Birgit Zimmermann

Rheumatoid arthritis progression mediated by activated synovial fibroblasts

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial hyperplasia and progressive joint destruction. Rheumatoid arthritis synovial fibroblasts (RASFs) are leading cells in joint erosion and contribute actively to inflammation. RASFs show an activated phenotype that is independent of the inflammatory environment and requires the combination of several factors. Although new aspects regarding RASF activation via matrix degradation products, epigenetic modifications, inflammatory factors, Toll-like receptor (TLR) activation and others have recently been uncovered, the primary pathophysiological processes in early arthritis leading to permanent activation are mostly unknown. Here, we review new findings regarding RASF activation and their altered behavior that contribute to matrix destruction and inflammation as well as their potential to spread RA.

Adiponectin-mediated changes in effector cells involved in the pathophysiology of rheumatoid arthritis

OBJECTIVE Rheumatoid arthritis (RA) is associated with increased production of adipokines, which are cytokine-like mediators that are produced mainly in adipose tissue but also in synovial cells. Since RA synovial fibroblasts (RASFs), lymphocytes, endothelial cells, and chondrocytes are key players in the pathophysiology of RA, this study was undertaken to analyze the effects of the key adipokine adiponectin on proinflammatory and prodestructive synovial effector cells. METHODS Lymphocytes were activated in part prior to stimulation. All cells were stimulated with adiponectin, and changes in gene and protein expression were determined by Affymetrix and protein arrays. Messenger RNA and protein levels were confirmed using semiquantitative reverse transcription-polymerase chain reaction (PCR), real-time PCR, and immunoassays. Intracellular signal transduction was evaluated using chemical signaling inhibitors. RESULTS Adiponectin stimulation of human RASFs predominantly induced the secretion of chemokines, as well as proinflammatory cytokines, prostaglandin synthases, growth factors, and factors of bone metabolism and matrix remodeling. Lymphocytes, endothelial cells, and chondrocytes responded to adiponectin stimulation with enhanced synthesis of cytokines and various chemokines. Additionally, chondrocytes released increased amounts of matrix metalloproteinases. In RASFs, adiponectin-mediated effects were p38 MAPK and protein kinase C dependent. CONCLUSION Our previous findings indicated that adiponectin was present in inflamed synovium, at sites of cartilage invasion, in lymphocyte infiltrates, and in perivascular areas. The findings of the present study indicate that adiponectin induces gene expression and protein synthesis in human RASFs, lymphocytes, endothelial cells, and chondrocytes, supporting the concept of adiponectin being involved in the pathophysiologic modulation of RA effector cells. Adiponectin promotes inflammation through cytokine synthesis, attraction of inflammatory cells to the synovium, and recruitment of prodestructive cells via chemokines, thus promoting matrix destruction at sites of cartilage invasion.

Rheumatoid synovial fibroblasts differentiate into distinct subsets in the presence of cytokines and cartilage

BackgroundWe investigated two distinct synovial fibroblast populations that were located preferentially in the lining or sub-lining layers and defined by their expression of either podoplanin (PDPN) or CD248, and explored their ability to undergo self-assembly and transmigration in vivo.MethodsSynovial fibroblasts (SF) were cultured in vitro and phenotypic changes following stimulation with interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and transforming growth factor (TGF)-β1 were examined. To examine the phenotype of SF in vivo, a severe combined immunodeficiency (SCID) human-mouse model of cartilage destruction was utilised.ResultsSF in the lining layer in rheumatoid arthritis (RA) expressed high levels of PDPN compared to the normal synovium, whereas CD248 expression was restricted to sub-lining layer cells. TNF-α or IL1 stimulation in vitro resulted in an increased expression of PDPN. In contrast, stimulation with TGF-β1 induced CD248 expression. In the SCID human-mouse model, rheumatoid SF recapitulated the expression of PDPN and CD248. Fibroblasts adjacent to cartilage expressed PDPN, and attached to, invaded, and degraded cartilage. PDPN+ CD248– SF preceded the appearance of PDPN– CD248+ cells in contralateral implants.ConclusionsWe have identified two distinct SF populations identified by expression of either PDPN or CD248 which are located within different anatomical compartments of the inflamed synovial membrane. These markers discriminate between SF subsets with distinct biological properties. As PDPN-expressing cells are associated with early fibroblast migration and cartilage erosion in vivo, we propose that PDPN-expressing cells may be an attractive therapeutic target in RA.

Migratory potential of rheumatoid arthritis synovial fibroblasts: Additional perspectives

Cell migration is a central part of physiological and pathophysiological processes including wound healing, immune defense, matrix remodeling and organ homeostasis. Different cell types have migratory potential including cells of the immune system and cells required in wound healing and tissue repair. These cells migrate locally through the tissue to the site of damage. The fibroblast is a central cell type of wound healing. In rheumatoid arthritis (RA), activated synovial fibroblasts (SFs) have the ability to invade joint cartilage, actively contributing to joint destruction in RA. Recently, RASFs have been shown to be able to migrate to non-affected areas and joints through the blood stream and to invade distant cartilage. RASFs most likely use similar mechanisms comparable to lymphocytes and tumor cells for long-distance and vascular trans-migration. Future experiments will address the goal to keep the transformed-appearing fibroblasts in the affected joints using therapeutical strategies that inhibit the pathophysiological changes of transformed-appearing RASFs but do not interfere with the physiological processes of ‘normal’ fibroblasts.

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.

Expression of extracellular RNA in synovial tissue and RNase activity in synovial fluid of rheumatoid arthritis patients

Background RNA is required for protein biosynthesis. However, extracellular RNA (exRNA) is also present within tissues and actively secreted by cells. Examples for the exRNA-activity are a pro-coagulative effect and an enhanced permeability of the blood-brain-barrier by exRNA. Furthermore, specific exRNAs are accumulated in the serum in cancer and these exRNA have actually been identified as tumour-markers. A pro-inflammatory effect of extracellular DNA due to tissue damage has been described in rheumatoid arthritis (RA). Therefore, the authors analysed the presence and the expression pattern of exRNA in the joint as well as the activity of RNase in the synovial fluid of RA patients. Methods Synovium from RA and OA patients (n=3 each) were stained with 4′,6-diamidin-2-phenylindole (DAPI) to locate DNA and SYTO RNASelect Green Fluorescent Stain to locate RNA in the tissue. A serial tissue section was stained with H&E to identify the lining layer and sublining. The expression pattern of RNA was analyed by comparing the RNA and DNA staining with the HE-staining on serial sections. In addition, the RNase activity of the synovium of different patient groups (RA: n=4; OA: n=3 and psoriatic arthritis (PsA): n=5) was measured using the Quant-iT RNA Assay Kit. Results RNA and DNA signals were detectable in all areas of the synovium. Merging the RNA and DNA signals showed a co-localisation of the signals within the nucleus of the cell. Interestingly, an intensive cytoplasmatic and exRNA signal could be observed in the lining layer of RA and OA patients with stronger signal intensity in the RA lining. Due to the increased thickness of the lining layer in RA patients, an increased amount of exRNA in RA patients when compared to OA was detectable. This signal was not co-localised with the DNA. A reduced RNase activity was measured in the synovial fluid of patients with RA (20.1±3.61) and PsA (21.3±2.5) in comparison to OA patients (35.8±4.5), reaching statistical significance when comparing PsA to OA patients (p=0,033). In addition, RNase activity was inversely correlated to CRP serum level (r=−0.43). Conclusion In OA and especially RA patients, exRNA is present in the synovial lining layer. RNase activity in the synovial fluid seems to be reduced in patients with chronic inflammation (RA and PSA) in contrast to OA. The increased amounts of exRNA could demonstrate a new pro-inflammatory mechanism active in chronic inflamed tissue that could be important for the perpetuation and chronification of inflammation.

OP0260 Adipokines Alter the Interaction of Rheumatoid Arthritis Synovial Fibroblasts with Endothelial Cells

Background As an endocrine organ, adipose tissue plays a crucial role in inflammatory processes. These can be mediated by adipokines, important factors secreted by adipose tissue, displaying pro- or anti-inflammatory effects. Rheumatoid arthritis (RA) is a chronic inflammatory joint disease, in which joints are consecutively affected. We previously showed that within the murine organism RA synovial fibroblasts (SF) are able to migrate from one site to another. This may contribute to the spreading of the disease in vivo. Due to the observation that in this model RASF migrate through the vasculature, the interaction between RASF and endothelial cells appears to be critical in this process. Objectives To assess whether adipokines affect the adhesion of RASF to endothelial cells (EC) or surface molecules expressed by EC (here: E-selectin) and to identify relevant adhesion molecules whose expression is altered by adipokines. Methods RASF and EC were stimulated with the adipokines adiponectin (Ad) (10 μg/ml), visfatin (Vis) (100 ng/ml) and resistin (Res) (20 ng/ml), and “therapeutically” with methotrexate (MTX) (1.5 μM) and the glucocorticoids prednisolone (PNL) (1 μM) and dexamethasone (DXM) (1 μM). The expression of selected adhesion molecules from RASF and EC was analyzed by real-time PCR. RASF adhesion to EC was studied under static conditions using a cell-to-cell binding assay, while RASF adhesion to E-selectin was studied under flow conditions (flow rates: 18.4/30.5/60.5 ml/h) in a dynamic adhesion assay as flow conditions are required for selectins to be obtain their active conformation. Results Under static conditions, the adipokines caused an increased adhesion of RASF to EC (Ad: 37%, Vis: 23%, Res: 32%; n=6), while PNL and MTX caused a minor decrease (-7% for both; n=4). DXM did not change RASF adhesion to EC under static conditions. Under flow conditions, visfatin increased RASF adhesion to E-selectin (28%/87%/29%; n=3 for each flow rate), while DXM decreased their adhesion ability (-33%/-35%/-41%; n=3 for each flow rate). mRNA expression of VCAM-1 in RASF (n=3) was reduced by stimulation with PLN (-3.3-fold) and DXM (-8.3-fold), respectively. TNF increased ICAM-1 mRNA expression (46.5 fold) and decreased P-selectin mRNA expression (-7.7-fold) in EC (n=3). Conclusions Adipokines increase the cellular expression of adhesion molecules on RASF as well as EC and strengthen their interaction. This increased adhesion of RASF to endothelial cells mediated by adipokines could therefore influence the migration of RASF and thus the spreading of RA in vivo. As glucocorticoids and MTX antagonized these effects, our experiments can also explain some of the clinical effects observed in patients. Disclosure of Interest None declared

A1.17 Analysis of two essential sub-processes of long distance migration of synovial fibroblasts from patients with rheumatoid arthritis

Background Synovial fibroblasts (SF) play a central role in cartilage destruction and angiogenesis in rheumatoid arthritis (RA). RASF can migrate from their implantation site through the vasculature to distant implanted cartilage in the SCID mouse model. Interaction of RASF with endothelial cells (EC) is required for RASF-mediated angiogenesis and migration. In this study, expression of the E-selectin ligand CD15s, interaction of RASF with EC via E-selectin as well as kinetics of vascular growth induced by RASF in the SCID mouse model were analysed. Methods Double staining in RA and osteoarthritis (OA) synovium for CD15s and vimentin as well as single staining for CD15s on serum stimulated RA- and OASF was performed. Flow assays were performed with RA- and OASF to analyse adhesion to selectin-coated capillaries as well as TNF-stimulated or latent HUVEC. In the SCID mouse model 1.5x105 RASF and healthy cartilage were implanted ipsilaterally and cartilage without RASF contralaterally. Implant images were taken, implants removed and angiogenesis analysed by CD31 staining. Neovascularisation next to and into the implants was determined at days 3-40. Results All RA tissues (n = 12) expressed CD15s. Of those, in 67% CD15s signals were co-localised with vimentin, mainly in the sublining (50%) but also inside vessels (33%). In addition solely RA serum increased CD15s expression in RASF (80%, n = 5) and 33% of OASF (n = 3). RASF (n = 6) adhered significantly stronger to E-selectin-coated capillaries than to P-selectin at all flow rates (e.g. 18.4 ml/h: E-sel. = 16.6 ± 4.4 vs. P-sel. = 1.0 ± 1.5, p = 0.00001; 30.5 ml/h: E-sel. = 8.0 ± 3.5 vs. P-sel. = 0.2 ± 0.4, p = 0.0003). RASF adhered also significantly stronger to E-selectin coating than OASF (n = 5; e.g. 30.5 ml/h: E-sel. = 0.9 ± 0.3, p = 0.0016). Adhesion of RASF (n = 3) to TNF-stimulated HUVEC was significantly increased compared to OASF at 18.4 ml/h (9.7 ± 2.2). Anti-CD31 implant staining revealed that angiogenesis started at day 9, increased over time and was similar at both sites. Tortuous and larger vessels in the murine skin close to implants were visible early (day 3-12) and decreased over time, especially ipsilaterally. Later, during neovascularisation (day 9-40), small vessels sprouted into the implant. Conclusion The data show that, RASF can express CD15s, the ligand for E-selectin and adhere stronger to E-selectin and to TNF-stimulated EC compared to OASF. In vivo, in the SCID mouse implants, RASF can actively induce neovascularisation illustrated by a distinct pattern in vessel formation. Thus, expression of CD15s and the ability to interact with EC could represent important steps for vascular transmigration relevant for RASF long distance migration.

SAT0555 Transmigration and Mobility of Rheumatoid Arthritis Synovial Fibroblasts is Dependent on Distinct Interaction Processes with Endothelial Cells

Background Synovial fibroblasts (SF) from patients with rheumatoid arthritis (RA) are involved in cartilage destruction. The behavior of RASF to migrate over long distances could be demonstrated in the SCID mouse model of RA: RASF migrate from the primary implantation site through the vasculature to distant implanted cartilage. For extravasation of RASF, up-regulation of adhesion molecules and binding of RASF to endothelial cells (EC) is crucial. Moreover, pro-angiogenic effects of RASF are known, which also require interaction with the vasculature. Objectives To elucidate the molecules involved in the binding process of RASF to EC. Methods Synovium of RA and osteoarthritis (OA) patients were double-stained for CD15s (E-selectin ligand) and vimentin (fibroblast marker). Serum stimulated RA- and OASF were stained for CD15s. Adhesion under dynamic conditions was analyzed by a laminar flow assay using RA- and OASF. Mobile SF, adherent and tethering on EC were quantified. The capillary slides were coated with E- or P-selectin (E-/P-S) or coated with 10ng/ml TNF-activated (aHUVEC) or untreated HUVEC. Static adhesion was analyzed with a cell-to-cell binding assay. SF were stained with Calcein-AM, resuspended in RPMI or DMEM, and incubated for 15 min on a monolayer of aHUVEC or HUVEC. Adherent SF were then quantified. Results The E-S ligand CD15s was expressed in all RA tissues (n=12) but only in 1/7 OA tissues. In 67% of the RA synovia, CD15s was co-localized with vimentin. Double stained cells were mainly located in the sublining (50%) but also inside vessels (33%) or both compartments (33%). CD15s expression in cultured SF was induced by RA serum in 80% of RASF (n=5) in contrast to 33% of OASF (n=3). Healthy serum did not induce CD15s. Adhesion of RASF (n=4) to E-S-coated capillaries was significantly higher than to P-S at all flow rates (e.g. 18.4 ml/h: E-S=15.3±2.5 vs. P-S=1.5±1.5; 30.5 ml/h: E-S=9.3±1.8 vs. P-S=0.3±0.3); Adhesion of RASF to E-S coating was significantly stronger than of OASF (n=3; e.g. 18.4 ml/h: E-S=2.1±0.2). Adhesion of RASF (n=3) to aHUVEC was also significantly increased at 18.4 ml/h (9.7±1.3) compared to OASF (2.1±0.4). In the static assay (n=3) a significantly increased adhesion was visible with RASF in RPMI on aHUVEC (122.1±10.3). Adhesion of RASF to HUVEC in RPMI (78.2±7.3) vs. aHUVEC in DMEM (77.1±6.5) was similar. Adhesion of RASF to HUVEC in DMEM (58.7±5.9) showed the lowest amount of adherent RASF. OASF showed a less adhesion compared to RASF (aHUVEC in RPMI: 55.0±3.1; HUVEC in RPMI: 47.2±5.1; aHUVEC in DMEM: 62.2±2.9; HUVEC in DMEM: 50.3±3.4). Conclusions The experiments revealed an increased adhesion behavior of RASF as compared to OASF. This effect was also visible in the dynamic adhesion assay using EC and most likely based on specific CD15 expression and subsequent interaction with endothelial cells. Thus, induction of CD15s and the ability to interact with EC could represent a key mechanism of RASF by which they migrate through and leave the vascular system at specific sites. Beside selectins, other adhesion molecules which are known to be increased in RASF are likely to participate in this initial interaction of SF with EC. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.2471

A9.16 Synovial Fibroblasts from Patients with Rheumatoid Arthritis Differentiate into Distinct Fibroblast Subsets in the Presence of Cartilage

Background Rheumatoid arthritis synovial fibroblasts (RASF) migrate to distant tissue sites and damage articular cartilage. Using novel markers of RASF subsets to identify lining and sub-lining layer RASF we investigated the ability of RASF to undergo self-assembly, transmigration and cartilage degradation in vivo. Methods Healthy human cartilage was co-implanted subcutaneously into the flank of SCID mice together with RASF. On the contralateral flank, cartilage was implanted without cells. After 60 days, implants and blood were analysed. Human cells were detected using immunohistochemistry for species-specific antibodies. For in vitro studies, RASF were isolated from patients with established RA and healthy controls and the expression of cellular markers were defined. Results RASF at the ipsilateral implant differentiated into distinct fibroblast subsets in the presence of cartilage. Cells proximal to cartilage expressed markers of a lining layer phenotype (GP38, FAP, VCAM-1 and Cadherin-11). These cells attached to, invaded and degraded cartilage. Cells more distal to cartilage expressed sub-lining layer phenotype markers including CD248 and CD90. Cells expressing CD248 and CD90 were never observed in the lining layer (proximal to cartilage) and never invaded cartilage. The development of this stromal architecture mirrored that observed in vivo in the inflamed synovial membrane. This stromal pattern of distinct lining layer and sub lining layer differentiation was recapitulated in the contralateral implant that contained only cartilage. In addition, we demonstrate that RASF in vitro can be directed towards either a lining layer (GP38, FAP, VCAM-1 and Cadherin-11) or sub-lining layer phenotype (CD248 and CD90) following cytokine treatment. The lining layer, but not sub lining cell phenotype is associated with increased cartilage degradation in vitro. Conclusions RASF have an activated cell phenotype ex vivo. In vitro and in vivo they display plasticity with the capacity to differentiate into distinct cell sub-populations that morphologically distinguish between the lining and sub-lining layer of the human joint. In vivo cell sub-population differentiation occurs locally at the site of engraftment and recapitulates the lining anatomy observed at the site of origin. This phenomenon is dependent on the release of tissue factors found present in the joint microenvironment following cartilage damage. This inflammatory microenvironment is required for cartilage destruction by RASF. Cellular therapies targeting RASF specific subsets are an unexplored but important therapeutic target to modulate inflammation and may provide an avenue to minimise joint damage in patients with RA.