Middle-Age Male Mice Have Increased Severity of Experimental Autoimmune Encephalomyelitis and Are Unresponsive to Testosterone Therapy
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免疫学杂志 2005年第4期
Abstract
Treatment with sex hormones is known to protect against experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. However, little is known about how age affects the course of EAE or response to hormone treatment. This study demonstrates striking differences between middle-age vs young C57BL/6 male mice in the clinical course of EAE and response to both testosterone (T4) and estrogen (E2) hormone therapy. Unlike young males that developed an acute phase of EAE followed by a partial remission, middle-age males suffered severe chronic and unremitting EAE that was likely influenced by alterations in the distribution and function of splenic immunocytes and a significant reduction in suppressive activity of CD4+CD25+ regulatory T cells in the spleen and spinal cord. Middle-age males had reduced numbers of splenic CD4+ T cells that were generally hypoproliferative, but enhanced numbers of splenic macrophages and MHC class II-expressing cells, and increased secretion of the proinflammatory factors IFN- and MCP-1. Surprisingly, middle-age males were unresponsive to the EAE-protective effects of T4 and had only a transient benefit from E2 treatment; young males were almost completely protected by both hormone treatments. T4 treatment of young males inhibited proliferation of myelin oligodendrocyte glycoprotein 35–55-specific T cells and secretion of TNF- and IFN-. The effects of T4 in vivo and in vitro were reversed by the androgen receptor antagonist, flutamide, indicating that the regulatory effects of T4 were mediated through the androgen receptor. These data are the first to define age-dependent differences in EAE expression and response to hormone therapy.
Introduction
Multiple Sclerosis (MS)3 is a chronic demyelinating disease of the human CNS of unknown etiology and pathogenesis, eventually leading to progressive disability (1). Multiple genes, as well as many environmental and immunological factors, are thought to contribute to the pathology of MS. Nevertheless, sex hormones are considered to be critical modulating factors in the susceptibility and progression of disease. Women tend to have stronger humoral and cellular immune responses and are disproportionately susceptible to autoimmune diseases, including rheumatoid arthritis (2), systemic lupus erythematosus (3), scleroderma (4), Sj?gren’s syndrome (5), Grave’s disease (4), and MS (6). Although the occurrence of MS in males is less frequent and occurs later in life than in females, the disease tends to be more severe, especially when testosterone (T4) levels are diminished (7, 8, 9, 10, 11, 12), suggesting that low androgen levels may be permissive for disease.
The gender difference in MS is similar in animals with experimental autoimmune encephalomyelitis (EAE), an animal model for MS (13, 14, 15). The clinical course of EAE was shown to be affected by castration and hormone administration (16, 17, 18, 19). For example, castration of male SJL mice increased EAE severity (20). Conversely, female SJL mice implanted with testosterone pellets developed less severe EAE compared with mice implanted with placebo pellets (17, 21). Finally, treatment with exogenous androgen was shown to be protective in gonadally intact C57BL/6 and SJL males (22). Although testosterone clearly has protective effects against EAE in young males, the relationship between T4 levels and EAE disease susceptibility has not been studied in older males. In fact, we initially reasoned that the mechanism of T4-induced effects might involve distinct pathways in young vs middle-age males. Testosterone can be converted intracellularly to either dihydrotestosterone via the enzyme 5-reductase, or to 17-estradiol (E2) via cytochrome P450 (cytochrome P450 aromatase). The former pathway is particularly potent in younger males and declines with age, while the latter pathway remains steady throughout life. In the current study, we set out to identify the predominant pathway by which testosterone impacts neuronal pathology in young vs aging males.
The data from this study demonstrated age-dependent differences in the immune response which result in striking differences in the development and progression of EAE in young vs middle-age males. In middle-age mice, we found that EAE was more severe; this was likely to be a consequence of higher levels of MCP-1 and IFN-, higher percentage of MHC class II molecules on splenocytes, and impaired T cell regulatory responses despite a reduced splenic response to stimuli and a decreased CD4+:CD11b+ ratio. Interestingly, we found that exogenous supplementation of testosterone had a beneficial effect only on young males. Our data indicated that testosterone selectively diminished secretion of TNF- and IFN- cytokines and proliferation of myelin oligodendrocyte glycoprotein (MOG) 35–55 peptide-specific T cells, suggesting that testosterone treatment promoted anti-inflammatory activity. The protective effect of testosterone treatment was blocked by flutamide, an androgen receptor (AR) antagonist, supporting the hypothesis that this protection is mediated via AR. Our study expands the understanding of the mechanisms by which hormones promote anti-inflammatory responses and provides a rationale for future hormone treatment in MS.
Materials and Methods
Mice
C57BL/6 males were obtained from The Jackson Laboratory at 6–8 wk of age (young) and 8 mo of age (middle-age). The mice were housed in the Animal Resource Facility at the Portland Veterans Affairs Medical Center, and experimental protocols were performed in accordance with institutional guidelines.
Antigens
Mouse MOG35–55 peptide (MEVGWYRSPFSRVVHLYRNGK) was synthesized using solid-phase techniques and purified by HPLC at the Beckman Institute, Stanford University (Palo Alto, CA).
Induction of EAE
C57BL/6 male mice were immunized by s.c. injections in the flank with 200 μg of MOG35–55 peptide in CFA containing 200 μg of heat-killed Mycobacterium tuberculosis (Difco) on day 0. Additionally, mice received i.p. injections of 75 ng/mouse pertussis toxin (Ptx; List Biological Laboratories) on days 0 and +2. The mice were assessed daily for clinical signs of EAE according to the following scale: 0, normal; 1, limp tail or mild hind limb weakness; 2, moderate hind limb weakness or mild ataxia; 3, moderately severe hind limb weakness; 4, severe hind limb weakness or mild forelimb weakness and moderate ataxia; 5, paraplegia with no more than moderate forelimb weakness; and 6, paraplegia with severe forelimb weakness or severe ataxia, moribund, or dead.
Castration
Orchidectomy was conducted under aseptic conditions with halothane anesthesia administered by face mask. The scrotum was prepped with betadine and draped. A longitudinal incision, 1 cm in length, was made over the median septum dividing the right and left cavities. The left testicular capsule was incised and the testicle was exteriorized. The spermatic cord was cauterized 1 cm above the head of the epididymis and amputated. The stump and all vessels were cauterized. The stump was then replaced into the testicular cavity. The right testicle was excised similarly. The wound was closed with 6.0 Vicryl.
Hormone treatment
Hormone-treated mice were implanted 1 wk after castration with 60-day release pellets containing 5 mg testosterone (T4) and/or flutamide or 17-estradiol (E2) (Innovative Research of America). Pellets were introduced s.c. in the scapular region behind the neck using a 12-gauge trochar as described by the manufacturer. After 1 wk postimplantation, mice were immunized as described above. Serum concentrations of hormones were monitored by RIA before and during the course of EAE in representative mice from each treatment group and consistently fell within the ranges provided by the manufacturer. In a previous study, we demonstrated that there was no significant difference in basal hormone levels in middle-age (mean ± SEM, 2.6 ± 0.2 pg/ml; range, 0.3–14.3 pg/ml; median, 0.95 pg/ml; n = 8) vs young (mean ± SEM, 3.7 ± 0.9 pg/ml; range, 0.4–16.2 pg/ml; median, 0.8 pg/ml; n = 10) B6 male mice. Implantation of 5 mg T4 pellets produced comparable levels of serum T4 in middle-age (mean ± SEM, 22.3 ± 4.0 pg/ml; range, 5.3–56.7 pg/ml; median, 29.7 pg/ml; n = 8) vs young (mean ± SEM, 16.6 ± 3.0 pg/ml; range, 4.2–49.6 pg/ml; median, 9.4 pg/ml; n = 9) mice.
Histological analysis of spinal cords
Mice with representative clinical disease were selected from young and middle-age groups. Spinal cords were isolated and fixed in 10% paraformaldehyde. Transverse paraffin sections of the spinal cords were stained with Luxol fast blue-periodic acid-Schiff reagent-hematoxylin. The slides were analyzed by light microscopy. Inflammatory cells were detected as an accumulation of dark-stained (hematoxylin-stained) nuclei.
Analysis of cell populations by FACS
Four-color (FITC, PE, PerCP, allophycocyanin) fluorescence flow cytometry analyses were performed to determine the phenotypes of splenocytes. Spleens were harvested and single-cell suspensions were obtained by mechanical disruption. Cells were washed with staining medium (PBS containing 0.1% NaN3 and 2% FCS) and stained with a combination of the following mAbs: CD3 (145-2C11), CD4 (GK1.5), CD8 (53-6.7), CD11b (M1/70), CD11c (HL-3), CD19 (1D3), CD25 (PC61), LFA-1 (I21/7), VLA-4 (PS/2), I-A/I-E (M5/114.15.2), NK1.1 (PK136), Pan-NK (DX5), and CD45 (BD Pharmingen) for 20 min on ice. After incubation with mAbs, cells were washed twice, resuspended in the staining medium, and analyzed with a FACSCalibur (BD Biosciences). Forward and side scatter parameters were chosen to identify lymphocytes. Data were analyzed using CellQuest software (BD Biosciences). For each experiment, cells were stained with appropriate isotype control Abs to establish background staining and to set quadrants before calculating the percentage of positive cells.
Cytokine determination by cytometric bead array (CBA)
Splenocytes were cultured at 4 x 106 cells/well in a 24-well, flat-bottom culture plate in stimulation medium with 25 μg/ml MOG35–55 peptide for 48 h. Supernatants were then harvested and stored at –70°C until tested for cytokines. The mouse inflammation CBA kit was used to detect simultaneously IL-12p40, TNF-, IFN-, MCP-1, IL-10, and IL-6 (BD Biosciences). Briefly, 50 μl of cell sample was mixed with 50 μl of the mixed capture beads and 50 μl of the mouse Th1/Th2 PE detection reagent. The tubes were incubated at room temperature for 2 h in the dark, followed by a wash step. The samples were then resuspended in 300 μl of wash buffer before acquisition on the FACSCalibur. The data were analyzed using CBA software (BD Biosciences). Standard curves were generated for each cytokine using the mixed bead standard provided in the kit, and the concentration of cytokine in the cell supernatant was determined by interpolation from the appropriate standard curve.
RT-PCR
For quantitative real-time PCR analysis, total RNA was extracted from spinal cords using a Total RNAeasy kit (Qiagen) according to the manufacturer’s instructions, and cDNA was prepared with 2.5 μM random hexamer primers. The sequence-specific primer was designed using Primer Express software (Applied Biosystems). The level of Foxp3 was quantified by real-time PCR using the ABI 7000 Sequence Detection System (Applied Biosystems). Amplification was performed in a total volume of 25 μl for 40 cycles and product was detected using SYBR Green I dye (Molecular Probes). Samples were run in triplicates and their relative expression levels were determined by normalizing expression of each target to L32. Expression level of normalized samples is displayed in relative expression units.
Treg cell suppression assay using FACS-sorted cells
Indicator (CD4+CD25–) and suppressor (CD4+CD25+) cell fractions were isolated from splenocytes. These cell fractions were sorted using a FACSVantage (BD Biosciences) after staining with anti-CD4FITC and anti-CD25PE Abs (BD Pharmingen) for 20 min at 4°C. On the forward vs side scatter plot, the sort regions were constrained to the lymphocyte population. Sorted cells were collected into serum-containing medium, washed, and assessed for Treg activity. The CD4+CD25– cells were plated in triplicate at 5.0 x 104 cells/well, whereas the CD4+CD25+ cell fractions were plated in triplicate at 5.0 x 104, 2.5 x 104, 1.25 x 104, and 0.5 x 104. Thus, the two cell fractions were cocultured at the following ratios: 1:0, 1:1, 1:0.5, 1:0.25, 1:0.1, and 0:1. Irradiated and T cell-depleted splenocytes were added to all wells as APC at 5.0 x 104 along with 1 μg of anti-CD3. After 3 days of culture, plates were harvested on glass fiber filters and assessed for uptake of the labeled thymidine by liquid scintillation. The percent suppression was plotted vs decreasing mixtures of indicator/suppressor cells and a regression line was calculated. I50 were determined as the ratio of indicator/suppressor cells that produced 50% suppression.
Statistical analyses
Differences in day of onset were assessed using the ANOVA test. Differences in peak scores and cumulative disease index (CDI) were assessed using the Mann-Whitney U test. Difference in disease incidence between two treatment groups was assessed by Fisher’s exact test. Student’s t test was used for unmatched data for analysis of significance. Values of p 0.05 were considered significant.
Results
Severity of EAE is increased in middle-age vs young C57BL/6 male mice
We compared the time course and severity of EAE following immunization with MOG35–55 peptide in middle-age male (8 mo) vs young male (8 wk) mice. As shown in Fig. 1A, all animals tested developed EAE with no mortality. In two separate experiments, middle-age males displayed a significantly later onset, but more severe disease scores compared with young males. As is illustrated for experiment 1 (Fig. 1B), mean EAE scores rapidly worsened in both young and middle-age mice during the early disease phase (days 13–20), but thereafter, young mice showed a significant clinical improvement compared with middle-age mice that maintained chronic and severe disease. Pathological examination of spinal cords from young (Fig. 2A) and middle-age (Fig. 2B) mice was performed during the chronic phase of disease (day 33 after immunization). Widespread perivascular inflammation was observed in both groups, although the middle-age males had a more pronounced cellular infiltration (Fig. 2B) reflective of more severe clinical disease.
FIGURE 1. Age-dependent differences in the disease course of EAE in C57BL/6 males. A, Clinical parameters of EAE. CDI and peak were significantly higher in middle-age (8 mo) mice as compared with their young (6–8 wk) counterparts. Additionally, middle-age mice had delayed onset of disease. Data are from two separate experiments. B, EAE course in young and middle-age C57BL/6 males. Male C57BL/6 mice were immunized with MOG35–55 peptide in CFA plus Ptx on days 0 and +2 and observed/scored daily over 33 days postimmunization. Data are from experiment 1. *, Significantly more severe EAE in middle-age males from days 21 to 33 postimmunization.
FIGURE 2. Inflammation is more pronounced in spinal cords of middle-age mice. C57BL/6 males were immunized with MOG35–55 peptide in CFA plus Ptx and sacrificed on day 33 postimmunization. Spinal cord tissue sections were stained with Luxol fast blue-periodic acid Schiff-hematoxylin stain to indicate mononuclear cell infiltration. A, Young mice; B, middle-age mice.
Phenotypic characterization of splenocytes from young and middle-age mice
Differences in the disease severity in young vs middle-age mice might be attributable to differences in immune cell populations. We used FACS analysis to characterize the phenotype of splenocytes (Fig. 3) and found that middle-age mice had a significantly lower percentage of CD3+ (17 ± 1 vs 25 ± 3) and CD4+ (9 ± 1 vs 19 ± 4) T cells compared with young mice, but a significantly higher percentage of CD11b+ macrophages (17 ± 2 vs 7 ± 3) and I-A/I-E+ (MHC class II-expressing) cells (26 ± 2 vs 14 ± 1). Healthy naive young and middle-age mice had levels of CD3+, CD4+, and CD11b+ cells that were similar to each other (CD3+ = 29 ± 1 vs 28 ± 4; CD4+ = 18 ± 2 vs 16 ± 3;CD11b = 10 ± 1 vs 9 ± 3, middle-age vs young mice, respectively) and comparable to those from young mice with EAE. Thus, changes in these cell populations in middle-age mice occurred concomitantly with development of disease. No differences were seen in any of the other cell surface markers tested, including CD8 (T cells), CD11c (dendritic cells), CD19 (B cells), NK1.1 and DX5 (NK cells), CD25 (IL-2R), LFA-1 (integrin), VLA-4 (adhesion molecule), and CD45 (tyrosine phosphatase).
FIGURE 3. Middle-age males have fewer CD3+ and CD4+ T cells and more CD11b+ cells and increased expression of MHC class II molecules compared with young mice. Splenocytes were isolated from MOG35–55 peptide-immunized middle-age and young C57BL/6 males at the termination of experiment (day 33 postimmunization). The cells were analyzed for expression of extracellular markers by FACS analysis as described in Materials and Methods. The expression of CD3, CD4, CD11b, and I-A/I-E on total splenocytes from sick middle-age and young mice is shown. Data presented are representative of three separate experiments and are expressed as percentage of total splenocyte population. *, Statistically significant differences (p < 0.05).
Reduced Treg activity in middle-age vs young male mice
The chronic and unremitting course of EAE observed in middle-age males might be influenced by reduced suppressive activity of CD4+CD25+ Treg cells. Thus, we compared the suppressive properties of CD4+CD25+ splenic T cells obtained from middle-age vs young males (day 33 after immunization). CD4+CD25+ suppressor cells and CD4+CD25– indicator cells were purified by flow cytometry and mixed at various ratios (suppressor (S):indicator (I) = 1:0, 1:1, 1:2, 1:4, 1:10, and 0:1) before stimulation with anti-CD3 Ab in the presence of APC (T cell-depleted splenocytes). Uptake of [3H]thymidine was determined after 3 days of culture to assess T cell proliferation. CD4+CD25+ T cells from young males (Fig. 4A) were significantly more suppressive than the comparable population from middle-age males (Fig. 4B), showing a reduced I50 value (2% vs 26%) reflective of enhanced suppression at S:I cell ratios of 1:4 (76% vs 47%) and 1:10 (72% vs 20%). Interestingly, this reduced suppression observed in CD4+CD25+ splenocytes from middle-age males did not result in significant changes in the expression of Foxp3, a specific marker for regulatory T cells, in total unfractionated spleen cells (data not shown). This result may be explained by a general shift in Foxp3 expression from CD4+CD25+ to CD4+CD25– splenocytes in naive middle-age vs young male mice (data not shown). However, Foxp3 was strongly expressed in spinal cord tissue of mice of both ages, but was significantly reduced in middle-age vs young male mice (Fig. 4C). Of importance, Foxp3 expression in spinal cord tissue from naive middle-age and young mice was very low (Fig. 3C), indicating that CD4+CD25+ Treg cells had migrated into the spinal cord during the course of EAE and were selectively enhanced in young mice. The reduced presence of Treg cells in the CNS of middle-age males may account for increased severity of clinical and histological EAE (Figs. 1 and 2). Functional assessment of Treg suppression by spinal cord CD4+CD25+ T cells was not feasible due to low numbers of recovered cells.
FIGURE 4. Middle-age mice with EAE have reduced functional suppression of CD4+CD25+ splenocytes and reduced expression of Foxp3 in spinal cords compared with young mice. Splenocytes were isolated from young and middle-age C57BL/6 males with EAE on day 33 postimmunization. A and B, Purified CD4+CD25+ suppressor cells (S) and CD4+CD25– indicator cells (I) from young and middle-age mice, respectively, were cultured at different ratios of S:I (1:0, 0.1:1, 0.25:1, 0.5:1, 1:1, and 0:1) in the presence of anti-CD3 mAb and allophycocyanin. Three days later, cell proliferation was determined by [3H]thymidine incorporation and I50 was calculated for both groups. C, Foxp3 mRNA expression was measured by RT-PCR technique in spinal cords isolated from young and middle-age male mice with EAE 33 days postimmunization as well as from healthy naive controls. Expression of Foxp3 was statistically higher in spinal cord of immunized young males as compared with middle-age males. No difference in Foxp3 expression was observed in splenocytes isolated from naive young and middle-age mice. Data are from one of three separate experiments. *, Statistically significant difference (p < 0.05) in Foxp3 expression.
Testosterone treatment significantly benefits only young males and is mediated through the AR
In the light of the recent reports about protective properties of androgens on the development and progression of autoimmune diseases, we sought to determine whether testosterone (T4) protection was age dependent. To avoid possible interference by endogenous androgens, we castrated the subject mice and allowed them to recover for 1 wk before implantation of pellets containing EAE-protective levels of T4 (5-mg pellets). One week after implantation, the mice were immunized with MOG35–55 peptide in CFA plus Ptx to induce EAE. As shown in Fig. 5, pretreatment with T4 almost totally prevented EAE in young males, but had no significant effect on EAE in middle-age males. The T4-induced protection in young males was almost completely reversed in the presence of the AR antagonist flutamide (Fig. 5, A and B), indicating the protective effect was mediated through the AR.
FIGURE 5. Effects of testosterone treatment alone or in combination with flutamide on the course of EAE in young and middle-age C57BL/6 males. Young and middle-age C57BL/6 males were castrated and allowed to recover for 1 wk before hormonal treatment. One week after hormone pellet implantation, mice were immunized with MOG35–55 peptide in CFA plus Ptx to induce EAE. The mice were scored daily for 30 days after immunization. A, Clinical parameters of EAE for all treated groups. CDI and peak were significantly lower and onset of disease was delayed in young mice treated with T4 compared with both other groups. Testosterone treatment was ineffective in older mice. B and C, Clinical course of EAE in young mice and middle-age mice, respectively.
Estrogen treatment benefits both young and middle-age male mice
We have established previously that supplemental estrogen (E2) can prevent EAE in most C57BL/6 female mice, and thus sought to determine whether there were discernable differences in E2 effects on EAE in young vs middle-age male mice. In contrast to the effects of treatment with T4, we found that estrogen treatment benefited both young and middle-age males, but to varying degrees. Young mice pretreated with E2 had reduced incidence and severity of EAE (Fig. 6, A and B). E2 treatment of middle-age males also delayed the onset and reduced the incidence and severity of EAE in the early phase of the disease, but the effect was transient and eventually most of the middle-age E2-treated males developed moderately severe EAE (Fig. 6, A and C).
FIGURE 6. Effects of estrogen treatment on the course of EAE in young and middle-age C57BL/6 males. A, Clinical parameters of EAE for all treated groups. Onset of EAE was delayed in young and middle-age estrogen-treated males as compared with controls. CDI and peak was significantly reduced in young estrogen-treated males as compared with young controls. Clinical course of EAE in young mice (B) and middle-age mice (C). *, Significant difference in mean daily scores for E2-treated vs placebo-treated groups.
Splenocytes from middle-age males and testosterone-treated splenocytes from young males exhibit reduced proliferative responses
We compared the ability of splenocytes from young and middle-age males to proliferate in response to MOG35–55 peptide. As is shown in Fig. 7A, proliferation responses to MOG35–55 peptide by splenocytes from middle-age mice were reduced by >70% when compared with responses of young males. This pattern indicated an age-dependent hyporesponsiveness characteristic of the MOG-specific T cell population. The reduced proliferation response observed in middle-age vs young males could reflect either a reduced number or hyporeactivity of splenic T cells. To address this question, we stimulated with anti-CD3 and Con A the same number of isolated splenic CD4+ T cells from middle-age and young males with added APC from young males (Fig. 7B). Under these conditions, we found that T cells from middle-age mice were indeed hyporesponsive on a per-cell basis.
FIGURE 7. Proliferation to MOG35–55 peptide, anti-CD3, and Con A was impaired in splenocytes from middle-age mice. Testosterone treatment reduced the proliferation response of MOG35–55-specific T cells from young mice. Splenocytes were isolated from young and middle-age males with EAE 33 days postimmunization and T cell proliferation to MOG35–55 peptide was determined by a standard [3H]thymidine uptake assay. Additionally, proliferation of purified T cells isolated from healthy naive young and middle-age mice was evaluated on a per-cell basis in the presence of Con A or anti-CD3 and allophycocyanin from young mice. Proliferation experiments were repeated at least three times and a representative experiment is presented. A, Reduced proliferation response (cpm) of splenic T cells from middle-age vs young in response to MOG35–55 peptide. B, Reduced proliferation response (cpm) to Con A and anti-CD3 of purified T cells from healthy naive young vs middle-age males. C, T4 treatment reduced the proliferation response (cpm) to MOG35–55 peptide of splenocytes from young but not middle-age mice with EAE, and the effect was prevented when mice were treated with T4 plus the AR antagonist flutamide. D, No inhibition of splenocyte proliferation response (cpm) to Con A was found in young mice with EAE after treatment with T4 or T4 plus flutamide. *, Statistically significant differences (p < 0.05).
To test the effects of testosterone treatment on T cell proliferation, we compared responses in young and middle-age males treated previously with T4, T4/flutamide, and control. Interestingly, T4 treatment suppressed proliferation of MOG35–55 peptide-specific T cells, but not that of other T cells, as determined by response to Con A (Fig. 7, C and D). The level of proliferation to MOG35–55 peptide in the group treated with T4/flutamide was equivalent to that of control mice (Fig. 7C), indicating that the mechanism of T4-induced suppression was mediated through the AR. In contrast to its suppressive effects on T cells from young mice, T4 treatment had no effect on splenocyte responses to MOG35–55 peptide in middle age mice (Fig. 7C), consistent with a lack of clinical effect. Of interest, E2 treatment only nominally inhibited (20%) splenocyte responses to MOG35–55 peptide in both young and middle-age mice and did not restore responses in middle-age males to levels observed in young mice (data not shown).
Direct relationship among cytokine production, aging, and testosterone treatment
The increased severity of EAE in middle-age mice may be related to an age-related increase in the levels of proinflammatory cytokines. Cytokine production was measured by the CBA technique in splenocytes cultured from young and middle-age male mice. As shown in Fig. 8, splenocytes from middle-age mice produced significantly higher quantities of the proinflammatory chemokine MCP-1 (Fig. 8A) and cytokine IFN- (Fig. 8B) compared with young mice. However, no differences were seen in the expression of other proinflammatory cytokines, TNF-, IL-6, and IL-12p40, or the anti-inflammatory cytokine IL-10 (data not shown). Testosterone treatment has been shown to exhibit potent anti-inflammatory effects in several experimental systems by reducing the expression of IFN- and increasing the expression of IL-10 (21, 23). A similar mechanism may underlie the efficacy of testosterone treatment in the reduction of EAE severity in young males. To test this hypothesis, we determined cytokine levels in T4-, T4/flutamide-, and placebo-treated young and middle-age animals. Testosterone treatment decreased production of TNF- and IFN- in young males (Fig. 8, C and D), but not middle-age males (data not shown). No differences were seen in the expression of MCP-1, IL-6, IL-10, and IL-12p40 (data not shown). This testosterone-dependent reduction in the expression of TNF- and IFN- in young males was reversed by flutamide, once again suggesting that the T4 inhibition was dependent upon signaling through the AR (Fig. 8, C and D).
FIGURE 8. Cytokine production from splenocytes of young and middle-age males. Testosterone treatment alters cytokine secretion in young males. Splenocytes were isolated from young and middle-age males with EAE 33 days postimmunization and cytokine production was determined by CBA. Splenocytes isolated from young mice produced less MCP-1 (A) and IFN- (B) compared with middle-age mice. Testosterone treatment specifically diminished production of TNF- (C) and IFN- (D) in young males. Data presented are from one of three individual experiments. *, Statistically significant difference (p < 0.05).
Discussion
The results presented above demonstrate striking differences between middle-age and young C57BL/6 male mice in the clinical course of EAE and response to both testosterone and estrogen hormone therapy. The severe chronic and unremitting EAE observed in middle-age males could not be attributed to differences in basal levels of serum T4, but was likely influenced by alterations in the distribution and function of splenic immunocytes and a significant reduction in suppressive activity of CD4+CD25+ Treg cells in the spleen and spinal cord. Middle-age males had reduced numbers of splenic CD4+ T cells that were generally hypoproliferative, but enhanced numbers of macrophages and MHC class II-expressing cells and increased secretion of the proinflammatory factors IFN- and MCP-1. Surprisingly, middle-age males were unresponsive to the EAE-protective effects of T4, but had a transient benefit from E2 treatment, unlike young males that were almost completely protected by both hormone treatments. T4 treatment of young males inhibited proliferation of MOG35–55-specific T cells and secretion of TNF- and IFN-, and effects of T4 in vivo and in vitro were reversed by the AR antagonist flutamide, indicating that the regulatory effects of T4 were mediated through the AR. In contrast, T4 treatment of middle-age males had no effect on splenocyte proliferation, chemokine, or cytokine responses.
Differences in the susceptibility and disease severity of MS in males vs females suggest that gonadal hormones may play an important role in the pathology of this disease. Differences between the sexes in MS may be attributable to the direct action of steroids on immune and nonimmune cells and to the fact that males and females have different circulatory profiles for these steroids. However, the mechanisms underlying the response of the immune system to gonadal hormones are still unclear.
The gender differences observed in MS in humans are paralleled by those found for EAE in the SJL strain of mice, with males being less susceptible to disease than females, suggesting that androgens may play a protective role in disease induction (15, 24). Castration of male mice increased the severity not only of EAE (23), but also other autoimmune diseases, including nonobese diabetes, thyroiditis, and adjuvant arthritis (17, 25, 26, 27). Furthermore, endogenous and exogenous androgens have been shown to reduce the incidence and severity of disease (21, 22, 23, 24, 28). Interestingly, abnormally low levels of testosterone were found in male mice with EAE and humans with MS (29, 30), suggesting a bidirectional interaction between disease state and sex hormone production. However, it is important to note that all those studies were done on young males. Testosterone levels are thought to decline naturally in aging men and animals, and this may change the male response to disease. However, our results were apparently not affected by a reduced basal level of T4 in middle-age mice, since no significant difference in serum levels of T4 was observed between young (3.7 ± 0.9 pg/ml) and middle-age (2.6 ± 0.2 pg/ml) males. To date, very few, if any, studies of MS or EAE have been done on aging males, leaving a wide gap in our knowledge about the consequences of androgen loss on autoimmune disease. In this study, we evaluated the differential effects of exogenous replacement of testosterone and its intracellular "metabolite" E2 on young vs sexually senescent male mice.
Our study reveals increased EAE severity for middle-age males of the C57BL/6 strain relative to younger males following immunization with the MOG35–55 peptide in CFA. Although development of EAE was delayed in middle-age males, they showed a statistically significant increase in EAE severity during the chronic phase of disease. In contrast, the severity of EAE in young males declined substantially in the chronic phase, suggesting that regulatory mechanisms are more effective in younger mice. Recently, there has been renewed interest in regulatory cells that influence the course of a number of autoimmune diseases. Regulatory CD4+CD25+ T cells have been shown to inhibit progression of several autoimmune diseases (31, 32, 33), including EAE (34), and to control type 1 diabetes in rats and mice (35). In the pathogenesis of MS, the balance between pathogenic and protective/regulatory cells is the key event for induction, development, and progression of disease. Defective immunoregulation was observed in relapsing and progressive MS patients (36, 37). We hypothesized that the severe and permissive EAE in middle-age mice was related to dysfunctional regulatory mechanisms, including a reduced ability of CD4+CD25+ cells to suppress ongoing disease.
Indeed, our in vitro study revealed that the ability of CD4+CD25+ cells to suppress proliferation of CD4+CD25– indicator cells was diminished on a per-cell basis in middle-age males compared with young males. This reduction in functional splenic Treg activity is highly consistent with the age-dependent functional decline reported for murine and human Treg cells (38, 39, 40, 41, 42, 43). The underlying reason for the observed decline in suppressive activity as a function of aging is unknown, but could be influenced by an age-dependent decline in thymic function or a shift in the suppressive T cell population from activated (CD25+) to a nonactivated (CD25–) state. Indeed, we observed reduced expression of the Foxp3 gene (a marker for Treg cells) in CD4+CD25+ splenocytes but enhanced expression of Foxp3 in CD4+CD25– splenocytes in naive middle-age vs young mice, perhaps explaining why we did not observe a significant change in net Foxp3 expression in unfractionated spleen cells in mice with EAE. Of importance, however, we found a marked decrease of Foxp3 expression in spinal cords of middle-age mice as compared with young counterparts, strongly suggesting a reduced migration of Treg cells into the CNS of middle-age mice with EAE that could account for increased severity of clinical and histological EAE.
Several other factors may also contribute to the delayed onset and increased severity of EAE in aging mice. It is well known that aging leads to a decline in normal function of CD4+ T cells (44, 45, 46). We observed decreased ratios of CD4+:CD11b+ and a reduced responsiveness to both Ag and mitogen stimuli in splenocytes isolated from middle-age mice. Moreover, splenocytes from aging mice produced higher levels of IFN- and MCP-1 cytokines. Undoubtedly, the differences in immunological parameters between young and middle-age males presented in this study have a critical impact on the development and progression of EAE in aging males. Based on our findings, we suggest that EAE in middle-age mice is largely mediated by CD11b+ macrophages, and the increased severity may be attributed to a proinflammatory environment created by the increased production of MCP-1 and IFN- and a failure to mount sufficient CD4+CD25+ Treg responses, particularly in the CNS.
Both androgens and estrogens have been found to regulate the adult immune system. Whitacre et al. (7) reported that low estrogen levels foster proinflammatory Th1 responses, whereas high estrogen levels or testosterone treatment favors the anti-inflammatory Th2 state. The cytokine secretion pattern of human CD4+ T cell clones was strongly influenced by high concentrations of estrogen (47, 48). Recently, we discovered that estrogen treatment caused a significant decrease in TNF- and, to a lesser degree, IFN- by T cells, macrophages, dendritic cells, and microglial cells (49, 50, 51, 52, 53, 54). Males were found to have lower secretion of IFN- than females, suggesting that androgen can induce a Th2 bias (55, 56). Furthermore, Bebo et al. (20) found that inflammatory infiltration of CD4+ T cells to the CNS in EAE occurred only after castration, suggesting that androgens suppress Th1 responses.
In the present study, we found that splenocytes isolated from testosterone-treated young mice produced less IFN- and TNF- compared with placebo-treated control mice. Additionally, testosterone treatment rendered splenocytes from young mice hyporesponsive to MOG35–55 peptide, but not mitogen (Con A) stimulation, suggesting a selective influence on the encephalitogenic population. It should be noted, however, that Con A stimulation appears to act via cell surface sugar residues rather than through the TCR, thus leaving open the possibility that T4 treatment may also inhibit TCR activation of other, non-MOG35–55 peptide-specific T cells. Concurrent flutamide treatment reversed the effect of testosterone on the clinical outcome, as well as its effect on cytokine release and proliferation, suggesting that the androgen receptor is involved in all testosterone-dependent responses. Despite the immunological differences between middle-age and young male mice described above, it was surprising to discover that testosterone treatment was beneficial only in young males. In contrast, our estrogen treatment ameliorated the symptoms of EAE in both young and middle-age mice, although its effects were transient in middle-age mice. It is noteworthy that the effects of E2 on castrated young male mice were essentially identical to the effects of E2 in ovariectomized young female mice (52). These findings suggest that testosterone and estrogen may exert their protective effects through very different pathways.
The reason behind the failure of testosterone to protect middle-age mice from development of EAE or to inhibit splenocyte responses to MOG peptide remains unknown. Recent evidence emphasizes that neuroendocrine aging in men becomes significant by the fourth decade of life (57, 58). In addition to lower production of androgens, sensitivity to androgens appears to wane with age by poorly understood mechanisms. In rodents, receptor distribution in spinal cord has not been well described, but AR protein is well represented in brain regions not linked to reproductive function, including cortex, hippocampus, and basal ganglia (59, 60), and in forebrain axons and dendrites (61). Effects of age on AR immunoreactivity or receptor affinity to agonist have not been well characterized at any level within the CNS. However, several studies suggest a decrease in AR high-affinity binding sites with age in mice and rats (62, 63, 64). Therefore, our results would be consistent with these observations: the waning response to testosterone in older vs younger males is due to depressed receptor signaling due to loss of binding sites. Alternatively, a T4 metabolite, rather than T4 itself, may be responsible for protection against EAE, and the predominant pathway for testosterone metabolism is known to be age dependent. In younger males, testosterone is largely converted to dihydrotestosterone, a highly potent activator of the AR; however, the importance of this pathway diminishes with age. In a secondary pathway, T4 is converted into E2. Although this secondary pathway is less prominent in young males, it is sustained throughout life, becoming the predominant mechanism during andropause.
These data are the first to define age-dependent differences in EAE expression and response to hormone therapy in males. Although treatment of MS in males with testosterone shows some promise, our study raises the possibility that the success of this approach might be age dependent, with lesser or no effects in older males. At a minimum, it would seem prudent to carry out exploratory studies on older male MS patients to assess the immunoregulatory potential of T4 before initiating a treatment trial.
Acknowledgments
We thank Eva Niehaus for assistance in preparing this manuscript.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by Grants AI42376, NS23221, and NS23444 from the National Institutes of Health and grants from the National Multiple Sclerosis Society, the Nancy Davis Center Without Walls, and the Department of Veterans Affairs.
The authors have no financial conflict of interest.
2 Address correspondence and reprint requests to Dr. Halina Offner, Neuroimmunology Research R&D-31, Portland VA Medical Center, 3710 SW US Veterans Hospital Road, Portland, OR 97239. E-mail address: offnerva@ohsu.edu
3 Abbreviations used in this paper: MS, multiple sclerosis; EAE, experimental autoimmune encephalomyelitis; MOG, myelin oligodendrocyte glycoprotein; AR, androgen receptor; Ptx, pertussis toxin; CBA, cytometric bead array; Treg, regulatory T; CDI, cumulative disease index; S:I, suppressor:indicator.
Received for publication August 27, 2004. Accepted for publication December 3, 2004.
References
Vyse, T. J., J. A. Todd. 1996. Genetic analysis of autoimmune disease. Cell 85:311.
Glynn, L. E.. 1967. Experimental model of rheumatoid arthritis. J. Belge Rheumatol. Med. Phys. 22:201.
Inman, R. D.. 1978. Immunologic sex differences and the female predominance in systemic lupus erythematosus. Arthritis Rheum. 21:849.
Rodnan, G. P., R. L. Myerowitz, G. O. Justh. 1980. Morphologic changes in the digital arteries of patients with progressive systemic sclerosis (scleroderma) and Raynaud phenomenon. Medicine 59:393.
Whaley, K., W. W. Buchanan. 1971. Recent advances in Sjogren’s syndrome. Mod. Trends Rheumatol. 2:139.
Birk, K., C. Ford, S. Smeltzer, D. Ryan, R. Miller, R. A. Rudick. 1990. The clinical course of multiple sclerosis during pregnancy and the puerperium. Arch. Neurol. 47:738
Whitacre, C. C., S. C. Reingold, P. A. O’Looney. 1999. A gender gap in autoimmunity. Science 283:1277.
Gray, A., H. A. Feldman, J. B. McKinlay, C. Longcope. 1991. Age, disease, and changing sex hormone levels in middle-aged men: results of the Massachusetts Male Aging Study. J. Clin. Endocrinol. Metab. 73:1016.
Morley, J. E., F. E. Kaiser, H. M. Perry, III, P. Patrick, P. M. Morley, P. M. Stauber, B. Vellas, R. N. Baumgartner, P. J. Garry. 1997. Longitudinal changes in testosterone, luteinizing hormone, and follicle-stimulating hormone in healthy older men. Metabolism 46:410.
Nankin, H. R., J. H. Calkins. 1986. Decreased bioavailable testosterone in aging normal and impotent men. J. Clin. Endocrinol. Metab. 63:1418.
Tenover, J. S., A. M. Matsumoto, S. R. Plymate, W. J. Bremner. 1987. The effects of aging in normal men on bioavailable testosterone and luteinizing hormone secretion: response to clomiphene citrate. J. Clin. Endocrinol. Metab. 65:1118
Vermeulen, A.. 1991. Clinical review 24: androgens in the aging male. J. Clin. Endocrinol. Metab. 73:221.
Bebo, B. F., Jr, A. A. Vandenbark, H. Offner. 1996. Male SJL mice do not relapse after induction of EAE with PLP 139–151. J. Neurosci. Res. 45:680.
Cua, D. J., D. R. Hinton, S. A. Stohlman. 1995. Self-antigen-induced Th2 responses in experimental allergic encephalomyelitis (EAE)-resistant mice: Th2-mediated suppression of autoimmune disease. J. Immunol. 155:4052.
Voskuhl, R. R., H. Pitchekian-Halabi, A. MacKenzie-Graham, H. F. McFarland, C. S. Raine. 1996. Gender differences in autoimmune demyelination in the mouse: implications for multiple sclerosis. Ann. Neurol. 39:724
Fox, H. S.. 1995. Sex steroids and the immune system. Ciba Found. Symp. 191:203
Fox, H. S.. 1992. Androgen treatment prevents diabetes in nonobese diabetic mice. J. Exp. Med. 175:1409.
Roubinian, J. R., N. Talal, J. S. Greenspan, J. R. Goodman, P. K. Siiteri. 1978. Effect of castration and sex hormone treatment on survival, anti-nucleic acid antibodies, and glomerulonephritis in NZB/NZW F1 mice. J. Exp. Med. 147:1568.
Jansson, L., T. Olsson, R. Holmdahl. 1994. Estrogen induces a potent suppression of experimental autoimmune encephalomyelitis and collagen-induced arthritis in mice. J. Neuroimmunol. 53:203.
Bebo, B. F., Jr, J. C. Schuster, A. A. Vandenbark, H. Offner. 1998. Gender differences in experimental autoimmune encephalomyelitis develop during the induction of the immune response to encephalitogenic peptides. J. Neurosci. Res. 52:420.
Dalal, M., S. Kim, R. R. Voskuhl. 1997. Testosterone therapy ameliorates experimental autoimmune encephalomyelitis and induces a T helper 2 bias in the autoantigen-specific T lymphocyte response. J. Immunol. 159:3.
Palaszynski, K. M., K. K. Loo, J. F. Ashouri, H. B. Liu, R. R. Voskuhl. 2004. Androgens are protective in experimental autoimmune encephalomyelitis: implications for multiple sclerosis. J Neuroimmunol. 146:144
Bebo, B. F., Jr, J. C. Schuster, A. A. Vandenbark, H. Offner. 1999. Androgens alter the cytokine profile and reduce encephalitogenicity of myelin-reactive T cells. J. Immunol. 162:35
Kim, S., S. M. Liva, M. A. Dalal, M. A. Verity, R. R. Voskuhl. 1999. Estriol ameliorates autoimmune demyelinating disease: implications for multiple sclerosis. Neurology 52:1230.
Ahmed, S. A., W. J. Penhale. 1982. The influence of testosterone on the development of autoimmune thyroiditis in thymectomized and irradiated rats. Clin. Exp. Immunol. 48:367
Fitzpatrick, F., F. Lepault, F. Homo-Delarche, J. F. Bach, M. Dardenne. 1991. Influence of castration, alone or combined with thymectomy, on the development of diabetes in the nonobese diabetic mouse. Endocrinology 129:1382.
Harbuz, M. S., Z. Perveen-Gill, S. L. Lightman, D. S. Jessop. 1995. A protective role for testosterone in adjuvant-induced arthritis. Br. J. Rheumatol. 34:1117.
Voskuhl, R. R., K. Palaszynski. 2001. Sex hormones in experimental autoimmune encephalomyelitis: implications for multiple sclerosis. Neuroscientist 7:258.
Wei, T., S. L. Lightman. 1997. The neuroendocrine axis in patients with multiple sclerosis. Brain 120:1067.
Foster, S. C., C. Daniels, D. N. Bourdette, B. F. Bebo, Jr. 2003. Dysregulation of the hypothalamic-pituitary-gonadal axis in experimental autoimmune encephalomyelitis and multiple sclerosis. J. Neuroimmunol. 140:78
Mason, D., F. Powrie. 1998. Control of immune pathology by regulatory T cells. Curr. Opin. Immunol. 10:649.
Maloy, K. J., F. Powrie. 2001. Regulatory T cells in the control of immune pathology. Nat. Immunol. 2:816.
Roncarolo, M. G., M. K. Levings. 2000. The role of different subsets of T regulatory cells in controlling autoimmunity. Curr. Opin. Immunol. 12:676.
Kohm, A. P., P. A. Carpentier, H. A. Anger, S. D. Miller. 2002. Cutting edge: CD4+CD25+ regulatory T cells suppress antigen-specific autoreactive immune responses and central nervous system inflammation during active experimental autoimmune encephalomyelitis. J. Immunol. 169:4712.(Agata Matejuk, Corwyn Hop)
Treatment with sex hormones is known to protect against experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. However, little is known about how age affects the course of EAE or response to hormone treatment. This study demonstrates striking differences between middle-age vs young C57BL/6 male mice in the clinical course of EAE and response to both testosterone (T4) and estrogen (E2) hormone therapy. Unlike young males that developed an acute phase of EAE followed by a partial remission, middle-age males suffered severe chronic and unremitting EAE that was likely influenced by alterations in the distribution and function of splenic immunocytes and a significant reduction in suppressive activity of CD4+CD25+ regulatory T cells in the spleen and spinal cord. Middle-age males had reduced numbers of splenic CD4+ T cells that were generally hypoproliferative, but enhanced numbers of splenic macrophages and MHC class II-expressing cells, and increased secretion of the proinflammatory factors IFN- and MCP-1. Surprisingly, middle-age males were unresponsive to the EAE-protective effects of T4 and had only a transient benefit from E2 treatment; young males were almost completely protected by both hormone treatments. T4 treatment of young males inhibited proliferation of myelin oligodendrocyte glycoprotein 35–55-specific T cells and secretion of TNF- and IFN-. The effects of T4 in vivo and in vitro were reversed by the androgen receptor antagonist, flutamide, indicating that the regulatory effects of T4 were mediated through the androgen receptor. These data are the first to define age-dependent differences in EAE expression and response to hormone therapy.
Introduction
Multiple Sclerosis (MS)3 is a chronic demyelinating disease of the human CNS of unknown etiology and pathogenesis, eventually leading to progressive disability (1). Multiple genes, as well as many environmental and immunological factors, are thought to contribute to the pathology of MS. Nevertheless, sex hormones are considered to be critical modulating factors in the susceptibility and progression of disease. Women tend to have stronger humoral and cellular immune responses and are disproportionately susceptible to autoimmune diseases, including rheumatoid arthritis (2), systemic lupus erythematosus (3), scleroderma (4), Sj?gren’s syndrome (5), Grave’s disease (4), and MS (6). Although the occurrence of MS in males is less frequent and occurs later in life than in females, the disease tends to be more severe, especially when testosterone (T4) levels are diminished (7, 8, 9, 10, 11, 12), suggesting that low androgen levels may be permissive for disease.
The gender difference in MS is similar in animals with experimental autoimmune encephalomyelitis (EAE), an animal model for MS (13, 14, 15). The clinical course of EAE was shown to be affected by castration and hormone administration (16, 17, 18, 19). For example, castration of male SJL mice increased EAE severity (20). Conversely, female SJL mice implanted with testosterone pellets developed less severe EAE compared with mice implanted with placebo pellets (17, 21). Finally, treatment with exogenous androgen was shown to be protective in gonadally intact C57BL/6 and SJL males (22). Although testosterone clearly has protective effects against EAE in young males, the relationship between T4 levels and EAE disease susceptibility has not been studied in older males. In fact, we initially reasoned that the mechanism of T4-induced effects might involve distinct pathways in young vs middle-age males. Testosterone can be converted intracellularly to either dihydrotestosterone via the enzyme 5-reductase, or to 17-estradiol (E2) via cytochrome P450 (cytochrome P450 aromatase). The former pathway is particularly potent in younger males and declines with age, while the latter pathway remains steady throughout life. In the current study, we set out to identify the predominant pathway by which testosterone impacts neuronal pathology in young vs aging males.
The data from this study demonstrated age-dependent differences in the immune response which result in striking differences in the development and progression of EAE in young vs middle-age males. In middle-age mice, we found that EAE was more severe; this was likely to be a consequence of higher levels of MCP-1 and IFN-, higher percentage of MHC class II molecules on splenocytes, and impaired T cell regulatory responses despite a reduced splenic response to stimuli and a decreased CD4+:CD11b+ ratio. Interestingly, we found that exogenous supplementation of testosterone had a beneficial effect only on young males. Our data indicated that testosterone selectively diminished secretion of TNF- and IFN- cytokines and proliferation of myelin oligodendrocyte glycoprotein (MOG) 35–55 peptide-specific T cells, suggesting that testosterone treatment promoted anti-inflammatory activity. The protective effect of testosterone treatment was blocked by flutamide, an androgen receptor (AR) antagonist, supporting the hypothesis that this protection is mediated via AR. Our study expands the understanding of the mechanisms by which hormones promote anti-inflammatory responses and provides a rationale for future hormone treatment in MS.
Materials and Methods
Mice
C57BL/6 males were obtained from The Jackson Laboratory at 6–8 wk of age (young) and 8 mo of age (middle-age). The mice were housed in the Animal Resource Facility at the Portland Veterans Affairs Medical Center, and experimental protocols were performed in accordance with institutional guidelines.
Antigens
Mouse MOG35–55 peptide (MEVGWYRSPFSRVVHLYRNGK) was synthesized using solid-phase techniques and purified by HPLC at the Beckman Institute, Stanford University (Palo Alto, CA).
Induction of EAE
C57BL/6 male mice were immunized by s.c. injections in the flank with 200 μg of MOG35–55 peptide in CFA containing 200 μg of heat-killed Mycobacterium tuberculosis (Difco) on day 0. Additionally, mice received i.p. injections of 75 ng/mouse pertussis toxin (Ptx; List Biological Laboratories) on days 0 and +2. The mice were assessed daily for clinical signs of EAE according to the following scale: 0, normal; 1, limp tail or mild hind limb weakness; 2, moderate hind limb weakness or mild ataxia; 3, moderately severe hind limb weakness; 4, severe hind limb weakness or mild forelimb weakness and moderate ataxia; 5, paraplegia with no more than moderate forelimb weakness; and 6, paraplegia with severe forelimb weakness or severe ataxia, moribund, or dead.
Castration
Orchidectomy was conducted under aseptic conditions with halothane anesthesia administered by face mask. The scrotum was prepped with betadine and draped. A longitudinal incision, 1 cm in length, was made over the median septum dividing the right and left cavities. The left testicular capsule was incised and the testicle was exteriorized. The spermatic cord was cauterized 1 cm above the head of the epididymis and amputated. The stump and all vessels were cauterized. The stump was then replaced into the testicular cavity. The right testicle was excised similarly. The wound was closed with 6.0 Vicryl.
Hormone treatment
Hormone-treated mice were implanted 1 wk after castration with 60-day release pellets containing 5 mg testosterone (T4) and/or flutamide or 17-estradiol (E2) (Innovative Research of America). Pellets were introduced s.c. in the scapular region behind the neck using a 12-gauge trochar as described by the manufacturer. After 1 wk postimplantation, mice were immunized as described above. Serum concentrations of hormones were monitored by RIA before and during the course of EAE in representative mice from each treatment group and consistently fell within the ranges provided by the manufacturer. In a previous study, we demonstrated that there was no significant difference in basal hormone levels in middle-age (mean ± SEM, 2.6 ± 0.2 pg/ml; range, 0.3–14.3 pg/ml; median, 0.95 pg/ml; n = 8) vs young (mean ± SEM, 3.7 ± 0.9 pg/ml; range, 0.4–16.2 pg/ml; median, 0.8 pg/ml; n = 10) B6 male mice. Implantation of 5 mg T4 pellets produced comparable levels of serum T4 in middle-age (mean ± SEM, 22.3 ± 4.0 pg/ml; range, 5.3–56.7 pg/ml; median, 29.7 pg/ml; n = 8) vs young (mean ± SEM, 16.6 ± 3.0 pg/ml; range, 4.2–49.6 pg/ml; median, 9.4 pg/ml; n = 9) mice.
Histological analysis of spinal cords
Mice with representative clinical disease were selected from young and middle-age groups. Spinal cords were isolated and fixed in 10% paraformaldehyde. Transverse paraffin sections of the spinal cords were stained with Luxol fast blue-periodic acid-Schiff reagent-hematoxylin. The slides were analyzed by light microscopy. Inflammatory cells were detected as an accumulation of dark-stained (hematoxylin-stained) nuclei.
Analysis of cell populations by FACS
Four-color (FITC, PE, PerCP, allophycocyanin) fluorescence flow cytometry analyses were performed to determine the phenotypes of splenocytes. Spleens were harvested and single-cell suspensions were obtained by mechanical disruption. Cells were washed with staining medium (PBS containing 0.1% NaN3 and 2% FCS) and stained with a combination of the following mAbs: CD3 (145-2C11), CD4 (GK1.5), CD8 (53-6.7), CD11b (M1/70), CD11c (HL-3), CD19 (1D3), CD25 (PC61), LFA-1 (I21/7), VLA-4 (PS/2), I-A/I-E (M5/114.15.2), NK1.1 (PK136), Pan-NK (DX5), and CD45 (BD Pharmingen) for 20 min on ice. After incubation with mAbs, cells were washed twice, resuspended in the staining medium, and analyzed with a FACSCalibur (BD Biosciences). Forward and side scatter parameters were chosen to identify lymphocytes. Data were analyzed using CellQuest software (BD Biosciences). For each experiment, cells were stained with appropriate isotype control Abs to establish background staining and to set quadrants before calculating the percentage of positive cells.
Cytokine determination by cytometric bead array (CBA)
Splenocytes were cultured at 4 x 106 cells/well in a 24-well, flat-bottom culture plate in stimulation medium with 25 μg/ml MOG35–55 peptide for 48 h. Supernatants were then harvested and stored at –70°C until tested for cytokines. The mouse inflammation CBA kit was used to detect simultaneously IL-12p40, TNF-, IFN-, MCP-1, IL-10, and IL-6 (BD Biosciences). Briefly, 50 μl of cell sample was mixed with 50 μl of the mixed capture beads and 50 μl of the mouse Th1/Th2 PE detection reagent. The tubes were incubated at room temperature for 2 h in the dark, followed by a wash step. The samples were then resuspended in 300 μl of wash buffer before acquisition on the FACSCalibur. The data were analyzed using CBA software (BD Biosciences). Standard curves were generated for each cytokine using the mixed bead standard provided in the kit, and the concentration of cytokine in the cell supernatant was determined by interpolation from the appropriate standard curve.
RT-PCR
For quantitative real-time PCR analysis, total RNA was extracted from spinal cords using a Total RNAeasy kit (Qiagen) according to the manufacturer’s instructions, and cDNA was prepared with 2.5 μM random hexamer primers. The sequence-specific primer was designed using Primer Express software (Applied Biosystems). The level of Foxp3 was quantified by real-time PCR using the ABI 7000 Sequence Detection System (Applied Biosystems). Amplification was performed in a total volume of 25 μl for 40 cycles and product was detected using SYBR Green I dye (Molecular Probes). Samples were run in triplicates and their relative expression levels were determined by normalizing expression of each target to L32. Expression level of normalized samples is displayed in relative expression units.
Treg cell suppression assay using FACS-sorted cells
Indicator (CD4+CD25–) and suppressor (CD4+CD25+) cell fractions were isolated from splenocytes. These cell fractions were sorted using a FACSVantage (BD Biosciences) after staining with anti-CD4FITC and anti-CD25PE Abs (BD Pharmingen) for 20 min at 4°C. On the forward vs side scatter plot, the sort regions were constrained to the lymphocyte population. Sorted cells were collected into serum-containing medium, washed, and assessed for Treg activity. The CD4+CD25– cells were plated in triplicate at 5.0 x 104 cells/well, whereas the CD4+CD25+ cell fractions were plated in triplicate at 5.0 x 104, 2.5 x 104, 1.25 x 104, and 0.5 x 104. Thus, the two cell fractions were cocultured at the following ratios: 1:0, 1:1, 1:0.5, 1:0.25, 1:0.1, and 0:1. Irradiated and T cell-depleted splenocytes were added to all wells as APC at 5.0 x 104 along with 1 μg of anti-CD3. After 3 days of culture, plates were harvested on glass fiber filters and assessed for uptake of the labeled thymidine by liquid scintillation. The percent suppression was plotted vs decreasing mixtures of indicator/suppressor cells and a regression line was calculated. I50 were determined as the ratio of indicator/suppressor cells that produced 50% suppression.
Statistical analyses
Differences in day of onset were assessed using the ANOVA test. Differences in peak scores and cumulative disease index (CDI) were assessed using the Mann-Whitney U test. Difference in disease incidence between two treatment groups was assessed by Fisher’s exact test. Student’s t test was used for unmatched data for analysis of significance. Values of p 0.05 were considered significant.
Results
Severity of EAE is increased in middle-age vs young C57BL/6 male mice
We compared the time course and severity of EAE following immunization with MOG35–55 peptide in middle-age male (8 mo) vs young male (8 wk) mice. As shown in Fig. 1A, all animals tested developed EAE with no mortality. In two separate experiments, middle-age males displayed a significantly later onset, but more severe disease scores compared with young males. As is illustrated for experiment 1 (Fig. 1B), mean EAE scores rapidly worsened in both young and middle-age mice during the early disease phase (days 13–20), but thereafter, young mice showed a significant clinical improvement compared with middle-age mice that maintained chronic and severe disease. Pathological examination of spinal cords from young (Fig. 2A) and middle-age (Fig. 2B) mice was performed during the chronic phase of disease (day 33 after immunization). Widespread perivascular inflammation was observed in both groups, although the middle-age males had a more pronounced cellular infiltration (Fig. 2B) reflective of more severe clinical disease.
FIGURE 1. Age-dependent differences in the disease course of EAE in C57BL/6 males. A, Clinical parameters of EAE. CDI and peak were significantly higher in middle-age (8 mo) mice as compared with their young (6–8 wk) counterparts. Additionally, middle-age mice had delayed onset of disease. Data are from two separate experiments. B, EAE course in young and middle-age C57BL/6 males. Male C57BL/6 mice were immunized with MOG35–55 peptide in CFA plus Ptx on days 0 and +2 and observed/scored daily over 33 days postimmunization. Data are from experiment 1. *, Significantly more severe EAE in middle-age males from days 21 to 33 postimmunization.
FIGURE 2. Inflammation is more pronounced in spinal cords of middle-age mice. C57BL/6 males were immunized with MOG35–55 peptide in CFA plus Ptx and sacrificed on day 33 postimmunization. Spinal cord tissue sections were stained with Luxol fast blue-periodic acid Schiff-hematoxylin stain to indicate mononuclear cell infiltration. A, Young mice; B, middle-age mice.
Phenotypic characterization of splenocytes from young and middle-age mice
Differences in the disease severity in young vs middle-age mice might be attributable to differences in immune cell populations. We used FACS analysis to characterize the phenotype of splenocytes (Fig. 3) and found that middle-age mice had a significantly lower percentage of CD3+ (17 ± 1 vs 25 ± 3) and CD4+ (9 ± 1 vs 19 ± 4) T cells compared with young mice, but a significantly higher percentage of CD11b+ macrophages (17 ± 2 vs 7 ± 3) and I-A/I-E+ (MHC class II-expressing) cells (26 ± 2 vs 14 ± 1). Healthy naive young and middle-age mice had levels of CD3+, CD4+, and CD11b+ cells that were similar to each other (CD3+ = 29 ± 1 vs 28 ± 4; CD4+ = 18 ± 2 vs 16 ± 3;CD11b = 10 ± 1 vs 9 ± 3, middle-age vs young mice, respectively) and comparable to those from young mice with EAE. Thus, changes in these cell populations in middle-age mice occurred concomitantly with development of disease. No differences were seen in any of the other cell surface markers tested, including CD8 (T cells), CD11c (dendritic cells), CD19 (B cells), NK1.1 and DX5 (NK cells), CD25 (IL-2R), LFA-1 (integrin), VLA-4 (adhesion molecule), and CD45 (tyrosine phosphatase).
FIGURE 3. Middle-age males have fewer CD3+ and CD4+ T cells and more CD11b+ cells and increased expression of MHC class II molecules compared with young mice. Splenocytes were isolated from MOG35–55 peptide-immunized middle-age and young C57BL/6 males at the termination of experiment (day 33 postimmunization). The cells were analyzed for expression of extracellular markers by FACS analysis as described in Materials and Methods. The expression of CD3, CD4, CD11b, and I-A/I-E on total splenocytes from sick middle-age and young mice is shown. Data presented are representative of three separate experiments and are expressed as percentage of total splenocyte population. *, Statistically significant differences (p < 0.05).
Reduced Treg activity in middle-age vs young male mice
The chronic and unremitting course of EAE observed in middle-age males might be influenced by reduced suppressive activity of CD4+CD25+ Treg cells. Thus, we compared the suppressive properties of CD4+CD25+ splenic T cells obtained from middle-age vs young males (day 33 after immunization). CD4+CD25+ suppressor cells and CD4+CD25– indicator cells were purified by flow cytometry and mixed at various ratios (suppressor (S):indicator (I) = 1:0, 1:1, 1:2, 1:4, 1:10, and 0:1) before stimulation with anti-CD3 Ab in the presence of APC (T cell-depleted splenocytes). Uptake of [3H]thymidine was determined after 3 days of culture to assess T cell proliferation. CD4+CD25+ T cells from young males (Fig. 4A) were significantly more suppressive than the comparable population from middle-age males (Fig. 4B), showing a reduced I50 value (2% vs 26%) reflective of enhanced suppression at S:I cell ratios of 1:4 (76% vs 47%) and 1:10 (72% vs 20%). Interestingly, this reduced suppression observed in CD4+CD25+ splenocytes from middle-age males did not result in significant changes in the expression of Foxp3, a specific marker for regulatory T cells, in total unfractionated spleen cells (data not shown). This result may be explained by a general shift in Foxp3 expression from CD4+CD25+ to CD4+CD25– splenocytes in naive middle-age vs young male mice (data not shown). However, Foxp3 was strongly expressed in spinal cord tissue of mice of both ages, but was significantly reduced in middle-age vs young male mice (Fig. 4C). Of importance, Foxp3 expression in spinal cord tissue from naive middle-age and young mice was very low (Fig. 3C), indicating that CD4+CD25+ Treg cells had migrated into the spinal cord during the course of EAE and were selectively enhanced in young mice. The reduced presence of Treg cells in the CNS of middle-age males may account for increased severity of clinical and histological EAE (Figs. 1 and 2). Functional assessment of Treg suppression by spinal cord CD4+CD25+ T cells was not feasible due to low numbers of recovered cells.
FIGURE 4. Middle-age mice with EAE have reduced functional suppression of CD4+CD25+ splenocytes and reduced expression of Foxp3 in spinal cords compared with young mice. Splenocytes were isolated from young and middle-age C57BL/6 males with EAE on day 33 postimmunization. A and B, Purified CD4+CD25+ suppressor cells (S) and CD4+CD25– indicator cells (I) from young and middle-age mice, respectively, were cultured at different ratios of S:I (1:0, 0.1:1, 0.25:1, 0.5:1, 1:1, and 0:1) in the presence of anti-CD3 mAb and allophycocyanin. Three days later, cell proliferation was determined by [3H]thymidine incorporation and I50 was calculated for both groups. C, Foxp3 mRNA expression was measured by RT-PCR technique in spinal cords isolated from young and middle-age male mice with EAE 33 days postimmunization as well as from healthy naive controls. Expression of Foxp3 was statistically higher in spinal cord of immunized young males as compared with middle-age males. No difference in Foxp3 expression was observed in splenocytes isolated from naive young and middle-age mice. Data are from one of three separate experiments. *, Statistically significant difference (p < 0.05) in Foxp3 expression.
Testosterone treatment significantly benefits only young males and is mediated through the AR
In the light of the recent reports about protective properties of androgens on the development and progression of autoimmune diseases, we sought to determine whether testosterone (T4) protection was age dependent. To avoid possible interference by endogenous androgens, we castrated the subject mice and allowed them to recover for 1 wk before implantation of pellets containing EAE-protective levels of T4 (5-mg pellets). One week after implantation, the mice were immunized with MOG35–55 peptide in CFA plus Ptx to induce EAE. As shown in Fig. 5, pretreatment with T4 almost totally prevented EAE in young males, but had no significant effect on EAE in middle-age males. The T4-induced protection in young males was almost completely reversed in the presence of the AR antagonist flutamide (Fig. 5, A and B), indicating the protective effect was mediated through the AR.
FIGURE 5. Effects of testosterone treatment alone or in combination with flutamide on the course of EAE in young and middle-age C57BL/6 males. Young and middle-age C57BL/6 males were castrated and allowed to recover for 1 wk before hormonal treatment. One week after hormone pellet implantation, mice were immunized with MOG35–55 peptide in CFA plus Ptx to induce EAE. The mice were scored daily for 30 days after immunization. A, Clinical parameters of EAE for all treated groups. CDI and peak were significantly lower and onset of disease was delayed in young mice treated with T4 compared with both other groups. Testosterone treatment was ineffective in older mice. B and C, Clinical course of EAE in young mice and middle-age mice, respectively.
Estrogen treatment benefits both young and middle-age male mice
We have established previously that supplemental estrogen (E2) can prevent EAE in most C57BL/6 female mice, and thus sought to determine whether there were discernable differences in E2 effects on EAE in young vs middle-age male mice. In contrast to the effects of treatment with T4, we found that estrogen treatment benefited both young and middle-age males, but to varying degrees. Young mice pretreated with E2 had reduced incidence and severity of EAE (Fig. 6, A and B). E2 treatment of middle-age males also delayed the onset and reduced the incidence and severity of EAE in the early phase of the disease, but the effect was transient and eventually most of the middle-age E2-treated males developed moderately severe EAE (Fig. 6, A and C).
FIGURE 6. Effects of estrogen treatment on the course of EAE in young and middle-age C57BL/6 males. A, Clinical parameters of EAE for all treated groups. Onset of EAE was delayed in young and middle-age estrogen-treated males as compared with controls. CDI and peak was significantly reduced in young estrogen-treated males as compared with young controls. Clinical course of EAE in young mice (B) and middle-age mice (C). *, Significant difference in mean daily scores for E2-treated vs placebo-treated groups.
Splenocytes from middle-age males and testosterone-treated splenocytes from young males exhibit reduced proliferative responses
We compared the ability of splenocytes from young and middle-age males to proliferate in response to MOG35–55 peptide. As is shown in Fig. 7A, proliferation responses to MOG35–55 peptide by splenocytes from middle-age mice were reduced by >70% when compared with responses of young males. This pattern indicated an age-dependent hyporesponsiveness characteristic of the MOG-specific T cell population. The reduced proliferation response observed in middle-age vs young males could reflect either a reduced number or hyporeactivity of splenic T cells. To address this question, we stimulated with anti-CD3 and Con A the same number of isolated splenic CD4+ T cells from middle-age and young males with added APC from young males (Fig. 7B). Under these conditions, we found that T cells from middle-age mice were indeed hyporesponsive on a per-cell basis.
FIGURE 7. Proliferation to MOG35–55 peptide, anti-CD3, and Con A was impaired in splenocytes from middle-age mice. Testosterone treatment reduced the proliferation response of MOG35–55-specific T cells from young mice. Splenocytes were isolated from young and middle-age males with EAE 33 days postimmunization and T cell proliferation to MOG35–55 peptide was determined by a standard [3H]thymidine uptake assay. Additionally, proliferation of purified T cells isolated from healthy naive young and middle-age mice was evaluated on a per-cell basis in the presence of Con A or anti-CD3 and allophycocyanin from young mice. Proliferation experiments were repeated at least three times and a representative experiment is presented. A, Reduced proliferation response (cpm) of splenic T cells from middle-age vs young in response to MOG35–55 peptide. B, Reduced proliferation response (cpm) to Con A and anti-CD3 of purified T cells from healthy naive young vs middle-age males. C, T4 treatment reduced the proliferation response (cpm) to MOG35–55 peptide of splenocytes from young but not middle-age mice with EAE, and the effect was prevented when mice were treated with T4 plus the AR antagonist flutamide. D, No inhibition of splenocyte proliferation response (cpm) to Con A was found in young mice with EAE after treatment with T4 or T4 plus flutamide. *, Statistically significant differences (p < 0.05).
To test the effects of testosterone treatment on T cell proliferation, we compared responses in young and middle-age males treated previously with T4, T4/flutamide, and control. Interestingly, T4 treatment suppressed proliferation of MOG35–55 peptide-specific T cells, but not that of other T cells, as determined by response to Con A (Fig. 7, C and D). The level of proliferation to MOG35–55 peptide in the group treated with T4/flutamide was equivalent to that of control mice (Fig. 7C), indicating that the mechanism of T4-induced suppression was mediated through the AR. In contrast to its suppressive effects on T cells from young mice, T4 treatment had no effect on splenocyte responses to MOG35–55 peptide in middle age mice (Fig. 7C), consistent with a lack of clinical effect. Of interest, E2 treatment only nominally inhibited (20%) splenocyte responses to MOG35–55 peptide in both young and middle-age mice and did not restore responses in middle-age males to levels observed in young mice (data not shown).
Direct relationship among cytokine production, aging, and testosterone treatment
The increased severity of EAE in middle-age mice may be related to an age-related increase in the levels of proinflammatory cytokines. Cytokine production was measured by the CBA technique in splenocytes cultured from young and middle-age male mice. As shown in Fig. 8, splenocytes from middle-age mice produced significantly higher quantities of the proinflammatory chemokine MCP-1 (Fig. 8A) and cytokine IFN- (Fig. 8B) compared with young mice. However, no differences were seen in the expression of other proinflammatory cytokines, TNF-, IL-6, and IL-12p40, or the anti-inflammatory cytokine IL-10 (data not shown). Testosterone treatment has been shown to exhibit potent anti-inflammatory effects in several experimental systems by reducing the expression of IFN- and increasing the expression of IL-10 (21, 23). A similar mechanism may underlie the efficacy of testosterone treatment in the reduction of EAE severity in young males. To test this hypothesis, we determined cytokine levels in T4-, T4/flutamide-, and placebo-treated young and middle-age animals. Testosterone treatment decreased production of TNF- and IFN- in young males (Fig. 8, C and D), but not middle-age males (data not shown). No differences were seen in the expression of MCP-1, IL-6, IL-10, and IL-12p40 (data not shown). This testosterone-dependent reduction in the expression of TNF- and IFN- in young males was reversed by flutamide, once again suggesting that the T4 inhibition was dependent upon signaling through the AR (Fig. 8, C and D).
FIGURE 8. Cytokine production from splenocytes of young and middle-age males. Testosterone treatment alters cytokine secretion in young males. Splenocytes were isolated from young and middle-age males with EAE 33 days postimmunization and cytokine production was determined by CBA. Splenocytes isolated from young mice produced less MCP-1 (A) and IFN- (B) compared with middle-age mice. Testosterone treatment specifically diminished production of TNF- (C) and IFN- (D) in young males. Data presented are from one of three individual experiments. *, Statistically significant difference (p < 0.05).
Discussion
The results presented above demonstrate striking differences between middle-age and young C57BL/6 male mice in the clinical course of EAE and response to both testosterone and estrogen hormone therapy. The severe chronic and unremitting EAE observed in middle-age males could not be attributed to differences in basal levels of serum T4, but was likely influenced by alterations in the distribution and function of splenic immunocytes and a significant reduction in suppressive activity of CD4+CD25+ Treg cells in the spleen and spinal cord. Middle-age males had reduced numbers of splenic CD4+ T cells that were generally hypoproliferative, but enhanced numbers of macrophages and MHC class II-expressing cells and increased secretion of the proinflammatory factors IFN- and MCP-1. Surprisingly, middle-age males were unresponsive to the EAE-protective effects of T4, but had a transient benefit from E2 treatment, unlike young males that were almost completely protected by both hormone treatments. T4 treatment of young males inhibited proliferation of MOG35–55-specific T cells and secretion of TNF- and IFN-, and effects of T4 in vivo and in vitro were reversed by the AR antagonist flutamide, indicating that the regulatory effects of T4 were mediated through the AR. In contrast, T4 treatment of middle-age males had no effect on splenocyte proliferation, chemokine, or cytokine responses.
Differences in the susceptibility and disease severity of MS in males vs females suggest that gonadal hormones may play an important role in the pathology of this disease. Differences between the sexes in MS may be attributable to the direct action of steroids on immune and nonimmune cells and to the fact that males and females have different circulatory profiles for these steroids. However, the mechanisms underlying the response of the immune system to gonadal hormones are still unclear.
The gender differences observed in MS in humans are paralleled by those found for EAE in the SJL strain of mice, with males being less susceptible to disease than females, suggesting that androgens may play a protective role in disease induction (15, 24). Castration of male mice increased the severity not only of EAE (23), but also other autoimmune diseases, including nonobese diabetes, thyroiditis, and adjuvant arthritis (17, 25, 26, 27). Furthermore, endogenous and exogenous androgens have been shown to reduce the incidence and severity of disease (21, 22, 23, 24, 28). Interestingly, abnormally low levels of testosterone were found in male mice with EAE and humans with MS (29, 30), suggesting a bidirectional interaction between disease state and sex hormone production. However, it is important to note that all those studies were done on young males. Testosterone levels are thought to decline naturally in aging men and animals, and this may change the male response to disease. However, our results were apparently not affected by a reduced basal level of T4 in middle-age mice, since no significant difference in serum levels of T4 was observed between young (3.7 ± 0.9 pg/ml) and middle-age (2.6 ± 0.2 pg/ml) males. To date, very few, if any, studies of MS or EAE have been done on aging males, leaving a wide gap in our knowledge about the consequences of androgen loss on autoimmune disease. In this study, we evaluated the differential effects of exogenous replacement of testosterone and its intracellular "metabolite" E2 on young vs sexually senescent male mice.
Our study reveals increased EAE severity for middle-age males of the C57BL/6 strain relative to younger males following immunization with the MOG35–55 peptide in CFA. Although development of EAE was delayed in middle-age males, they showed a statistically significant increase in EAE severity during the chronic phase of disease. In contrast, the severity of EAE in young males declined substantially in the chronic phase, suggesting that regulatory mechanisms are more effective in younger mice. Recently, there has been renewed interest in regulatory cells that influence the course of a number of autoimmune diseases. Regulatory CD4+CD25+ T cells have been shown to inhibit progression of several autoimmune diseases (31, 32, 33), including EAE (34), and to control type 1 diabetes in rats and mice (35). In the pathogenesis of MS, the balance between pathogenic and protective/regulatory cells is the key event for induction, development, and progression of disease. Defective immunoregulation was observed in relapsing and progressive MS patients (36, 37). We hypothesized that the severe and permissive EAE in middle-age mice was related to dysfunctional regulatory mechanisms, including a reduced ability of CD4+CD25+ cells to suppress ongoing disease.
Indeed, our in vitro study revealed that the ability of CD4+CD25+ cells to suppress proliferation of CD4+CD25– indicator cells was diminished on a per-cell basis in middle-age males compared with young males. This reduction in functional splenic Treg activity is highly consistent with the age-dependent functional decline reported for murine and human Treg cells (38, 39, 40, 41, 42, 43). The underlying reason for the observed decline in suppressive activity as a function of aging is unknown, but could be influenced by an age-dependent decline in thymic function or a shift in the suppressive T cell population from activated (CD25+) to a nonactivated (CD25–) state. Indeed, we observed reduced expression of the Foxp3 gene (a marker for Treg cells) in CD4+CD25+ splenocytes but enhanced expression of Foxp3 in CD4+CD25– splenocytes in naive middle-age vs young mice, perhaps explaining why we did not observe a significant change in net Foxp3 expression in unfractionated spleen cells in mice with EAE. Of importance, however, we found a marked decrease of Foxp3 expression in spinal cords of middle-age mice as compared with young counterparts, strongly suggesting a reduced migration of Treg cells into the CNS of middle-age mice with EAE that could account for increased severity of clinical and histological EAE.
Several other factors may also contribute to the delayed onset and increased severity of EAE in aging mice. It is well known that aging leads to a decline in normal function of CD4+ T cells (44, 45, 46). We observed decreased ratios of CD4+:CD11b+ and a reduced responsiveness to both Ag and mitogen stimuli in splenocytes isolated from middle-age mice. Moreover, splenocytes from aging mice produced higher levels of IFN- and MCP-1 cytokines. Undoubtedly, the differences in immunological parameters between young and middle-age males presented in this study have a critical impact on the development and progression of EAE in aging males. Based on our findings, we suggest that EAE in middle-age mice is largely mediated by CD11b+ macrophages, and the increased severity may be attributed to a proinflammatory environment created by the increased production of MCP-1 and IFN- and a failure to mount sufficient CD4+CD25+ Treg responses, particularly in the CNS.
Both androgens and estrogens have been found to regulate the adult immune system. Whitacre et al. (7) reported that low estrogen levels foster proinflammatory Th1 responses, whereas high estrogen levels or testosterone treatment favors the anti-inflammatory Th2 state. The cytokine secretion pattern of human CD4+ T cell clones was strongly influenced by high concentrations of estrogen (47, 48). Recently, we discovered that estrogen treatment caused a significant decrease in TNF- and, to a lesser degree, IFN- by T cells, macrophages, dendritic cells, and microglial cells (49, 50, 51, 52, 53, 54). Males were found to have lower secretion of IFN- than females, suggesting that androgen can induce a Th2 bias (55, 56). Furthermore, Bebo et al. (20) found that inflammatory infiltration of CD4+ T cells to the CNS in EAE occurred only after castration, suggesting that androgens suppress Th1 responses.
In the present study, we found that splenocytes isolated from testosterone-treated young mice produced less IFN- and TNF- compared with placebo-treated control mice. Additionally, testosterone treatment rendered splenocytes from young mice hyporesponsive to MOG35–55 peptide, but not mitogen (Con A) stimulation, suggesting a selective influence on the encephalitogenic population. It should be noted, however, that Con A stimulation appears to act via cell surface sugar residues rather than through the TCR, thus leaving open the possibility that T4 treatment may also inhibit TCR activation of other, non-MOG35–55 peptide-specific T cells. Concurrent flutamide treatment reversed the effect of testosterone on the clinical outcome, as well as its effect on cytokine release and proliferation, suggesting that the androgen receptor is involved in all testosterone-dependent responses. Despite the immunological differences between middle-age and young male mice described above, it was surprising to discover that testosterone treatment was beneficial only in young males. In contrast, our estrogen treatment ameliorated the symptoms of EAE in both young and middle-age mice, although its effects were transient in middle-age mice. It is noteworthy that the effects of E2 on castrated young male mice were essentially identical to the effects of E2 in ovariectomized young female mice (52). These findings suggest that testosterone and estrogen may exert their protective effects through very different pathways.
The reason behind the failure of testosterone to protect middle-age mice from development of EAE or to inhibit splenocyte responses to MOG peptide remains unknown. Recent evidence emphasizes that neuroendocrine aging in men becomes significant by the fourth decade of life (57, 58). In addition to lower production of androgens, sensitivity to androgens appears to wane with age by poorly understood mechanisms. In rodents, receptor distribution in spinal cord has not been well described, but AR protein is well represented in brain regions not linked to reproductive function, including cortex, hippocampus, and basal ganglia (59, 60), and in forebrain axons and dendrites (61). Effects of age on AR immunoreactivity or receptor affinity to agonist have not been well characterized at any level within the CNS. However, several studies suggest a decrease in AR high-affinity binding sites with age in mice and rats (62, 63, 64). Therefore, our results would be consistent with these observations: the waning response to testosterone in older vs younger males is due to depressed receptor signaling due to loss of binding sites. Alternatively, a T4 metabolite, rather than T4 itself, may be responsible for protection against EAE, and the predominant pathway for testosterone metabolism is known to be age dependent. In younger males, testosterone is largely converted to dihydrotestosterone, a highly potent activator of the AR; however, the importance of this pathway diminishes with age. In a secondary pathway, T4 is converted into E2. Although this secondary pathway is less prominent in young males, it is sustained throughout life, becoming the predominant mechanism during andropause.
These data are the first to define age-dependent differences in EAE expression and response to hormone therapy in males. Although treatment of MS in males with testosterone shows some promise, our study raises the possibility that the success of this approach might be age dependent, with lesser or no effects in older males. At a minimum, it would seem prudent to carry out exploratory studies on older male MS patients to assess the immunoregulatory potential of T4 before initiating a treatment trial.
Acknowledgments
We thank Eva Niehaus for assistance in preparing this manuscript.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by Grants AI42376, NS23221, and NS23444 from the National Institutes of Health and grants from the National Multiple Sclerosis Society, the Nancy Davis Center Without Walls, and the Department of Veterans Affairs.
The authors have no financial conflict of interest.
2 Address correspondence and reprint requests to Dr. Halina Offner, Neuroimmunology Research R&D-31, Portland VA Medical Center, 3710 SW US Veterans Hospital Road, Portland, OR 97239. E-mail address: offnerva@ohsu.edu
3 Abbreviations used in this paper: MS, multiple sclerosis; EAE, experimental autoimmune encephalomyelitis; MOG, myelin oligodendrocyte glycoprotein; AR, androgen receptor; Ptx, pertussis toxin; CBA, cytometric bead array; Treg, regulatory T; CDI, cumulative disease index; S:I, suppressor:indicator.
Received for publication August 27, 2004. Accepted for publication December 3, 2004.
References
Vyse, T. J., J. A. Todd. 1996. Genetic analysis of autoimmune disease. Cell 85:311.
Glynn, L. E.. 1967. Experimental model of rheumatoid arthritis. J. Belge Rheumatol. Med. Phys. 22:201.
Inman, R. D.. 1978. Immunologic sex differences and the female predominance in systemic lupus erythematosus. Arthritis Rheum. 21:849.
Rodnan, G. P., R. L. Myerowitz, G. O. Justh. 1980. Morphologic changes in the digital arteries of patients with progressive systemic sclerosis (scleroderma) and Raynaud phenomenon. Medicine 59:393.
Whaley, K., W. W. Buchanan. 1971. Recent advances in Sjogren’s syndrome. Mod. Trends Rheumatol. 2:139.
Birk, K., C. Ford, S. Smeltzer, D. Ryan, R. Miller, R. A. Rudick. 1990. The clinical course of multiple sclerosis during pregnancy and the puerperium. Arch. Neurol. 47:738
Whitacre, C. C., S. C. Reingold, P. A. O’Looney. 1999. A gender gap in autoimmunity. Science 283:1277.
Gray, A., H. A. Feldman, J. B. McKinlay, C. Longcope. 1991. Age, disease, and changing sex hormone levels in middle-aged men: results of the Massachusetts Male Aging Study. J. Clin. Endocrinol. Metab. 73:1016.
Morley, J. E., F. E. Kaiser, H. M. Perry, III, P. Patrick, P. M. Morley, P. M. Stauber, B. Vellas, R. N. Baumgartner, P. J. Garry. 1997. Longitudinal changes in testosterone, luteinizing hormone, and follicle-stimulating hormone in healthy older men. Metabolism 46:410.
Nankin, H. R., J. H. Calkins. 1986. Decreased bioavailable testosterone in aging normal and impotent men. J. Clin. Endocrinol. Metab. 63:1418.
Tenover, J. S., A. M. Matsumoto, S. R. Plymate, W. J. Bremner. 1987. The effects of aging in normal men on bioavailable testosterone and luteinizing hormone secretion: response to clomiphene citrate. J. Clin. Endocrinol. Metab. 65:1118
Vermeulen, A.. 1991. Clinical review 24: androgens in the aging male. J. Clin. Endocrinol. Metab. 73:221.
Bebo, B. F., Jr, A. A. Vandenbark, H. Offner. 1996. Male SJL mice do not relapse after induction of EAE with PLP 139–151. J. Neurosci. Res. 45:680.
Cua, D. J., D. R. Hinton, S. A. Stohlman. 1995. Self-antigen-induced Th2 responses in experimental allergic encephalomyelitis (EAE)-resistant mice: Th2-mediated suppression of autoimmune disease. J. Immunol. 155:4052.
Voskuhl, R. R., H. Pitchekian-Halabi, A. MacKenzie-Graham, H. F. McFarland, C. S. Raine. 1996. Gender differences in autoimmune demyelination in the mouse: implications for multiple sclerosis. Ann. Neurol. 39:724
Fox, H. S.. 1995. Sex steroids and the immune system. Ciba Found. Symp. 191:203
Fox, H. S.. 1992. Androgen treatment prevents diabetes in nonobese diabetic mice. J. Exp. Med. 175:1409.
Roubinian, J. R., N. Talal, J. S. Greenspan, J. R. Goodman, P. K. Siiteri. 1978. Effect of castration and sex hormone treatment on survival, anti-nucleic acid antibodies, and glomerulonephritis in NZB/NZW F1 mice. J. Exp. Med. 147:1568.
Jansson, L., T. Olsson, R. Holmdahl. 1994. Estrogen induces a potent suppression of experimental autoimmune encephalomyelitis and collagen-induced arthritis in mice. J. Neuroimmunol. 53:203.
Bebo, B. F., Jr, J. C. Schuster, A. A. Vandenbark, H. Offner. 1998. Gender differences in experimental autoimmune encephalomyelitis develop during the induction of the immune response to encephalitogenic peptides. J. Neurosci. Res. 52:420.
Dalal, M., S. Kim, R. R. Voskuhl. 1997. Testosterone therapy ameliorates experimental autoimmune encephalomyelitis and induces a T helper 2 bias in the autoantigen-specific T lymphocyte response. J. Immunol. 159:3.
Palaszynski, K. M., K. K. Loo, J. F. Ashouri, H. B. Liu, R. R. Voskuhl. 2004. Androgens are protective in experimental autoimmune encephalomyelitis: implications for multiple sclerosis. J Neuroimmunol. 146:144
Bebo, B. F., Jr, J. C. Schuster, A. A. Vandenbark, H. Offner. 1999. Androgens alter the cytokine profile and reduce encephalitogenicity of myelin-reactive T cells. J. Immunol. 162:35
Kim, S., S. M. Liva, M. A. Dalal, M. A. Verity, R. R. Voskuhl. 1999. Estriol ameliorates autoimmune demyelinating disease: implications for multiple sclerosis. Neurology 52:1230.
Ahmed, S. A., W. J. Penhale. 1982. The influence of testosterone on the development of autoimmune thyroiditis in thymectomized and irradiated rats. Clin. Exp. Immunol. 48:367
Fitzpatrick, F., F. Lepault, F. Homo-Delarche, J. F. Bach, M. Dardenne. 1991. Influence of castration, alone or combined with thymectomy, on the development of diabetes in the nonobese diabetic mouse. Endocrinology 129:1382.
Harbuz, M. S., Z. Perveen-Gill, S. L. Lightman, D. S. Jessop. 1995. A protective role for testosterone in adjuvant-induced arthritis. Br. J. Rheumatol. 34:1117.
Voskuhl, R. R., K. Palaszynski. 2001. Sex hormones in experimental autoimmune encephalomyelitis: implications for multiple sclerosis. Neuroscientist 7:258.
Wei, T., S. L. Lightman. 1997. The neuroendocrine axis in patients with multiple sclerosis. Brain 120:1067.
Foster, S. C., C. Daniels, D. N. Bourdette, B. F. Bebo, Jr. 2003. Dysregulation of the hypothalamic-pituitary-gonadal axis in experimental autoimmune encephalomyelitis and multiple sclerosis. J. Neuroimmunol. 140:78
Mason, D., F. Powrie. 1998. Control of immune pathology by regulatory T cells. Curr. Opin. Immunol. 10:649.
Maloy, K. J., F. Powrie. 2001. Regulatory T cells in the control of immune pathology. Nat. Immunol. 2:816.
Roncarolo, M. G., M. K. Levings. 2000. The role of different subsets of T regulatory cells in controlling autoimmunity. Curr. Opin. Immunol. 12:676.
Kohm, A. P., P. A. Carpentier, H. A. Anger, S. D. Miller. 2002. Cutting edge: CD4+CD25+ regulatory T cells suppress antigen-specific autoreactive immune responses and central nervous system inflammation during active experimental autoimmune encephalomyelitis. J. Immunol. 169:4712.(Agata Matejuk, Corwyn Hop)