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Endogenous estrogen regulation of inflammatory arthritis and cytokine expression in male mice predominantly via estrogen receptor ╨Ю┬▒.

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ARTHRITIS & RHEUMATISM
Vol. 62, No. 4, April 2010, pp 1017–1025
DOI 10.1002/art.27330
© 2010, American College of Rheumatology
Endogenous Estrogen Regulation of Inflammatory Arthritis and
Cytokine Expression in Male Mice, Predominantly via
Estrogen Receptor ␣
Y. H. Yang,1 D. Ngo,1 M. Jones,2 E. Simpson,2 K. H. Fritzemeier,3 and E. F. Morand1
Objective. A number of experimental observations
have associated elevated estrogen levels with amelioration of inflammation. The involvement of estrogen and
estrogen receptor (ER) isotypes in the regulation of
inflammation in males is not well understood. In this
study, we used specific ER␣ and ER␤ agonists in male
mice deficient in estrogen because of a deletion of
aromatase (aromatase-knockout [ArKO] mice) to investigate ER isotype utilization in estrogen regulation of
inflammation.
Methods. Lipopolysaccharide (LPS)-induced cytokine expression and antigen-induced arthritis (AIA)
were investigated in male ArKO and WT littermate
mice, as well as in response to selective agonists of ER␣
(16␣-LE2) and ER␤ (8␤-VE2). The therapeutic effect of
selective ER agonists was also examined in mice with
collagen-induced arthritis (CIA).
Results. Estrogen deficiency in ArKO mice was
associated with significant increases in LPS-induced
serum interleukin-6 (IL-6), tumor necrosis factor,
monocyte chemotactic protein 1, and interferon-␥ levels,
which were significantly abrogated by administration of
16␣-LE2, but not 8␤-VE2. In contrast, both 16␣-LE2
and 8␤-VE2 significantly increased LPS-induced IL-10
levels. Estrogen deficiency was also associated with
significant exacerbation of AIA and antigen-specific T
cell proliferation, which was reversed by administration
of either 16␣-LE2 or 8␤-VE2. ArKO mice showed increased antigen-specific T cell proliferation in response
to immunization with type II collagen (CII). Administration of 16␣-LE2, but not 8␤-VE2, significantly reduced the severity of CIA, which was associated with
inhibition of anti-CII–specific IgG.
Conclusion. These data indicate that endogenous
estrogen plays an essential inhibitory role in inflammation in male mice and that ER␣ is the dominant
receptor that mediates these effects.
Most autoimmune diseases, including rheumatoid arthritis (RA), systemic lupus erythematosus, multiple sclerosis, and Sjögren’s syndrome (SS), are more
common in females than in males. A significant body of
evidence implicates the influence of sex-specific factors,
including sex hormones, on inflammation (1,2). For
example, pregnancy has an ameliorating effect on RA,
scleroderma, and SS.
A shift from Th1 to Th2 immune response bias
has been proposed as a mechanism underlying the
improvement in putative Th1-mediated autoimmune
diseases during pregnancy (2). In support of this,
collagen-induced arthritis (CIA) in rats has been shown
to be exacerbated by ovariectomy in females or by
castration in males, and in both cases, correction of the
sex hormone deficiency reversed these effects (3). Treatment with physiologic levels of 17␤-estradiol was shown
to inhibit the development of CIA in DBA/1 mice (4),
whereas deprivation of endogenous estrogen by ovariectomy exacerbated disease severity and induced bone loss
in this model (5).
Highlighting the complexity of this area, serum
estradiol levels in male patients with RA are higher than
those in healthy male controls (6), while some studies
Supported in part by Bayer Schering Pharma AG, Germany,
and by the National Health and Medical Research Council, Australia
(program grant 494802).
1
Y. H. Yang, MD, PhD, D. Ngo, BSc (Hons), E. F. Morand,
MD, PhD: Monash University Department of Medicine and Monash
Medical Centre, Clayton, Victoria, Australia; 2M. Jones, PhD, E.
Simpson, PhD: Prince Henry’s Institute, Clayton, Victoria, Australia;
3
K. H. Fritzemeier, PhD: Bayer Schering Pharma AG, Berlin, Germany.
Address correspondence and reprint requests to Y. H. Yang,
MD, PhD, Monash University Department of Medicine, Monash
Medical Centre, Locked Bag No. 29, Clayton, Victoria 3168, Australia.
E-mail: yuan.yang@med.monash.edu.au.
Submitted for publication August 24, 2009; accepted in
revised form December 28, 2009.
1017
1018
suggest a protective effect of estrogen replacement
therapy on the incidence of RA in postmenopausal
women (7), although this was not confirmed in other
studies (8). In a study of complement receptor deficiency
in arthritis, female mice exhibited more severe disease,
which was further exacerbated by ovariectomy (9).
Notwithstanding the unresolved issues with respect to the immunomodulatory effects of estrogens in
females, even less is understood about these effects in
males. Male humans produce estrogen, and aromatase,
the enzyme required for conversion of androgens to
estrogen, is found in the testis (10). Subramanian et al
(11) recently reported suppressive effects of estrogen in
CIA in a study of male DBA/1 mice. Similarly, inhibitory
effects of 2-methoxyestradiol, an estrogen metabolite,
were recently reported in rats with adjuvant-induced
arthritis in a study of male rodents (12). Interestingly, an
estrogen receptor polymorphism has been associated
with the risk of gouty arthritis in humans in a study
predominantly of men (13). Together, these and other
findings indicate a major, but poorly understood, role of
estrogen in the modulation of immune and inflammatory responses in males.
Estradiol (17␤-estradiol) acts through 2 known
hormone receptors, estrogen receptors (ERs) ␣ and ␤,
which are expressed in distinct tissues and immune cells.
The ER isotype(s) responsible for the apparent inhibitory effects of endogenous estrogen in males are not
known. ER␣ has been reported to mediate the inhibitory effects of estrogens on experimental autoimmune
encephalomyelitis (14,15) and on adjuvant-induced arthritis in Lewis rats (16), and reduced local expression of
ER␣ in synovium samples from mice with CIA has been
reported (17), but these studies were limited to female
rodents.
Tools for the investigation of estrogen function in
inflammatory immune regulation have recently been
developed. Highly potent and selective ER ligands permit the examination of ER subtype effects in a variety of
settings (18). The aromatase-knockout (ArKO) mouse
lacks a functional Cyp19 gene that encodes aromatase
and, hence, cannot synthesize endogenous estrogens
(19). To determine the functional contribution of ER
isotypes to the regulation of inflammation by endogenous estrogen in male mice, models of innate and
adaptive immunity and arthritis were investigated in
male ArKO mice and wild-type (WT) littermate control
mice, and the responses to selective ER␣ and ER␤
agonists were determined.
YANG ET AL
MATERIALS AND METHODS
Animals. ArKO mice (129SV/J ⫻ C57BL/6J) were
generated by deleting 90% of exon 9 of the Cyp19 gene as
described by Fisher et al (20). WT and homozygous-null
offspring were generated by heterozygous matings. The genotype of the offspring was determined by polymerase chain
reaction analysis, as described by Robertson et al (21). The
animals were housed under specific pathogen–free conditions
and had unlimited access to drinking water and a commercial
mouse diet containing 15% of calories as fat, 20% of calories
as protein, and 65% of calories as carbohydrate (Glen Forrest
Stockfeeders, Glen Forrest, Western Australia, Australia).
Studies were performed in male mice between the ages of 8
and 10 weeks. Male DBA/1J (DBA) mice were obtained from
the Animal Resources Centre (Perth, Western Australia,
Australia). These studies were approved by the Monash Medical Centre Animal Ethics Committee.
Endotoxin-induced cytokines and treatment with ER
agonists. Male ArKO and WT littermate mice ages 8–10 weeks
were injected intraperitoneally with 10 mg/kg of lipopolysaccharide (LPS) from Escherichia coli O111:B4 (Sigma-Aldrich,
Castle Hill, New South Wales, Australia). Blood was collected
at the indicated times. In ER-replacement experiments, the
selective ER␣ agonist 16␣-LE2 (3 ␮g/kg), the selective ER␤
agonist 8␤-VE2 (100 ␮g/kg), 17␤-estradiol (3 ␮g/kg), or vehicle was administered to ArKO and WT littermate mice 2 hours
after LPS challenge. Serum cytokine levels were measure by
cytometric bead array (see below).
Antigen-induced arthritis (AIA) and ER replacement
in ArKO mice. AIA was established as described previously
(22,23). Briefly, male WT or ArKO C57BL/6 mice were
immunized with 200 ␮g of methylated bovine serum albumin
(mBSA; Sigma-Aldrich) in 200 ␮l of Freund’s complete adjuvant, which was injected subcutaneously into the flank. On day
7, mice received 100 ␮g of mBSA/0.1 ml of Freund’s complete
adjuvant injected intradermally at the base of the tail. Arthritis
was induced on day 21 by injection of 30 ␮g of mBSA in 10 ␮l
of sterile saline or sterile saline alone into knee joints.
To investigate the effects of ER␣ and ER␤ agonists,
mice were treated daily with 16␣-LE2 (3 ␮g/kg), 8␤-VE2 (100 ␮g/
kg) (both donated by Schering; Bayer Schering Pharma, Berlin,
Germany), 17␤-estradiol (3 ␮g/kg), or vehicle for 7 days,
beginning on day 21. Arthritis was analyzed histologically on
day 28 after the first immunization, as described elsewhere
(23). Briefly, sagittal sections (5 ␮m) of fixed and decalcified
knee joints were stained with Safranin O and counterstained
with fast green/iron hematoxylin. Histologic sections were
scored on a scale of 0–3 for each of the following 5 parameters:
synovitis, joint space exudate, soft tissue inflammation, cartilage degradation, and bone damage. A total score was generated from the sum of the scores for these parameters (maximum total score 15).
T cell activation. Antigen-specific T cell activation
was analyzed as described previously (23). Spleens and lymph
nodes were removed on day 28, and single-cell suspensions
were prepared in RPMI 1640 containing 5% fetal calf serum
(FCS) and 50 ␮M 2-mercaptoethanol. Cells (105) were cultured in triplicate in the presence or absence of mBSA
(10 ␮g/ml) or anti-CD3 (1 ␮g/ml) for 48 hours (37°C in an
ESTROGEN REGULATION OF ARTHRITIS AND CYTOKINES IN MICE VIA ER␣
atmosphere containing 5% CO2). T cell proliferative responses
were determined by measuring the incorporation of 3Hthymidine (0.5 ␮Ci/well) during the final 18 hours of culture.
Anti-mBSA antibody response. Serum concentrations
of anti-mBSA IgG on day 28 were determined by enzymelinked immunosorbent assay (ELISA) as previously described
(24). Briefly, polyvinyl microtiter plates were coated with
100 ␮l of mBSA (100 ␮g/ml) for 24 hours at 4°C. The plates
were blocked for 1 hour with 2% casein (Sigma-Aldrich) in
phosphate buffered saline with 0.05% Tween 20. Then, 100 ␮l
of diluted serum samples was added and incubated for 24
hours at 4°C. Biotinylated rabbit anti-mouse IgG (1:2,000
dilution) or anti–isotype-specific (IgG1 or IgG2a) antibodies
(all from Dako, Carpinteria, CA) were incubated for 2 hours.
The streptavidin–horseradish peroxidase conjugate (1:2,000
dilution) was incubated for 30 minutes and detected using the
tetramethylbenzidine peroxidase substrate with hydrogen peroxide (optical density determined at 450 nm).
Measurement of cytokines. Concentrations of cytokines in serum and culture supernatants were measured using
a commercially available cytometric bead array (CBA; BD
Biosciences, San Jose, CA). A mouse inflammation CBA kit
was used to detect IL-6, IL-10, monocyte chemotactic protein
1 (MCP-1), interferon-␥ (IFN␥), tumor necrosis factor (TNF),
and IL-12p40, as described in the manufacturer’s instructions. The samples were analyzed by flow cytometry using a
MoFlo flow cytometer from Cytomation (Fort Collins, CO).
The amount of each cytokine in the supernatant or serum was
interpolated from a standard curve. Results are expressed as
picograms per milliliter.
Collagen-induced arthritis. A total of 100 ␮g of native
bovine type II collagen (CII; Chondrex, Redmond, WA)
dissolved in 0.05M acetic acid was emulsified with Freund’s
complete adjuvant and injected subcutaneously into the base
of the tail of 7–9-week-old DBA/1, ArKO, and WT mice.
Mice were given subcutaneous boosters with collagen (100 ␮g)
in Freund’s incomplete adjuvant on day 21. Mice were monitored daily for clinical features of arthritis beginning after
day 22. Once arthritis was detected, DBA mice were treated
daily for 14 days with 16␣-LE2 (3 or 30 ␮g/kg), 8␤-VE2 (100
or 1,000 ␮g/kg), 17␤-estradiol (3 ␮g/kg), or vehicle. The
clinical severity of arthritis was graded as follows: each limb
was scored on a scale of 0–3, where 0 ⫽ no erythema or
swelling, 1 ⫽ slight swelling and erythema in at least some
digits, 2 ⫽ moderate swelling and erythema involving the
entire limb or multiple limbs, and 3 ⫽ pronounced swelling
leading to incapacitated limbs. Clinical evaluation was performed in a blinded manner by 2 investigators (YHY and DN).
CII-specific T cell activation was analyzed in ArKO
mice. Spleens and lymph nodes were removed on day 41, and
single-cell suspensions were prepared in RPMI 1640 containing 5% FCS and 50 ␮M 2-mercaptoethanol. Cells (105)
were cultured in triplicate in the presence or absence of CII
(50 ␮g/ml) or anti-CD3 (1 ␮g/ml) for 72 hours (37°C in an
atmosphere containing 5% CO2). The T cell proliferative
response was determined by measuring the incorporation of
3
H-thymidine (0.5 ␮Ci/well) during the final 18 hours.
Serum was collected from mice developing CIA, and
IgG and IgM antibody levels were measured by ELISA. The
96-well plates were coated overnight at 4°C with 10 ␮g/ml of
1019
Figure 1. Endotoxin-induced cytokines in vivo in male wild-type
(WT) and aromatase-knockout (ArKO) mice. WT and ArKO littermates ages 8–10 weeks were injected with 10 mg/kg of lipopolysaccharide (LPS), and cytokine levels were examined in serial blood samples
collected at the indicated times from time 0 to 4 hours (n ⫽ 9 per
group) (A–D) as well as at 24 hours (n ⫽ 5 per group) (E–H).
Concentrations of tumor necrosis factor (TNF), interleukin-6 (IL-6),
monocyte chemotactic protein 1 (MCP-1), and interferon-␥ (IFN␥)
were determined by cytometric bead array. Values are the mean and
SEM. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01 versus WT mice.
bovine CII and blocked with blocking buffer as described
above. Following overnight incubation of samples, peroxidaseconjugated goat anti-mouse antibody specific for IgG and IgM
was added, and color was developed as described above.
Statistical analysis. Data were analyzed using the
Mann-Whitney 2-sample rank test to determine the level of
significance of between-group differences in mean histologic
scores, or Student’s t-test was used for comparison of continuous variables. Results are expressed as the mean ⫾ SEM.
P values less than 0.05 were considered statistically significant.
1020
YANG ET AL
To examine the role of ER isotypes in the
inhibitory effects of endogenous estrogen, selective ER
agonists were administered to ArKO mice 2 hours after
LPS challenge, and serum cytokine levels were measured at 24 hours. Administration of 16␣-LE2 (ER␣
agonist) significantly suppressed the LPS-induced levels
of serum TNF and MCP-1 (Figures 2A and C), but
significantly increased the LPS-induced serum levels of
the antiinflammatory cytokine IL-10 (Figure 2E). There
was also a trend toward suppression of LPS-induced
IL-6 (P ⫽ 0.057) and IFN␥ by treatment with 16␣-LE2,
although these differences were not significant. In contrast, treatment of ArKO mice with 8␤-VE2 (ER␤
agonist) had no effect on the levels of LPS-induced
proinflammatory cytokines but was associated with significantly increased levels of IL-10 (Figure 2E).
Effects of endogenous estrogen on AIA. Exacerbation of AIA. We next examined the effects of endogenous estrogen deficiency on the expression of arthritis
in male mice. In comparison with saline-injected joints,
which were histologically normal (Figure 3A), the joints
Figure 2. Therapeutic effect of estrogen receptors (ERs) on lipopolysaccharide (LPS)-induced cytokines in male wild-type (WT) and
aromatase-knockout (ArKO) mice. Treatment with 16␣-LE2 (ER␣
agonist; 3 ␮g/kg) or 8␤-VE2 (ER␤ agonist; 100 ␮g/kg) was administered intraperitoneally 2 hours after LPS (10 mg/kg) administration.
Blood was collected at 24 hours, and concentrations of A, tumor
necrosis factor (TNF), B, interleukin-6 (IL-6), C, monocyte chemotactic protein 1 (MCP-1), D, interferon-␥ (IFN␥), and E, IL-10 were
determined by cytometric bead array. Values are the mean and SEM
of 5–8 mice per group. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01 versus WT mice.
∧ ⫽ P ⬍ 0.05; ∧∧ ⫽ P ⬍ 0.01 versus vehicle-treated ArKO mice.
RESULTS
Effects of endogenous estrogen on LPS-induced
cytokines. The effects of endogenous estrogen on the
innate immune response in male mice were examined by
evaluating LPS-induced serum cytokines in male WT
and ArKO mice. In WT mice, LPS treatment for 4 hours
or 24 hours induced significant increases in circulating
levels of IL-6, TNF, MCP-1, and IFN␥ (Figure 1). In
ArKO mice, LPS-induced increases in serum cytokine
levels were also significant. There was a significant
increase in serum TNF levels at 4 hours and 24 hours
(Figures 1A and E). Serum levels of IL-6, MCP-1, and
IFN␥ were also significantly higher in ArKO mice at 24
hours (Figures 1F–H). LPS-induced levels of IL-10 were
not significantly affected by estrogen deficiency (data
not shown).
Figure 3. Histologic manifestations of antigen-induced arthritis (AIA) in
male wild-type (WT) and aromatase-knockout (ArKO) mice. Arthritis
severity in the knee joints was assessed in Safranin O–stained sections
obtained on day 28. Shown are the histopathologic features of AIA in
joint sections from A, a normal mouse, B, a wild-type (WT) arthritic
mouse, and C, an ArKO arthritic mouse. Two regions from representative
joints are shown. S ⫽ synovium; C ⫽ articular cartilage; J ⫽ joint space;
P ⫽ pannus; E ⫽ exudate. (Original magnification ⫻ 50.)
ESTROGEN REGULATION OF ARTHRITIS AND CYTOKINES IN MICE VIA ER␣
Figure 4. Total arthritis scores and antigen-specific lymphocyte proliferation in male wild-type (WT) and aromatase-knockout (ArKO)
mice with antigen-induced arthritis (AIA). A, Total arthritis scores in
WT and ArKO mice on day 28. Arthritis was assessed using a 0–3 scale
for each of 5 histopathologic features, and the results were summed
(maximum possible score of 15 per mouse). Each data point represents
the total score in a single animal; horizontal lines show the mean. B,
Arthritis scores for each of the 5 individual histopathologic features of
AIA, as determined on day 28. C, Methylated bovine serum albumin
(mBSA)–induced antigen-specific T cell proliferation in lymph node
cells from WT and ArKO mice. D, Anti-CD3–induced T cell proliferation in lymph node cells from WT (open bars) and ArKO (solid bars)
mice. NS ⫽ not significant. E, Serum levels of anti-mBSA total IgG,
IgG1, and IgG2a in WT and ArKO mice. Values in B–E are the mean
and SEM of 13–16 mice per group in B–D and 7 mice per group in E.
ⴱ ⫽ P ⬍ 0.05 versus WT mice. ∧ ⫽ P ⬍ 0.05; ∧∧ ⫽ P ⬍ 0.01 versus
vehicle-treated controls (CT) in C and D.
of mBSA-injected WT control mice showed extensive
synovial lining hypercellularity, soft tissue inflammation,
joint space exudation, cartilage degradation, and bone
damage (Figure 3B). In comparison with WT animals,
ArKO mice exhibited significant exacerbation of arthritis severity, as reflected in a significantly increased total
histology score (P ⬍ 0.05) (Figures 3C and 4A). Examination of individual aspects of the synovial pathology
revealed significantly increased soft tissue inflammation
1021
and synovitis in ArKO mice (P ⬍ 0.05 for each comparison) and trends toward exacerbation of the joint
space exudate, cartilage degradation, and bone damage
(Figure 4B).
T cell activation and antibody response. Significant
antigen-induced proliferation was observed in lymph
node cells from WT (P ⬍ 0.05) and ArKO (P ⬍ 0.01)
mice. A significantly greater increase in antigen-induced
proliferation was observed in lymphocytes from ArKO
mice (P ⬍ 0.05) (Figure 4C). Levels of anti-CD3–
induced T cell proliferation were comparable in lymphocytes from ArKO mice and those from WT mice (Figure
4D). In naive spleen cells from both WT and ArKO
mice, anti-CD3 significantly induced IFN␥, TNF, and
IL-6 (data not shown), and there was no significant
difference in the cytokine responses to anti-CD3 treatment between cells from WT mice and cells from ArKO
mice. IL-10, MCP-1, and IL-12p40 were not detectable
in response to anti-CD3 (data not shown). There were
no significant differences in serum levels of anti-mBSA
total IgG, IgG1, or IgG2a between the ArKO mice and
the WT mice (Figure 4E).
Effect of ER agonist replacement in ArKO mice
with AIA. We next investigated the effect of selective ER
isotype agonist replacement in estrogen-deficient male
mice. The increased arthritis severity in ArKO mice was
significantly reduced in response to treatment with 16␣LE2 (Figure 5A), with inhibition of most histologic
features of AIA, including synovitis, soft tissue inflammation, exudate, and cartilage degradation (Figure 5B).
Similarly, treatment with 8␤-VE2 significantly reduced
the overall arthritis severity and individual aspects of
synovial pathology in ArKO mice (Figures 5A and C).
Unexpectedly, treatment with 16␣-LE2 was associated
with increased severity of clinical arthritis in WT mice.
Antigen-specific T cell proliferation was examined ex vivo in spleen cells from all treatment groups.
The significantly increased antigen-specific T cell proliferation in cells from ArKO mice was significantly suppressed by in vivo administration of 16␣-LE2, but not
8␤-VE2 (Figure 5D). In contrast, treatment of ArKO
mice with 16␣-LE2 or 8␤-VE2 had no effect on antigenspecific total IgG or IgG isotypes (data not shown), but
this was not unexpected, since the antigen-specific titers
of IgG were not affected by estrogen deficiency per se.
Effects of estrogen on CIA. CIA is characterized
by activation of Th1 adaptive immunity, autoantibody
production, and monocyte cytokine expression, and it is
considered the murine model that is most similar to
human RA. ArKO mice are generated on the C57BL/6
1022
YANG ET AL
Figure 5. Role of estrogen receptor (ER) agonists in antigen-induced arthritis (AIA) in male wild-type
(WT) and aromatase-knockout (ArKO) mice. Treatment with 17␤-estradiol (E2; 3 ␮g/kg), 16␣-LE2 (ER␣
agonist; 3 ␮g/kg), 8␤-VE2 (ER␤ agonist; 100 ␮g/kg), or vehicle was administered daily from day 21 to day
27. A, Total arthritis scores on day 28. B, Arthritis scores for each of the 5 individual histopathologic
features of AIA, as determined on day 28, in mice treated with 16␣-LE2 versus vehicle. C, Arthritis scores
for each of the individual histopathologic features of AIA, as determined on day 28, in mice treated with
8␤-VE2 versus vehicle. D, Antigen-specific T cell proliferation in spleen cells from WT and ArKO mice
treated with vehicle (controls [CT]) or methylated bovine serum albumin (mBSA) and in ArKO mice
treated with the ER␣ and ER␤ agonists. NS ⫽ not significant. Values are the mean and SEM of 13–19
mice per group. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01 versus WT mice in B and C. ∧ ⫽ P ⬍ 0.05 versus
vehicle-treated controls (CT) in D. ∧∧ ⫽ P ⬍ 0.01 versus ArKO mice in A.
background, a strain which is relatively resistant to CIA.
In response to collagen immunization, a 33% incidence
of clinically detectable arthritis was observed in both the
ArKO and the WT mice. However, a trend toward
increased disease severity was observed in ArKO mice as
compared with WT mice (Figure 6A). Consistent with
this, lymph node cells from ArKO mice exhibited significantly greater antigen-specific proliferation in response
to CII as compared with lymph node cells from WT mice
(Figure 6B).
Having demonstrated the effects of endogenous
estrogen on immune responses to collagen, we then
investigated the effects of selective ER agonists on CIA
in disease-susceptible male DBA/1J mice. Administration of 16␣-LE2 significantly and dose-dependently reduced arthritis severity in mice with CIA (Figure 6C).
High-dose 16␣-LE2 significantly inhibited arthritis severity from an early time point, whereas the inhibitory
effect of low-dose 16␣-LE2, while apparent earlier,
became statistically significant only after 15 days of
treatment. Consistent with previous reports, estrogen
treatment markedly and significantly inhibited CIA severity (Figure 6D). In contrast, there was no significant
therapeutic effect on CIA in response to 8␤-VE2 (Figure 6D). Serum anti-CII IgG, but not IgM, antibody
levels were significantly inhibited by administration of
ESTROGEN REGULATION OF ARTHRITIS AND CYTOKINES IN MICE VIA ER␣
1023
Figure 6. Effects of endogenous estrogen and estrogen receptor (ER) agonists on collagen-induced arthritis (CIA) in male wild-type (WT) and
aromatase-knockout (ArKO) mice. A and B, CIA was induced in WT and ArKO mice with type II collagen (CII) as described in Materials
and Methods, and the clinical severity of CIA (n ⫽ 6 per group) (A) and CII-specific T cell proliferation in lymph nodes (B) were determined.
C–H, CIA was induced in DBA/1 mice as described in Materials and Methods. DBA/1 mice were treated daily with a low dose (3 ␮g/kg; n ⫽ 12 mice)
or a high dose (30 ␮g/kg; n ⫽ 7 mice) of ER␣ agonist, with a low dose (100 ␮g/kg; n ⫽ 11 mice) or a high dose (1 mg/kg; n ⫽ 7 mice) of ER␤ agonist,
with 17␤-estradiol (E2) (3 ␮g/kg; n ⫽ 7 mice), or with vehicle (n ⫽ 15 mice) for 14 days beginning at the onset of arthritis. The effects of
treatment with the ER␣ agonist (C) or with the ER␤ agonist or E2 (D) on the arthritis scores for CIA were determined at the indicated times.
Serum levels of anti-CII IgG (E) or IgM (F) were measured in mice with CIA treated with each dose of the ER␣ agonist or the ER␤ agonist.
Serum levels of interleukin-10 (IL-10) were measured in mice with CIA treated with E2, high-dose ER␣ agonist, or high-dose ER␤ agonist (G).
Levels of interferon-␥ (IFN␥) in culture supernatants of splenocytes cultured ex vivo and treated with E2, high-dose ER␣ agonist, or high-dose
ER␤ agonist (H) were also determined. Values are mean and SEM. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01 versus WT mice (B) or versus vehicle treatment
(C–H).
high-dose ER␣ agonist (Figures 6E and F). No significant effect on anti-CII antibodies was observed in response to 8␤-VE2 administration.
Serum concentrations of IL-6, TNF, and IFN␥
were undetectable in mice with CIA regardless of treatment. Interestingly, circulating IL-10 was detectable and
the levels were increased in mice treated with estrogen
or with 16␣-LE2, although there was no significant
difference in comparison with controls (Figure 6G). In
contrast, 8␤-VE2 had no effect on the induction of IL-10
in mice with CIA. IFN␥ concentrations were examined
in supernatants of spleen cells from all treatment groups
cultured ex vivo with CII. Although increased levels of
antigen-induced IFN␥ were not observed in any group,
significantly increased basal concentrations of IFN␥
were observed in supernatants of spleen cells from mice
treated in vivo with 8␤-VE2 (P ⬍ 0.05) (Figure 6H).
DISCUSSION
Considerable evidence suggests that endogenous
estrogen plays a significant role as a modulator of
immune responses and autoimmune disease. Although
the reported effects of estrogen are variable, when
taken together, the available data suggest that endogenous estrogen has an inhibitory immunoregulatory
effect on autoimmune diseases such as RA. Understandably, most previous studies have focused on the effects
of endogenous estrogen in females. However, one-third
of patients with RA are male, and as noted above,
limited previous studies suggest that estrogen has inhibitory effects in male mice. Our data support an inhibitory effect of endogenous estrogen on the inflammatory
response in male mice and, moreover, demonstrate
significant differences in the roles of ER␣ and ER␤ in
these effects.
In the current study, estrogen deficiency in male
mice was associated with increased and/or prolonged
expression of serum proinflammatory cytokines TNF,
IL-6, MCP-1, and IFN␥ in response to in vivo administration of LPS, demonstrating an inhibitory effect of
endogenous estrogen on cytokine expression in male
mice. Significant reversal of increased LPS-induced se-
1024
rum cytokine concentrations in estrogen-deficient mice
was only observed in response to administration of
16␣-LE2, an ER␣ agonist. These observations are consistent with a previous report of significantly increased
LPS-induced TNF release in macrophages deficient in
ER␣ (25) and suggest that ER␣, not ER␤, mediates the
inhibitory effects of endogenous estrogen on proinflammatory cytokine production in innate immune responses. The possibility that the ER␣-dependent inhibitory effect of estrogen on levels of LPS-induced
proinflammatory cytokines is mediated via induction of
IL-10 is not supported by the data from the current
study, since IL-10 was significantly increased by both
ER␣ and ER␤ agonists.
The deficiency of endogenous estrogen in male
ArKO mice resulted in exacerbation of joint inflammation and antigen-specific T cell activation in the presence
of AIA. These are the first observations of a suppressive
effect of endogenous estrogen on arthritis in male mice.
Estrogen deficiency had no effect on serum antigenspecific total IgG, IgG1, and IgG2a, suggesting that the
exacerbation of AIA in estrogen-deficient male mice is
independent of humoral immune responses. In contrast,
evidence presented here that endogenous estrogen inhibited antigen-specific T cell proliferation suggests that
estrogen plays a critical role in the regulation of cellmediated immunity in male mice and in AIA.
These findings are consistent with a previous
report that in vivo administration of 2-methoxyestradiol
suppressed adjuvant-induced arthritis in male rats, accompanied by inhibition of T cell responses to recall
antigens and mitogens ex vivo (12). The receptors used
by estrogen in the mediation of these effects on immune
responses in male mice have not previously been studied. Administration of either the ER␣ or the ER␤
agonist was able to significantly reverse the increase in
AIA severity, but only the ER␣ agonist inhibited
antigen-induced T cell activation in ArKO mice. These
results suggest that the inhibitory effects of endogenous
estrogen on antigen-specific T cell activation is mediated
through ER␣ but that additional effects contribute to
the antiinflammatory effects of estrogen on arthritis in
male mice with AIA.
Endogenous estrogen was observed in the
present study to inhibit aspects of both innate and
adaptive immune responses, as measured by LPSinduced cytokines and antigen-induced T cell proliferation, but only restoration with an ER␣ agonist was
able to reverse both of these responses. The broader
range of effects of ER␣ suggested the possibility that
ER␣ mediates the reported effects of estrogen in the
YANG ET AL
CIA model of arthritis in male mice, since aspects of
both these pathways are operative in CIA. A trend
toward increased CIA severity in male ArKO mice
suggested that endogenous estrogen is an inhibitory
factor in immune responses in CIA in male mice. This
was supported by the increased CII-induced T cell
proliferative responses in estrogen-deficient male mice,
which was also consistent with the results we observed in
AIA, and these data further support the hypothesis that
endogenous estrogen modulates adaptive immune responses in male mice.
The severity of CIA in ArKO mice, which are
generated on a C57BL/6 background, was insufficient to
allow the study of ER␣ and ER␤ agonists, so we elected
to study the effects of ER␣ and ER␤ agonists as
compared with the effects of estrogen on CIA in male
DBA/1 mice. A significant inhibitory effect of ER␣
agonist administration on arthritis severity was observed, which was comparable to the effects of estrogen
administration. Conversely, administration of ER␣ agonist increased the severity of AIA in male WT C57BL/6
mice. A significant inhibitory effect of ER␣ agonist on
anti-CII autoantibody production was also observed,
accompanied by an increase in serum levels of IL-10. In
contrast, no significant effect of ER␤ agonist administration on CIA or anti-CII autoantibody production was
observed. The lack of effect of 8␤-VE2 on the expression
of monocyte cytokines such as TNF and MCP-1 may
contribute to its lack of effect in CIA.
In conclusion, the current data indicate that
endogenous estrogen mediates an inhibitory effect on
innate and adaptive immune responses and on the
development of experimental arthritis in male mice.
Although both ER␣ and ER␤ appear to be able to
transduce inhibitory signals of estrogen on antigenspecific T cell responses and AIA, ER␣ mediates endogenous estrogen effects on a broader range of events,
including proinflammatory cytokine production, and in
association, is responsible for estrogen effects on inflammation in CIA in male mice. This suggests that ER␣ is
the dominant receptor that mediates the inhibitory
effects of estrogen on inflammation in male mice.
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Dr. Yang had full access to all of the
data in the study and takes responsibility for the integrity of the data
and the accuracy of the data analysis.
Study conception and design. Yang, Jones, Simpson, Fritzemeier,
Morand.
ESTROGEN REGULATION OF ARTHRITIS AND CYTOKINES IN MICE VIA ER␣
Acquisition of data. Yang, Ngo.
Analysis and interpretation of data. Yang, Ngo, Morand.
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