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Endocrinology, doi:10.1210/en.2003-1536
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Endocrinology Vol. 145, No. 5 2384-2391
Copyright © 2004 by The Endocrine Society

Estrogen Deficiency Accelerates Murine Autoimmune Arthritis Associated with Receptor Activator of Nuclear Factor-{kappa}B Ligand-Mediated Osteoclastogenesis

Tomoko Yoneda, Naozumi Ishimaru, Rieko Arakaki, Masaru Kobayashi, Takashi Izawa, Keiji Moriyama and Yoshio Hayashi

Departments of Pathology (T.Y., N.I., R.A., M.K., T.I., Y.H.) and Orthodontics (T.Y., T.I., K.M.), Tokushima University School of Dentistry, Tokushima 770-8504 Japan

Address all correspondence and requests for reprints to: Dr. Yoshio Hayashi, Department of Pathology, Tokushima University School of Dentistry, 3 Kuramotocho, Tokushima 770-8504, Japan. E-mail: hayashi{at}dent.tokushima-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The aims of this study were to evaluate the in vivo effects of estrogen deficiency in MRL/lpr mice as a model for rheumatoid arthritis and to analyze the possible relationship between immune dysregulation and receptor activator of nuclear factor-{kappa}B ligand (RANKL)-mediated osteoclastogenesis. Experimental studies were performed in ovariectomized (Ovx)-MRL/lpr, Ovx-MRL+/+, sham-operated-MRL/lpr, and sham-operated-MRL+/+ mice. Severe autoimmune arthritis developed in younger Ovx-MRL/lpr mice until 24 wk of age, whereas these lesions were entirely recovered by pharmacological levels of estrogen administration. A significant elevation in serum rheumatoid factor, anti-double-stranded DNA, and anti-type II collagen was found in Ovx-MRL/lpr mice and recovered in mice that underwent estrogen administration. A high proportion of CD4+ T cells bearing RANKL was found, and an enhanced expression of RANKL mRNA and an impaired osteoprotegerin mRNA was detected in the synovium. An increase in both osteoclast formation and bone resorption pits was found. These results indicate that estrogen deficiency may play a crucial role in acceleration of autoimmune arthritis associated with RANKL-mediated osteoclastogenesis in a murine model for rheumatoid arthritis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT IS WELL KNOWN that sex steroids have significant impact on the development of autoimmune diseases in both humans and rodents. In particular, estrogen has been suggested to be responsible for the strong female preponderance of the human rheumatoid arthritis (RA), systemic lupus erythematosus, scleroderma, and Sjögren’s syndrome (1, 2, 3), but the role of estrogens in the female has not been fully characterized. RA is a chronic inflammatory disease characterized by invasive synovial hyperplasia leading to progressive joint destruction. Rheumatoid synovial cells are not only morphologically characterized by their transformed appearance (4) but also are phenotypically transformed to proliferate abnormally (5, 6). These cells invade bone and cartilage by producing an elevated amount of proinflammatory cytokines (7) and matrix metalloproteinases (MMPs) (8) and by inducing osteoclast (OC) formation and activation (9, 10).

OCs, the multinucleated cells exclusively responsible for bone resorption, have been observed to resorb bone actively at the site of invasion of the proliferated synovial membrane into the adjacent bone (11). The cell types responsible for bone resorption in RA have been characterized as authentic OCs (12), and it was reported that rheumatoid synovial fibroblasts are involved in bone destruction by inducing osteoclastogenesis (13). However, the exact mechanisms involved in the formation and activation of OCs in RA are still unclear.

Receptor activator of nuclear facto-{kappa}B ligand (RANKL) (14) is a regulator of the immune system and of bone development (15). RANKL is expressed on activated T cells (16), and a major target for RANKL in the immune system appears to be mature dendritic cells (DCs) that express a high level of RANKL receptor (RANK) (17). In vitro, RANKL promotes the survival of mature DCs, most likely by up-regulating the expression of Bcl-XL (18), and induces the production of proinflammatory cytokines, such as IL-1 and IL-6, and cytokines that stimulate and induce differentiation of T cells, such as IL-12 and IL-15 (19). Therefore, RANKL is likely to act as a positive-feedback regulator during productive T cell-DC interactions (20).

The MRL/lpr mouse strain was chosen to test the estrogenic action because it has a genetic predisposition to arthritis with characteristics similar to those of human RA including cell infiltration, pannus formation, bone and cartilage breakdown, and the presence of serum rheumatoid factor (RF) (21, 22, 23). The aim of this study was to analyze the in vivo effects of estrogen deficiency on the development of autoimmune arthritis in MRL/lpr mice and to evaluate the possible relationship with RANKL-mediated osteoclastogenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and treatment
MRL/Mp-lpr/lpr (MRL/lpr, age 4–24 wk; n = 108) and MRL+/+ mice (age 4–24 wk; n = 58) were purchased from Charles River Japan Inc. (Atsugi, Japan), and were fed under specific pathogen-free conditions. Female MRL/lpr mice and MRL+/+ mice (4 wk of age) were ovariectomized (Ovx) and compared with sham-operated (sham)-MRL/lpr and MRL+/+ mice. Six to 10 mice in each group were analyzed at 8, 12, 16, 20, and 24 wk of age. Ovx-MRL/lpr mice were administered im with 60 mg/kg·wk estrogen (Ovahormine depo; Teikoku Zouki Inc., Tokyo, Japan) in sesame oil or sc with 25 mg/kg·d testosterone (Wako Pure Chemical, Osaka, Japan) from 4–20 wk of age. Care of the mice was in accordance with institutional guidelines.

Histology and immunohistology
All organs were removed from the mice and fixed with 10% phosphate-buffered formalin, and ankles were further decalcified in 10% EDTA. Sections (4 µm in thickness) were stained with hematoxylin and eosin. Histological grading of inflammatory arthritis was done according to the methods by Edwards et al. (24) as follows: one point score indicates hyperplasia/hypertrophy of synovial cells; fibrosis/fibroplasia; proliferation of cartilage and bone; destruction of cartilage and bone; and/or mononuclear cell infiltrate. Immunohistological analysis was performed on freshly frozen sections (4 µm in thickness) by the biotin-avidin immunoperoxidase method using ABC reagent (Vector Laboratories Inc., Burlingame, CA). The monoclonal antibodies (mAb) used were biotinylated rat mAbs to CD4, CD8 (BD Biosciences, San Jose, CA), and mouse RANKL (IMGENEX, San Diego, CA).

Flow cytometric analysis
Spleen and inguinal lymph node (LN) cell suspensions were stained with antibodies (Ab) conjugated to phycoerythrin (anti-CD4, Cedarlane Laboratories Ltd., Ontario, Canada; B220, PharMingen, San Diego, CA), fluorescein isothiocyanate (anti-CD8, Cedarlane Laboratories; Thy1.2, PharMingen), and antimouse RANKL (IMGENEX) and analyzed with EPICS (Coulter, Miami, FL).

Measurement of anti-double-stranded DNA (dsDNA) Ab, RF, and type 2 collagen (CII) Ab levels
Anti-dsDNA Abs, RF, and anti-CII Ab were detected by ELISA as described previously (25, 26, 27). Briefly, flat-bottom plates (Nalge Nunc International, Roskilde, Denmark) were coated with 1.5 µg/ml of native calf thymus DNA (Life Technologies, Inc., Rockville, MD) in buffer containing 0.1 M sodium bicarbonate and 0.05 M citric acid at 4 C overnight. Serum samples were serially diluted (starting at 1/200) and added to the plates for a 1-h incubation at 37 C. After washing, peroxidase-conjugated goat antimouse IgG, or IgM (Southern Biotechnology Associates, Birmingham, AL), was added and incubated for 1 h at 37 C. Ab binding was visualized using orthophenylenediamine (Sigma, St. Louis, MO). For the measurement of IgG and IgM RF, human IgG and IgM (Chemicon International, Temecula, CA) were coated onto plates at 10 µg/ml in carbonate buffer, and the same procedures were followed as described above. For the measurement of serum Abs to CII, native bovine CII was dissolved in 0.1 M acetic acid at 1 mg/ml and diluted with 0.1 M sodium bicarbonate at 10 µg/ml (pH 9.6). The microtiter plate was coated with 100 µl of CII antigen solution. After washing three times, 100 µl per well of serum samples that had been serially diluted in PBS/Tween 20/1% BSA and control serum were added and incubated for 1 h at 37 C. After washing, peroxidase-conjugated goat antimouse IgG (at 1.4 µg/ml, 100 µl per well) (Organon Teknika, Durham, NC) was added and incubated for 1 h at 37 C. A total of 100 µl o-phenylenediamine (0.5 mg/ml) dissolved in 0.1 M citrate buffer (pH 5.0) containing 0.012% H2O2 was added, and the reaction was stopped using 8 N H2SO4 (20 µl per well).

Measurement of cytokine production
Cytokine production was tested by two-step sandwich ELISA using a mouse IL-2, IL-4, and interferon (IFN)-{gamma} kit (Genzyme, Cambridge, MA). In brief, culture supernatants from LN cells activated with immobilized anti-CD3 mAb (Cedarlane Laboratories) for 3 d were added to microtiter plates precoated with anti-IL-2, IL-4, and IFN-{gamma} capture Ab and incubated overnight at 4 C. After addition of biotinylated detecting Ab and incubation at room temperature for 45 min, avidin-peroxidase was added and incubated at room temperature for 30 min. Finally, 2,2'-azino-di-3-ethylbenzthiazoline sulfonate substrate containing H2O2 was added, and the colorimetric reaction was read at an absorbance of 405 nm using an automatic microplate reader (Bio-Rad Laboratories Inc., Hercules, CA). The concentrations of IL-2 (picograms per milliliter), IL-4 (picograms per milliliter), and IFN-{gamma} (picograms per milliliter) were calculated according to the standard curves produced by various concentrations of recombinant cytokines.

ELISA for murine osteoprotegerin (OPG)
An anti-murine OPG mAb (TECHNE Corp., Minneapolis, MN) was diluted with 0.1 M sodium bicarbonate (pH 9.6) solution to a concentration of 10 µg/ml, and the 100-µl aliquot was added to each well of 96-well plates. After incubation at 4 C overnight, the capture solution was removed by flicking the plates, and the wells were blocked with the blocking solution (300 µl per well) for 2 h at room temperature. Recombinant OPG (100 µl) (R&D System Inc., Minneapolis, MN) standard and a series of test samples were added to the wells, and the plates were incubated for 2 h at room temperature. The wells were then washed with the washing buffer, and 100 µl of peroxidase-labeled anti-OPG mAb was added to each well. After incubation for an additional 2 h, 100 µl of tetramethylbenzidine substrate reagent was added to each well. Tetramethylbenzidine stop buffer (100 µl) was added to each well, and absorbance at 450 nm of the wells was measured with a microplate reader (Bio-Rad Laboratories Inc.).

RT-PCR
The total RNA from LNs and synovial tissues was extracted as reported previously (28). The RNA was reverse-transcribed into cDNA. The cDNA reaction mixture was diluted with 90 µl of PCR buffer and mixed with 500 nM of the 5' and 3' primers, 0.1 mM deoxynucleotide triphosphate mix, 2 mM MgCl2, and 2 U thermostable Taq polymerase (PerkinElmer Cetus, Norwalk, CT). The cDNA was subjected to enzymatic amplification in a DNA thermal cycler (PerkinElmer Cetus) by using specific primers. PCR was carried out at 55 C annealing temperature for 30–35 cycles. The specific primers used were as follows: IL-1ß, TGA TGA GAA TGA CCT GTT CT and CTT CTT CAA AGA TGA AGG AAA; TNF-{alpha}, ATG AGC ACA GAA AGC ATG ATC and AGA TGA TCT GAG TGT GAG GG; IL-6, CTC TGC AAG AGA GAC TTC CAT and ATA GGC AAA TTT CCT GAT TAT A; IFN-{gamma}, CCT CAG ACT CTT TGA ACT CT and CAG CGA CTC CTT TTC CGC TT; IFN regulatory factor (IRF)-1, TCT GAG TGG CAT ATG CAG ATG GAC and GGT CAG AGA CCC AAA CTA TGG TCG; MMP-1, ATG GTG GGG ATG CCC ATT TT and CAG CAT CTA CTT TGT TGC C; MMP-2, GAG TTG GCA GTG CAA TAC CT and GCC ATC CTT CTC AAA GTT GT; MMP-3, GAA ATG CAG AAG TTC CTC GG and GAG TTC CAT AGA GGG ACT GA; MMP-9, CCA TGA GTC CCT GGC AG and AGT ATG TGA TGT TAT GAT G; TIMP (tissue inhibitor of metalloproteinase)-1, CTG GCA TCC TCT TGT TGC TA and AGG GAT CTC CAA GTG CAC AA; RANKL, GGG AAT TAC AAA GTG CAC CAG and GCC ATC CTT CTC AAA GTT GT; RANK, GTC TTC TGG AAC CAT CTT CTC C and CAC AGA CAA ATG CAA ACC TTG; OPG, TCA AGT GCT TGA GGG CAT AC and TGG AGA TCG AAT TCT GCT TG; estrogen receptor (ER)-{alpha}, AAT TCT GAC AAT CGA CGC CAG and GTG CTT CAA CAT TCT CCC TCC TC; ER-ß, TTC CCA GCA GCA CCG GTA ACC T and TCC CTC TTG GCG CTT GGA CTA; ß-actin, GTG GGC CGC TCT AGG CAC CA and CGG TTG GCC TTA GGG TTC AGG GGG. The amplified DNA reaction mixture was subjected to 1.7% agarose gel electrophoresis, and the amplified product was visualized by UV fluorescence after staining with ethidium bromide.

Tartrate-resistant acid phosphatase (TRAP) staining
Staining for TRAP was performed according to the modified method of Takahashi et al. (29). Sections were rinsed once with PBS (pH 7.4), air dried, fixed with 10% formalin in PBS (pH 5.4) for 5 min, and fixed with methanol-acetone (50:50; pH 5.4) for 30 sec. The coverslips were air-dried and stained for 15 min at room temperature in a 0.1 M sodium acetate buffer (pH 5.0) containing naphthol AS-MX phosphate (Sigma) as a substrate and fast red violet LB salt (Sigma) as a stain for the reaction product in the presence of 50 mM of sodium tartrate.

Assessment of bone resorption
Bone marrow cells (5 x 105) from Ovx- and sham-MRL/lpr mice were added to the wells of 96-well plates containing a slice of bovine cortical bone and incubated in a total volume of 200 µl {alpha}-MEM-fetal bovine serum as described previously (30). All cultures were maintained in the presence of dexamethazone (10–7 M, FUNAKOSHI Pharmcol., Tokyo, Japan) and 1{alpha},25-(OH)2D3 (10–8 M, Chugai Inc., Tokyo, Japan) for 10 d. Bone slices were assessed for bone resorptive activity, brushed with a rubber policeman to remove cells after observation under a microscope, and stained with Mayer’s hematoxylin. Bone resorption pits were quantified by densitometric analysis of images of the whole area of bone slices as previously described (30). Additionally, in vitro osteoclastogenesis was assayed using bone marrow-derived TRAP-positive cells with macrophage colony stimulating factor (5 ng/ml; PeproTech EC, London, UK), recombinant murine RANKL (10 ng/ml; PeproTech Inc., Rocky Hill, NJ), recombinant OPG (100 ng/ml; R&D Systems Inc.), and 17ß-estradiol (10–9 M), at indicated concentration after the estimation of dose responses.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of the Ovx on joint histopathology
Destructive autoimmune arthritis developed in young Ovx-MRL/lpr mice, not in young sham-MRL/lpr and Ovx-MRL+/+ mice, and these lesions aggravated with age from 12 until 24 wk of age. Histological analysis of the knee joints was performed at 8, 12, 16, 20, and 24 wk of age for all the experiments. Analysis of the histological results for the experiment, shown in Fig. 1AGo, indicates that the group that was treated with Ovx had significant higher subsynovial inflammation, synovial hyperplasia, pannus formation and cartilage erosion, bone destruction, and overall histopathology. Shown in Fig. 1BGo are photomicrographs taken from representative Ovx- and sham-MRL/lpr mice at 12 and 20 wk of age. The effects observed in Ovx-MRL/lpr mice included synovial hyperplasia, pannus formation, bone erosion, and infiltration of mononuclear cells into the subsynovial tissue. In contrast, mononuclear cell infiltration and bone and cartilage pathology was absent in sham-MRL/lpr mice until age 20 wk.



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  		FIG. 1. Effects of the Ovx on joint histopathology. Histological score of autoimmune arthropathy developed in younger Ovx-MRL/lpr mice compared with those in sham-MRL/lpr and Ovx-MRL+/+ mice until 24 wk of age (A). Histological evaluation of the knee joints was performed according to the methods by Edwards et al. (24 ) (*, P < 0.05; and **, P < 0.01, Student’s t test). Representative photomicrographs taken from Ovx- and sham-MRL/lpr mice at 12 (12W) and 20 wk (20W) of age (B). The histopathological effects observed in Ovx-MRL/lpr mice at age 20 wk included mononuclear cell infiltration into the subsynovial tissue (lower left), and synovial hyperplasia (lower right) (hematoxylin and eosin). In contrast, mononuclear cell infiltration and bone and cartilage pathology was absent in sham-MRL/lpr mice until 20 wk of age (middle left).

 
Recovery of autoimmune arthritis by estrogen administration
The destructive lesions in the knee joints in Ovx-MRL/lpr mice were inhibited by estrogen administration (10–9 M) at 12 and 20 wk of age (Fig. 2AGo). Testosterone administration in Ovx-MRL/lpr mice resulted in severe inflammatory lesions as the same levels. We confirmed gene expression in ER-{alpha} but not ER-ß in synovial tissues by RT-PCR analysis (Fig. 2BGo), indicating that estrogenic action to the synovial tissues might be directly affected through estrogen/ER-{alpha} binding in vivo. We detected increased levels of serum RF, anti-dsDNA, and anti-CII Abs in Ovx-MRL/lpr mice compared with those in sham mice, and these levels were entirely recovered in Ovx-MRL/lpr mice that underwent estrogen administration (Fig. 2CGo).



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  		FIG. 2. The destructive lesions in the knee joints in Ovx-MRL/lpr mice were inhibited by estrogen administration (10–9 M) at ages 12 and 20 wk (*, P < 0.05 and **, P < 0.01, Student’s t test) (A). Detection of gene expression in ER-{alpha} in the synovium but not in ER-ß by RT-PCR analysis (B). E2, 17ß-Estradiol. Serum RF, anti-dsDNA Abs, and anti-CII were significantly increased in Ovx-MRL/lpr mice compared with those in sham-MRL/lpr mice, and these changes were entirely recovered by the treatment with estrogen administration (10–9 M) at 12 and 20 wk of age (*, P < 0.05; and **, P < 0.01, Student’s t test) (C). Tes, Testosterone.

 
Effects of the Ovx on cytokine and MMP expression
Culture supernatants from anti-CD3 mAb-stimulated LN T cells obtained from Ovx-MRL/lpr mice at 20 wk of age contained high levels of IFN-{gamma}, whereas no difference in levels of IL-2 and IL-4 was observed by ELISA (Fig. 3AGo). We next analyzed the effects of the Ovx on various gene expressions in the synovial tissues. Increased expressions of cytokine genes including IL-1ß, IFN-{gamma}, TNF-{alpha}, IL-6, IRF-1, and ß-actin mRNA were detected in synovial tissues from Ovx-MRL/lpr mice at 20 wk of age, compared with those from sham-MRL/lpr mice by RT-PCR analysis (Fig. 3BGo). In addition, we found elevated gene expressions of MMP-3 and MMP-9 in synovial tissues from Ovx-MRL/lpr mice (Fig. 3CGo). These data suggest that estrogen deficiency induces various gene expressions directly responsible for tissue damage on the development of autoimmune arthritis.



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  		FIG. 3. Effects of the Ovx on expression of cytokines and MMPs. Culture supernatants from anti-CD3 mAb-stimulated LN T cells obtained from Ovx-MRL/lpr mice at age 20 wk contained high levels of IFN-{gamma} (**, P < 0.01, Student’s t test), whereas no different levels of IL-2 and IL-4 was observed by ELISA (A). Increased expressions of cytokine genes including IL-1ß, IFN-{gamma}, TNF-{alpha}, IL-6, IRF-1, and ß-actin were detected in synovium from Ovx-MRL/lpr mice at age 20 wk, compared with those from sham-mice by RT-PCR analysis (B). Enhanced gene expressions of MMP-3, and MMP-9 mRNA were found in synovium from Ovx-MRL/lpr mice at age 20 wk (C).

 
Effects of the Ovx on RANKL, RANK, and OPG expression
We analyzed the spleen and LN cells bearing RANKL by flow cytometry. A significant increase of Thy1.2+, and CD4+ T cells bearing RANKL in the spleen from Ovx-MRL/lpr mice was observed, compared with those from sham-MRL/lpr (Fig. 4AGo). We detected a large proportion of CD4+ T cells in LN bearing RANKL (89.7%) from Ovx-MRL/lpr mice as compared with those from sham-MRL/lpr mice (28.2%) (Fig. 4BGo). In addition, an enhanced RANKL mRNA and an impaired OPG mRNA were observed in LN and synovium from Ovx-MRL/lpr mice, compared with those in sham-mice by RT-PCR analysis (Fig. 4CGo). Indeed, a significant decrease in OPG concentration was found in synovial fluid of Ovx-MRL/lpr mice compared with those of sham-mice at 12 and 20 wk of age (Fig. 4DGo). A large proportion of infiltrating cells in synovium was positive for RANKL in Ovx-MRL/lpr mice (Fig. 4EGo).



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  		FIG. 4. A significant increase in splenic Thy1.2+ and CD4+ T cells bearing RANKL from Ovx-MRL/lpr mice at 20 wk of age was observed as compared with those from sham-MRL/lpr mice, whereas no remarkable change in CD8+ T cells bearing RANKL was found (A). A large proportion of CD4+ T cells in LN bearing RANKL (89.7%) from Ovx-MRL/lpr mice was detected on flow cytometry, as compared with that of sham-MRL/lpr mice (28.2%) (B). A prominently enhanced RANKL mRNA and an impaired OPG mRNA were observed in LN and synovium from Ovx-MRL/lpr mice by RT-PCR analysis (C). A significantly decreased OPG concentration was found in synovial fluid of Ovx-MRL/lpr mice at ages 12 (12w) and 20 wk (20w) (*, P < 0.05; **, P < 0.01, Student’s t test) (D). A large proportion of infiltrating cells in synovium was positive for RANKL in Ovx-MRL/lpr mice at 20 wk of age. Isotype-matched controls were all negative (E).

 
Effects of the Ovx on OC formation and bone resorption
As seen in Fig. 5AGo, numerous multinucleated (more than three nuclei), TRAP-positive OC-like cells were detected in the knee joints from both Ovx- and sham-MRL/lpr mice. A significant increase in number of OCs formed in the joints from Ovx-MRL/lpr mice was observed compared with those from sham-MRL/lpr mice (Fig. 5BGo). We next examined in vitro whether the TRAP-positive multinucleated cells (more than three nuclei) from bone marrow cells resorbed bovine bone slices (30). When the bone marrow cells were cultured with dexamethasone and 1{alpha},25-(OH)2D3, they differentiated into TRAP-positive multinucleated cells, and numerous resorption pits were formed on their surfaces (Fig. 5CGo). A significantly large number of resorption pits were formed using the bone marrow cells from Ovx-MRL/lpr mice compared with those from sham-MRL/lpr mice (Fig. 5DGo).



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  		FIG. 5. Effect of the Ovx on OC formation and bone resorption assay. TRAP-positive OC-like cells were detected in the knee joints from both Ovx- and sham-MRL/lpr mice (A). A significantly increased number of OCs formed in the joints from Ovx-MRL/lpr mice at ages 12 (12w) and 20 wk (20w) was observed compared with those from sham-MRL/lpr mice (B) (*, P < 0.05 and **, P < 0.01, Student’s t test). TRAP-positive multinucleated cells (more than three nuclei) from bone marrow cells were observed in both Ovx- and sham-MRL/lpr mice when cultured with dexamethasone (10–7 M) and 1{alpha},25-(OH)2D3 (10–8 M) (C). A significantly large number of resorption pits was formed using the bone marrow cells from Ovx-MRL/lpr mice compared with those from sham-MRL/lpr mice (*, P < 0.05 and **, P < 0.01, Student’s t test)(D).

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
RA is characterized by progressive joint damage that is mediated by several mechanisms (31). Although the etiology of RA remains unknown, joint damage results from the degradation of connective tissue by MMPs and the stimulation of osteoclastogenesis by activated CD4+ T cells (10). Sex hormones influence both humeral and cell-mediated immune response, and estrogen is one of potential factors in this immunological dimorphism (32, 33, 34). We have examined the effects of the Ovx on the development of autoimmune arthritis in animals that are susceptible to the development of human RA-like disease. Histology of autoimmune arthritis in Ovx-MRL/lpr mice showed severe destructive changes in the younger age examined. It is generally accepted that a severe inflammatory arthritis and systemic autoimmune disease, including glomerulonephritis and autoantibody production, develop in aged (>6-month-old) MRL/lpr mice (21, 22). We show here that estrogen deficiency caused a severe inflammatory arthritis in younger (<3-month-old) MRL/lpr mice, and these lesions were dramatically prevented by exogenous estrogen treatment. Although the mechanism in detail of estrogen activity still remains unclear, these findings suggest that estrogenic action has an important role during development of autoimmune arthritis in MRL/lpr mice. An estrogen deficiency by Ovx in murine RA model results in a significant increase of serum RF, anti-dsDNA Abs, and anti-CII Ab, and these changes were recovered by estrogen administration. A previous report has shown that treatment with low doses of ß-estradiol exerts a suppressive effect on both development of collagen arthritis as well as T cell-dependent immune reactivity toward type II collagen (35). It was also reported that an estrogen deficiency stimulates B cell development (36) and autoantibody production (36, 37, 38), and its increase by an estrogen deficiency has been mediated by cytokines such as IL-6, IFN-{gamma}, and TNF-{alpha} (39, 40, 41). A recent report has demonstrated that estrogen deficiency induces bone loss by increasing T cell proliferation through IFN-{gamma}-induced class II transactivator (42).

Bone resorption is regulated by the immune system, in which T cell expression of RANKL, which is essential for osteoclastogenesis, may contribute to pathological conditions, such as RA (43). However, it remains unclear whether activated T cells maintain bone homeostasis by counterbalancing the action of RANKL. In this study, a significant increase of CD4+ T cells bearing RANKL in the spleen and inguinal LN from Ovx-MRL/lpr mice was observed. Moreover, we detected an enhanced RANKL mRNA expression and RANKL+ infiltrating cells in synovium from Ovx-MRL/lpr mice. In contrast, an impaired OPG concentration was found in synovial fluid, in addition to a decreased OPG mRNA in synovium of Ovx-MRL/lpr mice. Although molecular mechanisms demonstrating that the appearance of increased RANKL expression in T cells and synovium is related to the development of the autoimmune response are obscure, a role for RANKL in bone resorption in RA is suggested by the identification of RANKL mRNA and protein in cultured synovial fibroblasts from patients with RA and in CD4+ and CD8+ T cells in RA synovial tissues (10). Additional evidence that RANKL plays a critical role in the pathogenesis of bone destruction in inflammatory arthritis comes from studies in the rat adjuvant arthritis model (44, 45). Moreover, it has been recently demonstrated that up-regulation of RANKL on bone marrow cells is an important determinant of increased bone resorption induced by estrogen deficiency (46). Treatment with OPG also prevented OC accumulation, whereas destruction of bone in untreated arthritic animals was accompanied by the accumulation of large numbers of TRAP+ OC-like cells (47). OPG treatment in an animal model of arthritis that is dependent on T cell activation has the potential for blocking not only the effects of RANKL on OC differentiation and activation but also the influence of RANKL on T cell-DC interactions (20).

OCs have a crucial role in the local bone destruction that occurs in association with chronic inflammatory diseases (48). Diseases such as RA have been associated with the accumulation of TNF-{alpha} and/or other proinflammatory cytokines such as IL-1 and IL-6, which likely mediate local bone destruction by stimulating OC activity (49). The results in the present study demonstrated an increased gene expression of cytokines including IL-1ß, TNF-{alpha}, IL-6, IFN-{gamma}, ß-actin, and IRF-1 in the synovium from Ovx-MRL/lpr mice. In addition, an elevated gene expressions of MMP-3 and MMP-9 mRNA were observed in the synovium. Moreover, we found a significant increase in number of OCs, and bone resorption pits formed in Ovx-MRL/lpr mice. It has been shown that estrogen prevents bone loss through multiple effects on bone marrow and bone cells, which result in decreased OC formation (50), increased OC apoptosis (51), and decreased capacity of mature OCs to resorb bone (52). Because it was also demonstrated by direct evidence that treatment with estrogens suppressed RANKL-mediated OC formation (53), it is possible that their contribution to the increased osteoclastogenesis and the bone loss has been induced by estrogen deficiency.

In conclusion, we have demonstrated that activation of CD4+ T cells bearing RANKL induced by an estrogen deficiency may play an important role on acceleration of autoimmune arthritis, and estrogenic action appears to influence joint destruction associated with RANKL-mediated osteoclastogenesis in a murine model for RA.


   Footnotes
 
This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.

Abbreviations: Ab, Antibody; CII, type 2 collagen; DC, dendritic cell; dsDNA, double-stranded DNA; ER, estrogen receptor; IFN, interferon; IRF, IFN regulatory factor; LN, lymph node; mAb, monoclonal antibody; MMP, matrix metalloproteinase; OC, osteoclast; OPG, osteoprotegerin; Ovx, ovariectomized; RA, rheumatoid arthritis; RANK, RANKL receptor; RANKL, receptor activator of nuclear factor-{kappa}B ligand; RF, rheumatoid factor; sham, sham-operated; TRAP, tartrate-resistant acid phosphatase.

Received November 12, 2003.

Accepted for publication January 8, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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