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1.  Mosaic chromosomal aberrations in synovial fibroblasts of patients with rheumatoid arthritis, osteoarthritis, and other inflammatory joint diseases 
Arthritis Research  2001;3(5):319-330.
Chromosomal aberrations were comparatively assessed in nuclei extracted from synovial tissue, primary-culture (P-0) synovial cells, and early-passage synovial fibroblasts (SFB; 98% enrichment; P-1, P-4 [passage 1, passage 4]) from patients with rheumatoid arthritis (RA; n = 21), osteoarthritis (OA; n = 24), and other rheumatic diseases. Peripheral blood lymphocytes (PBL) and skin fibroblasts (FB) (P-1, P-4) from the same patients, as well as SFB from normal joints and patients with joint trauma (JT) (n = 4), were used as controls. Analyses proceeded by standard GTG-banding and interphase centromere fluorescence in situ hybridization. Structural chromosomal aberrations were observed in SFB (P-1 or P-4) from 4 of 21 RA patients (19%), with involvement of chromosome 1 [e.g. del(1)(q12)] in 3 of 4 cases. In 10 of the 21 RA cases (48%), polysomy 7 was observed in P-1 SFB. In addition, aneusomies of chromosomes 4, 6, 8, 9, 12, 18, and Y were present. The percentage of polysomies was increased in P-4. Similar chromosomal aberrations were detected in SFB of OA and spondylarthropathy patients. No aberrations were detected in i) PBL or skin FB from the same patients (except for one OA patient with a karyotype 45,X[10]/46,XX[17] in PBL and variable polysomies in long-term culture skin FB); or ii) synovial tissue and/or P-1 SFB of normal joints or of patients with joint trauma. In conclusion, qualitatively comparable chromosomal aberrations were observed in synovial tissue and early-passage SFB of patients with RA, OA, and other inflammatory joint diseases. Thus, although of possible functional relevance for the pathologic role of SFB in RA, these alterations probably reflect a common response to chronic inflammatory stress in rheumatic diseases.
PMCID: PMC64845  PMID: 11549374
osteoarthritis; rheumatoid arthritis; spondylarthropathy; synovial fibroblasts; trisomy/polysomy 7
2.  Cytokine mRNA and protein expression in primary-culture and repeated-passage synovial fibroblasts from patients with rheumatoid arthritis 
Arthritis Research  2001;4(2):117-125.
Constitutive mRNA expression and secretion of proinflammatory and anti-inflammatory cytokines was comparatively analyzed in rheumatoid arthritis (RA) synovial fibroblasts (SFB), isolated from primary culture or derived by repeated passage; normal-skin fibroblasts were used as controls. First-passage RA-SFB (n = 3) secreted large amounts of IL-6 (15,800 ± 2,110 pg/ml; mean ± SEM), but only limited amounts of tumor necrosis factor (TNF)-α (22.1 ± 1.1 pg/ml) or IL-10 (35.7 ± 34.2 pg/ml; only one of three samples was positive). IL-1β, IL-15, and IL-18 were not detectable at the protein level and showed very low mRNA levels by semiquantitative RT-PCR. In repeated-passage RA-SFB (tenth passage), protein secretion was significantly lower for IL-6 (one-twentieth of the initial level) and TNF-α (two-thirds), and markedly reduced for IL-10 (one-quarter, with only one of three samples positive). While the decrease of IL-10 protein from first to tenth passage was paralleled by a corresponding decrease of mRNA, the relative mRNA levels for IL-6 and TNF-α were actually increased (20-fold and 300-fold, respectively), indicating post-transcriptional and/or post-translational regulation of these cytokines. Due to highly variable levels among individual patients, however, no significant differences were observed for any cytokine mRNA between primary-culture and repeated-passage RA-SFB (ninth passage). Likewise, no significant differences were detectable between RA-SFB and normal-skin fibroblasts (primary-culture and repeated-passage). By producing high amounts of IL-6 and limited amounts of TNF-α, RA-SFB may contribute to the (im)balance of proinflammatory and anti-inflammatory cytokines in the inflamed joint.
PMCID: PMC83845  PMID: 11879547
cytokines; inflammation; mRNA; rheumatoid arthritis; synovial fibroblasts
3.  CD14-Negative Isolation Enhances Chondrogenesis in Synovial Fibroblasts 
Tissue Engineering. Part A  2009;15(11):3261-3270.
Synovial membrane has been shown to contain mesenchymal stem cells. We hypothesized that an enriched population of synovial fibroblasts would undergo chondrogenic differentiation and secrete cartilage extracellular matrix to a greater extent than would a mixed synovial cell population (MSCP). The optimum doses of transforming growth factor beta 1 (TGF-β1) and insulin-like growth factor 1 (IGF-1) for chondrogenesis were investigated. CD14-negative isolation was used to obtain a porcine cell population enriched in type-B synovial fibroblasts (SFB) from an MSCP. The positive cell surface markers in SFB were CD90, CD44, and cadherin-11. SFB and MSCP were cultured in the presence of 20 ng/mL TGF-β1 for 7 days, and SFB were demonstrated to have higher chondrogenic potential. Further dose–response studies were carried out using the SFB cells and several doses of TGF-β1 (2, 10, 20, and 40 ng/mL) and/or IGF-1 (1, 10, 100, and 500 ng/mL) for 14 days. TGF-β1 supplementation was essential for chondrogenesis and prevention of cell death, whereas IGF-1 did not have a significant effect on the SFB cell number or glycosaminoglycan production. This study demonstrates that the CD14-negative isolation yields an enhanced cell population SFB that is more potent than MSCP as a cell source for cartilage tissue engineering.
PMCID: PMC2792095  PMID: 19382853
4.  Macrophage specificity of three anti-CD68 monoclonal antibodies (KP1, EBM11, and PGM1) widely used for immunohistochemistry and flow cytometry 
Annals of the Rheumatic Diseases  2004;63(7):774-784.
Objectives: To investigate the specificity of three anti-CD68 monoclonal antibodies (mAbs) for macrophages (Mφ) in immunohistochemistry (IHC) and flow cytometry (FACS).
Methods: IHC was performed on cryostat sections of rheumatoid arthritis (RA) and osteoarthritis (OA) synovial membranes using the anti-CD68 mAbs KP1, EBM11, and PGM1, and the fibroblast (FB) markers CD90 and prolyl 4-hydroxylase. Expression of CD68 was also analysed by FACS on the monocytic cell lines THP-1 and U937, as well as on synovial fibroblasts (SFB), skin FB, and gingival FB (both surface and intracellular staining).
Results: In IHC, there was an overlap between CD68 (mAbs KP1 and EBM11) and the FB markers CD90/prolyl 4-hydroxylase in the lining layer, diffuse infiltrates, and stroma of RA and OA synovial membranes. In FACS analysis of THP-1 and U937 cells, the percentage of cells positive for the anti-CD68 mAbs KP1 and EBM11 progressively increased from surface staining of unfixed cells, to surface staining of pre-fixed cells, to intracellular staining of the cells. Upon intracellular FACS of different FB, nearly all cells were positive for KP1 and EBM11, but only a small percentage for PGM1. In surface staining FACS, a small percentage of FB were positive for all three anti-CD68 mAbs.
Conclusion: An overlap between CD68 (mAbs KP1 or EBM11) and the FB markers CD90 or prolyl 4-hydroxylase may prevent unequivocal identification of Mφ in synovial tissue by IHC or in monocytic cells and FB upon intracellular FACS. This may be due to sharing of common markers by completely different cell lineages.
PMCID: PMC1755048  PMID: 15194571
5.  Constitutive upregulation of the transforming growth factor-β pathway in rheumatoid arthritis synovial fibroblasts 
Genome-wide gene expression was comparatively investigated in early-passage rheumatoid arthritis (RA) and osteoarthritis (OA) synovial fibroblasts (SFBs; n = 6 each) using oligonucleotide microarrays; mRNA/protein data were validated by quantitative PCR (qPCR) and western blotting and immunohistochemistry, respectively. Gene set enrichment analysis (GSEA) of the microarray data suggested constitutive upregulation of components of the transforming growth factor (TGF)-β pathway in RA SFBs, with 2 hits in the top 30 regulated pathways. The growth factor TGF-β1, its receptor TGFBR1, the TGF-β binding proteins LTBP1/2, the TGF-β-releasing thrombospondin 1 (THBS1), the negative effector SkiL, and the smad-associated molecule SARA were upregulated in RA SFBs compared to OA SFBs, whereas TGF-β2 was downregulated. Upregulation of TGF-β1 and THBS1 mRNA (both positively correlated with clinical markers of disease activity/severity) and downregulation of TGF-β2 mRNA in RA SFBs were confirmed by qPCR. TGFBR1 mRNA (only numerically upregulated in RA SFBs) and SkiL mRNA were not differentially expressed. At the protein level, TGF-β1 showed a slightly higher expression, and the signal-transducing TGFBR1 and the TGF-β-activating THBS1 a significantly higher expression in RA SFBs than in OA SFBs. Consistent with the upregulated TGF-β pathway in RA SFBs, stimulation with TGF-β1 resulted in a significantly enhanced expression of matrix-metalloproteinase (MMP)-11 mRNA and protein in RA SFBs, but not in OA SFBs. In conclusion, RA SFBs show broad, constitutive alterations of the TGF-β pathway. The abundance of TGF-β, in conjunction with an augmented mRNA and/or protein expression of TGF-β-releasing THBS1 and TGFBR1, suggests a pathogenetic role of TGF-β-induced effects on SFBs in RA, for example, the augmentation of MMP-mediated matrix degradation/remodeling.
PMCID: PMC2206335  PMID: 17594488
6.  Enhance and Maintain Chondrogenesis of Synovial Fibroblasts by Cartilage Extracellular Matrix Protein Matrilins 
Cartilage-specific extracellular matrix (ECM) proteins have been proposed to play key roles in modulating cellular phenotypes during chondrogenesis of mesenchymal stem cells. Matrilin (MATN) 1 and 3 are among the most up-regulated ECM proteins during chondrogenesis. The aim of this study was to analyze their roles in chondrogenesis of mesenchymal fibroblasts from synovium.
Primary synovial fibroblasts (SFBs) were purified from porcine synovium and incubated in pellet culture for 18 days. Chondrogenesis of SFB was analyzed by histological staining with safranin-O/fast green, and by quantifying glycosaminoglycans with dimethylmethylene blue assay. The mRNA levels of chondrogenic markers including collagen II, aggrecan, and Sox 9 were quantified by real-time RT-PCR, while the protein levels of Col II and matrilins were determined by western blot analysis.
SFBs underwent chondrogenesis after incubation with TGF-β1 for three days; however, this process was attenuated during the subsequent incubation period. Expression of a MATN1 or 3 cDNA maintained and further enhanced chondrogenesis of SFBs as shown by increased cartilaginous matrix areas, elevated amount of glycosaminoglycans, and stimulated expression of chondrogenic markers.
Our findings suggest a novel function for MATN1 and 3 to maintain and enhance chondrogenesis of mesenchymal fibroblasts initiated by TGF-β. Our results also support a critical role of cartilage-specific ECM proteins to modulate cellular phenotypes in the microenvironment during chondrogenic differentiation.
PMCID: PMC2596998  PMID: 18282772
Matrilin; Synovium; Mesenchymal stem cells; Chondrogenesis; TGF-β
7.  Predominant activation of MAP kinases and pro‐destructive/pro‐inflammatory features by TNF α in early‐passage synovial fibroblasts via TNF receptor‐1: failure of p38 inhibition to suppress matrix metalloproteinase‐1 in rheumatoid arthritis 
Annals of the Rheumatic Diseases  2007;66(8):1043-1051.
To examine the relative importance of tumour necrosis factor‐receptor 1 (TNF‐R1) and TNF‐R2 and their signalling pathways for pro‐inflammatory and pro‐destructive features of early‐passage synovial fibroblasts (SFB) from rheumatoid arthritis (RA) and osteoarthritis (OA).
Cells were stimulated with tumour necrosis factor (TNF)α or agonistic anti‐TNF‐R1/TNF‐R2 monoclonal antibodies. Phosphorylation of p38, ERK and JNK kinases was assessed by western blot; proliferation by bromodesoxyuridine incorporation; interleukin (IL)6, IL8, prostaglandin E2 (PGE2) and matrix metalloproteinase (MMP)‐1 secretion by ELISA; and MMP‐3 secretion by western blot. Functional assays were performed with or without inhibition of p38 (SB203580), ERK (U0126) or JNK (SP600125).
In RA‐ and OA‐SFB, TNFα‐induced phosphorylation of p38, ERK or JNK was exclusively mediated by TNF‐R1. Reduction of proliferation and induction of IL6, IL8 and MMP‐1 were solely mediated by TNF‐R1, whereas PGE2 and MMP‐3 secretion was mediated by both TNF‐Rs. In general, inhibition of ERK or JNK did not significantly alter the TNFα influence on these effector molecules. In contrast, inhibition of p38 reversed TNFα effects on proliferation and IL6/PGE2 secretion (but not on IL8 and MMP‐3 secretion). The above effects were comparable in RA‐ and OA‐SFB, except that TNFα‐induced MMP‐1 secretion was reversed by p38 inhibition only in OA‐SFB.
In early‐passage RA/OA‐SFB, activation of MAPK cascades and pro‐inflammatory/pro‐destructive features by TNFα is predominantly mediated by TNF‐R1 and, for proliferation and IL6/PGE2 secretion, exclusively regulated by p38. Strikingly, RA‐SFB are insensitive to p38 inhibition of MMP‐1 secretion. This indicates a resistance of RA‐SFB to the inhibition of pro‐destructive functions and suggests underlying structural/functional alterations of the p38 pathway, which may contribute to the pathogenesis or therapeutic sensitivity of RA, or both.
PMCID: PMC1954705  PMID: 17223661
TNF‐receptor; synovial fibroblast; p38 MAP kinase; interleukin; matrix metalloproteinase
8.  Apathogenic, intestinal, segmented, filamentous bacteria stimulate the mucosal immune system of mice. 
Infection and Immunity  1993;61(1):303-306.
Segmented filamentous bacteria (SFBs) are apathogenic autochthonous bacteria in the murine small intestine that preferentially attach to Peyer's patch epithelium. SFBs have never been cultured in vitro. We have studied the effects of SFBs on the immune system of the host. Mice monoassociated with SFBs were compared with germ-free mice and with mice without SFBs but with a specific-pathogen-free (SPF) gut flora. SFBs versus no microbial flora raised the number of lymphoid cells in the lamina propria of the ileal and cecal mucosa, raised the number of immunoglobulin A (IgA)-secreting cells in the intestinal mucosa, produced elevated IgA titers in serum and intestinal secretions, and enhanced the concanavalin A-induced proliferative responses of mesenteric lymph node cells. The SPF flora had effects similar to but less pronounced than those mediated by SFBs. The results indicate that SFBs stimulate the mucosal immune system to a greater extent than do other autochthonous gut bacteria.
PMCID: PMC302719  PMID: 8418051
9.  Hyaluronic acid production in vitro by synovial lining cells from normal and rheumatoid joints. 
Annals of the Rheumatic Diseases  1985;44(10):647-657.
Organ cultures and primary cell cultures were established from synovial tissue collected from patients with rheumatoid arthritis. Hyaluronic acid measured by the incorporation of [3H]glucosamine into the polysaccharide was found to be synthesised in the cultures immediately after transfer from in-vivo to in-vitro conditions. This was in contrast to the primary cultures established from cells isolated from normal joints. The latter cells did not synthesise any detectable hyaluronate. 90-100% of the cells in primary culture were found to be esterase positive, indicating their macrophage nature. The molecular weight of the hyaluronate produced by the pathological cells was low (approximately 50 000) compared with the molecular weight of hyaluronate found in joint fluid from normal or rheumatoid joints. Cell lines of fibroblasts established from rheumatoid joints and studied after four or seven passages also produced hyaluronate of low molecular weight. It is known that similar cell lines from normal joints produce a high molecular weight polymer.
PMCID: PMC1001731  PMID: 4051585
10.  Collagenases in human synovial fluid 
Journal of Clinical Investigation  1969;48(11):2104-2113.
An enzyme which degrades native collagen at neutral pH has been isolated from cultures of rheumatoid synovium in vitro, but little or no collagenolytic activity has been found in homogenates of fresh rheumatoid synovium. Similar to most other mammalian collagenases this synovial enzyme is readily inhibited by serum proteins. Proteins of synovial fluid are derived largely from serum and synovial fluid from noninflamed joints was found to inhibit synovial collagenase; the inhibitor was destroyed by trypsin, but not by hyaluronidase. Inhibitory activity was reduced in approximately one-half of the fluids from patients with rheumatoid arthritis. In a total of nine synovial fluids, collagenolytic activity was detectable. This activity was not present in constant amounts in synovial fluids aspirated at different times from the same patient and tended to vary inversely with the titer of inhibitory proteins. The collagenolytic activity in the synovial fluids from different patients was variably inhibited by serum proteins. Two distinct collagenases were detected in some rheumatoid synovial fluids and separated by gel filtration. One, labeled “B” enzyme, with an estimated molecular weight 20,000-25,000 resembled the collagenase obtained from synovial cultures. The other, labeled “A” enzyme degraded collagen fibrils as well as collagen in solution. Disc electrophoresis on acrylamide gels and electron microscopy of segment long spacing (SLS) aggregates of reaction products of the enzymes at 27°C demonstrated that both “A” and “B” enzymes cleaved collagen molecules at a point three-quarters from the amino terminal end of the molecule. Thus collagen degradation in rheumatoid arthritis could result from the operation of these two collagenases.
PMCID: PMC297464  PMID: 4309955
11.  Cell culture and passaging alters gene expression pattern and proliferation rate in rheumatoid arthritis synovial fibroblasts 
Rheumatoid 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.
RASF 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.
From 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.
The 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.
PMCID: PMC2911867  PMID: 20462438
12.  Development and Retranslational Validation of an In Vitro Model to Characterize Acute Infections in Large Human Joints 
BioMed Research International  2014;2014:848604.
Bacterial infections can destroy cartilage integrity, resulting in osteoarthritis. Goal was to develop an in vitro model with in vivo validation of acute joint inflammation. Inflammation in cocultivated human synovial fibroblasts (SFB), chondrocytes (CHDR), and mononuclear cells (MNC) was successively relieved for 10 days. Articular effusions from patients with (n = 7) and without (n = 5) postoperative joint infection in healthy patients (ASA 1-2) were used as model validation. Inflammation in vitro resulted in an enormous increase in IL-1 and a successive reduction in SFB numbers. CHDR however, maintained metabolic activity and proteoglycan synthesis. While concentrations of bFGF in vivo and in vitro rose consistently, the mRNA increase was only moderate. Concurring with our in vivo data, cartilage-specific IGF-1 steadily increased, while IGF-1 mRNA in the CHDR and SFB did not correlate with protein levels. Similarly, aggrecan (ACAN) protein concentrations increased in vivo and failed to correlate in vitro with gene expression in either the CHDR or the SFB, indicating extracellular matrix breakdown. Anabolic cartilage-specific BMP-7 with highly significant intra-articular levels was significantly elevated in vitro on day 10 following maximum inflammation. Our in vitro model enables us to validate early inflammation of in vivo cell- and cytokine-specific regulatory patterns. This trial is registered with MISSinG, DRKS 00003536.
PMCID: PMC4021994  PMID: 24877141
13.  Some in-vitro comparisons of synovial cells dispersed by trypsin from rheumatoid and nonrheumatoid synovium. 
Annals of the Rheumatic Diseases  1981;40(3):286-292.
Life spans, growth rate, glucose utilisation, response to hydrocortisone, and intracellular activity of lysosomal N-acetyl-beta-glucosaminidase of rheumatoid synovial cells in culture were compared with these properties in nonrheumatoid synovial cells. Except for a small group of RA cells derived from tissue explants, the cells were all isolated by trypsinisation of synovial tissue, either within intact joints or after synovectomy. Cell lines were established by passaging with trypsin. In a study of 56 nonrheumatoid and 24 rheumatoid synovial lines isolated during a 7-year period the latter were found to have a shortened mean life expectancy in culture, though there was wide variation between individual lines. This is in agreement with reported findings from untrypsinised explant-derived synovial lines. However, in the present study mean multiplication rates were identical for nonrheumatoid and rheumatoid synovial cells, and on clear differences could be demonstrated for the other properties studied. No correlation could be found between the life spans of synovial cell lines and the age of the cell donors, whether from rheumatoid or nonrheumatoid sources. Rheumatoid synovial cells isolated from intact joints were notable for especially high proportions of macrophage-like cells and suppression of fibroblasts. In most cases cell lines could not be established from these rheumatoid primary cultures, and in others the lines were short-lived. Early association with relatively high proportions of macrophage-like cells in rheumatoid cultures might thus be important in influencing the establishment and behaviour of synovial cell lines.
PMCID: PMC1000764  PMID: 7247472
14.  The genome of Th17 cell-inducing segmented filamentous bacteria reveals extensive auxotrophy and adaptations to the intestinal environment 
Cell host & microbe  2011;10(3):260-272.
Perturbations of the composition of the symbiotic intestinal microbiota can have profound consequences for host metabolism and immunity. In mice, segmented filamentous bacteria (SFB) direct the accumulation of potentially pro-inflammatory Th17 cells in the intestinal lamina propria. We present the genome sequence of SFB isolated from mono-colonized mice, which classifies SFB phylogenetically as a unique member of Clostridiales with a highly reduced genome. Annotation analysis demonstrates that SFB depends on its environment for amino acids and essential nutrients and may utilize host and dietary glycans for carbon, nitrogen, and energy. Comparative analyses reveal that SFB is functionally related to members of the genus Clostridium and several pathogenic or commensal “minimal” genera, including Finegoldia, Mycoplasma, Borrelia, and Phytoplasma. However, SFB is functionally distinct from all 1,200 examined genomes, indicating a gene complement representing biology relatively unique to its role as a gut commensal closely tied to host metabolism and immunity.
PMCID: PMC3209701  PMID: 21925113
15.  In vitro model for the analysis of synovial fibroblast-mediated degradation of intact cartilage 
Activated synovial fibroblasts are thought to play a major role in the destruction of cartilage in chronic, inflammatory rheumatoid arthritis (RA). However, profound insight into the pathogenic mechanisms and the impact of synovial fibroblasts in the initial early stages of cartilage destruction is limited. Hence, the present study sought to establish a standardised in vitro model for early cartilage destruction with native, intact cartilage in order to analyse the matrix-degrading capacity of synovial fibroblasts and their influence on cartilage metabolism.
A standardised model was established by co-culturing bovine cartilage discs with early-passage human synovial fibroblasts for 14 days under continuous stimulation with TNF-α, IL-1β or a combination of TNF-α/IL-1β. To assess cartilage destruction, the co-cultures were analysed by histology, immunohistochemistry, electron microscopy and laser scanning microscopy. In addition, content and/or neosynthesis of the matrix molecules cartilage oligomeric matrix protein (COMP) and collagen II was quantified. Finally, gene and protein expression of matrix-degrading enzymes and pro-inflammatory cytokines were profiled in both synovial fibroblasts and cartilage.
Histological and immunohistological analyses revealed that non-stimulated synovial fibroblasts are capable of demasking/degrading cartilage matrix components (proteoglycans, COMP, collagen) and stimulated synovial fibroblasts clearly augment chondrocyte-mediated, cytokine-induced cartilage destruction. Cytokine stimulation led to an upregulation of tissue-degrading enzymes (aggrecanases I/II, matrix-metalloproteinase (MMP) 1, MMP-3) and pro-inflammatory cytokines (IL-6 and IL-8) in both cartilage and synovial fibroblasts. In general, the activity of tissue-degrading enzymes was consistently higher in co-cultures with synovial fibroblasts than in cartilage monocultures. In addition, stimulated synovial fibroblasts suppressed the synthesis of collagen type II mRNA in cartilage.
The results demonstrate for the first time the capacity of synovial fibroblasts to degrade intact cartilage matrix by disturbing the homeostasis of cartilage via the production of catabolic enzymes/pro-inflammatory cytokines and suppression of anabolic matrix synthesis (i.e., collagen type II). This new in vitro model may closely reflect the complex process of early stage in vivo destruction in RA and help to elucidate the role of synovial fibroblasts and other synovial cells in this process, and the molecular mechanisms involved in cartilage degradation.
PMCID: PMC2688258  PMID: 19226472
16.  Adapted Boolean network models for extracellular matrix formation 
BMC Systems Biology  2009;3:77.
Due to the rapid data accumulation on pathogenesis and progression of chronic inflammation, there is an increasing demand for approaches to analyse the underlying regulatory networks. For example, rheumatoid arthritis (RA) is a chronic inflammatory disease, characterised by joint destruction and perpetuated by activated synovial fibroblasts (SFB). These abnormally express and/or secrete pro-inflammatory cytokines, collagens causing joint fibrosis, or tissue-degrading enzymes resulting in destruction of the extra-cellular matrix (ECM).
We applied three methods to analyse ECM regulation: data discretisation to filter out noise and to reduce complexity, Boolean network construction to implement logic relationships, and formal concept analysis (FCA) for the formation of minimal, but complete rule sets from the data.
First, we extracted literature information to develop an interaction network containing 18 genes representing ECM formation and destruction. Subsequently, we constructed an asynchronous Boolean network with biologically plausible time intervals for mRNA and protein production, secretion, and inactivation. Experimental gene expression data was obtained from SFB stimulated by TGFβ1 or by TNFα and discretised thereafter. The Boolean functions of the initial network were improved iteratively by the comparison of the simulation runs to the experimental data and by exploitation of expert knowledge. This resulted in adapted networks for both cytokine stimulation conditions.
The simulations were further analysed by the attribute exploration algorithm of FCA, integrating the observed time series in a fine-tuned and automated manner. The resulting temporal rules yielded new contributions to controversially discussed aspects of fibroblast biology (e.g., considerable expression of TNF and MMP9 by fibroblasts stimulation) and corroborated previously known facts (e.g., co-expression of collagens and MMPs after TNFα stimulation), but also revealed some discrepancies to literature knowledge (e.g., MMP1 expression in the absence of FOS).
The newly developed method successfully and iteratively integrated expert knowledge at different steps, resulting in a promising solution for the in-depth understanding of regulatory pathways in disease dynamics. The knowledge base containing all the temporal rules may be queried to predict the functional consequences of observed or hypothetical gene expression disturbances. Furthermore, new hypotheses about gene relations were derived which await further experimental validation.
PMCID: PMC2734845  PMID: 19622164
17.  Batch correction of microarray data substantially improves the identification of genes differentially expressed in Rheumatoid Arthritis and Osteoarthritis 
BMC Medical Genomics  2012;5:23.
Batch effects due to sample preparation or array variation (type, charge, and/or platform) may influence the results of microarray experiments and thus mask and/or confound true biological differences. Of the published approaches for batch correction, the algorithm “Combating Batch Effects When Combining Batches of Gene Expression Microarray Data” (ComBat) appears to be most suitable for small sample sizes and multiple batches.
Synovial fibroblasts (SFB; purity > 98%) were obtained from rheumatoid arthritis (RA) and osteoarthritis (OA) patients (n = 6 each) and stimulated with TNF-α or TGF-β1 for 0, 1, 2, 4, or 12 hours. Gene expression was analyzed using Affymetrix Human Genome U133 Plus 2.0 chips, an alternative chip definition file, and normalization by Robust Multi-Array Analysis (RMA). Data were batch-corrected for different acquiry dates using ComBat and the efficacy of the correction was validated using hierarchical clustering.
In contrast to the hierarchical clustering dendrogram before batch correction, in which RA and OA patients clustered randomly, batch correction led to a clear separation of RA and OA. Strikingly, this applied not only to the 0 hour time point (i.e., before stimulation with TNF-α/TGF-β1), but also to all time points following stimulation except for the late 12 hour time point. Batch-corrected data then allowed the identification of differentially expressed genes discriminating between RA and OA. Batch correction only marginally modified the original data, as demonstrated by preservation of the main Gene Ontology (GO) categories of interest, and by minimally changed mean expression levels (maximal change 4.087%) or variances for all genes of interest. Eight genes from the GO category “extracellular matrix structural constituent” (5 different collagens, biglycan, and tubulointerstitial nephritis antigen-like 1) were differentially expressed between RA and OA (RA > OA), both constitutively at time point 0, and at all time points following stimulation with either TNF-α or TGF-β1.
Batch correction appears to be an extremely valuable tool to eliminate non-biological batch effects, and allows the identification of genes discriminating between different joint diseases. RA-SFB show an upregulated expression of extracellular matrix components, both constitutively following isolation from the synovial membrane and upon stimulation with disease-relevant cytokines or growth factors, suggesting an “imprinted” alteration of their phenotype.
PMCID: PMC3528008  PMID: 22682473
Microarray analysis; Batch correction; Rheumatoid arthritis; Osteoarthritis; Collagen; Extracellular matrix
18.  Down regulation by iron of prostaglandin E2 production by human synovial fibroblasts 
Annals of the Rheumatic Diseases  1998;57(12):742-746.
OBJECTIVE—To examine the effect of iron on the prostaglandin (PG) E2 production by human synovial fibroblasts in vitro.
METHODS—Human synovial fibroblasts were isolated from synovial tissue of rheumatoid arthritis (RA) and osteoarthritis (OA) patients and cultured in medium. Synovial fibroblasts were stimulated by human recombinant interleukin (IL) 1β (0.1-10 ng/ml) with or without ferric citrate (Fe-citrate, 0.01-1 mM). The amount of PGE2 in the culture medium was measured by an enzyme linked immunosorbent assay.
RESULTS—The production of PGE2 by the synovial fibroblasts was increased by stimulation with IL1β at all concentrations tested. Fe-citrate but not sodium citrate (Na-citrate) down regulated the production of PGE2 by the synovial fibroblasts, both with and without stimulation by IL1β. Fe-citrate inhibited the spontaneous PGE2 production by the cells in a dose dependent manner, and a maximum inhibition by Fe-citrate was observed at the concentration of 0.1 mM with IL1β stimulation. The down regulation by iron was reversed by the co-addition of desferrioxamine (100 µg/ml), an iron chelator.
CONCLUSION—Iron down regulates the PGE2 production by synovial fibroblasts in vitro.

 Keywords: rheumatoid arthritis; joint inflammation; cyclooxygenase; cytokine; inflammatory mediators
PMCID: PMC1752513  PMID: 10070275
19.  Effects of carrageenin on human synovial cells in vitro: morphology, hyaluronic acid production, growth, and the lysosomal system. 
Annals of the Rheumatic Diseases  1979;38(3):295-298.
Some in-vitro effects of the arthritogenic polysaccharide carrageenin were studied on cells from human synovium. Synovial cells were isolated from intact human knee joints, and cell lines were developed by passaging with trypsin. Carrageenin was ingested by the cells but did not significantly affect cell growth, numbers of lysosomes, intracellular lysosomal enzyme activity (N-acetyl-beta-D-glucosaminidase), or release of lysosomal enzyme from cells. Carrageenin produced a reduction in net hyaluronic acid synthesis. It also induced a striking morphological change in a high proportion of synovial cells, characterised by increased spreading over the culture surface and apparent condensation of the cytoplasm into a pattern of ridges. Nonrheumatoid and rheumatoid synovial cells behaved similarly to one another.
PMCID: PMC1000453  PMID: 384933
20.  Cytotoxic activity of rheumatoid and normal lymphocytes against allogeneic and autologous synovial cells in vitro. 
Journal of Clinical Investigation  1976;58(3):613-622.
The possibility that lymphocytes from patients with rheumatoid arthritis (RA) might be sensitized to RA synovial cell antigens was investigated with a 51Cr release cytotoxicity assay. Peripheral blood lymphocytes from rheumatoid and normal donors were tested for cytotoxic activity against their own synovial cells and against allogeneic rheumatoid and nonrhemuatoid synovial cells. In the allogeneic studies, the degree of cytotoxicity was significantly influenced by the age in culture (passage number) of the synovial target cells (P less than 0.001). When the passage number of the target cells was considered in the analysis, rheumatoid lymphocytes were found to have greater cytotoxic activity than normal lymphocytes against young cultures (low passage number) of both RA and non-RA synovial cells (P = 0.0042). Differences in susceptibility to lysis between RA and non-RA synovial cells were more susceptible to both RA and normal lymphocyte-induced lysis than were non-RA synovial cells (P = 0.0048). No evidence of cytotoxicity was detected when lymphocytes from nine RA patients and two osteoarthritis patients were reacted against their own synovial cells. Although the data demonstrated an increased cytotoxic activity of peripheral blood lymphocytes from some RA patients against allogeneic synovial cells, the fact that this reactivity was seen against both non-RA and RA synovial cells and was not demonstrated against autologous synovial cells argues against the presence of an immunospecific response of RA lymphocytes to RA synovial cell antigens.
PMCID: PMC333220  PMID: 956390
21.  Rheumatoid synovial cells from intact joints. Morphology, growth, and polykaryocytosis. 
Annals of the Rheumatic Diseases  1977;36(4):293-301.
Synovial cell lines were isolated by instillation of trypsin or chymotrypsin into intact knee joints of patients with persistent rheumatoid effusions resistant to conventional therapy. Morphology and growth in the primary phase were compared with rheumatoid cells isolated from excised synovium and nonrheumatoid synovial cells obtained from intact joints of cadavers or amputated limbs. Cell populations from all sources included varying proportions of macrophage-like and fibroblast-like cells, with only 1-3% multinucleated cells. In medium supplemented with calf serum alone, rheumatoid cells from intact joints showed negligible changes in morphology. However, in the presence of nonrheumatoid, autologous rheumatoid or homologous rheumatoid serum a rapid increase occurred in size of the macrophage-like cells and numbers of polykaryocytes, including some giant syncytial cells. These effects were directly proportional to serum concentration and were identical in fresh or heat-inactivated serum. In most of these rheumatoid cell lines no multiplication occurred, regardless of serum type or concentration. In rheumatoid synovial cells from excised synovium, human serum induced both polykaryocytosis and rapid growth of fibroblasts. Nonrheumatoid synovial cells grew rapidly but few polykaryocytes developed, mostly with less than 6 nuclei. Evidence of viral infection in rheumatoid synovial cells was sought by electron microscopy after stimulation of polykaryocytosis by human serum. In one of the cultures many cells were found with intranuclear particles possessing characteristics of the adenovirus group.
PMCID: PMC1006689  PMID: 901027
22.  Spontaneous production of fibroblast-activating factor(s) by synovial inflammatory cells. A potential mechanism for enhanced tissue destruction 
A characteristic feature of rheumatoid arthritis is hyperplasia of the synovial lining cells and fibroblasts, the source of tissue-degrading mediators, in association with the appearance and persistence of lymphocytes in affected joints. Diseased synovial tissue obtained at arthroscopy from 10 of 12 rheumatoid arthritis patients was found to release a factor(s) that could stimulate quiescent fibroblasts to proliferate in vitro. Mononuclear cells isolated from this synovial tissue and from the synovial fluid spontaneously produced fibroblast- activating factor(s) (FAF). In contrast, synovial tissue from patients with noninflammatory joint disease did not release FAF. By gel filtration, FAF was detected in two peaks (40,000 and 15,000 mol wt) that were consistent with the previously described peripheral blood T lymphocyte- and monocyte-derived factors with identical activity. The mononuclear cells were predominantly OKT3+/Leu-1+ T lymphocytes and OKM1+ cells of monocyte/macrophage lineage that expressed HLA-DR antigens, suggesting prior activation of these cells. Mononuclear cells isolated from the peripheral blood of these patients did not spontaneously secrete FAF. Lymphocytes and monocytes from the site of synovial inflammation appear to be activated in situ to produce factors that may contribute to the hyperplasia and overgrowth of the synovial membrane in rheumatoid arthritis.
PMCID: PMC2187543  PMID: 3968518
23.  Segmented Filamentous Bacteria Are Potent Stimuli of a Physiologically Normal State of the Murine Gut Mucosal Immune System 
Infection and Immunity  1999;67(4):1992-2000.
Segmented filamentous bacteria (SFB) are autochthonous bacteria inhabiting the intestinal tracts of many species, including humans. We studied the effect of SFB on the mucosal immune system by monoassociating formerly germfree C3H/HeN mice with SFB. At various time points during 190 days of colonization, fragment cultures of small intestine and Peyer’s patches (PP) were analyzed for total immunoglobulin A (IgA) and SFB-specific IgA production. Also, phenotypic changes indicating germinal center reactions (GCRs) and the activation of CD4+ T cells in PP were determined by using fluorescence-activated cell sorter analyses. A second group of SFB-monoassociated mice was colonized with a gram-negative commensal, Morganella morganii, to determine if the mucosal immune system was again stimulated and to evaluate the effect of prior colonization with SFB on the ability of M. morganii to translocate to the spleen and mesenteric lymph nodes. We found that SFB stimulated GCRs in PP from day 6 after monoassociation, that GCRs only gradually waned over the entire length of colonization, that natural IgA production was increased to levels 24 to 63% of that of conventionally reared mice, and that SFB-specific IgA was produced but accounted for less than 1.4% of total IgA. Also, the proportion of CD4+, CD45RBlow T cells, indicative of activated cells, gradually increased in the PP to the level found in conventionally reared mice. Secondary colonization with M. morganii was able to stimulate GCRs anew, leading to a specific IgA antibody response. Previous stimulation of mucosal immunity by SFB did not prevent the translocation of M. morganii in the double-colonized mice. Our findings generally indicate that SFB are one of the single most potent microbial stimuli of the gut mucosal immune system.
PMCID: PMC96557  PMID: 10085047
24.  Mapping of proteins from cultured fibroblasts of synovial and subcutaneous origin by high resolution two-dimensional polyacrylamide gel electrophoresis. 
Annals of the Rheumatic Diseases  1985;44(5):302-306.
Nine different cell lines of human (A) rheumatoid and (B) normal synovial fibroblasts and of (C) normal skin fibroblasts were obtained from tissue explants and grown as monolayers. The cellular protein synthesis was studied by high resolution two-dimensional polyacrylamide gel electrophoresis. Between the fourth and 10th passage of the cells in culture apparently no consistent differences were found which could be ascribed to the origin of the cells, to the rheumatoid state of the synovial tissue, or to the increasing age of the cultured fibroblasts.
PMCID: PMC1001636  PMID: 4004359
25.  Synovial microparticles from arthritic patients modulate chemokine and cytokine release by synoviocytes 
Arthritis Research & Therapy  2005;7(3):R536-R544.
Synovial fluid from patients with various arthritides contains procoagulant, cell-derived microparticles. Here we studied whether synovial microparticles modulate the release of chemokines and cytokines by fibroblast-like synoviocytes (FLS). Microparticles, isolated from the synovial fluid of rheumatoid arthritis (RA) and arthritis control (AC) patients (n = 8 and n = 3, respectively), were identified and quantified by flow cytometry. Simultaneously, arthroscopically guided synovial biopsies were taken from the same knee joint as the synovial fluid. FLS were isolated, cultured, and incubated for 24 hours in the absence or presence of autologous microparticles. Subsequently, cell-free culture supernatants were collected and concentrations of monocyte chemoattractant protein-1 (MCP-1), IL-6, IL-8, granulocyte/macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF) and intracellular adhesion molecule-1 (ICAM-1) were determined. Results were consistent with previous observations: synovial fluid from all RA as well as AC patients contained microparticles of monocytic and granulocytic origin. Incubation with autologous microparticles increased the levels of MCP-1, IL-8 and RANTES in 6 of 11 cultures of FLS, and IL-6, ICAM-1 and VEGF in 10 cultures. Total numbers of microparticles were correlated with the IL-8 (r = 0.91, P < 0.0001) and MCP-1 concentrations (r = 0.81, P < 0.0001), as did the numbers of granulocyte-derived microparticles (r = 0.89, P < 0.0001 and r = 0.93, P < 0.0001, respectively). In contrast, GM-CSF levels were decreased. These results demonstrate that microparticles might modulate the release of chemokines and cytokines by FLS and might therefore have a function in synovial inflammation and angiogenesis.
PMCID: PMC1174949  PMID: 15899040

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