Hans Carlsten

Role of oestrogen receptors α and β in immune organ development and in oestrogen-mediated effects on thymus

Oestrogens affect the development and regulation of the immune system. To determine the role of oestrogen receptors α (ER‐α) and β (ER‐β) on the development of the immune system, male ER‐α (ERKO) and ER‐β (BERKO) mice, as well as αβ‐double knockout (DERKO) mice, were studied. Deletion of ER‐α led to hypoplasia of both thymus and spleen. Interestingly, a higher frequency of immature double CD4+ CD8+ thymocytes was found in ER‐α− mice compared with ER‐α+ 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‐α, but not ER‐β, is mandatory in males for development of full‐size thymus and spleen, whereas expression of ER‐β is required for E2‐mediated thymic cortex atrophy and thymocyte phenotype shift in females. A potential background for the above findings may be down‐regulated activity in the growth hormone/insulin‐like growth factor‐1 (GH/IGF‐1) axis in males lacking ER‐α and suppressed sensitivity of females lacking ER‐β to E2‐mediated suppression of IGF‐1.

Estrogen accelerates immune complex glomerulonephritis but ameliorates T cell-mediated vasculitis and sialadenitis in autoimmune MRL lpr/lpr mice

Estrogen is known to influence immune responses in healthy subjects in a dichotomous fashion. Thus, in number of previous studies we and others have demonstrated that B cell activities are augmented after exposure to estrogen whereas T cell reactivity is suppressed. Furthermore, it has been shown that this hormone has significant impact on the course of certain human and experimental autoimmune diseases. In this study we report that treatment with physiological doses of estradiol exerts dichotomous effects on different manifestations of the lupus disease in MRL/l mice. On one hand immune complex-mediated glomerulonephritis was significantly accelerated. This outcome was due to polyclonal B cell activation with increased production of antibodies to double-stranded DNA and formation of circulating immune complexes. In contrast, T cell-mediated lesions such as focal sialadenitis, renal vasculitis, and periarticular inflammation were all significantly ameliorated in MRL/l mice exposed to estrogen. Thus, we were able to demonstrate that, within one subject and even within one organ, administration of estrogen leads to differential outcome of SLE morbidity. We propose that the differential effect of estrogen on the manifestations of the autoimmune disease of MRL/l mice is due to its dichotomous effects on B and T cell-mediated immune responses.

Oestrogen is a potent disease accelerator in SLE‐prone MRL lpr/lpr mice

The influence of oestrogen on the lupus disease in MRL/1 mice has been investigated. Adult, castrated male and female MRL/1 mice were administered with s.c. injections of 3–2 μg of 17β‐oestradiol twice a week. The results clearly demonstrate that a relatively small dose of oestrogen is a potent accelerator of the lupus disease in this mouse strain. Thus, administration of oestrogen accelerates glomerulonephritis, lymphoproliferation and mortality. Our results also indicate that oestrogen exerts a dual effect on the immune system of MRL/1 mice by depression of antigen‐specific and mitogen‐induced T cell responses as well as enhancement of polyclonal Bcell activation and autoantibody formation. In addition, even short‐term administration of oestrogen in the preclinical phase of the disease resulted in long‐lasting effects as evaluated by reduced longevity and aggravation of renal disease.

Radiographic joint destruction in postmenopausal rheumatoid arthritis is strongly associated with generalised osteoporosis

Objectives: To investigate determinants of joint destruction and reduced bone mineral density (BMD) in postmenopausal women with active rheumatoid arthritis (RA) not treated with bisphosphonates or hormone replacement therapy and to evaluate if there are common markers of erosive disease and bone loss. Methods: BMD was measured using dual x ray absorptiometry and joint damage was examined by x ray examination according to the Larsen method in 88 patients with RA. Associations between BMD and Larsen score, and between demographic and disease related variables, including proinflammatory cytokines, HLA-DR4 epitopes, and markers of bone and cartilage turnover, were examined bivariately by simple and multiple linear regression analyses. Results: 49/88 (56%) patients had osteoporosis in at least one site. Reduced BMD and increased joint destruction were associated with: at the forearm and femoral neck, high Larsen score, low weight, and old age (R2=0.381, p<0.001; R2=0.372, p<0.001, respectively); at the total hip, low weight, high Larsen score, and dose of injected glucocorticosteroids (R2=0.435, p<0.001); at the lumbar spine, low weight, reduced cartilage oligomeric matrix protein, and increased carboxyterminal propeptide of type I procollagen (R2=0.248, p<0.001). Larsen score was associated with long disease duration and increased C reactive protein (CRP) (R2=0.545, p<0.001). Conclusions: Osteoporosis is common in postmenopausal patients with RA. Low weight and high Larsen score were strongly associated with BMD reduction. Increased CRP and long disease duration were determinants of erosive disease in postmenopausal women with RA. These findings indicate common mechanisms of local and generalised bone loss in RA.

The role of the G protein-coupled receptor GPR30 in the effects of estrogen in ovariectomized mice

In vitro studies suggest that the membrane G protein-coupled receptor GPR30 is a functional estrogen receptor (ER). The aim of the present study was to determine the possible in vivo role of GPR30 as a functional ER primarily for the regulation of skeletal parameters, including bone mass and longitudinal bone growth, but also for some other well-known estrogen-regulated parameters, including uterine weight, thymus weight, and fat mass. Three-month-old ovariectomized (OVX) GPR30-deficient mice (GPR30(-/-)) and wild-type (WT) mice were treated with either vehicle or increasing doses of estradiol (E(2); 0, 30, 70, 160, or 830 ng.mouse(-1).day(-1)). Body composition [bone mineral density (BMD), fat mass, and lean mass] was analyzed by dual-energy-X ray absorptiometry, while the cortical and trabecular bone compartments were analyzed by peripheral quantitative computerized tomography. Quantitative histological analyses were performed in the distal femur growth plate. Bone marrow cellularity and distribution were analyzed using a fluorescence-activated cell sorter. The estrogenic responses on most of the investigated parameters, including increase in bone mass (total body BMD, spine BMD, trabecular BMD, and cortical bone thickness), increase in uterine weight, thymic atrophy, fat mass reduction, and increase in bone marrow cellularity, were similar for all of the investigated E(2) doses in WT and GPR30(-/-) mice. On the other hand, E(2) treatment reduced longitudinal bone growth, reflected by decreased femur length and distal femur growth plate height, in the WT mice but not in the GPR30(-/-) mice compared with vehicle-treated mice. These in vivo findings demonstrate that GPR30 is not required for normal estrogenic responses on several major well-known estrogen-regulated parameters. In contrast, GPR30 is required for a normal estrogenic response in the growth plate.

Immune responses and bone loss: the estrogen connection.

Summary:  In addition to its effects on sexual differentiation and reproduction, estrogen has important impact on the immune system and on bone. It has also been evident that the effects of estrogen on bone to a large extent are mediated via its action on immune cells. Estrogen has a dichotomous impact on the immune system by downregulation of inflammatory immune responses but simultaneous upregulation of immunoglobulin production. Consequently, immune‐mediated diseases in humans and in animal models are modulated by estrogen. Estrogen deficiency after ovariectomy in mice and after menopause in women is associated with significant bone loss. In rheumatic diseases such as rheumatoid arthritis (RA), osteoporosis is frequent, and in patients with postmenopausal RA, the degree of bone loss is dramatically increased. Hormone replacement therapy (HRT) in murine and human arthritis has beneficial effects on bone loss, as expected, but it also ameliorates inflammation and inflammation‐triggered joint destruction. Long‐term use of HRT has been associated with increased risk of breast cancer, thrombosis, and possibly also stroke. Accordingly, there is great need for new activators of estrogen receptors (ERs) selectively reproducing only the beneficial effects of estrogen. To achieve this aim, better knowledge of the mechanisms of how activation of ER‐α and ER‐β modulates the immune system and bone at the cellular and molecular levels is necessary.

Osteoporosis in ankylosing spondylitis - prevalence, risk factors and methods of assessment

IntroductionOsteoporosis can be a complication of ankylosing spondylitis (AS), but diagnosing spinal osteoporosis can be difficult since pathologic new bone formation interferes with the assessment of the bone mineral density (BMD). The aims of the current study were to investigate prevalence and risk factors for reduced BMD in a Swedish cohort of AS patients, and to examine how progressive ankylosis influences BMD with the use of dual-energy x-ray absorptiometry (DXA) of the lumbar spine in different projections.MethodsMethods of assessment were questionnaires, back mobility tests, blood samples, lateral spine radiographs for syndesmophyte grading (mSASSS), DXA of the hip, radius and lumbar spine in anteroposterior (AP) and lateral projections with estimation of volumetric BMD (vBMD).ResultsAS patients (modified New York criteria), 87 women and 117 men, mean age 50 ± 13 years and disease duration 15 ± 11 years were included. According to World Health Organization (WHO) criteria 21% osteoporosis and 44% osteopenia was diagnosed in patients > = 50 years. Under age 50 BMD below expected range for age was found in 5%. Interestingly lateral lumbar DXA showed significantly lower BMD and revealed significantly more cases with osteoporosis as compared with AP DXA. Lumbar vBMD was not different between sexes, but women had significantly more lumbar osteoporosis measured with AP DXA (P < 0.001). Men had significantly higher mSASSS (P < 0.001). Low BMD was associated with high age, disease duration, mSASSS, Bath Ankylosing Spondylitis Metrology Index (BASMI), inflammatory parameters and low body mass index (BMI). Increasing mSASSS correlated significantly with decreasing lateral and volumetric lumbar BMD, while AP lumbar BMD showed tendency to increase.ConclusionsOsteoporosis and osteopenia is common in AS and associated with high disease burden. Lateral and volumetric lumbar DXA are more sensitive than AP DXA in detecting osteoporosis and are less affected by syndesmophyte formation.

Oestrogen receptor specificity in oestradiol-mediated effects on B lymphopoiesis and immunoglobulin production in male mice

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.

Ethanol prevents development of destructive arthritis

Environmental factors are thought to play a major role in the development of rheumatoid arthritis. Because the use of ethanol is widespread, we assessed the role of ethanol intake on the propensity to develop chronic arthritis. Collagen type II-immunized mice were given water or water containing 10% (vol/vol) ethanol or its metabolite acetaldehyde. Their development of arthritis was assessed, as well as the impact of ethanol on leukocyte migration and activation of intracellular transcription factors. Mice exposed daily to this dose of ethanol did not display any liver toxicity, and the development of erosive arthritis was almost totally abrogated. In contrast, the antibody-mediated effector phase of collagen-induced arthritis was not influenced by ethanol exposure. Also, the major ethanol metabolite, acetaldehyde, prevented the development of arthritis. This antiinflammatory and antidestructive property of ethanol was mediated by (i) down-regulation of leukocyte migration and (ii) up-regulation of testosterone secretion, with the latter leading to decreased NF-κB activation. We conclude that low but persistent ethanol consumption delays the onset and halts the progression of collagen-induced arthritis by interaction with innate immune responsiveness.

Beneficial effect of the inosine monophosphate dehydrogenase inhibitor mycophenolate mofetil on survival and severity of glomerulonephritis in systemic lupus erythematosus (SLE)-prone MRLlpr/lpr mice

The aim of the present study was to evaluate the therapeutic effect of mycophenolate mofetil (MMF) on the course of disease in SLE‐prone MRLlpr/lpr mice. Three‐months‐old mice displaying clinical symptoms of glomerulonephritis were given MMF (100 mg/kg per day) orally via the drinking water. Control mice received i.p. injections of cyclophosphamide (CYC) (1.8 mg/mouse per week) or saline. Survival, albuminuria and haematuria, immunoglobulin levels and anti‐dsDNA antibodies in serum, frequencies of immunoglobulin‐producing B lymphocytes and glomerular deposits of immunoglobulin and C3 were analysed. The results showed that MMF treatment significantly prolonged survival and reduced the occurrence of albuminuria and haematuria in MRLlpr/lpr mice. In addition, the number of immunoglobulin‐producing B cells and serum levels of IgG and IgG anti‐dsDNA antibodies were reduced after MMF and CYC treatment. MMF treatment significantly reduced the extent of deposition of C3 in glomeruli. We conclude that the reduced severity of glomerulonephritis following treatment of lupus‐prone mice with MMF was as efficacious as that of CYC. These results warrant clinical trials of MMF in SLE patients with glomerulonephritis.