Stephanie Lefèvre

Synovial fibroblasts spread rheumatoid arthritis to unaffected joints

Active rheumatoid arthritis originates from few joints but subsequently affects the majority of joints. Thus far, the pathways of the progression of the disease are largely unknown. As rheumatoid arthritis synovial fibroblasts (RASFs) which can be found in RA synovium are key players in joint destruction and are able to migrate in vitro, we evaluated the potential of RASFs to spread the disease in vivo. To simulate the primary joint of origin, we implanted healthy human cartilage together with RASFs subcutaneously into severe combined immunodeficient (SCID) mice. At the contralateral flank, we implanted healthy cartilage without cells. RASFs showed an active movement to the naive cartilage via the vasculature independent of the site of application of RASFs into the SCID mouse, leading to a marked destruction of the target cartilage. These findings support the hypothesis that the characteristic clinical phenomenon of destructive arthritis spreading between joints is mediated, at least in part, by the transmigration of activated RASFs.

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.

Cell culture and passaging alters gene expression pattern and proliferation rate in rheumatoid arthritis synovial fibroblasts

IntroductionRheumatoid arthritis synovial fibroblasts (RASF) are key players in synovial pathophysiology and are therefore examined extensively in various experimental approaches. We evaluated, whether passaging during culture and freezing has effects on gene expression and cell proliferation.MethodsRASF were passaged for up to 8 passages. RNA was isolated after each passage and cDNA arrays were performed to evaluate the RNA expression pattern during passaging. In addition, doubling time of the cells was also measured.ResultsFrom passages 2-4, mRNA expression did not change significantly. Gene expression in RASF started to change in passages 5-6 with 7-10% differentially expressed genes. After passages 7-8, more than 10% of the genes were differentially expressed. The doubling rate was constant for up to 5 passages and decreased after passages 6-8. After freezing, gene expression of the second passage is comparable to gene expression prior to freezing.ConclusionsThe results of this study show, that experiments, which examine gene expression of RASF and shall reflect or imitate an in vivo situation, should be limited to early culture passages to avoid cell culture effects. It is not necessary to stop culturing SF after a few passages, but to keep the problems of cell culture in mind to avoid false positive results. Especially, when large-scale screening methods on mRNA level are used. Of note, freezing does not affect gene expression substantially.

Visfatin/Pre-B-cell Colony-enhancing Factor (PBEF), a Proinflammatory and Cell Motility-changing Factor in Rheumatoid Arthritis

Background: The adipokine visfatin exerts proinflammatory effects in synovial fibroblasts of patients with rheumatoid arthritis (RA). Results: Visfatin induces high amounts of chemokines, creating a microenvironment of enhanced fibroblast motility. Conclusion: Visfatin is therefore very capable of contributing to the inflammatory state of RA. Significance: Enlightening visfatin pathophysiology may lead to possible therapeutic targeting in the future. Adipokines such as adiponectin and visfatin/pre-B-cell colony-enhancing factor (PBEF) have been recently shown to contribute to synovial inflammation in rheumatoid arthritis (RA). In this study, we evaluated the pathophysiological implication of visfatin/PBEF in the molecular patterns of RA synovial tissue, focusing on RA synovial fibroblasts (RASFs), key players in RA synovium. Expression of visfatin/PBEF in synovial fluid and tissue of RA patients was detected by immunoassays and immunohistochemistry. RASFs were stimulated with different concentrations of visfatin/PBEF over varying time intervals, and changes in gene expression were evaluated at the RNA and protein levels using Affymetrix array, real-time PCR, and immunoassays. The signaling pathways involved were identified. The influence of visfatin/PBEF on fibroblast motility and migration was analyzed. In RA synovium, visfatin/PBEF was predominantly expressed in the lining layer, lymphoid aggregates, and interstitial vessels. In RASFs, visfatin/PBEF induced high amounts of chemokines such as IL-8 and MCP-1, proinflammatory cytokines such as IL-6, and matrix metalloproteinases such as MMP-3. Phosphorylation of p38 MAPK was observed after visfatin/PBEF stimulation, and inhibition of p38 MAPK showed strong reduction of visfatin-induced effects. Directed as well as general fibroblast motility was increased by visfatin/PBEF-induced factors. The results of this study indicate that visfatin/PBEF is involved in synovial fibroblast activation by triggering fibroblast motility and promoting cytokine synthesis at central sites in RA synovium.

The therapeutic use of osmotic minipumps in the severe combined immunodeficiency (SCID) mouse model for rheumatoid arthritis

Objectives: The viral gene transfer of interleukin 1 receptor antagonist (IL1ra) and interleukin 10 (IL10) into rheumatoid arthritis (RA) synovial fibroblasts (RASFs) has shown protective effects on cartilage destruction in the severe combined immunodeficiency (SCID) mouse model of RA. Nevertheless, side effects of viral transduction are possible and a number of cytokines or cytokine inhibitors are not available encoded in viral vehicles. As the production of viruses coding for bioactive proteins is cost and time intensive, we established an in vivo long-term release model using osmotic minipumps in the SCID mouse model for RA. Methods: Isolated RASFs were cultured for four passages and coimplanted together with human cartilage and an Alzet osmotic miniature pump model 2004, containing 200 μl of IL10 and IL1ra for 40 days in SCID mice. Implants were removed after 40 days and evaluated histologically. The actual rates of IL10 and IL1ra in murine serum were measured by ELISA. Results: Release of IL10 and IL1ra by the pumps was effective as both could be measured in significant amounts in the serum of the mice. IL10 and IL1ra release showed protective effects towards the coimplanted cartilage, similar to the adenovirally IL10/IL1ra-transduced RASFs. The mean (SD) invasion scores for the implants with the osmotic pumps were: invasion 0.7 (0.5), degradation 0.5 (0.3) (all parameters significant vs controls, p<0.05). Conclusions: The results demonstrate that the combination of osmotic pumps with the SCID mouse model for RA can be used as approach for application and evaluation of cartilage-protective molecules. Furthermore, the effect of cartilage-protective cytokines is independent of the type of application.

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.

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.

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-β, 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-β 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.

Interactions between rheumatoid arthritis synovial fibroblast migration and endothelial cells

Leukocytes travel within the circulation and enter connective tissues by interactions with endothelium of postcapillary venules mediated by cell adhesion molecules, summarized as the leukocyte adhesion cascade. In the severe combined immunodeficient (SCID) mouse model, rheumatoid arthritis (RA) synovial fibroblasts (SF) migrated to distant cartilage through the vasculature. Therefore, RASF adhesion toward endothelial cells (EC) and E‐ and P‐selectins were analyzed. Cell‐to‐cell binding assays between SF and EC were performed. Interactions of SF with tumor necrosis factor α (TNFα)‐activated EC or selectins were analyzed in flow adhesion assays. Immunohistochemistry for E‐selectin ligand CD15s was performed. CD15s induction in RASF by human serum or media was evaluated. Wild‐type and E‐/‐/P‐/‐Selectin‐SCID mice were used for inverse‐wrap surgery. After laser‐mediated microdissection, real‐time PCR for E‐/P‐selectin/vascular cell adhesion molecule 1 was performed. Adhesion between SF/EC under static conditions was highest in Roswell Park Memorial Institute‐cultured RASF to TNFαα‐activated human umbilical vein endothelial cells (2.25‐fold) and RASF adhesion was higher toward venous than arterial EC (Dulbeccos modified eagle medium P = 0.0419, RPMI P = 0.0119). In flow chamber assays, RASF adhesion to E‐selectin was higher than to P‐selectin (e.g. 0.9 dyn cm−2 P = 0.0001). Osteoarthritis synovial fibroblasts showed lower rolling/adhesion properties (e.g. 0.5 dyn cm−2, P = 0.0010). RASF adhesion to TNFαα‐activated EC was increased (e.g. 0.9 dyn cm−2, P = 0.0061). CD15s induction in RASF was strongest in RA serum. Vimentin/CD15s double‐positive cells were detectable. In E‐/P‐selectin‐deficient mice, contralateral invasion was reduced (P = 0.023). E‐ and P‐selectin, and vascular cell adhesion molecule 1 expression in EC of implants was confirmed. Our data indicate that the milieu within vessels induces CD15s which enables RASF to interact with E‐selectin/EC under flow. Therefore, RASF may migrate to distant sites and leave the vasculature similarly to leukocytes.

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.