, by what mechanism does this hormone function? The effects of melatonin on immune function may be direct via melatonin receptors located on immune tissue, including WBC (Liu and Pang, 1992; Calvo et al., 1995; Skwarlo-Sonta, 1996). In addition, mammalian research suggests several possible indirect mechanisms of immunomodulation by melatonin. The most widely accepted theory is that melatonin is acting through the opioid peptides (Maestroni et al., 1988; Maestroni and Conti, 1991). There is also evidence that melatonin may be acting through other endocrine hormones, most notably the gonadal, thyroid, and adrenal hormones (Poon and Pang, 1996). The results from the present study do not support a possible mechanism but certainly suggest a difference of sensitivity between the two branches of immunity. As seen in the results from experiment 2, there was a clear melatonin dose response of immuno-enhancement and the response generally occurred stepwise with increasing doses. However, a clearly defined
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Moore and Siopes
peak response occurred only in the antibody response but not in the cellular hypersensitivity response. This may indicate a difference in sensitivity to melatonin between the humoral and cellular components of the immune response. Poon and Pang (1996) noted that within human lymphocytes, but not granulocytes, sensitivity and maximal binding to melatonin are reduced upon prolonged melatonin injections. In the present study, the maximal cellular immune response increased 202% with exogenous melatonin whereas the same response increased 138% using only 8:16LD, as compared to LL. This suggests that exogenous melatonin may provide an additional enhancement above and beyond that available naturally, even in short daily photoperiods. This is of interest because existing literature with mammals indicates an immuno-enhancement by melatonin only under conditions of stress or existing immunosuppression. From the present study it is clear that daily LD cycles have an immuno-enhancing effect on cellular and humoral components of the avian immune response. Furthermore, because supplemented levels of melatonin have an immuno-reconstituting effect in LL, melatonin may be a mediator of the LD vs LL immune response differences. Practical indications include manipulation of the daily LD cycle as a potentially valuable tool for enhancing the immune system and welfare of captive birds or perhaps using melatonin as a supplement to boost the immune system.
ACKNOWLEDGMENT The authors sincerely appreciate the technical assistance provided by Robert Neely.
REFERENCES Bentley, G. E., Demas, G. E., Nelson, R. J., and Ball, G. F. (1998). Melatonin, immunity and cost of reproductive state in male European starlings. Proc. R. Soc. Lond. 265, 1191–1195. Binkley, S., Stephens, J., Reibman, J., and Reilly, K. (1977). Regulation of pineal rhythms in chickens: Photoperiod and dark-time sensitivity. Gen Comp. Endocrinol. 32, 411– 416. Blom, J. M. C., Gerber, J. M., and Nelson, R. J. (1994). Day length affects immune cell numbers in deer mice: Interactions with age, sex, and prenatal photoperiod. Am. Physiol. Soc. 267, R596 –R601.
Melatonin and Immunity in Quail
Calvo, J. R., Rafii-El-Idrissi, M., Pozo, D., and Guerrero, J. M. (1995). Immunomodulatory role of melatonin: Specific binding sites in human and rodent lymphoid cells. J. Pineal Res. 18, 119 –126. Champney, T. H., and McMurray, D. N. (1991). Spleen morphology and lymphoproliferative activity in short photoperiod exposed hamsters. In “Role of Melatonin and Pineal Peptides in Neuroimmunomodulation” (F. Fraschini and R. J. Reiter, Eds.), pp. 219 – 213. Plenum Press, New York, NY. Caroleo, M. C., Nistico, G., and Doria, G. (1992). Effect of melatonin on the immune system. Pharmacol. Res. 26, 34 –37. Demas, G. E., Klein, S. L., and Nelson, R. J. (1996). Reproductive and immune responses to photoperiod and melatonin are linked in Peromyscus subspecies. J. Comp. Physiol. 179, 819 – 825. Demas, G. E., and Nelson, R. J. (1996). Photoperiod and temperature interact to affect immune parameters in adult male deer mice. J. Biol. Rhythms. 11, 94 –102. Giannessi, F., Bianchi, F., Dolfi, A., and Lupetti, M. (1992). Changes in the chicken bursa of Fabricius and immune response after treatment with melatonin. In Vivo 6, 507–512. Haus, E., and Smolensky, M. H. (1999). Biological rhythms in the immune system. Chronobiol. Int. 16, 581– 622. Kirby, J. D., and Froman, D. P. (1991). Research note. Evaluation of humoral and delayed hypersensitivity responses in cockrels reared under constant light or a twelve hour light: Twelve hour dark photoperiod. Poult. Sci. 70, 2375–2378. Kumar, V., and Follett, B. K. (1993). The circadian nature of melatonin secretion in Japanese quail (Coturnix coturnix japonica). J. Pineal Res. 14, 192–200. Lewis, P. D., Morris, T. R., and Perry, G. C. (1995). Lighting and mortality rates in domestic fowl. Br. Poult. Sci. 37, 295–300. Liebmann, P. M., Wolfer, A., Felsner, P., Hofer, D., and Schauenstein, K. (1997). Melatonin and the immune system. Int. Arch. Allergy Immunol. 112, 203–211. Liu, Z. M., and Pang, S. F. (1992). [ 125I]Iodomelatonin-binding sites in the bursa of Fabricius of birds: Binding characteristics, subcellular distribution, diurnal variations and age studies. J. Endocrinol. 138, 51–57. Maestroni, G. J. M. (1993). The immunoneuroendocrine role of melatonin. J. Pineal Res. 14, 1–10. Maestroni, G. J. M., Conti, A., and Pierpaoli, W. (1988). Role of the pineal gland in immunity: Melatonin antagonizes the immunosuppressive effect of acute stress via an opatergic mechanism. Immunology 63, 465– 469. Maestroni, G. J. M., Conti, A., and 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. J. Neuroimmunol. 13, 19 –30. Maestroni, J. M., and Conti, A. (1991). Anti-stress role of the melatonin-immuno-opioid network: Evidence for a physiological mechanism involving T cell-derived, immuno-reactive beta-endorphin and met-enkephalin binding to thymic opiod receptors. Int. J. Neurosci. 61, 289 –298. Mahmoud, I., Salman, S. S., and Al-Khateeb, A. (1994). Continuous darkness and continuous light induce structural changes in the rat thymus. J. Anat. 185, 143–149.
103 Mocchegiani, E., Bulian, D., Santarelli, L., Tibaldi, A., Muzzioli, M., Pierpaoli, W., and Fabris, N. (1994). The immuno-reconstituting effect of melatonin or pineal grafting and its relation to zinc pool in aging mice. J. Neuro-immunol. 53, 189 –201. Natt, M. P., and Herrick, C. A. (1952). A new blood diluent for counting the erythrocytes and leucocytes of the chicken. Poult. Sci. 31, 735–738. Ottinger, M. A., and Brinkley, H. J. (1979). Testosterone and sex related physical characteristics during the matruation of the male Japanese quail (Coturnix coturnix japonica). Biol. Reprod. 20, 905– 909. Nelson, R. J., and Demas, G. E. (1996). Seasonal changes in immune function. Q. Rev. Biol. 71, 524 –526. Nelson, R. J., and Blom, J. M. C. (1994). Photoperiodic effects on tumor development and immune function. J. Biol. Rhythms 9, 233–249. Nelson, R. J., Demas, G. E., Klein, S. L., and Kriegsfeld, L. J. (1995). The influence of season, photoperiod, and pineal melatonin on immune function. J. Pineal Res. 19, 149 –165. Pevet, P. (1998). Me´latonine et rythmes biologiques. The´rapie 53, 411– 420. Pioli, C., Caroleo, M. C., Nistico, G., and Doria, G. (1993). Melatonin increases antigen presentation and amplifies specific and non specific signals for T-cell proliferation. Int. J. Immunopharmacol. 15, 463– 468. Poon, A. M. S., and Pang, S. F. (1996). Pineal melatonin–immune system interaction. In “Melatonin: A Universal Photoperiodic Signal with Diverse Actions” (P. L. Tang, S. F. Pang, and R. J. Reiter, Eds.), pp. 73–74. Karger, Basel. Reiter, R. J. (1988). Neuroendocrinology of melatonin. In “Melatonin: Clinical Perspectives” (A. Miles, D. R. S. Philbrick, and C. Thompson, Eds.), pp. 1–28. Oxford Univ. Press, New York, NY. Rodriguez, A. B., and Lea, R. W. (1994). Effect of pinealectomy upon the nonspecific immune response of the ring-dove (streptopelia risoria). J. Pineal Res. 16, 159 –166. Rodriguez, A. B., Marchena, J. M., Nogales, G., Duran, J., and Barriga, C. (1999). Correlation between the circadian rhythm of melatonin, phagocytosis, and superoxide anion levels in ring dove heterophils. J. Pineal Res. 26, 35– 42. Rodriguez, A. B., Nogales, G., Marchena, J. M., Ortega, E., and Barriga, C. (1999). Suppression of both basal and antigen-induced lipid peroxidation in ring dove heterophils by melatonin. Biochem. Pharmacol. 58, 1301–1306. Rodriguez, A. B., Nogales, G., Ortega, E., and Barriga, C. (1998). Melatonin controls superoxide anion level: Modulation of superoxide dismutase activity in ring dove heterophils. J. Pineal Res. 24, 9 –14. Rodriguez, A. B., Ortega, E., Lea, R. W., and Barriga, C. (1997). Melatonin and the phagocytic process of heterophils from the ring dove (Streptopelia risoria). Mol. Cell. Biochem. 168, 185–190. Rosolowska-Huszcz, D., Thaela, M.-J., Jagura, M., Stepie, D., and Skwarlo-Sota, K. (1991). Pineal influence on the diurnal rhythm of nonspecific immunity indices in chickens. J. Pineal Res. 10, 190 – 195. SAS Institute (1990). “SAS/STAT威 User’s Guide, Version 6, 4th ed., Vol. 2. SAS Institute Inc., Cary, NC.
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104 Sever, J. L. (1961). Application of a microtechnique to viral serological investigations. J. Immunol. 88, 320 –329. Stinson, R., McCorkle, F., Mashaly, M., Taylor, R., Martin, D., and Glick, B. (1980). The effects of diurnal rhythms on immune parameters in new hampshire chickens. Int. Arch. Allergy Appl. Immun. 61, 220 –226. Skwarlo-Sonta, K. (1996). Functional connections between the pineal gland and immune system. Acta Neurobiol. 56, 341–357. Skwarlo-Sonta, K., Thaela, M.-J., Gluchowska, B., Stepien, D., and Jagura, M. (1991). Effect of dose and time of melatonin injections on the diurnal rhytm of immunity in chicken. J. Pineal Res. 10, 30 –35. Skwarlo-Sonta, K., Thaela, M.-J., Midura, M., Lech, B., Gluchowska, D., Drela, N., Kozlowska, E., and Kowalczyk, R. (1992). Exogenous melatonin modifies the circadian rhythm but does not in-
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crease the level of some immune parameters in the chicken. J. Pineal Res., 27–34. Stadecker, M. J., Lukic, M., Dvorak, A., and Leskowitz, S. (1977). The cutaneous basophil response to phytohemagglutinin in chickens. J. Immunol. 118, 1564 –1568. Underwood, H., Binkley, S., Siopes, T., and Mosher, K. (1984). Melatonin rhythms in the eyes, pineal bodies, and blood of japanese quail (Coturnix coturnix japonica). Gen. Comp. Endocrinol. 56, 70 – 81. Underwood, H., and Edmonds, K. (1995). The circadian rhythm of thermoregulation in japanese quail. III. Effects of melatonin administration. J. Biol. Rhythms 10, 284 –298. Underwood, H., and Siopes, T. (1985). Melatonin rhythms in quail: Regulation by photoperiod and circadian pacemakers. J. Pineal Res. 2, 133–143.
Effects of Lighting Conditions and Melatonin Supplementation on the Cellular and Humoral Immune Responses in Japanese Quail Coturnix coturnix japonica C. B. Moore and T. D. Siopes 1 Department of Poultry Science, Box 7608, North Carolina State University, Raleigh, North Carolina 27695-7608 Accepted April 3, 2000
The humoral immune response increased to a peak at the 5.0 g/ml dose while the cellular immune response increased across all dose levels. From the present study it was clear that quail placed in daily light-dark cycles (LD), possessing a diurnal rhythm of melatonin, had significantly elevated immune responses as compared to those birds in LL. Furthermore, melatonin supplemented to birds exposed to LL was immuno-enhancing. This suggests that melatonin may be a mediator of the differences seen between LD and LL lighting conditions and may have important immune modulating properties. © 2000
Two experiments were conducted to determine the effects of lighting conditions and melatonin supplementation on the cellular and humoral immune responses in Japanese quail. The first experiment was designed to evaluate differing light regimes as immune modulators in both adult and juvenile quail. The cellular and humoral immune responses were determined for three lighting conditions; short days (8:16LD), long days (16:8LD), and constant light (LL). In the second experiment, melatonin was administered in varying doses to adult quail placed in LL. The doses used in this experiment were 0.0, 0.5, 5.0, and 50.0 g/ml melatonin given in the drinking water for 16 h per day for 2 weeks. The cellular and humoral immune responses were evaluated after 1 week of melatonin treatment. In both experiments, 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 calculated 7 days postintravenous injection with a Chukar red blood cell suspension. In the adult birds of experiment 1, both the 8:16LD and 16:8LD treatments produced similar cellular and humoral immune responses but these responses were significantly greater than those observed in LL. The juvenile birds held under 8:16LD also had significantly greater cellular and humoral immune responses as compared to juvenile birds held in LL. In experiment 2, there was a clear melatonin dose response on immune function in LL. 1
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Key Words: melatonin; photoperiod; immune response; Japanese quail. It is well established that melatonin biosynthesis and secretion in essentially all vertebrates are affected by environmental light. The enzymatic processes leading to melatonin biosynthesis are suppressed by light and enhanced by darkness (Reiter, 1988). Thus, animals respond to increased daylengths (decreased dark period) by reducing the duration of nocturnal melatonin secretion (Kumar and Follett, 1993; Underwood and Siopes, 1985) while circulating melatonin levels may be completely suppressed to basal levels in constant light (LL) (Ralph et al., 1975; Siopes, unpublished). In recent years, researchers have found an association between the duration of environmental light and some immune parameters of mammals. Most of this
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research in mammals has indicated that in short photoperiods both the cellular and the humoral components of the immune system were enhanced as compared to mammals kept in long photoperiods (Champney and McMurray, 1991; Mahmoud et al., 1994; Blom et al., 1994; Nelson and Blom, 1994; Demas et al., 1996; Demas and Nelson, 1996). Furthermore, the pineal hormone melatonin may be the mediator of these photoperiod-immune effects due to its direct and indirect effects on immune function (Maestroni et al., 1986; Caroleo et al., 1992; Maestroni, 1993; Nelson et al., 1995; Liebmann et al., 1997). There are few reports about melatonin as an immune modulator for avian species and there is no literature establishing a short-day immuno-enhancing effect. It has been suggested that melatonin injections do not have an immunostimulatory effect on immune indices in the chicken (Skwarlo-Sonta, 1991; SkwarloSonta, 1992). However, pinealectomized ring doves have reduced immune responses as compared to sham-operated birds, suggesting an immuno-enhancing effect for melatonin (Rodriguez et al., 1994). Furthermore, a link between melatonin and certain nonspecific immune responses has been established in the ring dove (Rodriguez et al., 1997, 1999). Melatonin has also been shown to have some useful antioxidant properties in this species (Rodriguez et al., 1998, 1999). Also, exogenous melatonin boosted cellular immune function in photostimulated male starlings (Bentley et al., 1998). However, melatonin has been shown to reduce primary and secondary immune responses in the chicken (Giannessi et al., 1992). Therefore, unlike the extensive mammalian literature, the avian literature is sparse and inconsistent about a role for lighting conditions or melatonin in immune function. Most of the literature for photoperiod effects on the avian immune system is indirect, reflected by growth, performance, and reproductive changes (Lewis and Morris, 1995). However, Kirby and Froman (1991) reported a suppressed cellular immunity and secondary antibody response in immature cockerels reared under LL as compared to 12:12LD (light-dark) daily cycles. Therefore the objective of our research was to determine if changes in lighting conditions could alter immune function in the Japanese quail and, if so, to determine if melatonin is involved. This was accomplished by establishing a LD vs LL treatment difference in immune function and then determining if
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Moore and Siopes
melatonin was the mediator of this effect, by use of a replacement therapy technique. We hypothesized an immuno-inhibition in LL, due to suppressed levels of melatonin, and immuno-enhancement as melatonin was increased artificially (replacement) or naturally (light-dark cycle).
MATERIALS AND METHODS Husbandry Adult and juvenile Japanese quail were used in the study. All adult birds were females and greater than 5 weeks of age whereas the mixed-sex juvenile quail were 21 days old at the start of the experiments. Japanese quail are considered mature and produce sperm or ovulate by 5 to 6 weeks of age on long daylength. Males mature in advance of females and testicular weights and testosterone remain very low until 21 days of age (Ottinger and Brinkley, 1979). Birds were randomly selected for each treatment group and placed in one of three light-controlled boxes which were housed in a room maintained at 23 ⫾ 3°C and constant darkness (DD). The lighting conditions used in the study were 8:16LD (light:dark), 16:8LD (light:dark), and constant light (LL). The daily lights-on time was 0800 for both LD cycle treatment groups. Four 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.
Experiment 1 This experiment was designed to evaluate lighting condition as an immune modulator in adult and juvenile birds. Three different lighting conditions were used, short days (8:16LD), long days (16:8LD), and constant light (LL), to provide long, short, and no daily dark period, respectively. It was expected that LL continually suppressed circulating melatonin to basal levels whereas the dark periods in the LD cycles provided increased daily melatonin levels in proportion to the length of the dark period.
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Fifteen female adult birds were selected for each lighting condition and remained in this treatment for 3 weeks prior to immune response evaluation. As a consequence, the birds on short days had regressed ovaries whereas those on long days had fully developed ovaries and were ovulating. Likewise, 15 unsexed juvenile birds, 14 days posthatch, received 1 week of exposure to 8:16LD and LL treatments. At 21 days of age the gonads remained undeveloped and immune challenges were started. All birds remained on their respective lighting protocols during the period of immune response evaluation. The cutaneous basophil hypersensitivity reaction to phytohemagglutinin (PHA-P), a lectin from Phaseolus Vulgaris (Sigma Chemical Co., St. Louis, MO), was measured in the wing web of each bird to evaluate the cellular immune response (Stadecker et al., 1977). An intradermal injection of PHA-P was given in the wing web of each bird and at the same time of day (1400) in all treatment groups. The dermal swelling response was measured as the percentage increase in wing-web thickness at the injection site 24 h post-PHA-P injections. 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. The primary antibody response was measured 7 days following the CRBC injections using a microagglutination assay (Sever, 1961). Both the CRBC injections and the primary antibody titers were performed at the same time of day as the cellular immune response (1400). In addition, the total and differential white blood cell (WBC) counts of the adult quail were counted using a method provided by Natt and Herrick (1952). Differential counts of 100 leukocytes per blood smear were made following staining of the smear with a Diff-Quik stain set (Baxter Scientific, McGaw Park, IL) which is a modified WrightGiemsa stain. Total WBC and differentials were measured at four time intervals: 0, 2, 4, and 7 days postCRBC. Blood was obtained from the brachial vein to perform the WBC counts during the middle of the dark phase, in the LD groups, with use of a dim, site-localized light and for a maximum exposure of 3 min. Blood was collected from birds in the LL group at the same time of day. The juvenile birds were euthanized by carbon monoxide asphyxiation 7 days
postimmunization (28 days of age) and gonadal weights were obtained.
Experiment 2 This experiment was conducted to determine if exogenous melatonin administered to simulate daily plasma melatonin patterns typical in short photoperiods (8:16LD) could enhance the cellular and humoral immune responses of birds maintained in LL. Forty adult female birds were placed in two light-controlled boxes. These birds were allowed to adjust to LL for 2 weeks before the treatments were started. Four treatment groups (n ⫽ 10) were formed with 0.0, 0.5, 5.0, and 50 g/ml of melatonin given in the drinking water while the birds remained in LL. It has been reported that 100 g/ml melatonin given in the drinking water to birds placed in DD produced approximately 4 times the physiological levels of melatonin as compared to the normal peak nighttime levels seen in birds entrained to LD 12:12 (Underwood and Edmonds, 1995; Underwood et al., 1984). Since quail have a robust melatonin rhythm in DD, this must be due to additive effects of the exogenous and endogenous melatonin. Therefore, although plasma melatonin concentrations were not measured in the present study, the melatonin dose range used should result in plasma melatonin levels approximating physiological levels. In order to simulate melatonin plasma profiles typically observed on an 8:16LD photoperiod, the melatonin doses were administered for 16 h (1700 to 0900) and substituted with tap water for the remaining 8 h. Doses of melatonin were prepared daily by dissolving the appropriate amount in 1 ml of 95% ethanol and diluting to 1 L with distilled water. A control group (0.0 g/ml melatonin) was treated similarly but received a distilled water/ethanol solution (diluent) instead of melatonin at the same time of day as the treatment groups. After 1 week of melatonin supplementation, the cellular and humoral immune responses were evaluated by PHA-P and primary antibody responses, respectively, as described in experiment 1. The total and differential WBC counts were determined at three time intervals; 0, 3, and 7 days post-CRBC injection in the control group and 5.0 g/ml melatonin treatment group only. As in experiment 1, all injections and measurements were made at the same time of day in
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TABLE 1 Effect of Daily Lighting Conditions on Immune Responses in Adult and Juvenile Quail Cellular response 1
Humoral response 2
Photoperiod
Adult
Juvenile
Adult
Juvenile
8:16LD 16:8LD LL RMSE 3 N (Range) P⬎F
137.8 a 112.6 a 102.8 b 28.6 11–13 0.0001
230.7 a — 196.1 b 76.3 14–16 0.048
2.81 a 2.93 a 2.08 b 0.36 11–13 0.0001
2.65 a — 1.85 b 0.33 12–14 0.0001
a,b
Means within a column and with no common superscript differ significantly (P ⱕ 0.05). 1 Cellular data are expressed as a percentage change in wing web thickness, 24 h postphytohemagglutinin (PHA-P) injection. 2 Humoral data are expressed as a log 2 of the primary antibody titer. 3 Root-mean-square error.
each treatment group (1400). Melatonin or distilled water/ethanol administration was continued throughout all immune tests.
Statistical Analyses All data were analyzed by one-way analysis of variance (ANOVA) using the General Linear Model procedure of the SAS institute (SAS Institute, 1990). Repeated-measures ANOVA was used to assess time effects on white blood cell counts. Significant differences among means were estimated using least-square means. Statements of statistical significance are based on P ⱕ 0.05.
humoral immune response compared to LL. In all juvenile birds the mean gonadal weights at the end of the test (28 days of age) were 57 and 23 mg for ovaries and 125 and 8 mg for testes in LL and 8:16LD, respectively. Furthermore, there was no cloacal gland foam in the birds placed in LL, which is dependent on testosterone. The total WBC counts of adult quail under each lighting condition tested are expressed in Table 2. Prior to immunization with antigen, there was a significant elevation in total WBCs within the 8:16LD treatment group compared to the 16:8LD or LL groups, which were similar. At 2 and 4 days postimmunization, the two LD treatment groups had similar numbers of white blood cells and both were elevated compared to the LL group. There was a significant time effect as well as treatment main effect ⫻ time interaction. The data in Table 3 are the differential WBC counts before antigen stimulation and represent lighting effects on the differential counts. No significant differences occurred among treatments after inoculation compared to preinoculation; accordingly, only the results prior to inoculation (Day 0 of Table 2) are presented. The lymphocyte populations were significantly greater and heterophils significantly less for birds in the 8:16LD or 16:8LD treatments, compared to those in LL. Therefore, the heterophil/lymphocyte ratio was significantly increased within those birds kept
TABLE 2 Effect of Daily Lighting Conditions on Total White Blood Cell Counts in Adult Quail Following Immunization Total white blood cell (cells/mm 3 ⫻ 10 4) 1 Days
RESULTS Experiment 1 The data in Table 1 represent the cellular and humoral immune responses of adult and juvenile birds in each light treatment. Within the adult bird data, it was clear that both the 8:16LD and the 16:8LD groups had similar responses to PHA-P and CRBC and both were greater compared to the LL group. The juvenile bird responses were consistent with the adult data. Therefore LD treatments have a greater cellular and
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Photoperiod
0
2
4
7
8:16LD 16:8LD LL RMSE 2 N (Range) P⬎F
5.19 a 4.18 b 4.17 b 0.486 11–13 0.0039
6.34 a 6.46 a 4.66 b 0.401 11–13 0.0001
6.41 a 6.15 a 4.14 b 0.250 11–13 0.0001
3.88 a 3.93 a 3.06 a 0.197 10–12 0.162
a,b
Means within a column with no common superscript differ significantly, (P ⱕ 0.05). 1 Counts were taken at four time intervals: 0, 2, 4, and 7 days postantigenic challenge. Repeated-measures ANOVA for Days 0 to 7: Probability (P ⱖ F) for treatment ⫽ 0.0001; time ⫽ 0.0001; time ⫻ treatment ⫽ 0.0001. Pooled SEM ⫽ 0.34. 2 Root-mean-square error.
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TABLE 3 Effect of Daily Lighting Conditions on the Mean Differential White Blood Cell Counts in Adult Quail prior to Immunization
on the red blood cell count, therefore these data were not presented.
Differential cell count (%) 1 Photoperiod
Lymphocyte
Heterophil
H/L ratio
8:16LD 16:8LD LL RMSE 2 Pr ⬎ F
79 a 70 a 60 b 5.362 0.0001
13 a 19 a 35 b 5.585 0.0001
0.165 a 0.271 a 0.583 b 0.129 0.0001
a,b Means within a column with no common superscript differ significantly (P ⱕ 0.05). 1 Differential counts of 100 leukocytes per blood smear; N ⫽ 15. 2 Root-mean-square error.
in LL compared to the LD treatments. The white blood cell populations other than lymphocytes and heterophils were not different among the treatment groups. Also, there was no effect of lighting condition
Experiment 2 Figure 1 illustrates the cellular and humoral immune responses of quail placed in LL and given different oral doses of melatonin. All melatonin doses resulted in elevated cellular and humoral immune responses compared to the control group. The cellular immune response generally increased with the dose of melatonin and no clearly defined peak response occurred over the dose range used. The humoral immune response to oral melatonin doses significantly increased across all melatonin treatments compared to the control. A significant elevation occurred between each treatment except the 5.0 and 50.0 g/ml treatment groups.
FIG. 1. Cellular immune response (top) and primary antibody response (bottom) of adult Japanese quail given different doses of melatonin. All birds were in constant light (LL) throughout the experiment. The PHA-P-induced response (top) was measured 1 week following initiation of melatonin treatments. Immunizations were given at this time and titers (bottom) were determined 7 days postimmunization. (Top) N ⫽ 15 per treatment, RMSE ⫽ 45.2, P ⫽ 0.012. (Bottom) N ⫽ 6 – 8 per treatment, RMSE ⫽ 0.38, P ⫽ 0.0014. Means with no common superscript differ significantly (P ⱕ 0.05). Control group ⫽ 0.0 g/ml melatonin.
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DISCUSSION
TABLE 4 Mean Total and Differential White Blood Cell Count in Adult Quail Held in Constant Light and Following 7 Days Melatonin Treatment Total WBC (cells/mm ⫻ 10 ) Days postimmunization 3
4 1
Treatment
0
3
7
0.0 g/ml 5.0 g/ml RMSE 3 P⬎F
2.68 a 4.41 b 0.372 0.0001
3.59 a 8.02 b 0.486 0.0001
4.78 a 5.41 a 0.291 0.078
Differential WBC (%) 2 Cell type Treatment
Lymphocyte
Heterophil
H/L ratio
0.0 g/ml 5.0 g/ml RMSE 3 P⬎F
59 a 71 b 3.692 0.0001
28 a 17 b 3.861 0.0001
0.475 a 0.239 b 0.073 0.0001
a,b
Means within a column and with no common superscript differ significantly (P ⱕ 0.05). 1 Counts were taken at three time intervals: 0, 3, and 7 days postantigenic challenge. N (range) ⫽ 8 –9. 2 Differential counts of 100 leukocytes per blood smear, prior to immunization: N (range) ⫽ 8 –9. 3 Root-mean-square error.
Before antigen stimulation, and after 7 days of melatonin treatment (Day 0 postimmunization), the total WBC counts were elevated within the melatonin treatment group compared to the untreated control group (Table 4). On Day 3 postantigenic challenge, the melatonin group was still significantly elevated from controls. By the seventh day postimmunization, the WBC counts were not significantly different between treatment groups. The differential WBC in the two treatment groups was not significantly different following CRBC antigen stimulation as compared to preimmunization. Therefore, only the preimmunization results (Day 0) are given (Table 4). The melatonin treatment significantly enhanced the lymphocyte percentage while the heterophil percentage was significantly reduced. Accordingly, the heterophil/lymphocyte ratio was significantly elevated in the control group compared to the melatonin treatment. Also, melatonin treatment had no effect on red blood cell numbers therefore these data were not presented.
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From the results in experiment 1, it was clear that differing lighting conditions had similar qualitative effects on immune responses in both adult and juvenile Japanese quail. Both the cellular and humoral immune responses were greater when birds were placed in daily LD treatments as compared to LL. Clearly, the responses were not age dependent and were not dependent on state of sexual development because gonadal weights of the juvenile birds were similar among treatments and all were clearly immature in development. In addition, the adult immune responses in 16:8LD and LL were different yet both had similar ovarian status, that is, both were laying eggs. Our results are consistent with the report by Kirby and Froman (1991) in which 12:12LD photoperiods enhanced the phytohemagglutinin and secondary antibody responses of immature cockerels compared to LL treatment. We are not aware of any similar reports using adult birds. However, Bentley et al. (1998) reported that immune function was suppressed in adult starlings photostimulated with long days (18:6LD). In the mammalian literature, Blom et al. (1994) and Demas and Nelson (1996) reported an enhanced immune response in adult deer mice placed in short photoperiods. It was interesting that the boost in cellular immunity was greater in the juvenile birds as compared to the adult birds. This likely is related to the presence of immune tissue such as the thymus that subsequently regresses in the adult. Mahmoud et al. (1994) reported that photoperiod influences thymus morphology in rats and suggested an involvement by melatonin. The immuno-enhancing effect of LD vs LL light treatments in the present study was strong enough to be observed in the total number of white blood cells, with and without antigen challenge (Table 2). Interestingly, not only did LL treatment suppress WBC as compared to the LD treatments before antigen stimulation, but also birds in LL did not significantly increase WBC following an antigenic challenge, as did those birds in the light-dark cycles. This of course could mean decreased ability to resist certain antigenic challenges and result in adverse physiological consequences. The preimmunization WBC increase, in LD versus LL-treated birds, was associated with a preim-
Melatonin and Immunity in Quail
munization rise in the number of lymphocytes and a fall in the number of heterophils for birds placed in LD cycles. Therefore, the heterophil/lymphocyte ratio was lower in the LD treatments than for those birds placed in LL. An elevated H/L ratio is well established as a general indicator of stress, suggesting that LL was more stressful than long or short daily photoperiods. This is consistent with the adverse effects of LL on immune responses. Experiment 1 was designed to roughly mimic the range of natural lighting conditions experienced by birds during changes in temperate zone seasons. That is, the 8:16LD (short day) group was indicative of a “winter-like” condition while the 16:8LD (long day) group was more “summer-like.” It has been reported that seasonal changes in photoperiod may alter the immune responses of birds and ultimately the health of an overall population. Immunity is enhanced in the wintertime (short days) when the animal is subjected to many environmental stresses such as hypothermia and malnutrition (Nelson and Demas, 1996). The fact that there was no significant difference seen between fixed short and long photoperiods in the present study indicated that other factors might be involved in the reported seasonal variations of immune responses or that certain quantitative and/or qualitative characteristics of the light exposure may be important. Interestingly, Bentley et al. (1998) have reported that changes in reproductive state, and not simply a change in photoperiod, had a significant effect on the immune response of adult starlings. Although the results from experiment 1 demonstrated that LD cycles increase immune responses as compared to LL, a possible mechanism for this effect was not addressed. Certainly, the presence or absence of gonadal steroids does not seem to be an essential factor because the immuno-enhancing effect of LD cycles was seen in both adult and juvenile birds. This is in agreement with Blom et al. (1994) using adult deer mice. Likewise immuno-enhancement in 16:8LD as compared to LL occurred in the presence of fully functional ovaries. From research with mammals it has been suggested that differing photoperiods influence immune responses by altering the amount of circulating melatonin (Nelson et al., 1995). Also, melatonin supplementation can have immuno-enhancing effects in immunocompromised mammals (Maestroni, 1993; Piolo et al., 1993).
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In Japanese quail, we may assume that melatonin is completely suppressed to basal levels in LL and increases with an increased duration of the dark phase (Kumar and Follett, 1991). Because there can be substantial circulating melatonin present in LD treatment but only minimal (basal) levels present in LL, it would be consistent with melatonin as a possible mediator of the LD treatment-induced immuno-enhancement exhibited in experiment 1. Furthermore, although there was no significant difference seen between the 8:16LD and the 16:8LD treatments, the longer photoperiod was intermediate in every immune parameter measured. There certainly seems to be a trend toward increased photoperiods (decreased scotophase) causing a decreased immune response. Although this lends support to melatonin being a mediator of these effects, there is little evidence of this within the avian literature, which is sparse and contradictory. Experiment 2 was done to evaluate melatonin as a mediator of the LD effects on immune responses. The results indicated that exogenous melatonin given as a simulation of the plasma profile of melatonin typical in 8:16LD, to quail placed in LL, has an immunoreconstituting effect. This is consistent with the mammalian literature in that immuno-enhancement in mammals due to exogenous administration of melatonin is almost always seen as a reconstituting effect in immunocompromised or stressed animals (Maestroni et al., 1988; Maestroni and Conti, 1991; Caroleo et al., 1992; Mocchegiani et al., 1994). The reduced immune responses and increased heterophil/lymphocyte ratios (stress) occurring in our birds in LL effectively “immunocompromised” them, which could then be alleviated by melatonin supplements. These results provide evidence for melatonin functioning as a stimulator of both the cellular and the humoral immune response in quail and we are suggesting that melatonin plays a role in mediating the effects of LD cycles on immune responses. Since melatonin levels are directly proportional to the duration of the dark period, stepwise increases in the dark period would result in stepwise increases in plasma melatonin. Therefore all of our treatment groups had different degrees of melatonin secretion with 8:16LD and LL being the highest and lowest, respectively. There was certainly an immune response difference between the LD and LL treatments and a consistent, but not statistically significant, difference was ob-
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served between 8:16LD and 16:8LD. Thus, both increases in the daily dark period or increasing amounts of melatonin should result in increasing immune function, and this was generally observed in our study. It is known that a circadian rhythm in immune response occurs in mammals and birds (Haus and Smolensky, 1999; Rodriguez et al., 1999). Certainly birds placed in different LD cycles can have out-ofphase immune function rhythms. Furthermore, the LL treatment may abolish the melatonin rhythm along with perhaps the immune rhythm. Therefore a potential problem may arise if measurements of certain acute responses are made at a single time point during the day and compared between treatment groups. However, the PHA-P and primary antibody responses are prolonged responses measured after a 24-h and 7-day period, respectively. Circadian variations in immune responses should not be significant under these conditions. Also, it has been suggested that the T cell (PHA-P)- and B cell (CRBC)-mediated immune responses are not significantly effected by varying the injection time during a 24-h period (Stinson et al., 1980). Furthermore, there is evidence that the pineal gland via its hormone melatonin controls the diurnal rhythm of certain immune parameters in mammals and birds (Rosolowska et al., 1991; Pevet, 1998; Skwarlo-Sonta, 1996). If melatonin indeed has important immune properties, by what mechanism does this hormone function? The effects of melatonin on immune function may be direct via melatonin receptors located on immune tissue, including WBC (Liu and Pang, 1992; Calvo et al., 1995; Skwarlo-Sonta, 1996). In addition, mammalian research suggests several possible indirect mechanisms of immunomodulation by melatonin. The most widely accepted theory is that melatonin is acting through the opioid peptides (Maestroni et al., 1988; Maestroni and Conti, 1991). There is also evidence that melatonin may be acting through other endocrine hormones, most notably the gonadal, thyroid, and adrenal hormones (Poon and Pang, 1996). The results from the present study do not support a possible mechanism but certainly suggest a difference of sensitivity between the two branches of immunity. As seen in the results from experiment 2, there was a clear melatonin dose response of immuno-enhancement and the response generally occurred stepwise with increasing doses. However, a clearly defined
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Moore and Siopes
peak response occurred only in the antibody response but not in the cellular hypersensitivity response. This may indicate a difference in sensitivity to melatonin between the humoral and cellular components of the immune response. Poon and Pang (1996) noted that within human lymphocytes, but not granulocytes, sensitivity and maximal binding to melatonin are reduced upon prolonged melatonin injections. In the present study, the maximal cellular immune response increased 202% with exogenous melatonin whereas the same response increased 138% using only 8:16LD, as compared to LL. This suggests that exogenous melatonin may provide an additional enhancement above and beyond that available naturally, even in short daily photoperiods. This is of interest because existing literature with mammals indicates an immuno-enhancement by melatonin only under conditions of stress or existing immunosuppression. From the present study it is clear that daily LD cycles have an immuno-enhancing effect on cellular and humoral components of the avian immune response. Furthermore, because supplemented levels of melatonin have an immuno-reconstituting effect in LL, melatonin may be a mediator of the LD vs LL immune response differences. Practical indications include manipulation of the daily LD cycle as a potentially valuable tool for enhancing the immune system and welfare of captive birds or perhaps using melatonin as a supplement to boost the immune system.
ACKNOWLEDGMENT The authors sincerely appreciate the technical assistance provided by Robert Neely.
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