Estrogen exerts a variety of important physiological effects, which have been suggested to be mediated via the two known estrogen receptors (ERs), alpha and beta. Three-month-old ovariectomized mice, lacking one or both of the two estrogen receptors, were given estrogen subcutaneously (2.3 micro g/mouse per day) and the effects on different estrogen-responsive parameters, including skeletal effects, were studied. We found that estrogen increased the cortical bone dimensions in both wild-type (WT) and double ER knockout (DERKO) mice. DNA microarray analysis was performed to characterize this effect on cortical bone and it identified four genes that were regulated by estrogen in both WT and DERKO mice. The effect of estrogen on cortical bone in DERKO mice might either be due to remaining ERalpha activity or represent an ERalpha/ERbeta-independent effect. Other effects of estrogen, such as increased trabecular bone mineral density, thymic atrophy, fat reduction and increased uterine weight, were mainly ERalpha mediated.

Estrogens are important for the male skeleton. Osteoprotegerin (OPG), receptor activator of NF-kappa B ligand (RANKL), interleukin-6 (IL-6), IL-1 and tumor necrosis factor alpha (TNFalpha) have been suggested to be involved in the skeletal effects of estrogen. We treated orchidectomized mice with estradiol for 2 weeks and observed a 143% increase in the trabecular bone mineral density of the distal metaphysis of femur that was associated with a decreased OPG/RANKL mRNA ratio in vertebral bone. A similar decreased OPG/RANKL ratio was also seen after estrogen treatment of ovariectomized female mice. The effect of estrogen receptor (ER) inactivation on the OPG/RANKL ratio was dissected by using intact male mice lacking ER alpha (ERKO), ER beta (BERKO) or both receptors (DERKO). The expression of OPG was increased in ERKO and DERKO but not in BERKO male mice, resulting in an increased OPG/RANKL ratio. Furthermore, serum levels of IL-6 and tartrate-resistant acid phosphatase 5b (TRAP 5b) were decreased in ERKO and DERKO, but not in BERKO male mice. These results demonstrate that ER alpha, but not ER beta, is involved in the regulation of the vertebral OPG/RANKL ratio, serum levels of IL-6 and TRAP 5b in male mice.

Summary: Oestrogen treatment down‐regulates B lymphopoiesis in the bone marrow of mice. Meanwhile it up‐regulates immunoglobulin production. To understand better the oestrogen action on bone marrow male mice lacking oestrogen receptor α (ERα; ERKO mice), lacking ERβ (BERKO mice), lacking both receptors (DERKO mice) or wild‐type (wt) littermates were castrated and treated for 2·5 weeks with 30 μg/kg 17β‐oestradiol (E₂) or vehicle oil as controls. The B lymphopoiesis in the bone marrow was examined by flow cytometry and mature B‐cell function was studied using an ELISPOT assay enumerating the B cells in bone marrow and spleen that were actively producing immunoglobulins. In wt mice the frequency of B‐lymphopoietic (B220⁺) cells in the bone marrow decreased from 15% to 5% upon E₂ treatment. In ERKO and BERKO mice significant reduction was seen but not of the same magnitude. In DERKO mice no reduction of B lymphopoiesis was seen. In addition, our results show that E₂ mediated reduction of different steps in B lymphopoiesis require only ERα or both receptors. In wt and BERKO mice E₂ treatment resulted in significantly increased levels of B cells actively producing immunoglobulin, while in ERKO and DERKO mice no such change was seen. Similar results were found in both bone marrow and spleen. In conclusion our results clearly show that both ERα and ERβ are required for complete down‐regulation of B lymphopoiesis while only ERα is needed to up‐regulate immunoglobulin production in both bone marrow and spleen.

Introduction: The incidence and progression of many autoimmune diseases are sex-biased, which might be explained by the immunomodulating properties of endocrine hormones. Treatment with estradiol potently inhibits experimental autoimmune arthritis. Interleukin-17-producing T helper cells (Th17) are key players in several autoimmune diseases, particularly in rheumatoid arthritis. The aim of this study was to investigate the effects of estrogen on Th17 cells in experimental arthritis. Methods: Ovariectomized DBA/1 mice treated with 17β-estradiol (E2) or placebo were subjected to collagen-induced arthritis (CIA), and arthritis development was assessed. Th17 cells in joints and lymph nodes were studied by flow cytometry. Lymph node Th17 cells were also examined in ovariectomized estrogen receptor α–knockout mice (ERα^−/−) and wild-type littermates, treated with E2 or placebo and subjected to antigen-induced arthritis. Results: E2-treated mice with established CIA showed reduced severity of arthritis and fewer Th17 cells in joints compared with controls. Interestingly, E2-treated mice displayed increased Th17 cells in lymph nodes during the early phase of the disease, dependent on ERα. E2 increased the expression of C-C chemokine receptor 6 (CCR6) on lymph node Th17 cells as well as the expression of the corresponding C-C chemokine ligand 20 (CCL20) within lymph nodes. Conclusions: This is the first study in which the effects of E2 on Th17 cells have been characterized in experimental autoimmune arthritis. We report that E2 treatment results in an increase of Th17 cells in lymph nodes during the early phase of arthritis development, but leads to a decrease of Th17 in joints during established arthritis. Our data suggest that this may be caused by interference with the CCR6-CCL20 pathway, which is important for Th17 cell migration. This study contributes to the understanding of the role of estrogen in the development of autoimmune arthritis and opens up new fields for research concerning the sex bias in autoimmune disease.