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Rheumatic diseases encompass a diverse group of chronic disorders that commonly affect musculoskeletal structures. Osteoarthritis (OA) and rheumatoid arthritis (RA) are the two most common, leading to considerable functional limitations and irreversible disability when patients are unsuccessfully treated. Although the specific causes of many rheumatic conditions remain unknown, it is generally accepted that immune mechanisms and/or uncontrolled inflammatory responses are involved in their etiology and symptomatology. In this regard, the bidirectional communication between neuroendocrine and immune system has been demonstrated to provide a homeostatic network that is involved in several pathological conditions. Adipokines represent a wide variety of bioactive, immune and inflammatory mediators mainly released by adipocytes that act as signal molecules in the neuroendocrine-immune interactions. Adipokines can also be synthesized by synoviocytes, osteoclasts, osteoblasts, chondrocytes and inflammatory cells in the joint microenvironment, showing potent modulatory properties on different effector cells in OA and RA pathogenesis. Effects of adiponectin, leptin, resistin and visfatin on local and systemic inflammation are broadly described. However, more recently, other adipokines, such as progranulin, chemerin, lipocalin-2, vaspin, omentin-1 and nesfatin, have been recognized to display immunomodulatory actions in rheumatic diseases. This review highlights the latest relevant findings on the role of the adipokine network in the pathophysiology of OA and RA.

Due to their role in inflammatory metabolic diseases, we hypothesised that free fatty acids (FFA) are also involved in inflammatory joint diseases. To test this hypothesis, we analysed the effect of FFA on synovial fibroblasts (SF), human chondrocytes and endothelial cells. We also investigated whether the toll-like receptor 4 (TLR4), which can contribute to driving arthritis, is involved in FFA signalling. Rheumatoid arthritis SF, osteoarthritis SF, psoriatic arthritis SF, human chondrocytes and endothelial cells were stimulated in vitro with different FFA. Immunoassays were used to quantify FFA-induced protein secretion. TLR4 signalling was inhibited extracellularly and intracellularly. Fatty acid translocase (CD36), responsible for transporting long-chain FFA into the cell, was also inhibited. In rheumatoid arthritis synovial fibroblasts (RASF), FFA dose-dependently enhanced the secretion of the proinflammatory cytokine IL-6, the chemokines IL-8 and MCP-1, as well as the matrix-degrading enzymes pro-MMP1 and MMP3. The intensity of the response was mainly dependent on the patient rather than on the type of disease. Both saturated and unsaturated FFA showed similar effects on RASF, while responses to the different FFA varied for human chondrocytes and endothelial cells. Extracellular and intracellular TLR4 inhibition as well as fatty acid transport inhibition blocked the palmitic acid-induced IL-6 secretion of RASF. The data show that FFA are not only metabolic substrates but may also directly contribute to articular inflammation and degradation in inflammatory joint diseases. Moreover, the data suggest that, in RASF, FFA exert their effects via TLR4 and require extracellular and intracellular access to the TLR4 receptor complex.

Objectives Systemic sclerosis (SSc) is a rare disease requiring multicentre collaboration to reveal comprehensive details of disease-related causes for morbidity and mortality. Methods The European League Against Rheumatism (EULAR) Scleroderma Trials and Research (EUSTAR) group initiated a database to prospectively gather key data of patients with SSc using a minimal essential dataset that was reorganised in 2008 introducing new items. Baseline visit data of patients who were registered between 2004 and 2011 were analysed using descriptive statistics. Results In June 2011, 7655 patients (2838 with diffuse cutaneous (dc) and 4481 with limited cutaneous (lc) SSc who fulfilled the American College of Rheumatology diagnostic criteria had been registered in 174 centres, mainly European. The most prominent hallmarks of disease were Raynaud's phenomenon (96.3%), antinuclear antibodies (93.4%) and a typical capillaroscopic pattern (90.9%). Scleroderma was more common on fingers and hands than on any other part of the skin. Proton pump inhibitors (65.2%), calcium channel blockers (52.7%), and corticosteroids (45.3%) were most often prescribed. Among the immunosuppressant agents, cyclophosphamide was used more often in dcSSc than in lcSSc. Conclusions The EUSTAR database provides an abundance of information on the true clinical face of SSc that will be helpful in improving the classification of SSc and its subsets and for developing more specific therapeutic recommendations.

Adipokines are adipose tissue-derived factors not only playing an important role in metabolism but also influencing other central processes of the body, such as inflammation. In autoimmune diseases, adipokines are involved in inflammatory pathways affecting different cell types. Many rheumatic diseases belong to the group of autoimmune diseases, for example rheumatoid arthritis (RA) and psoriatic arthritis. Due to the autoimmune responses, a chronic inflammatory milieu develops, which affects the whole body, including adipose tissue. Metabolic alterations such as obesity influence inflammatory responses in autoimmune diseases. Adipokines are bioactive mediators mainly produced by adipose tissue. Due to alterations of systemic adipokine levels, their role as biomarkers with diagnostic potential has been suggested in the context of rheumatic diseases. In the affected joints of RA patients, different synoviocytes but also osteoclasts, osteoblasts, and chondrocytes produce several adipokines, contributing to the unique inflammatory microenvironment. Adipokines have been shown to be potent modulatory effectors on different cell types of the immune system but also local cells in synovial tissue, cartilage, and bone. This review highlights the most recent findings on the role of adipokines in the pathophysiology of inflammatory arthritis with a distinct focus on RA in the quickly developing research field.

Obesity-in which free fatty acid (FFA) levels are chronically elevated-is a known risk factor for different rheumatic diseases, and obese patients are more likely to develop osteoarthritis (OA) also in non-weight-bearing joints. These findings suggest that FFA may also play a role in inflammation-related joint damage and bone loss in rheumatoid arthritis (RA) and OA. Therefore, the objective of this study was to analyze if and how FFA influence cells of bone metabolism in rheumatic diseases. When stimulated with FFA, osteoblasts from RA and OA patients secreted higher amounts of the proinflammatory cytokine interleukin (IL)-6 and the chemokines IL-8, growth-related oncogene α, and monocyte chemotactic protein 1. Receptor activator of nuclear factor kappa B ligand (RANKL), osteoprotegerin, and osteoblast differentiation markers were not influenced by FFA. Mineralization activity of osteoblasts correlated inversely with the level of FFA-induced IL-6 secretion. Expression of the Wnt signaling molecules, axin-2 and β-catenin, was not changed by palmitic acid (PA) or linoleic acid (LA), suggesting no involvement of the Wnt signaling pathway in FFA signaling for osteoblasts. On the other hand, Toll-like receptor 4 blockade significantly reduced PA-induced IL-8 secretion by osteoblasts, while blocking Toll-like receptor 2 had no effect. In osteoclasts, IL-8 secretion was enhanced by PA and LA particularly at the earliest time point of differentiation. Differences were observed between the responses of RA and OA osteoclasts. FFA might therefore represent a new molecular factor by which adipose tissue contributes to subchondral bone damage in RA and OA. In this context, their mechanisms of action appear to be dependent on inflammation and innate immune system rather than Wnt-RANKL pathways.

The long-distance migration of rheumatoid arthritis synovial fibroblasts (RASFs) in the severe combined immunodeficiency (SCID) mouse model of rheumatoid arthritis (RA) suggests that an interaction between RASFs and endothelial cells (EC) is critical in this process. Our objective was to assess whether immunomodulatory factors such as adipokines and antirheumatic drugs affect the adhesion of RASFs to ECs or the expression of surface molecules. Primary ECs or human umbilical vein endothelial cell (HUVEC) and primary RASFs were stimulated with adiponectin (10 μg/mL), visfatin (100 ng/mL), and resistin (20 ng/mL) or treated with methotrexate (1.5 and 1,000 μM) and the glucocorticoids prednisolone (1 μM) and dexamethasone (1 μM), respectively. The expression of adhesion molecules was analyzed by real-time polymerase chain reaction. The interaction of both cell types was analyzed under static (cell-to-cell binding assay) and dynamic conditions (flow-adhesion assay). Under static conditions, adipokines increased mostly binding of RASFs to EC (adiponectin: 40%, visfatin: 28%, tumor necrosis factor α: 49%). Under flow conditions, visfatin increased RASF adhesion to HUVEC (e.g., 0.5 dyn/cm : 75.2%). Reduced adhesion of RASFs to E-selectin was observed after treatment with dexamethasone (e.g., 0.9 dyn/cm : -40%). In ECs, tumor necrosis factor α (TNF-α) increased expression of intercellular adhesion molecule 1 (20-fold) and vascular cell adhesion molecule 1 (77-fold), whereas P-selectin was downregulated after stimulation with TNF-α (-6-fold). The adhesion of RASFs to EC was increased by visfatin under static and flow conditions, whereas glucocorticoids were able to decrease adhesion to E-selectin. The process of migration and adhesion of RASFs to ECs could be enhanced by adipokines via adhesion molecules and seems to be targeted by therapeutic intervention with glucocorticoids.

Objectives Several clinical studies have suggested the adipocytokine adiponectin is involved in the progression of rheumatoid arthritis (RA). From this point of view, adiponectin might present a new therapeutic target. However, as adiponectin also exerts beneficial effects in the human organism, a strategy that would allow its detrimental effects to be abolished while maintaining the positive effects would be highly favourable. To elucidate such a strategy, the authors analysed whether the different adiponectin isoforms induce diverging effects, especially with regard to rheumatoid arthritis synovial fibroblasts (RASF), a central cell type in RA pathogenesis capable of invading into and destroying cartilage. Methods Affymetrix microarrays were used to screen for changes in gene expression of RASF. Messenger RNA levels were quantified by real-time PCR, protein levels by immunoassay. The migration of RASF and primary human lymphocytes was analysed using a two-chamber migration assay. Results In RASF, the individual adiponectin isoforms induced numerous genes/proteins relevant in RA pathogenesis to clearly different extents. In general, the most potent isoforms were the high molecular weight/middle molecular weight isoforms and the globular isoform, while the least potent isoform was the adiponectin trimer. The chemokines secreted by RASF upon adiponectin stimulation resulted in an increased migration of RASF and lymphocytes. Conclusion The results clearly suggest a pro-inflammatory and joint-destructive role of all adiponectin isoforms in RA pathophysiology, indicating that in chronic inflammatory joint diseases the detrimental effects outweigh the beneficial effects of adiponectin.

Breast-fed preterm infants often show a better outcome, partly ascribed to the benefit of insulin-like growth factors (IGFs) and their binding proteins (IGFBP). We compared IGF-I, IGF-II, IGFBP-2 and IGFBP-3 levels, measured by radioimmunoassays in milk samples from 30 mothers of preterm (<31 weeks) and from 19 mothers of term (>37 weeks) infants at days 7 and 21 postpartum. Proteolysis of IGFBP-2 within mother's milk and digestion of (125)I-IGF-II and (125)I-IGFBP-2 by gastric juice from neonates were assessed by electrophoretic techniques. Mean concentrations did not differ between preterm and term milk: IGF-I (2.8 +/- 0.2 vs. 2.3 +/- 0.1 ng/ml), IGF-II (12.0 +/- 0.4 vs. 12.2 +/- 0.5 ng/ml), IGFBP-3 (100.0 +/- 5.1 vs. 80.0 +/- 5.8 ng/ml), but did so for IGFBP-2 (3,144 +/- 172 vs. 2,428 +/- 188 ng/ml, p < 0.02). Immunoblots revealed 42% (p < 0.05) more IGFBP-2 fragments of 14 and 25 kDa in preterm milk. Incubation with gastric juice caused cleavage of (125)I-IGFBP-2 and partial cleavage of (125)I-IGF-II. Mutual complexation protected IGF-II and IGFBP-2 from cleavage, suggesting that both are likely to arrive in the bowel in an intact form to exert promotive effects. The results provide further evidence that IGFBP-2 and IGF-II in breast milk are relevant factors for the early development of preterm infants.

Activin A and follistatin belong to an anti-inflammatory auto-regulatory cycle. Patients with rheumatoid arthritis (RA) have increased activin A levels in the synovial fluid and tissue. During inflammation, activin A is released systemically, then inducing its antagonist follistatin. This negative feedback is active in different cell types but not RA synovial fibroblasts (SF). Fibroblasts interact with endothelial cells in inflamed tissues inducing angiogenesis. Evaluation of the role of activin A and follistatin on RASF and endothelial cell interactions. RA synovium was used for RASF isolation, HUVEC were commercially obtained. RASF and HUVEC were stimulated in mono- and coculture. Direct: RASF together with HUVEC; indirect: inserts with HUVEC separated by a membrane from RASF. Stimuli: activin A 15ng/ml, follistatin 500ng/ml, IL-1β 1ng/ml. Proliferation was analyzed by BrdU assay. RASF were Calcein-AM stained. Cells were transferred to 24-well plates after 18h stimulation. After adhesion for 1h, non-adherent cells were removed by shaking 3x for 5 min. Afterwards, fluorescent cells were quantified. For the flow-adhesion assay, HUVEC were cultured on rattail collagen coated capillary slides. HUVEC and RASF were stimulated for 4h with TNFα, TNFα+activin A or TNFα+follistatin. After stimulation, 2x10^6 RASF were resuspended in 20ml medium and sent through the capillaries. Two 1min videos were recorded (18.4ml/h, 30.5ml/h). Settings: TNFα-stimulated RASF on HUVEC stimulated wit TNFα or activin A+TNFα or follistatin+ TNFα. For migration assays, 2% FCS medium with 1x10^5 cells were placed in inserts (8µm membrane) into wells with 10% FCS (control: 2%FCS vs. 2%FCS) and stimulated with, IL-1, IL-1+activinA and IL-1+follistatin. After 16h, migrated cells were quantified. For scratch-assay 4x10^5 cells were cultured overnight, then cells were scratched and stimulated, afterwards 3 pictures per scratch were taken at start, after 10h and every 1.5h. Cells migrating into the scratch area were quantified. IL-1 induced activin A in RASF and HUVEC in all settings. IL-1-induced activin A release was reduced by follistatin in HUVEC monoculture and both cocultures compared to IL-1 alone but not in RASF monoculture. IL-1-induced IL-6 release was reduced by activin A in HUVEC and indirect coculture but not in RASF monoculture and direct coculture. Follistatin did not alter IL-6 responses. IL-1 induced VEGF in RASF but not in HUVEC and was not altered by activin A. Short-term adhesion showed no significant influence of activin A or follistatin (n=4). Flow adhesion assay showed reduced adherence/rolling of RASF on HUVEC stimulated with TNFα and activin A (n=4). Migration assays showed that IL-1 decreased migration but without significant difference between the induced effects mediated by IL-1+activinA and IL-1+follistatin (n=4). Scratch assay showed increased migration in dicrect coculture with greater difference between stimulated and unstimulated cells in RASF monoculture and indirect coculture (n=4). Proliferation was not altered by activin A or follistatin. In direct and indirect coculture of HUVEC with RASF the effect on HUVEC was dominant leading to reduced IL-1-induced activin A release. However, the IL-1-induced IL-6 release in RASF or HUVECs was decreased by activin A in HUVEC monoculture and indirect coculture but not during cell-contact between both cell types. The direct interaction of RASF with HUVEC seems to prevent the reducing activin A effect on IL-6 release in HUVECs. Activin A seems to not to have an impact on short-term cell adhesion but first observations show, that activin A alters selectin-mediated adhesion under flow conditions. The migration assay shows that IL-1-induced effects on cell migration were enhanced by activin A and follistatin. Migration assay shows that IL-1-induced decrease on migration more prominent in indirect coculture and RASF monoculture than in direct coculture although in gap migration in the scratch assay was highest in direct coculture. None declared

BackgroundThe role of fat-bone interactions in the pathogenesis of osteoporosis is poorly understood. An inverse relationship between bone marrow adipose tissue and bone mineral density during ageing and in osteoporosis is well documented. Adipokines (eg, visfatin, resistin and leptin) are adipocyte-derived factors with immunomodulatory properties and they might influence the differentiation of bone marrow-derived mesenchymal stem cells (MSC) into osteoblasts and adipocytes. The aim was to analyse the presence of adipokines in the bone marrow cavity and their effects on MSC differentiation.MethodsSpongiosa from femoral heads containing bone marrow were collected (hip replacement of osteoarthritis patients or after osteoporotic femoral neck fracture). MSC were cultured in adipogenic and osteogenic media with/without adipokines. For the transfer and differentiation of MSC on cancellous bone, bone fragments were purified and sterilised. mRNA expression of adipokines, bone marker genes, TIMPs and MMPs of stimulated MSC and of bone samples were evaluated by real time PCR. Matrix mineralization was investigated using Alizarin red S staining. Proinflammatory factors were measured by ELISA.ResultsVisfatin and leptin level were increased in osteoporotic bone vs. non-osteoporotic bone (n=14). Visfatin induced the secretion of proinflammatory factors (IL-6, IL-8, MCP-1) during both, osteogenic and adipogenic differentiation but not leptin or resistin. Visfatin markedly increased mineralization during osteogenic differentiation, whereas the Coll.1-expression was significantly downregulated (eg, d21: -4.6-fold). In contrast to resistin and leptin visfatin also reduced the expression of MMP2, MMP13, RunX2, TIMP1 and TIMP2 (eg, d21: -2.4-fold/-3.18 fold/-5.85 fold/ -3.2 fold/-4.3 fold respectively) in osteogenic differentiated cells. In contrast to osteogenesis, visfatin significantly increased MMP13 expression (eg, d21: 104-fold) during adipogenic differentiation under standard cell culture conditions, however visfatin induced MMP13-Expression was markedly reduced during differentiation on purified autologous cancellous bone.ConclusionVisfatin and leptin were elevated in osteoporotic bone tissue. Visfatin-mediated increase of matrix mineralization and reduction of Coll.1 expression could enhance bone fragility and contribute to the pathogenesis of osteoporosis. Visfatin might impair bone remodelling at the adipose tissue/bone interface through enhancing secretion of proinflammatory factors and dysregulated MMP- and TIMP-expression during MSC-differentiation.