We assessed whether citrullinated fibronectin would alter matrix degradation, a pathologic function of syno- vial fibroblasts in rheumatoid arthritis. We found no dif- ference in the number of degrading cells in the presence of citrullinated versus untreated fibronectin. This was surprising to us. We had predicted either an increase in the number of degrading cells given the increased arthri- togenicity of citrullinated collagen and fibrinogen, or a decrease in degrading cells due to the importance of fibronectin in matrix metalloprotease production and invadopodia stability. We performed additional experi- ments that showed no alteration in invasion through Matrigel invasion chambers incubated with PAD com- pared with untreated chambers (data not shown), but in those experiments multiple matrix proteins were prob- ably citrullinated and the specific role of citrullinated fibronectin could not be addressed. However, those results are in agreement with the findings of our focal degradation experiments on fibronectin. Citrullination of fibronectin therefore appears to affect some synovialfibroblast behaviors but not all.
We also found that FAK inhibitors reduced synovial fibro- blast migration, but that FAK protein itself did not appear to be required. This is surprising because FAK is required for migration in other cell types, such as macrophages, although in the macrophage experiments described in , transwells were not coated with fibronectin, which might lead to an al- tered need for FAK. It is possible that residual amounts of FAK after deletion were sufficient to allow migration. How- ever, FAK depletion appeared more robust (Figure 4) than FAK inhibition (Figure 1). Another possibility is that cells may compensate for the loss of FAK better than inhibition of FAK, because, in the case of inhibition, the FAK molecule is still present and cannot necessarily be replaced by a similar protein. One final explanation for the discrepancy between the FAK inhibitors and FAK depletion is that FAK inhibi- tors also inhibit Pyk2 and several cyclin-dependent kinases . Perhaps inhibition of at least one of these other mole- cules is necessary in addition to inhibition of FAK itself to reduce synovialfibroblast migration in transwell experi- ments. Such off-target effects of the FAK inhibitors may contribute to their efficacy in cancer and may also make them efficacious in rheumatoid arthritis, despite the fact that depletion of FAK alone appears to have no effect in synovialfibroblast migration or arthritis.
The effects of bacterial products on selected synovialfibroblast functions were studied. Extracts of commonly encountered microorganisms were prepared by sonic or mechanical disruption. “Purified” endotoxins were prepared from selected organisms, and in some cases were purchased commercially. Normal fibroblasts were derived from synovial connective tissue obtained from amputations or arthrotomy. The cells were grown as a monolayer on glass and were nourished by a semisynthetic nutrient medium.
The synovialfibroblast has emerged as a pivotal effector cell in the inflamed joint, based on its ability to degrade the extracellular matrix and to provide chemotactic and activa- tion signals to resident parenchymal cells and infiltrating immunocytes. In vitro studies have demonstrated that cul- tured synovial fibroblasts display unique properties that set them apart from fibroblasts isolated from different anatomic sites. These cells, most importantly, release an impressive array of cytokines and growth factors, which have the capacity to stimulate and, in some cases, dampen the inflammatory response. However, the impact of these effector molecules on the pathobiology of synovi- tis must be viewed in the context of a cytokine network involving complex cellular interactions both locally and sys- temically. Exploring the interaction between monocytes and SF may yield valuable insights given the close apposi- tion of these cells in the synovial lining and the key role of TNF in the early phases of synovitis. Moreover, therapeutic strategies that inhibit SF effector pathways responsible for angiogenesis, pro-inflammatory cytokine release and matrix degradation should significantly diminish joint inflammation and prevent bone resorption.
synthesis of collagens and fibronectins are also increased by a monocyte factor. In the present study we found that the fibroblastlike cells expressed major histocompatibility complex class II (Ia-like) antigens after initial dispersion from the synovial membrane. Monocyte lineage antigens were detected on some round cells in early passage, but no T lymphocytes were identified in established cultures. There was loss of Ia expression on the fibroblastlike cells with age and passage in culture. The addition of the lymphokine, gamma interferon (recombinant), induced class II antigen (DR and DS/DQ) expression in early or late passage cells in a time- and dose-dependent manner and required protein synthesis. Furthermore, the adherent synovial fibroblastlike cells continued to be Ia-positive when examined as long as 10 d after the removal of gamma interferon. Ia expression was also induced by gamma interferon in normal skin fibroblasts. Synovial cells that could be induced to express Ia also bound a monoclonal antibody to type […]
In an attempt to replace, or at least reduce, the number of ani- mal experiments, several co-culture models of cartilage destruction have been established to date. Besides differ- ences in the co-cultured cell types and their purity (whole syn- ovial membranes, pools of synovial macrophages, fibroblasts, T- and B-cells, or polymorphic neutrophilic leucocytes), most notably the type of cartilage (-like) matrix varied widely. The types of cartilage ranged from artificial, cell-free matrix substi- tutes based on collagen/peptide matrices  or extracted car- tilage components (reconstituted from milled cartilage)  to in vitro generated, cell-containing matrices (derived from the three-dimensional (3D) culture of chondrocytes) . In artifi- cial matrices, however, the matrix structure barely resembles the natural structure and properties of native cartilage con- cerning zonal architecture, density, rigidity and composition of matrix constituents. In the case of in vitro models with isolated chondrocytes, on the other hand, cells may de-differentiate from their chondrogenic phenotype (even in 3D culture) and a re-differentiation of the expanded chondrocytes may be diffi- cult to achieve, especially in long-term cultures.
some proliferator-activated receptor gamma (Pparγ) and retinoic acid receptor gamma (Rxrγ), as well as NR target genes in syn- ovial tissues of protected rats, suggesting a new antiinflammatory and homeostatic role for NRs. Although the vitamin D re- ceptor (VDR) (also an NR) itself was not differentially expressed, there was an overrepresentation of VDR target genes among the genes with increased expres- sion in synovial tissues from arthritis- protected rats, suggesting increased VDR activity. Furthermore, genes implicated in the hydroxylation and synthesis of alter- native vitamin D active metabolites such as Cyp27a1, Cyp2j3 and Cyp11a1 (6,7) were also expressed in increased levels in the tissues of the protected rats, suggesting that the increased expression of target genes could be related at least in part to increased local production of VDR agonists (5).
to the membrane. Upon righting the chambers, dilutions of synovial fluids collected from patients with RA (or control agents) were added to the top wells and the chambers were incubated overnight at 37°C. PBS served as a negative con- trol, whereas recombinant human basic fibroblast growth fac- tor (bFGF; R&D Systems, Minneapolis, MN, USA) served as a positive control. The next morning, non-migrated cells were detached with a cotton swab, membranes were removed, fixed in methanol, and stained with Diff-Quik (Dade Behring, Deer- field, IL, USA). Checkerboard analysis was performed in a sim- ilar manner, except that the concentrations of RA SF were varied in the upper and lower chambers. Dilutions of RA SFs (1:100, 1:75 or 1:50) were added to the cell suspension in the bottom wells as well as on the opposite side of the membrane, when appropriate. Each condition was analyzed in quadrupli- cate and migrated cells from membranes mounted on glass slides were quantified in three representative high power fields. Quantification of high powered fields was accom- plished by analyzing photographs of chemotaxis spots taken with a Nikon Coolpix E5000 (5.0 megapixel) camera mounted on a Nikon Eclipse TS100 inverted microscope. Chemotaxis data appeared normally distributed based on examination of histogram plots, and statistical analysis was performed using a Student's t-test.
classes. Fibronectin guides fibroblast migration as an immobilized substrate and attractant in the leading edge of the pannus (haptotaxis) . The extra domain-A fibronectin isoform is associated with the activated, trans- formed state of type B lining cells . The interaction between connecting sequence-1 fibronectin (or vascular cell adhesion molecule-1) and α4β1 (very late activation antigen-4) may play a role in the proliferation of synovial lining and lymphocyte migration . EMMPRIN (M r ≈ 58 000) is an integral plasma membrane glycoprotein of the pericellular matrix belonging to the immunoglobulin superfamily, previously referred to as tumor-cell derived collagenase stimulatory factor. It is identical to the M6 leukocyte activation antigen, and highly homologous to rat OX-47 or CE9, mouse basigin or gp42, and chicken HT-7 or neurothelin molecules. Reciprocal immunoprecipitation, cell surface crosslinking and immunofluorescence co- localization experiments demonstrated that EMMPRIN can form a complex with integrins α 3 β 1 and α 6 β 1, which may play a role in the synovial membrane . Many other ECM–fibroblast interactions are of potential relevance in the synovial membrane (Table 3) [46–61].
arthritis, supporting a central role for this factor in inflam- matory bone loss. Our findings demonstrate that the effect of inflammation on DKK1 synthesis is not direct, but instead depends on an increase in local glucocorticoid levels through the induction of the 11b-HSD1 enzyme (illustrated schematically in Figure 5). High levels of gluco- corticoids are linked with a range of conditions that detri- mentally affect bone through uncoupling bone formation from resorption, but this is the first study to show a link between effects of glucocorticoids on synovial tissue and uncoupling of bone metabolism through a paracrine effect. Glucocorticoids also cause a coordinated change in pro- duction of Wnt signalling modulators by synovial fibro- blasts that extends beyond DKK1 with an increase in several antagonists and a suppression of agonists. These findings are in accordance with the effects of glucocorti- coids on Wnt production by osteoblasts , a cell type closely related developmentally to the synovialfibroblast.
process with the appearance of small clefts which extend and coalesce to form the synovial cavity . Cells of the interzone then give rise to the synovium, as well as other joint structures, including articular cartilage, me- nisci and ligaments [14,15]. However, whether every sin- gle cell in the synovium originates from the joint interzone is not known. Macrophages and endothelial cells are unlikely to descend from the joint interzone and instead are most likely to derive from the bone mar- row . With regards fibroblasts, we could postulate a dual origin, with FLSs of the lining being progeny of the joint interzone and the fibroblasts of the sublining pos- sibly deriving from the bone marrow or, more generally, blood-borne fibroblasts. In this regard, third passage primary FLS cultures established from normal synovial joints of mice carrying green-fluorescent protein (GFP)- positive bone marrow comprised approximately 1% of GFP-positive (bone marrow-derived) fibroblast-like cells . Distinct origins of the synovialfibroblast popula- tions may be the basis of functional differences and would strengthen the notion that the FLSs of the lining and the fibroblasts of the sublining are distinct cell types. The modern technologies of lineage tracing will shed light on the origins of the fibroblasts in the synovium.
As an E3 ubiquitin ligase on the ER membrane, SYN functions as an ER-associated degradation system in both yeast and mammals [8,9]. The biological functions of SYN were analyzed in SYN transgenic mice and heterozygous knockout mice because the homozygous mice are embryonic-lethal . Interestingly, the expression level of SYN correlates signifi- cantly with the onset of arthropathy: Increased SYN expres- sion causes synovium overgrowth and spontaneous arthropathy, whereas reduced SYN expression (heterozygous mutant mice) is associated with resistance to CIA . Our finding that the suppression of SYN inhibits IL-1 β -induced synovialfibroblast proliferation provides a direct rationale for SYN as a potential target for the treatment of RA. It will be extremely interesting to investigate the effects of gene thera- peutic delivery of dominant-negative SYN or its siRNA (small interfering RNA) on the development of arthritis in animals such as DBA mice with CIA.
Previous studies have reported that mesenchymal stem cells (MSC) may be isolated from the synovial membrane by the same protocol as that used for synovialfibroblast cultivation, suggesting that MSC correspond to a subset of the adherent cell population, as MSC from the stromal compartment of the bone marrow (BM). The aims of the present study were, first, to better characterize the MSC derived from the synovial membrane and, second, to compare systematically, in parallel, the MSC-containing cell populations isolated from BM and those derived from the synovium, using quantitative assays. Fluorescent-activated cell sorting analysis revealed that both populations were negative for CD14, CD34 and CD45 expression and that both displayed equal levels of CD44, CD73, CD90 and CD105, a phenotype currently known to be characteristic of BM-MSC. Comparable with BM-MSC, such MSC-like cells isolated from the synovial membrane were shown for the first time to suppress the T-cell response in a mixed lymphocyte reaction, and to express the enzyme indoleamine 2,3-dioxygenase activity to the same extent as BM-MSC, which is a possible mediator of this suppressive activity. Using quantitative RT-PCR these data show that MSC-like cells from
Synovial tissues were obtained at the time of total knee joint replacement from six patients with RA; these patients were female, aged (mean ± standard deviation) 67.3 ± 9.1 years, and their serum C-reactive protein levels were 3.4 ± 2.4 mg/ dl. Of the six RA patients, five women took prednisolone, four women took methotrexate, two women took bucillamine, and one woman took leflunomide. Signed consent forms were obtained prior to the operation, and the experimental protocol was approved in advance by the Ethics Committee of Tokyo Medical and Dental University. RA was diagnosed according to the criteria of the American College of Rheumatology . RA fibroblast-like synovial cells (FLS) were prepared from the synovial tissues as described previously , and were cul- tured in DMEM with heat-inactivated 10% FCS (Sigma- Aldrich). After incubation with or without DHMEQ for 24 h, RA-FLS were collected and lysed with RIPA lysis buffer (Upstate, Lake Placid, NY, USA). After debris was eliminated by centrifugation, 5 μg proteins in the supernatant were sepa- rated by 10% SDS-PAGE under reducing conditions, and were transferred to a polyvinylidene difluoride membrane. The membranes were blocked with 4% Block Ace (Snow Brand Milk Products, Sapporo, Japan) in PBS containing 0.1% Tween 20 overnight, were incubated with anti-RANKL mono- clonal antibody 70513 (R&D Systems) or with anti-β-actin monoclonal antibody AC-15 (Sigma-Aldrich) in PBS contain- ing 0.1% Tween 20 with 0.4% Block Ace for 1 hour, and were
First, we studied expression of ED-A fibronectin in OA. We found formation of ED-A fibronectin in αSMA positive myofibroblasts isolated from OA synovial tissue, induction of ED-A fibronectin production by TGFβ, and co-localization of ED-A fibronectin and αSMA in the OA synovium. This is in line with previous findings sug- gesting that ED-A fibronectin is expressed in TGFβ in- duced myofibroblast differentiation , and that TGFβ is increased in OA synovial fluid . Further, the role of a general wound healing response in OA is supported by findings of increased fibronectin secretion after blunt cartilage trauma in a bovine model of OA , increased fibrosis and joint stiffening in OA , and by the pro- tective effect of blocking TGFβ in experimental OA in mice .
Studies of synovial tissue may also play an important role in the development of rational therapies in which biotech- nology products are used to influence defined patho- genetic mechanisms . The design of optimal treatment regimens for interventions with agents such as monoclonal antibodies, soluble receptors, cytokines and peptides can be facilitated by information regarding the actual achieve- ment of the biological effect at the site of inflammation. Such studies will also provide insight into the mode of action of such agents. Additionally, analysis of serial biopsy samples during treatment may provide useful alternative end points for both joint inflammation and joint destruc- tion. This approach could lead to a rapid screening method that would require relatively low numbers of patients to predict the effects of novel antirheumatic strategies. Studies of the relation between a defined modification of inflammation and the clinical course could also produce information about the pathogenesis of rheumatic diseases.
monocytes/macrophages. At the inflammatory site, IL-1 is a potent inducer of the production of prostaglandin E2 (PGE2) and metalloproteinases on fibroblast-like cells, thus triggering tissue damage. The biological activity of IL-1 is counterbalanced by two types of inhibitors: the IL-1 receptor antagonist (IL-1Ra) which competitively binds IL-1 receptor without
In addition, to determine whether fut1 was expressed in RA synovial fibroblasts, qPCR was performed. We found that expression of fut1 mRNA in nonstimulated RA synovial fibroblasts was significantly higher than in nonstimulated NL synovial fibroblasts (3.5 ± 0.7 fold in- creased (Figure 2E). Expression of fut1 mRNA in TNF-α stimulated RA synovial fibroblasts was also significantly elevated compared to that in TNF-α stimulated NL syn- ovial fibroblasts (4.6 ± 0.8-fold increased). To further validate and distinguish cellular fut1 staining in RA ST, we performed dual immunofluorescence staining on RA ST fibroblasts and macrophages. In Figure 2F, the left panel is fut1 staining in RA ST (green). The middle panel is cadherin-11 (fibroblast marker) staining in RA ST (red). The right panel is the merge of the previous two panels. The arrow indicates fut1- and cadherin-11- positive cells respectively (yellow), indicating that fut1 is expressed on fibroblasts in RA ST. In Figure 2G, the left panel is fut1 staining in RA ST (green). The middle panel is CD68 (macrophage marker) staining in RA ST (red). The right panel is the merge of these two panels. The arrows indicate fut1 on CD68-positive cells (yellow), indicating that fut1 is expressed on macrophages in RA ST. The blue background is DAPI staining. IgG control staining was performed and showed no fluorescence staining.