Modulation of Dopamine Receptors on Osteoblasts as a Possible Therapeutic Strategy for Inducing Bone Formation in Arthritis.

Rheumatoid arthritis (RA) is associated with systemic osteoporosis, which leads to severe disability and low quality of life. Current therapies target osteoclasts to reduce bone degradation, but more treatment options would be required to promote bone protection by acting directly on osteoblasts (OB). Recently, the local production of dopamine in inflamed joints of RA has been observed. Thus, in this project, we aimed to determine the implication of the neurotransmitter dopamine in the bone formation process in RA. Dopamine receptors (DR) in the human bone tissue of RA or osteoarthritis (OA) patients were examined by immunohistochemistry. DR in isolated human osteoblasts (OB) was analyzed by flow cytometry, and dopamine content was evaluated by ELISA. Osteoclasts (OC) were differentiated from the PBMCs of healthy controls (HC) and RA patients. Isolated cells were treated with specific dopamine agonists. The effect of dopamine on mineralization was evaluated by Alizarin red staining. Cytokine release in supernatants was measured by ELISA. Osteoclastogenesis was evaluated with TRAP staining. OC markers were analyzed via real-time PCR and bone resorption via staining of resorption pits with toluidine blue. All DR were observed in bone tissue, especially in the bone remodeling area. Isolated OB maintained DR expression, which allowed their study in vitro. Isolated OB expressed tyrosine hydroxylase, the rate-limiting enzyme for dopamine production, and contained dopamine. The activation of D2-like DR significantly increased bone mineralization in RA osteoblasts and increased osteoclastogenesis but did not alter the expression of OC markers nor bone resorption. DR were found in the bone remodeling area of human bone tissue and dopamine can be produced by osteoblasts themselves, thus suggesting a local autocrine/paracrine pathway of dopamine in the bone. D2-like DRs are responsible for bone mineralization in osteoblasts from RA patients without an increase in bone resorption, thus suggesting the D2-like DR pathway as a possible future therapeutic target to counteract bone resorption in arthritis.

Compensation of Adiponectin-Induced Adenosine Monophosphate-Activated Protein Kinase and p38 Mitogen-Activated Protein Kinase Signaling in Rheumatoid Arthritis Synovial Fibroblasts.

Rheumatoid arthritis (RA) is a chronic inflammatory disorder marked by synovitis, ultimately leading to cartilage and bone destruction. In RA, adiponectin levels are increased in serum and synovial fluid. Adiponectin belongs to the adipokines, a group of highly bioactive substances secreted by adipocytes and other cell types. It has been shown to induce the production of proinflammatory and prodestructive factors by human RA synovial fibroblasts (RASF), suggesting a role in the pathophysiology of the disease. Although adenosine monophosphate-activated protein kinase (AMPK) and p38 mitogen-activated protein kinase (MAPK) are known to be involved in adiponectin signaling in RASF, no literature is available about whether the different adiponectin isoforms affect AMPK and p38 MAPK signaling in the same manner. In this study, we elucidated the signaling mechanisms in RASF, activated in response to selective stimulation with the 2 biologically most potent adiponectin isoforms, and possible approaches to inhibit adiponectin-mediated effects in RASF. All adiponectin isoforms induced p38 MAPK and AMPK phosphorylation to various degrees. Blocking AMPK activation increased p38 MAPK phosphorylation, while blocking p38 MAPK activation increased AMPK phosphorylation, both independent of the effect of adiponectin. Neither AMPKα1 nor AMPKα2 knockdown reduced interleukin (IL)-6/IL-8 release. Targeting transforming growth factor-activated kinase 1 (TAK1), a signaling molecule upstream of p38 MAPK, reduced the IL-6/IL-8 release. Taken together, our study showed that, in the case of adiponectin isoforms, inhibiting the p38 MAPK or the AMPK signaling pathway individually is not sufficient, probably due to compensatory interactions between these pathways. TAK1 might provide an alternative approach by ameliorating the proinflammatory effects of adiponectin in RA. Our results do not suggest that targeting individual adiponectin isoforms specifically in RA would provide a benefit over targeting adiponectin as a whole. However, whether targeting individual adiponectin isoforms would allow minimizing the loss of the beneficial effects of adiponectin within the metabolic and cardiovascular system still needs further investigation.

Dopamine induces in vitro migration of synovial fibroblast from patients with rheumatoid arthritis.

Preventing synovial fibroblast (SF) migration into the adjacent cartilage is a desirable therapeutic target in rheumatoid arthritis (RA). As previous studies demonstrated that RASF and SF from osteoarthritis (OA) patients express dopamine receptors (DR), aim of the present study was to investigate the impact of dopamine on mobility of fibroblasts from patients with chronic arthritides. Synovial tissue and fibroblasts were obtained from RA and OA patients. Immunohistochemistry was performed for all DR-subtypes in the invasion zone. Migration- and motility-assays were performed under DR-stimulation. Cytokines were evaluated using ELISA. Expression of DRs was evaluated by flow cytometry, and DR activation was measured by xCELLigence real-time analysis. All DRs were expressed in RA invasion zone. Migration and motility of RASF and OASF were increased after DR stimulation in patients ≤ 75 years old. Synovial fibroblasts from older RA patients (> 75 years old) expressed lower levels of D1-, D2- and D4-DR than patients ≤ 75 years old. DR activation was not altered in older patients. Our results suggest a possible involvement of dopamine on migration of fibroblasts from arthritis patients. Therefore, the synovial dopaminergic pathway might represent a potential therapeutic target to interfere with progressive joint damage in RA patients.

Adipokines and Inflammation Alter the Interaction Between Rheumatoid Arthritis Synovial Fibroblasts and Endothelial Cells.

Objective: 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. Methods: 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). Results: 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/cm2: 75.2%). Reduced adhesion of RASFs to E-selectin was observed after treatment with dexamethasone (e.g., 0.9 dyn/cm2: -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). Conclusion: 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.

Free Fatty Acids in Bone Pathophysiology of Rheumatic Diseases.

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 ß-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.

[Osseous Tuberculosis - interdisciplinary treatment from diagnostics to microsurgical defect reconstruction - Case report and review of the literature and proposal of a therapeutic algorithm]. / Knochentuberkulose ­ interdisziplinäre Therapie von der Diagnosestellung bis zur plastisch- chirurgischen Deckung.

Tuberculosis is a central global health problem with an incidence of 10 million new cases per year and more than one million deaths per year. Contrary to this, osseous tuberculosis represents an extremely rare entity of tuberculosis. Osseous tuberculosis is challenging beginning with the correct diagnosis, adequate surgical as well infectiological treatment as well as extremity reconstruction. Facing increased migration and therefore increasing numbers of cases of tuberculosis in western countries, the question of a reliable diagnosis, therapy and protective measures in dealing with those patients is becoming increasingly important for Central Europe.In the present case, a 49-year-old female patient from Pakistan, the first presented to our institution with a clinical picture of an exanthema at the level of the upper ankle joint with radiological signs of osteolysis. Pathological and molecular pathological diagnostics revealed the presence of an infection caused by Mycobacteria tuberculosis complex. In the initial phase over 6 weeks, a 4-fold therapy with isoniazid (INH), rifampicin (RMP), pyrazinamide (PZA) and ethambutol (EMB) was administered in accordance with the WHO guidelines, followed by 2-fold therapy with INH and RMP for 12 months in the subsequent continuity phase.14 months later, the patient was re-admitted to hospital because of a recurrent abscess. Therefore tuberculostatic therapy as a quadruple combination of INH, RMP, PZA and EMB was initiated for 6 weeks and as a double combination of INH and RMP for a total of one year.After the abscess had been eradicated, the joint was immobilized by ankle arthrodesis and the deep necrosis of the right ankle was finally reconstructed with allergenic bone grafts and a free microvascular M. gracilis flap.In the case presented here, successful treatment was possible via an interdisciplinary treatment consisiting of infectiology, orthopaedic surgery as well as plastic surgery specialists. Osseous tuberculosis could be eradicated and the bony defect could be reconstructed together with resulting soft tissue defect ultimately preserving of the extremity. In the context of this case study, a comprehensive overview of the current literature is described and a therapy algorithm is proposed due to the increasing relevance of this entity.

The activin-follistatin anti-inflammatory cycle is deregulated in synovial fibroblasts.

BACKGROUND: Activin A and follistatin exhibit immunomodulatory functions, thus affecting autoinflammatory processes as found in rheumatoid arthritis (RA). The impact of both proteins on the behavior of synovial fibroblasts (SF) in RA as well as in osteoarthritis (OA) is unknown. METHODS: Immunohistochemical analyses of synovial tissue for expression of activin A and follistatin were performed. The influence of RASF overexpressing activin A on cartilage invasion in a SCID mouse model was examined. RASF and OASF were stimulated with either IL-1ß or TNFα in combination with or solely with activin A, activin AB, or follistatin. Protein secretion was measured by ELISA and mRNA expression by RT-PCR. Smad signaling was confirmed by western blot. RESULTS: In human RA synovial tissue, the number of activin A-positive cells as well as its extracellular presence was higher than in the OA synovium. Single cells within the tissue expressed follistatin in RA and OA synovial tissue. In the SCID mouse model, activin A overexpression reduced RASF invasion. In human RASF, activin A was induced by IL-1ß and TNFα. Activin A slightly increased IL-6 release by unstimulated RASF, but decreased protein and mRNA levels of follistatin. CONCLUSION: The observed decrease of cartilage invasion by RASF overexpressing activin A in the SCID mouse model appears to be mediated by an interaction between activin/follistatin and other local cells indirectly affecting RASF because activin A displayed certain pro-inflammatory effects on RASF. Activin A even inhibits production and release of follistatin in RASF and therefore prevents itself from being blocked by its inhibitory binding protein follistatin in the local inflammatory joint environment.

Targeting Activated Synovial Fibroblasts in Rheumatoid Arthritis by Peficitinib.

Background: Synovial fibroblasts (SF) play a major role in the pathogenesis of rheumatoid arthritis (RA) and develop an aggressive phenotype destroying cartilage and bone, thus termed RASF. JAK inhibitors have shown to be an efficient therapeutic option in RA treatment, but less is known about the effect of JAK inhibitors on activated RASF. The aim of the study was to examine the effects of JAK inhibitors on activated RASF. Methods: Synovium of RA patients was obtained during knee replacement surgeries. Synoviocytes were isolated and pretreated with JAK inhibitors. Pro-inflammatory cytokines and matrix degrading proteinases were measured by ELISA in supernatant after stimulation with oncostatin M or IL-1ß. The proliferation of RASF was measured by BrdU incorporation. Cell culture inserts were used to evaluate cell migration. For adhesion assays, RASF were seeded in culture plates. Then, plates were extensively shaken and adherent RASF quantified. Cell viability, cytotoxicity and apoptosis were measured using the ApoTox-Glo™ Triplex and the CellTox™ Green Cytotoxicity Assay. Results: Tofacitinib and baricitinib decreased the IL-6 release of RASF stimulated with oncostatin M. JAK inhibition attenuated the IL-6 release of IL-1ß activated and with soluble IL-6 receptor treated RASF. In contrast, only peficitinib and filgotinib decreased the IL-6 release of RASF activated with IL-1ß. Peficitinib decreased also the MMP-3, CXCL8, and CXCL1 release at 5 µM. Moreover, peficitinib was the only JAK inhibitor suppressing proliferation of activated RASF at 1 µM. Peficitinib further decreased the migration of RASF without being cytotoxic or pro-apoptotic and without altering cell adhesion. Conclusions: JAK inhibitors effectively suppress the inflammatory response induced by oncostatin M and by transsignaling of IL-6 in RASF. Only peficitinib modulated the IL-1ß-induced response of RASF and their proliferation in vitro at concentrations close to reported Cmax values of well tolerated doses in vivo. In contrast to filgotinib, peficitinib also highly suppressed RASF migration showing the potential of peficitinib to target RASF.

Radiocarpal fusion and midcarpal resection interposition arthroplasty: long-term results in severely destroyed rheumatoid wrists.

BACKGROUND: The aim of this retrospective study is to evaluate distal resection interposition arthroplasty of the wrist as a tool to restore mobility as well as to restore stability in severely destroyed wrist joints. METHODS: Thirty-four wrists in 28 rheumatoid arthritis patients were included. The mean follow-up time was 9 years after surgical treatment with clinical and radiological examination. The results were accessed based on a modification of Clayton ́s scoring system as well as a functional questionnaire. RESULTS: 71% patients were satisfied with pain, function and activities of daily life. Better results were reported by patients with a young age, early surgical intervention, a shorter duration of the disease, and lesser involvement of other joints. CONCLUSIONS: The results for radiocarpal arthrodesis were comparable to those of synovectomy or arthrodesis of the wrist. The results after total wrist joint arthroplasty varies probably as the result of different patient groups, implant types and evolution of prosthetic designs, and are not comparable with the present study.

Tetraspanin CD82 affects migration, attachment and invasion of rheumatoid arthritis synovial fibroblasts.

Tetraspanins function as membrane adaptors altering cell-cell fusion, antigen presentation, receptor-mediated signal transduction and cell motility via interaction with membrane proteins including other tetraspanins and adhesion molecules such as integrins. CD82 is expressed in several malignant cells and well described as tumour metastasis suppressor. Rheumatoid arthritis (RA) is based on persistent synovial inflammation and joint destruction driven to a large extent by transformed-appearing activated synovial fibroblasts (SF) with an increased migratory potential. OBJECTIVE: CD82 is upregulated in RA synovial fibroblasts (RASF) compared with osteoarthritis (OA) SF as well as within RA compared with OA synovial lining layer (LL) and the role of CD82 in RASF was evaluated. METHODS: CD82 and integrin immunofluorescence was performed. Lentiviral CD82 overexpression and siRNA-mediated knockdown was confirmed (realtime-PCR, Western blot, immunocytochemistry). RASF migration (Boyden chamber, scrape assay), attachment towards plastic/Matrigel, RASF-binding to endothelial cells (EC) and CD82 expression during long-term invasion in the SCID-mouse-model were evaluated. RESULTS: CD82 was induced by proinflammatory stimuli in SF. In RA-synovium, CD82 was expressed in RASF close to blood vessels, LL, sites of cartilage invasion and colocalised with distinct integrins involved in tumour metastasis suppression but also in RA-synovium by RASF. CD82 overexpression led to reduced RASF migration, cell-matrix and RASF-EC adhesion. Reduced CD82 expression (observed in the sublining) increased RASF migration and matrix adhesion whereas RASF-EC-interaction was reduced. In SCID mice, the presence of CD82 on cartilage-invading RASF was confirmed. CONCLUSION: CD82 could contribute to RASF migration to sites of inflammation and tissue damage, where CD82 keeps aggressive RASF on site.

Pilot study for the registry of complications in rheumatic diseases from the German Society of Surgery (DGORh): evaluation of methods and data from the first 1000 patients.

OBJECTIVE: Most patients suffering with rheumatic diseases who undergo surgical treatment are receiving immune-modulating therapy. To determine whether these medications affect their outcomes a national registry was established in Germany by the German Society of Surgery (DGORh). Data from the first 1000 patients were used in a pilot study to identify relevant corisk factors and to determine whether such a registry is suitable for developing accurate and relevant recommendations. DESIGN AND PARTICIPANTS: Data were collected from patients undergoing surgical treatments with their written consent. A second consent form was used, if complications occurred. During this pilot study, in order to obtain a quicker overview, risk factors were considered only in patients with complications. Only descriptive statistical analysis was employed in this pilot study due to limited number of observed complications and inhomogeneous data regarding the surgery and the medications the patients received. Analytical statistics will be performed to confirm the results in a future outcome study. RESULTS: Complications occurred in 26 patients and were distributed equally among the different types of surgeries. Twenty one of these patients were receiving immune-modulating therapy at the time, while five were not. Infections were observed in 2.3% of patients receiving and in 5.1% not receiving immunosuppression. CONCLUSIONS: Due to the low number of cases, inhomogeneity in the diseases and the treatments received by the patients in this pilot study, it is not possible to develop standardised best-practice recommendations to optimise their care. Based on this observation we conclude that in order to be suitable to develop accurate and relevant recommendations a national registry must include the most important and relevant variables that impact the care and outcomes of these patients.

Disease-Specific Effects of Matrix and Growth Factors on Adhesion and Migration of Rheumatoid Synovial Fibroblasts.

In rheumatoid arthritis (RA), cartilage and bone matrix are degraded, and extracellular matrix (ECM) proteins, acting as cellular activators, are liberated. Similar to ECM proteins, matrix-bound chemokines, cytokines, and growth factors (GFs) influence functional properties of key cells in RA, especially synovial fibroblasts. The role of these molecules on attachment, migration, and proinflammatory and prodestructive activation of RASFs was analyzed. Adhesion/migration of RASFs were examined under GF-enriched (GF+) or -reduced (GF-) conditions with or without addition of matrix-associated GFs, TGF-ß, and platelet-derived GF to GF- or culture supernatants. Fibroblast adhesion and alterations in proinflammatory/prodestructive properties (e.g., IL-6/matrix metalloproteinase 3-release) in response to matrix-associated molecules were compared. Effects of GF+, GF-, and other ECM components on human RASF-mediated cartilage invasion were examined in the SCID mouse model. RASF adhesion under GF- conditions was significantly lower compared with GF+ conditions (6.8- versus 8.3-fold). This effect was specific for RA because control cells showed opposite effects (e.g., osteoarthritis synovial fibroblasts [SF]; GF- versus GF+: 10.7- versus 8-fold). Addition of TGF-ß to GF- increased RASF attachment (12.7-fold) compared with other matrices and components. RASF adhesion to GF+ matrix resulted in the strongest IL-6 and matrix metalloproteinase-3 release, and was even more pronounced compared with supplementation of single GFs. In vivo, GF- matrix decreased RASF-mediated cartilage invasion compared with GF+ matrix. ECM components and especially GFs when bound within ECM actively enhance RASF attraction and cartilage adhesion. This observation was specific for RASFs as a reverse behavior was observed for controls.

Response of human rheumatoid arthritis osteoblasts and osteoclasts to adiponectin.

OBJECTIVES: Adiponectin is an effector molecule in the pathophysiology of rheumatoid arthritis, e.g. by inducing cytokines and matrix degrading enzymes in synovial fibroblasts. There is growing evidence that adiponectin affects osteoblasts and osteoclasts although the contribution to the aberrant bone metabolism in rheumatoid arthritis is unclear. Therefore, the adiponectin effects on rheumatoid arthritis-derived osteoblasts and osteoclasts were evaluated. METHODS: Adiponectin and its receptors were examined in bone tissue. Primary human osteoblasts and osteoclasts were stimulated with adiponectin and analysed using realtime polymerase chain-reaction and immunoassays. Effects on matrix-production by osteoblasts and differentiation and resorptive activity of osteoclasts were examined. RESULTS: Immunohistochemistry of rheumatoid arthritis bone tissue showed adiponectin expression in key cells of bone remodelling. Adiponectin altered gene expression and cytokine release in osteoblasts and increased IL-8 secretion by osteoclasts. Adiponectin inhibited osterix and induced osteoprotegerin mRNA in osteoblasts. In osteoclasts, MMP-9 and tartrate resistant acid phosphatase expression was increased. Accordingly, mineralisation capacity of osteoblasts decreased whereas resorptive activity of osteoclasts increased. CONCLUSIONS: The results confirm the proinflammatory potential of adiponectin and support the idea that adiponectin influences rheumatoid arthritis bone remodelling through alterations in osteoblast and osteoclast.

Expression of adipokines in osteoarthritis osteophytes and their effect on osteoblasts.

OBJECTIVE: Osteophyte formation in osteoarthritis (OA) is mediated by increased osteoblast activity, which is -in turn- regulated by the Wnt signaling pathway. Obesity is regarded a risk factor in OA, yet little is known about the interaction between adipose tissue-derived factors, the adipokines, and bone formation, although adipokines are associated with the pathogenesis of OA. Therefore, the effect of adipokines on bone and cartilage forming cells and osteophyte development was analyzed. METHODS: Human OA osteophytes were histologically characterized and adipokine expression was evaluated by immunohistochemistry. Osteoblasts and chondrocytes were isolated from OA tissue and stimulated with adiponectin, resistin, or visfatin. Cytokine and osteoblast/chondrocyte markers were quantified and activation of Wnt and p38 MAPK signaling was analyzed. RESULTS: Adiponectin, resistin, and visfatin were expressed in OA osteophytes by various articular cell types. Stimulation of OA osteoblasts with adiponectin and of OA chondrocytes with visfatin led to an increased release of proinflammatory mediators but not to osteoblast differentiation or activation. Additionally, visfatin increased matrix degrading factors in chondrocytes. Wnt signaling was not altered by adipokines, but adiponectin induced p38 MAPK signaling in osteoblasts. CONCLUSION: Adipokines are present in OA osteophytes, and adiponectin and visfatin increase the release of proinflammatory mediators by osteoblasts and chondrocytes. The effects of adiponectin were mediated by p38 MAPK but not Wnt signaling in osteoblasts. Therefore, the results support the idea that adipokines do not directly influence osteophyte development but the proinflammatory conditions in OA.

Influence of Extracellular RNAs, Released by Rheumatoid Arthritis Synovial Fibroblasts, on Their Adhesive and Invasive Properties.

Extracellular RNA (exRNA) has been characterized as a molecular alarm signal upon cellular stress or tissue injury and to exert biological functions as a proinflammatory, prothrombotic, and vessel permeability-regulating factor. In this study, we investigated the contribution of exRNA and its antagonist RNase1 in a chronic inflammatory joint disease, rheumatoid arthritis (RA). Upon immunohistochemical inspection of RA, osteoarthritis (OA), and psoriatic arthritis synovium, exRNA was detectable only in the RA synovial lining layer, whereas extracellular DNA was detectable in various areas of synovial tissue. In vitro, exRNA (150-5000 nt) was released by RA synovial fibroblasts (RASF) under hypoxic conditions but not under normoxia or TNF-α treatment. RNase activity was increased in synovial fluid from RA and OA patients compared with psoriatic arthritis patients, whereas RNase activity of RASF and OASF cultures was not altered by hypoxia. Reduction of exRNA by RNase1 treatment decreased adhesion of RASF to cartilage, but it had no influence on their cell proliferation or adhesion to endothelial cells. In vivo, treatment with RNase1 reduced RASF invasion into coimplanted cartilage in the SCID mouse model of RA. We also analyzed the expression of neuropilins in synovial tissue and SF, as they may interact with vascular endothelial growth factor signaling and exRNA. The data support the concepts that the exRNA/RNase1 system participates in RA pathophysiology and that RASF are influenced by exRNA in a prodestructive manner.

Differentiation of osteophyte types in osteoarthritis - proposal of a histological classification.

OBJECTIVE: Osteoarthritis is not only characterized by cartilage degradation but also involves subchondral bone remodeling and osteophyte formation. Osteophytes are fibrocartilage-capped bony outgrowths originating from the periosteum. The pathophysiology of osteophyte formation is not completely understood. Yet, different research approaches are under way. Therefore, a histological osteophyte classification to achieve comparable results in osteophyte research was established for application to basic science research questions. METHODS: The osteophytes were collected from knee joints of osteoarthritis patients (n=10, 94 osteophytes in total) after joint replacement surgery. Their size and origin in the respective joint were photo-documented. To develop an osteophyte classification, serial tissue sections were evaluated using histological (hematoxylin and eosin, Masson's trichrome, toluidine blue) and immunohistochemical staining (collagen type II). RESULTS: Based on the histological and immunohistochemical evaluation, osteophytes were categorized into four different types depending on the degree of ossification and the percentage of mesenchymal connective tissue. Size and localization of osteophytes were independent from the histological stages. CONCLUSION: This histological classification system of osteoarthritis osteophytes provides a helpful tool for analyzing and monitoring osteophyte development and for characterizing osteophyte types within a single human joint and may therefore contribute to achieve comparable results when analyzing histological findings in osteophytes.

Activated human B cells induce inflammatory fibroblasts with cartilage-destructive properties and become functionally suppressed in return.

BACKGROUND: Cross-talk between synovial fibroblasts (SF) and immune cells is suggested to play a crucial role in inflammation and chronification of rheumatoid arthritis (RA). The contribution of B cells in this process is poorly defined. METHODS: Here, primary B cells from healthy donors were polyclonally activated and cocultured with SF of non-synovitic origin from patients with osteoarthritis. RESULTS: In B-SF cocultures the concentrations of interleukin 6 (IL-6) and IL-8 increased manifold compared with single cultures even under physical separation and remained stable for several days after B-cell removal. Intracellular staining confirmed SF as key producers of IL-6 and IL-8, and B cells as main producers of tumour necrosis factor alpha (TNFα) and IL-1ß. Blocking experiments with a combination of anti-TNFα-antibodies and rIL-1RA significantly reduced SF cytokine production by up to 90%, suggesting that B-cell-derived TNFα and IL-1ß were crucial mediators of SF activation. Interestingly, B-cell cytokine production, CD25 expression and proliferation decreased in cocultures by at least 50%, demonstrating a negative regulatory loop towards the activated B cells. Inhibition of activin receptor-like kinase 5, a crucial component of the tumour growth factor ß (TGFß) signalling pathway, partly restored B-cell proliferation, suggesting a contribution of SF-derived TGFß in B-cell suppression. Besides cytokines, B-cell-activated SF also upregulated secretion of matrix metalloproteases such as MMP-3, thereby acquiring potential tissue destructive properties. This was confirmed by their invasion into human cartilage in the severe combined immunodeficiency mouse fibroblast invasion model in vivo. CONCLUSIONS: Interaction with activated B cells leads to conversion of non-arthritic SF into SF with a proinflammatory and aggressive RA-like phenotype, thereby suggesting a new, so far unrecognised role for B cells in RA pathogenesis.