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Endocrinology Vol. 147, No. 4 1717-1724
Copyright © 2006 by The Endocrine Society

Sex-Dependent Effect of Melatonin on Systemic Erythematosus Lupus Developed in Mrl/Mpj-Faslpr Mice: It Ameliorates the Disease Course in Females, whereas It Exacerbates It in Males

Antonio J. Jimenez-Caliani, Silvia Jimenez-Jorge, Patrocinio Molinero, Jose M. Fernandez-Santos, Ines Martin-Lacave, Amalia Rubio, Juan M. Guerrero and Carmen Osuna

Departments of Medical Biochemistry and Molecular Biology (A.J.J.-C., S.J.-J., P.M., A.R., J.M.G., C.O.) and Normal and Pathologic Cytology and Histology (J.M.F.-S., I.M.-L.), University of Seville School of Medicine and Virgen Macarena Hospital, Seville 41009, Spain

Address all correspondence and requests for reprints to: Dr. Carmen Osuna, Department of Medical Biochemistry and Molecular Biology, University of Seville School of Medicine, Avda Sánchez-Pizjuán 4, 41009 Seville, Spain. E-mail: cosuna{at}us.es.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In this study, the effect of chronic administration of melatonin on MRL/MpJ-Faslpr mice has been studied. These mice spontaneously develop an autoimmune disease that has many features resembling human systemic lupus erythematosus. In fact, histological studies showed that all female mice and most male mice exhibited glomerular abnormalities, arteritic lesions, and cellular interstitial inflammatory infiltrate ranging from mild to severe patterns. Treatment with melatonin improved the histological pattern in females and worsened it in males. Moreover, female mice treated with melatonin showed a diminution of titers of total serum IgG, IgM, and anti-double-stranded DNA and anti-CII autoantibodies; a decrease in proinflammatory cytokines (IL-2, IL-6, interferon-{gamma}, TNF-{alpha}, and IL-1ß), an increase in antiinflammatory cytokines (IL-10), and a decrease in nitrite/nitrate. In male mice, treatment with melatonin exhibited the opposite effect, worsening all the immunological parameters with an elevation of titers of autoantibodies and a prevalence of proinflammatory vs. antiinflammatory cytokines. Similar results were obtained when lymphocytes from spleen and lymph nodes were cultured. Again, melatonin treatment in females decreased proinflammatory cytokines and increased antiinflammatory cytokines produced by lymphocytes; in males, the effect was the opposite. These findings suggest that melatonin action in MRL/MpJ-Faslpr mice is gender dependent, probably through modulation and inhibition of sex hormones.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DISTURBANCES IN THE neuro-immune-endocrine system have been described in murine models of autoimmune diseases including systemic lupus erythematosus (SLE) (1). The onset of SLE in MRL+/+ and MRL-lpr/lpr mice correlates with alterations in hypothalamic-pituitary-adrenal function (2), and in MRL/MP-faslpr mice, altered circadian rhythms of the stress hormone and melatonin response have been described. Thus, MRL/MP-faslpr mice do not react appropriately to changes of the light/dark cycle, circadian melatonin rhythms seem to uncouple from the light/dark cycle, and plasma corticosterone levels are elevated during the resting phase (3). On the contrary, melatonin can also regulate the immune system (4) and can play a role in autoimmune diseases such as rheumatoid arthritis (5, 6) and Crohn’s disease (7).

Melatonin, which was assumed to be mainly synthesized in the pineal gland, is presently considered a molecule that is synthesized in many other tissues, including the immune system (8, 9, 10) where it acts as an intracrine, autocrine, and paracrine substance (11). Both normal human lymphocytes and Jurkat human leukemic T cells synthesize and release large amounts of melatonin, contributing to the regulation of its own IL-2 production (12, 13, 14). Melatonin also regulates interferon-{gamma} (IFN-{gamma}) production in type 1 helper T lymphocytes (Th1) and production of TNF-{alpha}, IL-1ß, and nitric oxide (NO) in monocytes (15).

To investigate the role of melatonin in autoimmune diseases, we have used the MRL/MpJ-Faslpr (MRL-lpr/lpr) mouse, a widely accepted and valuable model of SLE. This mouse strain spontaneously develops a generalized autoimmune disease that is similar to human SLE and is characterized by immune complex-mediated glomerulonephritis, lymphadenopathy, arthritis, vasculitis, and autoantibody production (16, 17). MRL-lpr/lpr mice are homozygous for the autosomal recessive lymphoproliferation (lpr) mutation, which inactivates the fas gene, resulting in defective apoptosis of lymphocytes and progressive accumulation of abnormal double-negative T cells in peripheral lymphoid tissues (18). Disease expression is regulated and accelerated by the lpr mutation. However, other factors and background genes clearly influence the severity and disease manifestations in the MRL-lpr/lpr mouse strain (19, 20). The pathogenesis of MRL-lpr/lpr mice largely requires CD4+{alpha}ß+ T cells, which provide help to autoreactive B cells (21, 22). The roles that individual Th1 and Th2 cells subsets play in this process remain unclear. Likewise, in MRL-lpr mice, disease has been associated with both Th1 cytokines such as IFN-{gamma} and the Th2 cytokine IL-10 (23, 24).

The aim of this work was to study the effects of melatonin on an experimental autoimmune model, MRL-lpr mice, and to ascertain the relationships between gender and melatonin-induced response.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice
MRL/MpJ-lprFas (MRL-lpr) mice, originally purchased from The Jackson Laboratory (Bar Harbor, ME), were maintained in the animal facilities of the Center of Animal Experimentation and Reproduction, Seville University. All animals were kept under controlled and standardized conditions. Standardized food and drinking water were provided ad libitum. The experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, National Academy of Science, Bethesda, MD).

Experimental protocols
Female and male MRL-lpr mice (14–16 wk old) were separated and randomized into four groups. Two groups, one female and the other male, received 30 mg melatonin/kg body weight in drinking water for 1 month and were designated FM (female + MEL) and MM (male + MEL) groups. Because melatonin was dissolved in ethanol, two additional groups, one female and the other male, received the same ethanol concentration (1%) in drinking water for 1 month and were designated FC (female + control) and MC (male + control) groups. MRL-lpr mice are an immunocompromised strain. For this reason, melatonin was not injected daily, but administered in drinking water. One month before melatonin administration, the amount of drinking water consumed by each animal as well its body weight were measured. After that, the concentration of melatonin in drinking water was manipulated so that the dose ended up being 30 mg melatonin/kg body weight. Drinking water with or without melatonin was daily supervised for all groups. The numbers of mice per group were: FC, 10 mice; FM, 10 mice; MC, 13 mice; and MM, 10 mice. After 1 month, animals were weighed and killed by decapitation. Blood was collected and centrifuged at 3000 x g for 15 min. Serum was aliquoted and frozen at –80 C until assayed.

Histological evaluation
After death, kidneys were removed, fixed in neutral buffered 4% formaldehyde, and embedded in paraffin. Despite the fact that intragroup differences were eventually minimal, to avoid disparity due to the area subjected to histological analysis, the kidneys were sagittally sectioned at 5 µm, five different equidistant levels were observed, and an overall evaluation for each animal was achieved. Sections were stained with hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS). Morphological changes in kidney sections were evaluated at the light microscopic level by two different observers who were unaware of the treatment group. The degree of severity of histological lesions characteristic of lupus nephritis was evaluated as absent (–), mild (+), moderate (2+), and severe (2++). According to Nose et al. (25), a mouse was considered positive for glomerulonephritis when it showed at least proliferative lesions and/or crescent lesions in more than 50% of 20 or more renal glomeruli. In addition to the glomerular changes described, arteritic lesions were observed in the mouse kidneys. A diagnosis of vasculitis was considered when one or more arteritic lesions in two sections of each kidney were observed, involving at least the destruction of the external lamina of the arterial wall.

Spleen cell and lymph node cell preparation
Spleens and axilar lymph nodes were aseptically collected free of connective tissue and weighed. Spleen and lymph node fragments were removed separately by passing them through a nylon mesh; then the lymph node cells and splenocytes were obtained by centrifugation. The contaminating red blood cells in splenocytes were lysed by Ficoll-Hypaque density gradient (26); those in lymph node were lysed in cold 0.2% NaCl. The isolated cells (splenocytes and axilar lymph node cells) were washed with cold 0.15 M NaCl and finally resuspended in RPMI 1640 medium containing 5% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. Cell viability was determined by the trypan blue exclusion test and was always greater than 95%.

Culturing spleen and axilar lymph node cells for in vitro production of cytokines
Spleen and axilar lymph node cells, at a final concentration of 5 x 106 cells/ml, were cultured in flat-bottom microtiter plates (Nunc, Copenhagen, Denmark) in RPMI 1640 culture medium containing 5% fetal calf serum with phytohemagglutinin (PHA; 8 µg/ml; 48 h), or lipopolysaccharide (LPS; 10 µg/ml; 24 h; Sigma-Aldrich Corp., St. Louis, MO) in a CO2 incubator. Cell suspensions were centrifuged at 10,000 x g for 1 min. Cell-free supernatants were stored at –80 C for cytokine determination.

Determination of cytokines in culture supernatants and serum
The cytokine concentrations in culture supernatants and serum were determined by ELISA using mouse cytokine kits purchased from BD Pharmingen (OptEIA Set, San Diego, CA). The kit protocols were followed. Briefly, Nunc-Maxisorb 96-well ELISA plates were coated with specific antimouse capture antibodies in a coating buffer and incubated at 4 C overnight. The following day, capture antibodies were aspirated, the plates were washed with 0.01 M PBS (pH 7.3) and 0.05% Tween 20. The plates were incubated with blocking buffer (PBS-3% BSA) for 2 h at room temperature. Appropriately diluted samples or standards (final volume, 100 µl) were added to test wells in triplicate and incubated for 3 h at room temperature. The wells were washed, and the secondary antibodies were added for 1-h incubation. After washes, 100 µl substrate (3,3',5,5'-tetramethylbenzidine and hydrogen peroxide solution) were added to the wells. The reaction was stopped by the addition of 50 µl HCl. The intensity of the yellow color was read at 450 nm in a microplate reader. The mean absorbances of standards and samples were calculated. A standard curve was constructed to quantify the concentration of specific cytokines in the samples.

Quantitation of serum Ig levels
Serum IgM and IgG levels were determined by sandwich ELISA according to a previous report (27). Microtiter plates were coated with 1 µg/ml goat antimouse Ig heavy and light chain (H + L; Zymed Laboratories, San Francisco, CA) for determination of IgM or with 2 µg/ml goat antimouse Ig (H + L) for determination of IgG at 4 C overnight and blocked with 3% BSA in PBS. Sera were added at 10,000- and 500,000-fold dilutions for quantification of IgM and IgG, respectively. After washing, horseradish peroxidase-conjugated goat antimouse Ig antibody (Calbiochem, San Francisco, CA) was added. After washing, the plates were incubated with 3,3',5,5'-tetramethylbenzidine substrate and H2O2. The reaction was stopped with HCl, then absorbance at 490 nm was determined in microplate reader. The total IgM and IgG concentrations were calculated with reference to the calibration curve generated with mouse Ig reference serum.

Quantification of serum anti-double-stranded DNA (anti-dsDNA) antibody levels
Anti-dsDNA antibodies of IgM and IgG were also determined by sandwich ELISA (27). Microtiter plates were coated with 50 µg/ml poly-L-lysine (Sigma-Aldrich Corp.) in PBS. After washing, the plates were incubated with 2 µg/ml calf thymus DNA (Sigma-Aldrich Corp.) in PBS at 37 C overnight to dry. After washing, horseradish peroxidase-conjugated antibodies against mouse IgM and IgG (Calbiochem) were used as a secondary antibody. Sera were diluted 200- and 2000-fold for quantification of IgM and IgG anti-dsDNA Abs, respectively. IgM and IgG dsDNA antibody concentrations were calculated with reference to the calibration curve generated with mouse Ig reference serum.

Quantification of serum antitype II collagen antibody
Antitype II collagen antibodies of IgM and IgG in serum were also measured by sandwich ELISA according to a previous report (28). Peroxidase-conjugated goat antimouse Ig IgG (H + L) (Calbiochem) was used for the detection of rat antitype II collagen antibodies. Serum samples were diluted to 1:2560. IgM and IgG antitype II collagen antibody concentrations were calculated with reference to the calibration curve generated with mouse Ig reference serum.

Measurement of nitrite/nitrate
Nitrite plus nitrate production, an indicator of NO synthesis, was measured in serum samples as previously described (29). In brief, samples were diluted 1:5 in PBS, and the nitrate in the samples was reduced to nitrite by incubation with nitrate reductase and reduced nicotinamide adenine phosphate dinucleotide at room temperature for 3 h. The nitrite concentration in the samples was measured by the Griess reaction, adding 100 µl Griess reagent (0.1% naphthylethylendiamine dihydrochloride in H2O and 1% sulfanilamide in 5% concentrated H3PO4; 1:1, vol/vol) to 100-µl samples. The OD at 550 nm was measured using an ELISA microplate reader. Nitrate concentrations were calculated by comparison with a standard curve of sodium nitrate.

Statistical analysis
Groups and treatments were compared with a parametric two-way ANOVA. If results were statistically significant, post hoc comparisons between treatment and disease were tested using a multiple comparison test, the Student-Newman-Keuls method. Homogeneity of variances between groups and treatment was previously checked, and when needed, logarithm transformations of data were performed. All results were reported as statistically significant for P < 0.05. Statistical analyses and calculations were performed with the statistical package SPSS version 12.0 (SPSS, Inc., Chicago, IL).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Histological changes in female and male mice treated, or not, with melatonin were compared at the light microscopic level. As shown in Table 1Go, all female mice and most male mice presented glomerular abnormalities ranging from mild to severe patterns. Both global and segmental proliferative lesions, characterized by increases in mesangial matrix and cellularity, were observed. In severely affected glomeruli, obliteration of the capillary lumina, advanced sclerosis, caryorrhesis, fibrinoid necrosis, and glomerular collapse were apparent. Besides those changes, numerous glomeruli presented crescentic lesions in different degrees of progression (Fig. 1Go, A1–A3). In female mice, the pattern was unaltered after melatonin treatment. Nevertheless, in male mice, the administration of melatonin changed the glomerulonephritic lesion from moderate to severe, and cellular crescents from mild to moderate (Table 1Go). Despite this apparent worsening of renal histology in male mice after melatonin treatment, the histopathological features of glomeruli in female mice treated, or not, with melatonin were, in general, more pronounced than in males.


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  		TABLE 1. Renal scores for histopathological features in female and male MRL/lpr mice, non treated or treated with melatonin for 1 month

 

Figure 1
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  		FIG. 1. Histopathological changes in renal tissues of male and female MRL/MpJ-Faslpr mice, treated, or not, with melatonin, compared with normal areas found in some of the same kidneys. A1, Glomerulus from an apparently normal renal area observed in a male mouse to demonstrate its normal appearance with the PAS technique (magnification, x400). A2, Mesangiopathic changes in a glomerulus from a nontreated male mouse, associated with global mesangial proliferation, mesangial deposits partially obliterating the capillary lumina, and an organizing cellular crescent formation on the right (PAS; magnification, x400). A3, Glomerulonephritis in a melatonin-treated male mouse characterized by conspicuous mesangial deposits of hyaline materials, karyorrhetic debris, and a crescentic lesion obliterating the capsular space (PAS; magnification, x400). B1, Artery located at the corticomedullary level from a normal renal area to demonstrate its normal appearance with the H&E technique (magnification, x200). B2, Vasculitis of renal artery from a nontreated female mouse, characterized by accumulation of mononuclear cells, destruction of external elastic lamina, and hyperplastic changes in the vascular wall (H&E; magnification, x200). B3, Artery from a melatonin-treated female mouse showing accumulation of mononuclear cells around the intact vascular wall (H&E; magnification, x200). C1, Severe inflammatory infiltrate in the cortical interstitium from a nontreated female mouse (PAS; magnification, x200). C2, Moderate inflammatory cell infiltration in the cortical interstitium from a melatonin-treated female mouse (PAS; magnification, x200). C3, This micrograph illustrates pyelonephritis in a melatonin-treated female mouse at the pelvicalyceal level (H&E; magnification, x40). The images shown are representative of each experimental group.

 
In addition to the glomerular changes described, arteritic lesions were observed in mouse kidneys. A diagnosis of vasculitis was considered when one or more arteritic lesions in two sections of each kidney were observed, involving at least the destruction of the external lamina of the arterial wall (17). Following this criterion, all female and some male nontreated mice presented this kind of vascular lesion. This histological pattern was moderate for females and mild for males. The lesions were also characterized by the accumulation of mononuclear cells around the blood vessels and the presence of hyperplastic changes in the vascular wall. After melatonin treatment, there was a general improvement in female mice compared with the nontreated female group. Thus, the vascular walls remained in all cases completely intact, although an accumulation of mononuclear cells around the arteries persisted (Fig. 1Go, B1–B3). In contrast, after melatonin treatment in male mice, the number of arteries affected with vasculitis increased, reaching a degree similar to that in the nontreated female mice group (Table 1Go).

In all animals the presence of cellular interstitial inflammatory infiltrates throughout the renal cortex was observed, with a severe pattern for females and a moderate pattern for males. A quite florid paravenular lymphoplasmacytic infiltrate was displayed in some cases. The more conspicuous interstitial infiltrate was presented by the kidneys belonging to the nontreated female group, but it was also observed in all male mice. However, after melatonin administration, the female mice, in contrast to the male mice, showed a considerable reduction of cellular infiltration (Fig. 1Go, C1–C2). Furthermore, all mice, independently of sex or treatment, presented pyelonephritis to variable degrees (Fig. 1Go, C3).

The comparison of total IgM and IgG levels between sex and treatment groups showed a significant result for sex (P < 0.001) and the interaction between sex and treatments (P < 0.001), but not for treatment (Fig. 2Go). Figure 2AGo shows lower total IgG levels in control males compared with control females (by Student-Newman-Keuls test, P < 0.05). However, among mice treated with melatonin, total IgM and IgG levels were lower in females than in males (by Student-Newman-Keuls test, P < 0.05).


Figure 2
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  		FIG. 2. Serum levels of total IgM and IgG (A), IgM and IgG anti-dsDNA antibodies (B), and IgM and IgG anticollagen type II antibodies (C) in MRL-lpr mice. Sera were obtained from female (F) or male (M) mice after 1 month of treatment with vehicle (FC or MC) or melatonin (FM or MM). Data are presented as the mean ± SEM of all animals per group. a, P < 0.05 vs. FC; b, P < 0.05 vs. MC.

 
Anti-DNAds antibody results are displayed in Fig. 2BGo, and anticollagen type II antibody results are shown in Fig. 2CGo. Comparing IgG anti-dsDNA antibodies, only the interaction effect between sex and treatment was statistically significant (P < 0.05). For IgM, both the main effect of sex (P < 0.001) and the interaction effect (P < 0.001) were statistically significant.

For IgG anticollagen type II antibodies, differences in data were statistically significant by sex (P < 0.001) and the interaction effect (P < 0.001). For IgM data, statistically significant differences by sex (P < 0.05) and the interaction (P < 0.05) were found. Both IgM and IgG antibody titers among females were lower in melatonin-treated females than in controls (by Student-Newman-Keuls test, P < 0.05). In males, levels were higher in the melatonin group than in controls (by Student-Newman-Keuls test, P < 0.05).

TNF-{alpha}, IL-1ß, IL-6, IFN-{gamma}, IL-10, and IL-2 were measured in sera from female and male mice (Fig. 3Go). TNF-{alpha}, IL-1ß, and IL-6 (Fig. 3Go, A–C) were chosen as cytokines that in nature mediate immunity; IFN-{gamma} and IL-10 (Fig. 3Go, D and E) were measured as cytokines that regulate inflammation of immune origin. IL-2 (Fig. 3FGo) was measured as a cytokine that regulates lymphocytary activation, proliferation, and differentiation. The comparison of cytokine levels (TNF-{alpha}, IL-1ß, IL-6, IFN-{gamma}, IL-10, and IL-2) by sex and treatment was statistically significant for the main effect of sex (P < 0.001 for TNF-{alpha}; P < 0.05 for the others) and the interaction between the two factors (P < 0.05), but not for treatment. Post hoc comparisons were significant for the control group, with higher levels of proinflammatory cytokines (IL-1ß and IL-6), IFN-{gamma}, and IL-2 in females than in control males (by Student-Newman-Keuls test, P < 0.05). Among females, levels were lower in the treated group than in controls (by Student-Newman-Keuls test, P < 0.05). On the contrary, these cytokines were increased in treated males compared with the control group (by Student-Newman-Keuls test, P < 0.05). Levels of IL-10, an antiinflammatory cytokine, were higher in the male group than the female group (by Student-Newman-Keuls test, P < 0.05). In this case, the administration of melatonin raised IL-10 levels in females while reducing them in males (by Student-Newman-Keuls test, P < 0.05).


Figure 3
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  		FIG. 3. Serum cytokine levels of TNF-{alpha} (A), IL-1ß (B), IL-6 (C), IFN-{gamma} (D), IL-10 (E), and IL-2 (F) in MRL-lpr mice. Sera were obtained from female (F) or male (M) mice after 1 month of treatment with vehicle (FC or MC) or melatonin (FM or MM). Data are presented as the mean ± SEM of all animals per group. a, P < 0.05 vs. FC; b, P < 0.05 vs. MC.

 
For nitrite/nitrate levels, two-way ANOVA test showed significant differences between sex (P < 0.001) and the interaction effect (P < 0.05). As shown in Fig. 4Go, levels were similar in both sexes within control groups. However, in the melatonin group, levels of nitrite/nitrate were lower in females and higher in males (by Student-Newman-Keuls test, P < 0.05).


Figure 4
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  		FIG. 4. Levels of nitrite/nitrate in serum of MRL-lpr mice. Sera were obtained from female (F) or male (M) mice after 1 month of treatment with vehicle (FC or MC) or melatonin (FM or MM). Data are presented as the mean ± SEM of all animals per group. a, P < 0.05 vs. FC; b, P < 0.05 vs. MC.

 
Cytokine production by spleen and stimulated lymph node cells was measured. The analysis of cytokine production data from spleen cells showed that the main effect of sex was statistically significant for all cytokines (P < 0.001 for TNF-{alpha} and IL-10; P < 0.05 for IL-1ß, IL-6, and IFN-{gamma}). A main effect of treatment was significant for TNF-{alpha} (P < 0.001) and IL-6 (P < 0.05). Interaction effects of sex and treatment were statistically significant (P < 0.001) for all analyzed cytokines.

Moreover, the comparison of cytokine production by lymph node cells found statistically significant interactions for TNF-{alpha} and IL-10 (P < 0.05) and for IL-1ß, IL-6, and IFN-{gamma} (P < 0.001). Similar to spleen cell results, differences by sex were significant for all cytokines (P < 0.001 for IL-1ß; P < 0.05 for the others). Comparisons by treatment showed a significant difference for IL-1ß (P < 0.05).

In both cell types, TNF-{alpha}, IL-1ß, IL-6, and IFN-{gamma} levels (Fig. 5Go) were significantly higher in control males than in control females (by Student-Newman-Keuls test, P < 0.05). In both cell types, treatment with melatonin increased TNF-{alpha}, IL-1ß, IL-6, and IFN-{gamma} levels in females, but decreased them significantly in males (by Student-Newman-Keuls test, P < 0.05). In contrast, IL-10 levels (Fig. 5DGo), which were similar among control groups, decreased in females after the administration of melatonin and increased in males (by Student-Newman-Keuls test, P < 0.05).


Figure 5
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  		FIG. 5. Levels of TNF-{alpha} (A), IL-1ß (B), IL-6 (C), IFN-{gamma} (D), and IL-10 (E) in spleen and lymph node cells from MRL-lpr mice stimulated with LPS. Spleens and lymph nodes were obtained from female (F) or male (M) mice after 1 month of treatment with vehicle (FC or MC) or melatonin (FM or MM). Data are presented as the mean ± SEM of all animals per group. In spleen cells: a, P < 0.05 vs. FC; b, P < 0.05 vs. MC. In lymph nodes: a, P < 0.05 vs. FC; b, P < 0.05 vs. MC.

 
We also studied the production of cytokines (IFN-{gamma}, IL-10, and IL-2) by spleen and lymph node cells in vitro stimulated with PHA. Similar results were found. Thus, on spleen lymph node cells, differences by sex were significant for all cytokines (P < 0.05 for IFN-{gamma} and IL-2; P < 0.001 for IL-10). Interactions were significant for all cytokines on spleen (P < 0.05) and lymph node cells (P < 0.001 for IFN-{gamma}; IL-10 and P < 0.05 for IL-2). No significant effect of treatment was found in either type of cell.

In contrast to nonstimulated cells, IFN-{gamma} and IL-2 levels (Fig 6Go, A and B) were lower in control males than in control females in both cell types (by Student-Newman-Keuls test, P < 0.05). In melatonin-treated females IFN-{gamma} and IL-2 levels diminished compared with their controls (by Student-Newman-Keuls test, P < 0.05), whereas in treated males, these levels were augmented compared with their controls (by Student-Newman-Keuls test, P < 0.05). IL-10 levels (Fig. 6CGo) were higher in control males than in control females (by Student-Newman-Keuls test, P < 0.05). The comparison between treated and nontreated groups resulted in a greater production of IL-10 in melatonin-treated females than in control females and a lesser production of IL-10 in melatonin-treated males than in control males (by Student-Newman-Keuls test, P < 0.05).


Figure 6
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  		FIG. 6. Levels of IFN-{gamma} (A), IL-10 (B), and IL-2 (C) in spleen and lymph node cells from MRL-lpr mice stimulated with PHA. Spleens and lymph nodes were obtained from female (F) or male (M) mice after 1 month of treatment with vehicle (FC or MC) or melatonin (FM or MM). Data are presented as the mean ± SEM of all animals per group. In spleen cells: a, P < 0.05 vs. FC; b, P < 0.05 vs. MC. In lymph node cells: a, P < 0.05 vs. FC; b, P < 0.05 vs. MC.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Differences between males and females in the incidence and prevalence of autoimmune disease have been reported in the literature. Thus, the female to male susceptibility ratio is 9:1 for SLE, 2:1 to 4:1 for rheumatoid arthritis, and 9:1 for Sjögren’s syndrome. Sex hormones have being proposed as a possible cause of this sexual dimorphism. A protective role of androgen and/or a deleterious role of estrogen have been suggested (30). Autoimmune disease has been described to be more severe in female MRL-lpr mice; besides, female MRL-lpr animals experience early mortality compared with males (16). In fact, histological studies showed that all female mice and most male mice exhibited glomerular abnormalities, arteritic lesions, and cellular interstitial inflammatory infiltrate, ranging from mild to severe patterns. Treatment with melatonin improved the histological pattern in females and worsened it in males.

Total IgM and IgG as well as levels of IgM and IgG autoantibodies were analyzed in males and females treated, or not, with melatonin. The female control group showed higher total IgG levels than the male control group. This fact correlates with the superior humoral immunity described in females (30). The administration of melatonin in females reduced humoral response with respect to controls. However, in melatonin-treated males the humoral response was higher than in controls. Considering levels of IgM and IgG anti-dsDNA antibodies as a disease marker (22), we may ensure that administration of melatonin ameliorates the course of the disease in females, but magnifies it in males.

When different cytokines were measured, we found no differences in inflammatory cytokines (TNF-{alpha}, IL-1ß, and IL-6) comparing male and female mice. Similar results were observed after NO determination. After melatonin administration, inflammatory capacity diminished in females, but increased in males, compared with their respective controls. An elevated inflammatory status has been reported to be involved in disease manifestations such as nephritis, vasculitis, and arthritis (16). This inflammatory status is caused by an enhanced state of macrophage activation with elevated levels of TNF-{alpha}, IL-1ß, IL-6, and NO that are crucial until the appearance of glomerulonephritis in mice (31, 32).

Over the past 2 decades, there have been many reports that IFN-{gamma} affects all major cellular players of the immune system, including macrophages and T, B and natural killer cells. In this way, IFN-{gamma} was also determined in serum. Our results show that control females had higher IFN-{gamma} levels than control males, and the administration of melatonin produced the same effect as previously observed, i.e. a decrease in this cytokine in females and an increase in males. It has been reported that IFN-{gamma} enhances the production of IgG2a and IgG3, accelerates glomerulonephritis in MRL/lpr mice (33), and is required for lupus development, presumably by increasing major histocompatibility complex expression and autoantigen presentation to otherwise quiescent nontolerant antiself T cells and also by promoting local immune and inflammatory processes (34, 35). IL-10 and antiinflammatory cytokine were measured, and opposite results were obtained. There is evidence that IL-10 may down-modulate murine lupus through inhibition of pathogenic Th1 cytokine responses (36). Thus, IL-10 levels were lower in females than in control males, increasing after melatonin administration in females and diminishing compared with their respective controls in males. This event might have a beneficial effect on mice, because there is evidence that IL-10 may down-modulate murine lupus through inhibition of Th1 cytokine production and the proliferation of CD4+ cells (37).

In contrast, the data obtained in vitro demonstrated that the cells stimulated with PHA had a cytokine production pattern similar to that in serum. Control females showed a prevalent proinflammatory response (high IFN-{gamma} levels and low IL-10 levels), whereas control males showed a prevalent antiinflammatory response (low IFN-{gamma} levels and high IL-10 levels). Melatonin treatment changed the type of response, i.e. decreased the inflammatory response in females and increased it in males. We thought that it was the antiinflammatory action of IL-10, increased by melatonin, that drove the change in the immune response. However, when cells were stimulated with LPS, we obtained a cytokine production pattern different from that in serum or PHA-stimulated cells. Thus, an antiinflammatory response predominated in melatonin-treated females and control males.

The dual effect of melatonin on SLE depending on gender may be explained by the role of melatonin as mediator of reproductive physiology through inhibition of GnRH liberation (38, 39). In females, the administration of melatonin produced a diminution of estrogen production. It is well known that estrogen promotes the development of SLE (40, 41), producing an increase in inflammatory cytokines and autoantibodies (42, 43, 44). Therefore, the administration of melatonin could be reducing estrogen production, and subsequently, there could be decreases in inflammatory cytokines, autoantibody production, and levels of IFN-{gamma} and IL-2. This tendency was observed in our study.

Testosterone has been suggested to have the opposite action on the immune system (45, 46, 47). Therefore, the opposite results obtained depending on the sex of the animal might be explained by the modification of sex hormone levels by melatonin. However, sex hormone determination was not performed in the present work, because gender differences in these mice were unexpected results. Additional experiments should be performed to better ascertain the relationship between sex hormones and melatonin.

In conclusion, we have demonstrated that the administration of melatonin has a dual effect, depending on gender, in MRL-lpr mice in the early stages of disease. In females, melatonin may have a beneficial effect, with low levels of inflammatory cytokines, diminution of total IgM and IgG levels, and a diminished concentration of autoantibodies. However, the administration of melatonin to males has the opposite effect, with high levels of inflammatory cytokines that injure kidneys and elevation of autoantibody titers. These results may have significant implications for the pathogenesis and treatment of human SLE. However, additional experiments are necessary to better understand the mechanisms for melatonin action on autoimmune diseases. Furthermore, to ascertain the relationship between melatonin action and mouse gender, experiments should be performed involving sex hormone determination.


   Acknowledgments
 
We thank Dr. Juan Ramón Lacalle for helpful statistical analysis.


   Footnotes
 
This work was supported by grants from Ministerio de Ciencia y Tecnología, (PM98/0157) of Spain.

First Published Online December 22, 2005

Abbreviations: ds, Double stranded; H&E, hematoxylin and eosin; H + L, heavy and light chain; IFN-{gamma}, interferon-{gamma}; LPS, lipopolysaccharide; NO, nitric oxide; PAS, periodic acid-Schiff; PHA, phytohemagglutinin; SLE, systemic lupus erythematosus; Th, helper T cell.

Received June 3, 2005.

Accepted for publication December 13, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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