Grit Krumbholz

Induction of osteoclastogenesis and bone loss by human autoantibodies against citrullinated vimentin

Autoimmunity is complicated by bone loss. In human rheumatoid arthritis (RA), the most severe inflammatory joint disease, autoantibodies against citrullinated proteins are among the strongest risk factors for bone destruction. We therefore hypothesized that these autoantibodies directly influence bone metabolism. Here, we found a strong and specific association between autoantibodies against citrullinated proteins and serum markers for osteoclast-mediated bone resorption in RA patients. Moreover, human osteoclasts expressed enzymes eliciting protein citrullination, and specific N-terminal citrullination of vimentin was induced during osteoclast differentiation. Affinity-purified human autoantibodies against mutated citrullinated vimentin (MCV) not only bound to osteoclast surfaces, but also led to robust induction of osteoclastogenesis and bone-resorptive activity. Adoptive transfer of purified human MCV autoantibodies into mice induced osteopenia and increased osteoclastogenesis. This effect was based on the inducible release of TNF-α from osteoclast precursors and the subsequent increase of osteoclast precursor cell numbers with enhanced expression of activation and growth factor receptors. Our data thus suggest that autoantibody formation in response to citrullinated vimentin directly induces bone loss, providing a link between the adaptive immune system and bone.

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.

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, Massons 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.

A8.16 The Role of Adipocytokines in Osteoarthritis Osteophyte Formation

Background and Objectives Although obesity is an established risk factor in osteoarthritis (OA), there is limited information about the role of adipose tissue derived factors in bone formation. Adipocytokines such as adiponectin, resistin, and visfatin, are known to be associated with the pathogenesis of rheumatoid arthritis (RA) and OA. Adipocytokines are locally produced in RA and OA joints by osteoblasts, osteoclasts, and chondrocytes. In contrast to their joint-destructive effects in RA, the role of adipocytokines in OA bone remodelling and osteophyte formation is unclear. Therefore, the adipocytokine expression during osteophyte development and in cells of bone formation was analysed as well as their effect on these cells. Methods Osteophytes, cartilage, and osteoblasts were obtained from OA patients during joint replacement surgery. Serial sections of bone tissue were stained (Masson trichrome, TRAP) and scored from grade one (no ossification, mainly connective tissue and cartilage) to five (ossified mineralised osteophytes, <10% connective tissue, ossified remodelling zones). Immunohistochemistry against alkaline phosphatase, collagen-type II, adiponectin, resistin, and visfatin was performed. OA osteoblasts were stimulated with adiponectin or resistin and immunoassays for IL-6, IL-8, and MCP-1 were performed. Results All adipocytokines were detectable in cultured osteoblasts and all osteophyte grades. In non ossified osteophytes (grade 1), especially adiponectin and to a lower extent resistin and visfatin were detectable in connective tissue fibroblasts. In ossified osteophytes (grade 2–5), resistin and visfatin and to a lower extend adiponectin were expressed by osteoblasts and resistin and visfatin by osteoclasts. In all osteophyte grades adiponectin was detectable in blood vessels and visfatin was found in about 50% of the chondrocytes. Osteoblast stimulation with adiponectin increased the release of the inflammatory mediators IL-6 (2.6-fold), IL-8 (4.9-fold), and MCP-1 (2.1-fold). In contrast, resistin led to a non-significant decrease of these factors. The osteoblast populations showed individual differences in the baseline expression of the analysed factors and in their responsiveness to adipocytokines. Conclusions The adiponectin and visfatin expression in osteophyte connective tissue and cartilage suggests their involvement in early osteophyte development. Resistin and visfatin in osteoblasts and osteoclasts in ossified osteophytes indicates a role in osteophyte formation at later stages. The stimulation of osteoblasts with adiponectin induces the release of inflammatory mediators. Therefore, the analysed adipocytokines most likely are involved in osteophyte formation at different stages and correspondingly affect cells of cartilage and bone formation to a different extent. Funded by the ANCYLOSS project of the German Ministry of Research and Education (BMBF).

A1.42 Adiponectin: modulation of bone remodelling in rheumatoid arthritis

Introduction Adiponectin is a strong effector molecule in the pathophysiology of RA, for example by contributing to the production of proinflammatory chemokines and matrix degrading enzymes synthesised by RA synovial fibroblasts. In addition, there is growing evidence that adiponectin influences bone metabolism. Our immunohistochemical analysis of RA bone tissue showed a co-localization of adiponectin with key cells of bone remodelling, i.e. osteoblasts and osteoclasts. Moreover, immunocytochemistry and RT-PCR confirmed the expression of adiponectin receptors by RA osteoblasts and osteoclasts. In this study, we therefore focussed on the influence of adiponectin on RA osteoblast and osteoclast activity, their differentiation as well as the immunomodulatory properties of adiponectin. Methods Human osteoblasts and osteoclasts were isolated from bone tissue and blood samples of RA patients. OA patients and healthy donors served as control. Osteoblasts and osteoclasts were treated with adiponectin (10 µg/ ml). Adiponectin-mediated effects on osteoblasts and osteoclasts were analysed using realtime PCR and immunoassays. Effects of adiponectin on matrix-production by osteoblasts as well as differentiation and resorptive activity of osteoclasts were examined using Alizarin-Red S-, TRAP- and von Kossa-staining. Results Stimulation with adiponectin induced the secretion of pro-inflammatory cytokines in RA osteoblasts (e.g. IL-6: 5.2 fold, p = 0.001; MCP-1: 7.6 fold, p = 0.2). Especially pre-osteoclasts responded with an increase in IL-8 production (12.9-fold, p = 0.041) in comparison to mature osteoclasts (2.6-fold, p = 0.06, n = 8). Gene expression analysis revealed an induction of OPG and an inhibition of osterix in RA osteoblasts by adiponectin (OPG: 2.7-fold; p = 0.04; osterix: -1.55-fold; p = 0.004). In osteoclasts, the expression of MMP-9 and TRAP was increased (MMP-9: 1.9-fold, p = 0.03; TRAP: 1.5-fold, p = 0.052). Moreover, differentiation of PBMCs in the presence of adiponectin resulted in an increased number of TRAP positive cells (131.2% ± 60.7, p = 0.3, n = 5) and a significantly increased resorptive activity (134% ± 16.3, p = 0.016; n = 5). In contrast, functional examinations of osteoblasts did not reveal differences with regard to mineralisation capacity in the presence of adiponectin. Conclusions On one hand, the present study shows an increased release of cytokines after adiponectin treatment by osteoblasts and osteoclasts and thereby confirms the pro-inflammatory potential of adiponectin in RA. On the other hand, our findings revealed adiponectin-mediated alterations in gene expression, activity and differentiation of human osteoblasts and osteoclasts in RA. This supports the idea of adiponectin being involved in the pathophysiologic modulation of RA effector cells with regard to the process of bone remodelling in RA. Acknowledgement Funded by the German Research Society (SPP1468, IMMUNOBONE, NE1174/6-1).

OP0023 Analysis of the Effect of Adipokines on WNT Signaling in Osteoarthritis Osteoblasts

Background Osteoarthritis (OA) is primarily characterized by cartilage and bone erosion as well as the formation of osteophytes. These processes are mediated by an increased osteoblast activity, which is -in turn- regulated by the Wnt signaling pathway. Obesity is a recognized risk factor in OA, yet little is known about the interaction between adipose tissue-derived factors including adipokines and bone formation. Histological analysis of OA osteophytes showed the presence of adiponectin, resistin and visfatin in osteophytes. In addition, OA osteoblasts respond to adipokines in a specific manner, e.g. adiponectin induces inflammatory mediators in these cells. These effects can be further modulated by activation of the Wnt-signaling pathway. Hence, in this context, the effects of adiponectin, resistin and visfatin on Wnt signaling in OA osteoblasts were analyzed. Methods Osteoblasts were isolated from OA bone tissue obtained during joint replacement surgery. Cultured osteoblasts were stimulated with adiponectin, resistin and visfatin. Additionally, a co-stimulation with Wnt3a or DKK1 was performed. The Wnt signaling pathway of stimulated vs. non-stimulated osteoblasts was analyzed for adipokine-mediated effects using immunoassays for IL-6, DKK1 and sclerostin, and by real-time PCR for axin2. Results Stimulation of OA osteoblasts with adiponectin, resistin or visfatin did not alter the release of the Wnt inhibitor DKK1 compared to the unstimulated controls. The level of the secreted Wnt inhibitor sclerostin was below the detection level of the immunoassay for both, adipokine-stimulated osteoblasts as well as for unstimulated control cells. Also, the expression of axin2, a direct target gene of the Wnt signaling showed no significant differences in OA osteoblasts after adipokine-stimulation compared to the unstimulated control. A co-stimulation with adiponectin, resistin or visfatin in combination with Wnt3a or DKK1 had no synergistic influence on the Wnt-mediated IL-6 release of OA osteoblasts. Conclusions The adipokines adiponectin, resistin and visfatin are present in osteoblasts in OA osteophytes, and adipokines such as adiponectin increase the release of inflammatory mediators by OA osteoblasts. However, the observed adipokine-mediated effects do not include induction of Wnt-signaling. Therefore, the involvement of adipokines in OA osteophyte formation via osteoblasts appear to be independent of Wnt signaling. Acknowledgements Funded by the ANCYLOSS project of the German Ministry of Research and Education (BMBF). Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3701

A8.18 Visfatin/PBEF in Bone Remodelling of Rheumatoid Arthritis

Objectives Rheumatoid arthritis (RA) is associated with increased production of the adipocytokine visfatin in synovial fluid and tissue of RA patients. Visfatin promotes the synthesis of pro-inflammatory and matrix-degrading effector molecules in RA synovial fibroblasts. Moreover, an immunohistochemical analysis of RA bone tissue showed a co-localisation of visfatin with key cells of bone remodelling (osteoblasts, osteoclasts) but the role of this adipokine in processes of bone remodelling in RA is unclear. In this study, we focussed on visfatin and its influence on RA osteoblast and osteoclast activity and differentiation as well as on its immunomodulatory properties. Methods Human osteoblasts and osteoclasts were isolated from bone tissue and blood samples of RA patients and stimulated with visfatin. Visfatin-mediated effects on osteoblasts and osteoclasts were analysed on the transcriptional and translational level using realtime polymerase chain reaction and immunoassays. Additionally, effects of visfatin on matrix-production of osteoblasts as well as differentiation and resorption activity of osteoclasts were examined by Alizarin-Red S-, TRAP- and von Kossa-staining. Results Stimulation with visfatin induced the secretion of pro-inflammatory cytokines (e.g. IL-6: 5-fold increase; IL-8: up to 100-fold) in RA osteoblasts. Additionally, quantitative realtime PCR showed several genes being differentially expressed in osteoblasts after stimulation with visfatin (e.g. alkaline phosphatase, OPG, Osterix). In contrast, osteoclasts only weakly respond to visfatin. A regulation on translational level was observed with regard to the production of the cytokines IL-6 and IL-8, showing a moderate increase. Conclusions The results of the present study indicate that visfatin influences the activity as well as the differentiation of human osteoblasts in RA by modulating the expression of genes being involved in matrix production and osteoblast phenotype development. These results support the idea of visfatin affecting bone metabolism in RA. Furthermore, the finding of cytokine-induction in osteoblasts and osteoclasts in RA confirm the pro-inflammatory potential of visfatin in RA. Acknowledgement Funded by the German Research Society (SPP1468, IMMUNOBONE, NE1174/6–1).

FRI0039 VISFATIN/PBEF in bone remodelling of rheumatoid arthritis

Objectives Rheumatoid arthritis (RA) is associated with increased production of adipocytokines, which are cytokine-like mediators. Increased levels of the adipokine visfatin were found in synovial fluid and tissue of RA patients. Moreover, visfatin promotes the synthesis of pro-inflammatory and matrix-degrading effector molecules in RA synovial fibroblasts. However, the role of visfatin in bone remodelling of RA is unclear. In this study, we focussed on visfatin and its influence on RA osteoblast activity and differentiation as well as on its immunomodulatory properties on RA osteoblasts. Methods For expression analysis of visfatin, bone tissue obtained from RA patients during joint replacement surgery, was analyzed. Serial sections of decalcified and deparaffinised bone tissue were used to identify the expression sites of visfatin in articular remodelling. (Immuno)histochemistry was performed by using anti-visfatin and anti human ALP (alkaline phosphatase) antibodies in addition to Masson- and TRAP-staining. Human osteoblasts isolated from bone tissue of RA patients were stimulated with visfatin. Visfatin-mediated effects on human osteoblasts were analyzed on the transcriptional and translational level using quantitative realtime polymerase chain reaction (qRT-PCR) and immunoassays. Furthermore, the cells were stimulated with the pro-inflammatory cytokine IL-1β and gene expression of visfatin was examined by qRT-PCR. Results Immunohistochemical staining of RA bone tissue showed a co-localization of visfatin with key cells of bone remodelling (osteoblasts, osteoclasts). Stimulation with visfatin induced the secretion of pro-inflammatory cytokines (e.g. IL-6: 5-fold increase; IL-8: up to 100-fold) in RA osteoblasts. Additionally, quantitative realtime PCR showed several genes being differentially expressed in osteoblasts after stimulation with Visfatin (e.g. alkaline phosphatase, osteocalcin, OPG). Furthermore, the stimulation with IL-1β led to a 5-fold increase of Visfatin gene expression in RA osteoblasts. Conclusions The results of the present study indicate that visfatin influences the activity as well as the differentiaton of human osteoblasts in RA by modulating the expression of genes being involved in matrix production and osteoblast phenotype development. These results, together with the finding of increased visfatin expression in osteoblasts after IL-1β treatment, support the idea of visfatin affecting bone metabolism in RA. Acknowledgement: Funded by the German Research Society (SPP1468, IMMUNOBONE, NE1174/6-1). Disclosure of Interest None Declared

Expression of adipocytokines at sites of bone remodelling in rheumatoid arthritis

Background Rheumatoid arthritis (RA) is associated with increased production of adipocytokines, which are cytokine-like mediators. Increased levels of the adipokines adiponectin and resistin in synovial fluids of RA patients as well as their activating/inhibitory effect on cells of bone remodeling (eg, osteoblasts, osteoclasts) raise the question of immunomodulatory properties of these cytokines towards bone resorption. In this study, the expression of adiponectin and resistin in joint compartments and cells at the sites of bone remodelling in RA was analysed. Methods Bone tissue was obtained from RA patients during joint replacement surgery. Serial sections of decalcified and deparaffinised bone tissue were prepared. Immunohistochemistry was used to identify the localisation of adipokines at sites of bone remodelling in articular bone and for identification of cells expressing the adipokines adiponectin and resistin. Additionally, immunohistochemical staining was carried out by using antihuman ALP (alkaline phosphatase) antibodies for identifying osteoblasts and TRAP staining to identify osteoclasts. For histological overview, a Masson Trichrome staining was performed. Results Adiponectin and resistin were detectable in all joints of RA patients at sites of bone remodelling. An expression of adiponectin and resistin by osteoblasts and osteoclasts could be observed. Both adipokines were co-localised with osteoblasts at the border of newly formed, non-mineralised bone tissue. Interestingly, a correlation of the expression intensity of resistin with the expression of ALP in osteoblasts could be observed, indicating that active osteoblasts produce increased amounts of resistin. Conclusions The results indicate that resistin and adiponectin are expressed by osteoblasts and osteoclasts at sites of bone erosion. Therefore, these cell types are able to respond to resistin and adiponectin in bone tissue, which potentially further contribute to the differentiation and activation of these cell types. Acknowledgement Funded by the German Research Society (SPP1468, IMMUNOBONE, NE1174/6-1).