SUMMARYOestrogens affect the development and regulation of the immune system. To determine the role of oestrogen receptors a (ER-a) and b (ER-b) on the development of the immune system, male ER-a (ERKO) and ER-b (BERKO) mice, as well as ab-double knockout (DERKO) mice, were studied. Deletion of ER-a led to hypoplasia of both thymus and spleen. Interestingly, a higher frequency of immature double CD4 + CD8 + thymocytes was found in ER-a ± mice compared with ER-a + mice. Female oophorectomized BERKO mice given oestradiol (E2) displayed a similar degree of thymic atrophy compared with the wild-type strain but showed only limited involution of thymus cortex and no alteration of thymic CD4/CD8 phenotype expression. Our data demonstrate that expression of ER-a, but not ER-b, is mandatory in males for development of full-size thymus and spleen, whereas expression of ER-b is required for E2-mediated thymic cortex atrophy and thymocyte phenotype shift in females. A potential background for the above ®ndings may be down-regulated activity in the growth hormone/insulin-like growth factor-1 (GH/IGF-1) axis in males lacking ER-a and suppressed sensitivity of females lacking ER-b to E2-mediated suppression of IGF-1.

Estrogen exerts a variety of important physiological effects, which have been suggested to be mediated via the two known estrogen receptors (ERs), and . Three-monthold ovariectomized mice, lacking one or both of the two estrogen receptors, were given estrogen subcutaneously (2·3 µg/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 ER activity or represent an ER /ER -independent effect. Other effects of estrogen, such as increased trabecular bone mineral density, thymic atrophy, fat reduction and increased uterine weight, were mainly ER mediated.

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Estrogens are important for the male skeleton. Osteoprotegerin (OPG), receptor activator of NF-B ligand (RANKL), interleukin-6 (IL-6), IL-1 and tumor necrosis factor (TNF ) 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 (ERKO), ER (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 , but not ER , is involved in the regulation of the vertebral OPG/RANKL ratio, serum levels of IL-6 and TRAP 5b in male mice.

SUMMARYOestrogen 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 a (ERa; ERKO mice), lacking ERb (BERKO mice), lacking both receptors (DERKO mice) or wild-type (wt) littermates were castrated and treated for 2Á5 weeks with 30 mg/kg 17b-oestradiol (E 2 ) 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 þ ) cells in the bone marrow decreased from 15% to 5% upon E 2 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 E 2 mediated reduction of different steps in B lymphopoiesis require only ERa or both receptors. In wt and BERKO mice E 2 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 ERa and ERb are required for complete down-regulation of B lymphopoiesis while only ERa is needed to up-regulate immunoglobulin production in both bone marrow and spleen.

Rho family proteins are prenylated by geranylgeranyltransferase type I (GGTase-I), which normally target proteins to membranes for GTP-loading. However, conditional deletion of GGTase-I in mouse macrophages increases GTP-loading of Rho proteins, leading to enhanced inflammatory responses and severe rheumatoid arthritis. Here we show that heterozygous deletion of the Rho family gene Rac1 , but not Rhoa and Cdc42 , reverses inflammation and arthritis in GGTase-I-deficient mice. Non-prenylated Rac1 has a high affinity for the adaptor protein Ras GTPase-activating-like protein 1 (Iqgap1), which facilitates both GTP exchange and ubiquitination-mediated degradation of Rac1. Consistently, inactivating Iqgap1 normalizes Rac1 GTP-loading, and reduces inflammation and arthritis in GGTase-I-deficient mice, as well as prevents statins from increasing Rac1 GTP-loading and cytokine production in macrophages. We conclude that blocking prenylation stimulates Rac1 effector interactions and unleashes proinflammatory signaling. Our results thus suggest that prenylation normally restrains innate immune responses by preventing Rac1 effector interactions.

BackgroundThe identification of biomarkers that predict optimal and individual choices of treatment for patients with rheumatoid arthritis gains increasing attention. The purpose of this study was to investigate if the proto-oncogene survivin might aid in treatment decisions in early rheumatoid arthritis.MethodsSerum survivin levels were measured in 302 patients who completed the Swedish pharmacotherapy (SWEFOT) trial at baseline, 3, 12, and 24 months. Survivin levels > 0.45 ng/mL were considered positive. Based on the survivin status, core set outcomes measuring disease activity, functional disability, as well as global health and pain were evaluated after methotrexate (MTX) monotherapy at 3 months, and at 12 and 24 months of follow-up. Treatment of non-responders was randomly intensified with either a combination of disease-modifying antirheumatic drugs (triple therapy: MTX, sulfasalazine, and hydroxychloroquine) or by adding antibodies against tumor necrosis factor (anti-TNF).ResultsAntirheumatic treatment resulted in an overall decrease of serum survivin levels. Survivin-positive patients at baseline who initially responded to MTX had a higher risk of disease re-activation (OR 3.21 (95 % CI 1.12–9.24), P = 0.032) and failed to improve in their functional disability (P = 0.018) if having continued on MTX monotherapy compared to survivin-negative patients. Ever-smokers who were survivin-positive were less likely to respond to MTX than those who were survivin-negative (OR 1.91 (1.01–3.62), P = 0.045). In survivin-positive patients, triple therapy led to better improvements in disease activity than did MTX + anti-TNF. At 24 months, survivin-positive patients randomized to anti-TNF had a higher risk of active disease than those randomized to triple therapy (OR 3.15 (1.09–9.10), P = 0.037).DiscussionWe demonstrate for the first time that survivin is a valuable serologic marker that can distinguish drug-specific clinical responses in early rheumatoid arthritis through the pragmatic clinical setting of the care-based SWEFOT trial. Although treatment response cannot solely be attributable to survivin status, per protocol sensitivity analyses confirmed the superior effect of triple therapy on survivin-positive patients.ConclusionsSurvivin-positive patients have poor outcomes if treated with MTX monotherapy. A decrease of survivin levels during treatment is associated with better clinical responses. For survivin-positive patients who fail MTX, triple therapy is associated with better outcomes than anti-TNF therapy.Trial registrationWHO database at the Karolinska University Hospital: CT20080004; ClinicalTrials.gov: NCT00764725, registered 1 October 2008.

Raloxifene is a selective estrogen receptor modulator approved for the prevention of osteoporosis in postmenopausal women. It is selective by having estrogen-agonistic effects on bone, vessels and blood lipids while it is antagonistic on mammary and uterine tissue. Our aim was to study the agonistic and antagonistic properties of the raloxifene analogue LY117018 (LY) on uterus, bone, B lymphopoiesis and B cell function.Oophorectomized and sham-operated animals were treated with s.c. injections of equipotent anti-osteoporotic doses of 17 -estradiol (E2) (0·1 mg/kg) or LY (3 mg/kg) or vehicle as controls. Effects on bone mineral density (BMD) were studied using peripheral quantitative computed tomography, uterine weight was examined, B lymphopoiesis was examined using flow cytometry and B cell function in bone marrow and spleen was studied by the use of an ELISPOT assay.E2 and LY had similar effects on BMD and bone marrow B lymphopoiesis, while LY had a clear antagonistic effect on endogenous estrogen in uterine tissue and no stimulating effect on the frequency of Ig-producing B cells in sham-operated animals. Our results are discussed in the context of estrogen receptor biology, relations between the immune system and bone metabolism and also with respect to the estrogen-mediated effects on rheumatic diseases.