Treatment with a tissue-selective oestrogen complex does not affect disease pathology but reduces pre-BI cells in lupus-prone mice.

OBJECTIVE: Systemic lupus erythematosus (SLE or lupus) is an autoimmune disease characterized by B-cell dysfunction, production of autoantibodies, and immune complex formation. Lupus is overrepresented in females, indicating that sex hormones play a role in the pathophysiology. Treatment with a tissue-selective oestrogen complex (TSEC) containing conjugated oestrogens and the selective oestrogen receptor modulator bazedoxifene (BZA) protects against postmenopausal vasomotor symptoms and osteoporosis, but its impact on organ damage in lupus is not fully understood. METHOD: We used ovariectomized MRL/lpr mice, treated with two different physiological doses of 17ß-oestradiol-3-benzoate (E2), BZA, or TSEC (E2 plus BZA), to assess early and late B-cell development and to determine histological disease manifestations in the kidneys and salivary glands. RESULTS: TSEC treatment reduced the frequency of the pre-BI population in bone marrow to levels equivalent to treatment with physiological doses of E2 alone but did not affect any of the other examined B-cell populations. Our earlier studies indicated that TSEC treatment did not aggravate disease development in ovariectomized MRL/lpr mice, while protecting against trabecular bone loss. Here, we follow up on our previous study and show that neither ovariectomy alone nor TSEC treatment of ovariectomized MRL/lpr mice influenced perivascular lymphocyte infiltration to the kidneys or salivary glands. CONCLUSION: TSEC does not aggravate a mouse model of lupus, when given in doses that protect against postmenopausal lupus-associated bone loss. This indicates that further investigations into TSEC as a treatment for osteoporosis or vasomotor symptoms in postmenopausal women with SLE are warranted.

Factors associated with changes in volumetric bone mineral density and cortical area in men with ankylosing spondylitis: a 5-year prospective study using HRpQCT.

Patients with ankylosing spondylitis (AS) have impaired volumetric bone mineral density (vBMD) assessed with high-resolution peripheral computed tomography (HRpQCT). This first longitudinal HRpQCT study in AS shows that cortical and trabecular vBMD decreased at tibia and that signs of inflammation were associated with cortical bone loss at tibia and radius. INTRODUCTION: Patients with ankylosing spondylitis (AS) have reduced volumetric bone mineral density (vBMD) in the peripheral skeleton assessed with high-resolution peripheral quantitative computed tomography (HRpQCT). The aims were to investigate longitudinal changes in vBMD, cortical area, and microarchitecture and to assess factors associated with changes in vBMD and cortical area in men with AS. METHODS: HRpQCT of radius and tibia was performed in 54 men with AS at baseline and after 5 years. Univariate and multivariable linear regression analyses were used. RESULTS: At tibia, there were significant decreases exceeding least significant changes (LSC) in cortical and trabecular vBMD, mean (SD) percent change -1.0 (1.9) and -2.7 (5.0) respectively (p<0.001). In multivariable regression analyses, increase in disease activity measured by ASDAS_CRP from baseline to follow-up was associated with decreases in cortical vBMD (ß -0.86, 95% CI -1.31 to -0.41) and cortical area (ß -1.66, 95% CI -3.21 to -0.10) at tibia. At radius, no changes exceeded LSC. Nonetheless, increase in ASDAS_CRP was associated with decreases in cortical vBMD, and high time-averaged ESR was associated with decreases in cortical area. Treatment with TNF inhibitor ≥ 4 years during follow-up was associated with increases in cortical vBMD and cortical area at tibia, whereas exposure to bisphosphonates was associated with increases in cortical measurements at radius. No disease-related variables or treatments were associated with changes in trabecular vBMD. CONCLUSION: The findings in this first longitudinal HRpQCT study in patients with AS strengthen the importance of controlling disease activity to maintain bone density in the peripheral skeleton.

Osteoporosis in a murine model of postmenopausal lupus.

BACKGROUND/OBJECTIVE: Postmenopausal women with systemic lupus erythematosus have an increased risk of osteoporosis and associated fractures. Their increased osteoporosis risk is probably caused by a high level of inflammation, use of glucocorticoids, impaired kidney function, and early menopause as these are known risk factors for osteoporosis. Due to these risk factors and the lack of safe and effective treatments, new therapies for the treatment of osteoporosis in this group of patients are needed. Ovariectomized MRL/lpr mice constitute a well-established model for studies of postmenopausal systemic lupus erythematosus; however, it is not clear to what extent this experimental model is associated with the development of osteoporosis. Thus, the aim of this study was to characterize the skeleton of ovariectomized MRL/lpr mice to determine the suitability of this model in studies of prospective new therapies for osteoporosis in postmenopausal systemic lupus erythematosus patients. METHODS: Skeletal parameters were measured in MRL/lpr mice and MRL/++ control mice, using peripheral quantitative computed tomography, high-resolution micro-computed tomography and biomechanical analyses. mRNA expression of bone-remodeling markers was measured by quantitative polymerase chain reaction and serological markers of lupus disease were evaluated using ELISA. RESULTS: Total bone mineral density was reduced in MRL/lpr mice compared with MRL/++ mice and MRL/lpr mice had reduced cortical and trabecular bone thickness compared with MRL/++ mice. In line with the low bone mass of MRL/lpr mice, gene expression analysis of cortical bone from these mice indicated an increased osteoclast activity as well as a decreased osteoblastogenesis and osteoblast activity, compared with MRL/++ mice. CONCLUSION: Ovariectomized MRL/lpr mice constitute a valuable experimental model for studies of osteoporosis development in postmenopausal systemic lupus erythematosus and this model is thus suitable for future studies of osteoporosis treatment in systemic lupus erythematosus.

Hepatocyte growth factor is a potential biomarker for osteoproliferation and osteoporosis in ankylosing spondylitis.

We explored relations between serum hepatocyte growth factor (HGF), disease activity, osteoproliferation, and bone mineral density (BMD) in ankylosing spondylitis (AS), in comparison with healthy controls. HGF was increased especially in male AS patients and smokers and associated with both lower BMD and more chronic radiographic changes in the spine. INTRODUCTION: Ankylosing spondylitis (AS) is characterized by both osteoproliferation and increased bone loss. Biomarkers are requested to predict the processes. The aims of this study were to compare serum levels of hepatocyte growth factor (HGF), matrix metalloproteinase-3 (MMP-3), and vascular endothelial growth factor (VEGF) in AS patients with healthy controls (HC) and to explore the associations with disease activity, osteoproliferation, and bone mineral density (BMD). METHODS: Serum from AS patients (modified NY-criteria) and HC was analyzed for HGF, MMP-3, and VEGF with ELISA. Disease activity parameters were collected. Osteoproliferation was assessed with modified Stoke Ankylosing Spondylitis Spine Score (mSASSS) and BMD was measured in femoral neck. RESULTS: Totally, 204 AS patients and 80 sex and age matched HC were included. Serum HGF was higher in the AS patients compared with the HC, whereas serum MMP-3 and VEGF were not. Serum HGF was also higher in smokers and in the male AS patients positively correlated with age, BASMI, and mSASSS, and negatively correlated with BMD. The biomarkers were all positively associated with ESR, CRP, and WBC. In multiple linear regression analysis serum HGF remained associated with higher mSASSS and lower BMD, after adjusting for age, sex, CRP, smoking, and body mass index. CONCLUSIONS: Serum HGF was increased in male AS patients and associated with higher mSASSS and lower BMD. In addition, serum HGF was positively associated with risk factors for osteoproliferation such as age, CRP and smoking. HGF could be a potential biomarker of importance for the bone metabolism in AS. TRIAL REGISTRATION: NCT00858819.

Genetic variants in CARD8 but not in NLRP3 are associated with ankylosing spondylitis.

OBJECTIVES: The NOD-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome is important for interleukin-1beta (IL-1ß) processing as part of an innate immune response. Caspase recruitment domain family, member 8 (CARD8) is an inhibitor of nuclear factor kappa B (NF-κB) and possibly also a part of the NLRP3 inflammasome. The objective of this study was to evaluate one single nucleotide polymorphism (SNP) in CARD8 and three SNPs in NLRP3 in ankylosing spondylitis (AS) susceptibility and disease phenotype. METHOD: We recruited 492 AS patients from Southern Sweden fulfilling the modified New York criteria for AS, and assessed phenotypic characteristics from medical records and questionnaires. Patients with psoriasis or clinically overt inflammatory bowel disease (IBD) were excluded, as were patients without human leucocyte antigen B27 (HLA-B27). Three NLRP3 SNPs (rs35829419, rs4353135, and rs10733113) and one SNP in CARD8 (rs2043211) were genotyped by commercially available TaqMan assays, and the results compared at genotype and allele levels to those of 793 population-based controls. In a subgroup of the patients (n = 169), faecal calprotectin was assessed as a marker of subclinical intestinal inflammation. RESULTS: The minor allele (A) of CARD8-C10X (rs2043211) was associated with a decreased risk of AS in a dominant model [odds ratio (OR) 0.74, 95% confidence interval (CI) 0.54-0.94, p = 0.012] and at the allelic level (OR 0.81, 95% CI 0.68-0.97, p = 0.02), but was not associated with levels of faecal calprotectin. There was no association regarding NLRP3 SNPs and AS susceptibility, and none of the investigated SNPs were associated with iritis, anti-tumour necrosis factor (anti-TNF) therapy, or peripheral joint involvement. CONCLUSION: In a Swedish population, the minor allele of CARD8-C10X is associated with a decreased risk of AS, but not with levels of faecal calprotectin or disease phenotype.

Decreased serum levels of TGF-ß1 are associated with renal damage in female patients with systemic lupus erythematosus.

INTRODUCTION: Transforming growth factor ß1 (TGF-ß1) has a large role in the control of autoimmunity. TGF-ß1 production by lymphocytes is reduced in systemic lupus erythematosus (SLE). Decreased levels of TGF-ß1 might associate to disease susceptibility, activity and organ damage in SLE. However, the correlation between TGF-ß1 levels and severity of renal damage in SLE has not been examined. METHODS: The present study was undertaken to assess the serum levels of total and active TGF-ß1 in 150 female patients with SLE and 31 healthy women. Simple and multiple regression analyses between TGF-ß1 levels and the diseases-related variables were performed in patients with SLE. RESULTS: Serum levels of both total and active TGF-ß1 were significantly reduced in patients with SLE compared with levels in healthy controls (p < 0.01). Total TGF-ß1 levels correlated positively with white blood cell, platelet counts, calculated glomerular filtration rate (GFR), and active TGF-ß1 level, and inversely with erythrocyte sedimentation rate (ESR). In multiple regression analysis, ESR and platelet counts remained determinants of total TGF-ß1. Total TGF-ß1 levels were lower in patients with high disease activity (SLEDAI > 10) and severe organ damage (SLICC > 3). Significantly lower levels of total TGF-ß1 were found in patients with severe renal damage, i.e. lower TGF-ß1 in patients with 24-h urine protein over 3.5 g than in those with below 3.5 g (p < 0.05); lower TGF-ß1 in patients with GFR less than 50 ml/min than in those with over 50 ml/min (p < 0.05). In contrast, active TGF-ß1 only correlated with platelet counts. There was no association between renal damage and the levels of active TGF-ß1. CONCLUSION: This study demonstrates significantly reduced serum levels of both total and active TGF-ß1 in women with SLE compared with healthy women. Total TGF-ß1 levels are correlated negatively with ESR and positively with blood platelets. Total TGF-ß1 levels were lower in SLE patients with high disease activity and severe organ damage. Importantly, the severity of the renal damage was associated with decreased serum levels of total TGF-ß1, suggesting that TGF-ß1 might be involved in pathogenesis of renal damage caused by lupus nephritis.

Effects of oestradiol and raloxifene on the induction and effector phases of experimental postmenopausal arthritis and secondary osteoporosis.

Oestradiol and the selective oestrogen receptor modulator (SERM) raloxifene have been shown to ameliorate collagen-induced arthritis (CIA) in rats and in mice. One aim was to investigate if raloxifene exerts its anti-arthritic and anti-osteoporotic effects during the induction or effector phase of arthritis. A second aim was to analyse if raloxifene activates the oestrogen response element (ERE) to produce its immune-modulator effects. CIA or collagen-antibody-induced arthritis (CAIA) was induced in ovariectomized DBA/1-mice. CIA was used for evaluation of treatment during the induction, and CAIA for the effector phase of arthritis and osteoporosis development. Raloxifene, oestradiol or vehicle was administered 5 days/week. The clinical disease was evaluated continuously. Bone marrow density (BMD) was analysed with peripheral quantitative computer tomography, paws were collected for histological examination, and sera were analysed for markers of bone and cartilage turnover and proinflammatory cytokines. Transgenic luciferase (Luc)-ERE mice were immunized with collagen (CII), and after 10 days injected once with raloxifene, oestradiol or vehicle before termination. Spleens were analysed for luciferase activity to measure ERE activation. Treatment with oestradiol or raloxifene during the induction phase of CIA failed to affect arthritis. Raloxifene did not hamper disease activity in CAIA, whereas oestradiol delayed the onset and ameliorated the severity. Both raloxifene and oestradiol preserved BMD in CAIA. CII-immunization increased the oestradiol-induced ERE activation in spleen, and raloxifene activated the ERE at about 25% the intensity of oestradiol. Further experiments are needed to elucidate the exact mechanisms behind this finding.

Health-related quality of life in systemic lupus erythematosus and its association with disease and work disability.

OBJECTIVES: To determine the health-related quality of life (HRQOL) and its relationship to disease variables, vertebral fractures, and employment status in female patients with systemic lupus erythematosus (SLE). METHODS: HRQOL was assessed with the Swedish version of the Medical Outcomes Study (MOS) 36-Item Short Form Survey (SF-36) in female patients (n=163) and in age- and sex-matched controls (n=1045). Associations between the SF-36 score and demographics, disease variables, prevalent vertebral fractures, and employment status were analysed. RESULTS: The SLE patients, aged 20 to 82 years, scored significantly lower than the controls on all SF-36 subscales. Patients with vertebral fractures were older, had greater disease damage, and lower physical functioning (PF) than patients without fractures. Of the SLE patients of working age (n=142), 54% worked full or part time. These patients scored their HRQOL significantly higher (better) than patients not working. Being able to work was significantly associated with low age and high scores in PF and role physical (RP): the area under the receiver operating characteristic (ROC) curve for these variables was 0.82, confidence interval 0.75-0.89. CONCLUSIONS: HRQOL is substantially lower in SLE than in the general population but working ability indicates better health. We encourage further research regarding the effects on HRQOL by preventive actions taken against work disability in SLE.

Interleukin 17A: a Janus-faced regulator of osteoporosis.

Interleukin (IL)-17A is a well-described mediator of bone resorption in inflammatory diseases, and postmenopausal osteoporosis is associated with increased serum levels of IL-17A. Ovariectomy (OVX) can be used as a model to study bone loss induced by estrogen deficiency and the role of IL-17A in osteoporosis development has previously been investigated using various methods to inhibit IL-17A signaling in this model. However, the studies show opposing results. While some publications reported IL-17A as a mediator of OVX-induced osteoporosis, others found a bone-protective role for IL-17 receptor signaling. In this study, we provide an explanation for the discrepancies in previous literature and show for the first time that loss of IL-17A has differential effects on OVX-induced osteoporosis; with IL-17A being important for cortical but not trabecular bone loss. Interestingly, the decrease in trabecular bone after OVX in IL-17A knock-out mice, was accompanied by increased adipogenesis depicted by elevated leptin levels. Additionally, the bone marrow adipose tissue expanded, and the bone-turnover decreased in ovariectomized mice lacking IL-17A compared to ovariectomized WT mice. Our results increase the understanding of how IL-17A signaling influences bone remodeling in the different bone compartments, which is of importance for the development of new treatments of post-menopausal osteoporosis.

Growth factors and interleukin-6 across the lung circulation in pulmonary hypertension.

The aim of our study was to assess the levels of growth factors and interleukin (IL)-6 across the pulmonary circulation in patients with pulmonary arterial hypertension (PAH) and correlate them with clinical and haemodynamic data and outcome. Simultaneous arterial and pulmonary arterial blood samples in patients with PAH (n = 44) and controls (n = 20) were obtained during right heart catheterisation. Vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF)-BB, transforming growth factor (TGF)-beta1 and IL-6 were measured using ELISA. Arterial median (interquartile range) values for VEGF, PDGF-BB, TGF-beta1 and IL-6 were significantly higher in patients (377 (218-588) versus 9.0 pg.mL(-1); 1,955 (1,371-2,519) versus 306 (131-502) pg.mL(-1); 26.42 (11.3-41.1) versus 7.0 (1.8-18.4) ng.mL(-1); and 3.98 (0.7-8.1) versus 0.7 pg.mL(-1), respectively; p<0.001 for all variables). There was a consistent step-up of VEGF, PDGF-BB and TGF-beta1 across the lungs in PAH patients (p<0.001, p = 0.002 and p<0.001, respectively), whereas in controls, arterial and pulmonary arterial serum levels of IL-6 and growth factors were similar (statistically nonsignificant). In multivariate analysis, increased IL-6 levels predicted mortality (hazard ratio 1.08 (95% confidence interval 1.02-1.15); p = 0.012). Our findings indicate increased release and/or decreased clearance of growth factors at the lung vascular level, which may contribute to vascular remodelling in PAH.

Long-term anti-arthritic and anti-osteoporotic effects of raloxifene in established experimental postmenopausal polyarthritis.

Both oestrogen deficiency and the inflammatory disease contribute to the generalized bone loss seen in postmenopausal rheumatoid arthritis (RA). Oestradiol and the selective oestrogen receptor modulator raloxifene have been shown to ameliorate the disease in collagen-induced arthritis (CIA), a well-established animal model for human RA. The aim of this study was to investigate whether raloxifene-treatment would be beneficial in long-term treatment of established CIA, both regarding anti-arthritic and anti-osteoporotic properties. Female dilute brown agouti mice were ovariectomized and CIA was induced. Raloxifene or vehicle treatment was administered 5 days per week, and the clinical arthritis score was evaluated continuously. At termination, bone mineral density was analysed, paws were collected for histological examination and sera were analysed for markers of bone and cartilage turnover, as well as antibodies to type II collagen and levels of interleukin (IL)-6. Treatment with raloxifene is beneficial in long-term treatment of established CIA. It hampers the disease severity and frequency, protects the joints from destruction and protects against the development of osteoporosis. The proinflammatory cytokine IL-6 was down-regulated in raloxifene-treated mice compared with controls. The serum levels of antibodies to collagen were not affected by raloxifene-treatment. Long-term treatment with raloxifene has both anti-arthritic and anti-osteoporotic effects in established experimental postmenopausal polyarthritis.

Resistin in serum is associated with higher levels of IL-1Ra in post-menopausal women with rheumatoid arthritis.

OBJECTIVES: The aim of this study was to investigate associations between serum levels of resistin, an adipokine and markers of inflammation, bone metabolism, plasma lipids and kidney function in post-menopausal RA patients and to evaluate if HRT during 2 yrs affected resistin levels. METHODS: Eighty-eight women were randomly allocated to receive HRT, vitamin D(3) and calcium or vitamin D(3) and calcium alone. Serum levels of resistin, IL-1beta, IL-1 receptor antagonist (IL-1Ra), IL-6, IL-6 soluble receptor, TNF-alpha were measured by ELISA, markers of bone metabolism, carboxyterminal cross-linked telopeptide of type I collagen (ICTP) and carboxyterminal propeptide of type I procollagen by RIA, ESR, CRP, Hb, creatinine and lipids by standard laboratory techniques, BMD and total lean mass (TLM) by DXA and joint destruction by Larsen score. Resistin was also measured in 42 healthy control women. RESULTS: There was no difference in resistin concentration between patients and healthy controls. Resistin was significantly correlated with IL-1Ra, CRP, TNF-alpha, ICTP, glucocorticosteroids and Larsen score and inversely with BMD, hip and with TLM. In multiple regression analysis, IL-1Ra, TLM and use of corticosteroids remained determinants of resistin. Patients treated with HRT displayed significant increase in resistin compared with controls in the first but not the second year. CONCLUSIONS: Resistin was associated with increased inflammation, particularly by the acute-phase reactant IL-1Ra antagonizing IL-1beta, joint destruction, glucocorticosteroids and with reduced BMD and TLM. These findings suggest resistin being a significant mediator in the inflammatory process in RA. Further studies examining the mechanisms behind the relation between resistin and IL-1Ra are encouraged. HRT does not seem to have important long-term effect on resistin.

The impact of hormone replacement therapy on humoral and cell-mediated immune responses in vivo in post-menopausal women with rheumatoid arthritis.

It is well known that oestrogen has immunomodulatory properties. We have previously shown disease ameliorating effects of hormone replacement therapy (HRT) in post-menopausal women with rheumatoid arthritis (RA). The aim of this study was to investigate the effects of HRT and the patients inflammatory state on humoral and cell-mediated immune responses. Eighty-eight post-menopausal RA women were allocated to receive HRT (oestradiol and noretisterone acetate), vitamin D3 and calcium or vitamin D3 and calcium alone in a 2-year randomized controlled trial. Immunoglobulins (IgM, IgG and IgA) in serum were measured by nephelometry and rheumatoid factor (RF) concentration by enzyme-linked immunosorbent assay. Immunization with influenza vaccine was performed to quantitate humoral response to recall antigen and tuberculin skin test with purified protein derivative (PPD) to test T-cell-mediated immune response. These immune related measures were correlated with demographic and disease-related variables. HRT during 2 years did not alter concentrations of Ig, RF, IgM response to influenza vaccine or the PPD reaction. The increase in IgM against influenza vaccine was significantly positively correlated with signs of disease activity; C-reactive protein, disease activity score 28 and inversely with haemoglobin. In contrast, PPD reactivity was inversely associated with disease activity. In conclusion, long-term HRT in RA does not influence Ig or autoantibody concentrations in serum and has no significant impact on humoral and cell-mediated immune responses to recall antigens. Interestingly, high disease activity was associated to increased humoral but decreased cell-mediated immune responses irrespectively of hormone treatment.

17Beta-estradiol expands IgA-producing B cells in mice deficient for the mu chain.

Oestrogen is not only a sex hormone but also an important regulator of the immune system. Expression of the heavy chain of IgM (mu) is essential for B-cell differentiation. However, a small number of IgA-positive B cells can be found in mice lacking the mu chain (muMT-/-). The aim of this study was to investigate the effects of oestrogen on this alternative B-cell pathway in muMT-/- mice. Our results clearly demonstrate that oestrogen increases the frequency of IgA-producing B cells in muMT-/- mice in both bone marrow and spleen cells. We also show that mature IgM-producing B cells are not required for oestrogen-mediated suppression of granulocyte-mediated inflammation or thymic involution. In conclusion, we demonstrate that 17beta-estradiol benzoate increases the frequency of IgA-producing B cells in muMT-/- mice, suggesting that oestrogen can influence the alternative B-cell pathway found in muMT-/- mice.

ERα expression in T lymphocytes is dispensable for estrogenic effects in bone.

Estrogen treatment has positive effects on the skeleton, and we have shown that estrogen receptor alpha (ERα) expression in cells of hematopoietic origin contributes to a normal estrogen treatment response in bone tissue. T lymphocytes are implicated in the estrogenic regulation of bone mass, but it is not known whether T lymphocytes are direct estrogen target cells. Therefore, the aim of this study was to determine the importance of ERα expression in T lymphocytes for the estrogenic regulation of the skeleton using female mice lacking ERα expression specifically in T lymphocytes (Lck-ERα-/-) and ERαflox/flox littermate (control) mice. Deletion of ERα expression in T lymphocytes did not affect bone mineral density (BMD) in sham-operated Lck-ERα-/- compared to control mice, and ovariectomy (ovx) resulted in a similar decrease in BMD in control and Lck-ERα-/- mice compared to sham-operated mice. Furthermore, estrogen treatment of ovx Lck-ERα-/- led to an increased BMD that was indistinguishable from the increase seen after estrogen treatment of ovx control mice. Detailed analysis of both the appendicular (femur) and axial (vertebrae) skeleton showed that both trabecular and cortical bone parameters responded to a similar extent regardless of the presence of ERα in T lymphocytes. In conclusion, ERα expression in T lymphocytes is dispensable for normal estrogenic regulation of bone mass in female mice.

Identification of target cells for the genomic effects of estrogens in bone.

Estrogen has bone protective effects, but the exact mechanism behind these effects remains unclear. The aim of the present study was to identify the primary target cells in bone for the classical genomic effects of estrogens in vivo. For this purpose we have used reporter mice with a luciferase gene under the control of three estrogen-responsive elements (EREs), enabling detection of in vivo activation of gene transcription. Three-month-old ovariectomized mice were treated with a single dose (50 mug/kg) 17beta-estradiol (E2). Luciferase activity was analyzed in several tissues and in different bone marrow-derived lymphocyte enriched/depleted preparations using MacsMouse CD19 (for B lymphocytes) or CD90 (for T lymphocytes) MicroBeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Histological characterization of cells with high luciferase content was performed using immunohistochemistry. Both cortical bone and bone marrow displayed a rapid (within 1 h) and pronounced E2-induced increase in luciferase activity. The luciferase activity in total bone marrow and in bone marrow depleted of lymphocytes was increased six to eight times more than in either B-lymphocyte or T-lymphocyte enriched cell fractions 4 h after the E2 injection, demonstrating that mature lymphocytes are not major direct targets for the genomic effect of estrogens in bone. Immunohistochemistry identified clear luciferase staining in hypertrophic growth plate chondrocytes, megakaryocytes, osteoblasts, and lining cells, whereas no staining was seen in proliferative chondrocyte. Although most of the osteocytes did not display any detectable luciferase staining, a subpopulation of osteocytes both in cortical and trabecular bone stained positive for luciferase. In conclusion, hypertrophic growth plate chondrocytes, megakaryocytes, osteoblasts, lining cells, and a subpopulation of osteocytes were identified to respond to estrogen via the classical ERE-mediated genomic pathway in bone. Furthermore, our findings indicate that possible direct estrogenic effects on the majority of osteocytes, not staining positive for luciferase, on proliferative chondrocytes and on mature lymphocytes are mediated by non-ERE actions.

Liver-derived IGF-I is permissive for ovariectomy-induced trabecular bone loss.

INTRODUCTION: Estrogen deficiency results in trabecular bone loss, associated with T-cell proliferation in the bone marrow. Insulin-like growth factor I (IGF-I) is involved in the regulation of both bone metabolism and lymphopoiesis. A major part of serum IGF-I is derived from the liver. The aim of the present study was to investigate the role of liver-derived IGF-I for ovariectomy (ovx)-induced trabecular bone loss. MATERIALS AND METHODS: Mice with adult liver-specific IGF-I inactivation (LI-IGF-I-/-) and wild type mice (WT) were either ovx or sham operated. After 5 weeks, the skeletal phenotype was analyzed by pQCT and microCT. The bone marrow cellularity was analyzed using FACS technique, and mRNA levels were quantified using real-time PCR. RESULTS: Ovx resulted in a pronounced reduction in trabecular bone mineral density (-52%, P < 0.001), number (-45%, P < 0.01) and thickness (-13%, P < 0.01) in WT mice while these bone parameters were unaffected by ovx in LI-IGF-I-/- mice. Furthermore, ovx increased the number of T-cells in the bone marrow of the femur in WT but not in LI-IGF-I-/- mice. Interleukin 7 (IL-7) has been reported to stimulate the formation and function of osteoclasts by inducing the expression of receptor activator of NF-kappaB ligand (RANKL) on T-cells. IL-7 mRNA levels and the RANKL/osteoprotegerin ratio in bone were increased by ovx in WT but not in LI-IGF-I-/- mice. CONCLUSIONS: Liver-derived IGF-I is permissive for ovx-induced trabecular bone loss. Our studies indicate that IGF-I might exert this permissive action by modulation of the number of T-cells and the expression of IL-7, which in turn is of importance for the RANKL/OPG ratio and consequently osteoclastogenesis in the bone marrow.

Estrogenic agonism and antagonism of the soy isoflavone genistein in uterus, bone and lymphopoiesis in mice.

The isoflavone genistein (Gen) is a naturally occurring phytoestrogen found in high concentrations in soy. The biological effects of Gen have been extensively studied. The immunomodulating properties of Gen are, however, less well investigated and the results are contradictory. Our aim was to study possible estrogen agonistic and antagonistic properties of Gen in uterus, bone, lymphopoiesis and B-cell function by comparing effects in castrated and intact female mice, respectively. Oophorectomized (OVX) and sham-operated mice were treated with s.c. doses of 17beta-estradiol (E2) (0.16 mg/kg), Gen (50 mg/kg), or vehicle (olive oil) as control. Effects on bone mineral density (BMD) were studied using peripheral quantitative computerized tomography, uterine and thymus weights were examined, lymphopoiesis in thymus and bone marrow was analyzed using flow cytometry, and the frequency of immunoglobulin-producing B cells in bone marrow and spleen was studied using an ELISPOT assay. Gen was clearly antagonizing endogenous estrogen in sham-operated female mice as shown by inhibiting the uterine weight and by increasing the frequency of B lymphopoietic cells in bone marrow. The only agonistic effect of Gen was shown by increased BMD in OVX mice. Our results are discussed in the context of estrogen receptor biology.

Raloxifene- and estradiol-mediated effects on uterus, bone and B lymphocytes in mice.

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 17beta-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.