Comparative morphofunctional analysis of fibroblast-like synoviocytes in human rheumatoid arthritis and mouse collagen-induced arthritis

DOI: https://doi.org/10.21203/rs.3.rs-129415/v1

Abstract

Background

Fibroblast-like synoviocytes (FLS) play a prominent role in rheumatoid synovitis and degradation of the extracellular matrix through the production of inflammatory cytokines and metalloproteinases (MMPs). Since animal models are frequently used for elucidating the disease mechanism and therapeutic development, it is relevant to compare ultrastructural characteristics and functional responses by human and mouse FLS. The objective of this study is to compare ultrastructural characteristics, IL-6 and MMP-3 production, and the activation of intracellular pathways in FLS from patients with RA (RA-FLS) and mice with collagen-induced arthritis (CIA-FLS). The objective of the study was to compare ultrastructural characteristics, Interleukin-6 (IL-6) and Metalloproteinase-3 (MMP-3) production and the activation of intracellular pathways in Fibroblast like synoviocytes (FLS) cultures obtained from patients with Rheumatoid Arthritis (RA) and from mice with collagen-induced arthritis.

Methods

FLSs were obtained from RA patients (RA-FLSs) (n = 8) and mice with collagen-induced arthritis (CIA-FLSs) (n = 4). Morphology was assessed by transmission and scanning electron microscopy. IL-6 and MMP-3 production was measured by ELISA, and activation of intracellular signaling pathways (NF-κB and MAPK: p-ERK1/2, p-P38 and p-JNK) was measured by Western blotting in cultures of RA-FLSs and CIA-FLSs stimulated with tumor necrosis factor - alpha (TNF-α) and IL-1β.

Results

RA-FLS and CIA-FLS cultures exhibited rich cytoplasm, rough endoplasmic reticula and prominent and well-developed Golgi complexes. Transmission electron microscopy demonstrated the presence of lamellar bodies, which are cytoplasmic structures related to surfactant production, in FLSs from both sources. Increased levels of pinocytosis and numbers of pinocytotic vesicles were observed in RA-FLSs (p < 0.05). Basal production of MMP-3 and IL-6 was present in RA-FLSs and CIA-FLSs. Regarding the production of MMP-3 and IL-6 and the activation of signaling pathways, the present study demonstrated a lower response to IL-1β by CIA-FLSs than by RA-FLSs.

Conclusion

There were differences between RA-FLSs and CIA-FLSs in their ultrastructural morphologies and functional responses. The differences shown in our study indicate that the adoption of an RA-FLS human model is a better alternative than the CIA-FLS animal model for in vitro studies of RA etiopathogenesis and new therapeutic targets.

Background

Rheumatoid arthritis (RA) is the most common inflammatory rheumatic disease. Destructive damage of the joints, resulting in physical disability, is the ultimate result of bone and cartilage damage mediated by the production of chemokines and matrix components by synoviocytes [1]. The etiopathogenesis of RA involves an intricate network of cellular interactions in the synovial membrane characterized by the proliferation of cells in the synovial lining, resulting in hyperplasia, pannus formation, and tissue destruction [1, 2].

Fibroblast-like synoviocytes (FLSs) are mesenchymal-derived cells responsible for providing support, nourishment and lubrication to the joint tissue. FLSs one of the most prominent cells in inflamed tissue in RA [2]. In the chronic inflammatory milieu of the rheumatoid synovium, these cells become autonomous, hyperplastic, and invasive and produce large amounts of proinflammatory cytokines, chemokines, and matrix-degrading enzymes [2]. Among these products, metalloproteinases (MMPs) are of fundamental importance in the pathophysiology of RA by promoting bone and cartilage degradation [3, 4] Stromelysin (MMP-3) is one of the most important factors in RA [5, 6]. In addition, FLSs produce interleukin 6 (IL-6), an important cytokine in the inflammatory cascade, leading to the interaction of FLS and other immune cells in the synovium and exacerbating inflammatory processes [7–10]. Previous studies of FLSs from patients with RA indicated that both IL-6 and MMP production increased after IL-1 and TNF-α stimulation and were dependent on the NF-κB and MAPK pathways (ERK, JNK and p38), ultimately contributing to the pathogenesis of RA [11–16].

Animal models have contributed to improved understanding of human disease and provide a useful tool for therapeutic testing. However, discrepant results from animal and human studies are worrisome, and efforts to evaluate morphological and functional similarities between animal and human cells are required. One of the most commonly used experimental models to study RA is murine collagen-induced arthritis (CIA). This animal model is characterized by peripheral symmetrical joint involvement that shares many histopathological features with human RA, including synovitis, pannus formation, cartilage and bone erosion [17, 18]. Furthermore, as in patients with RA, IL-6 and MMP-3 also play key roles in this model [19–21]. However, we could only find one study showing the effect of IL-1β and TNF-α in FLSs in CIA [20].

Primary FLS cultures from CIA and RA patients have been used as an in vitro exchange model to study the responses of these cells to different stimuli and pharmacological drugs. There have been no studies that compared the morphological ultrastructures and responses of cultures of FLSs derived from patients with RA (RA-FLS) and mice with CIA (CIA-FLS). Our objective was to compare RA-FLS with CIA-FLS cultures regarding their ultrastructural characteristics as well as their functional responses, such as the signaling pathways activated and the production of IL-6 and MMP-3 after stimulation with TNF-α and IL-1β.

Methods

Sample collection and culture of RA patient cells

Synovial fluid was obtained from eight (n = 8) RA patients according to the American College of Rheumatology (ACR) criteria [22]. The mean age of the patients was 52.62 years (range: 37–66 years). Samples of synovial fluid were collected and centrifuged at 1200 revolutions per minute (rpm) for 10 minutes. The pellet was resuspended in complete medium (DMEM high glucose plus 10% fetal bovine serum [FBS, Gibco, Life Technologies, USA], 1% streptomycin(S)/penicillin(P) and amphotericin B [Gibco, Life Technologies, USA] and 1% nonessential amino acids [Gibco, Life Technologies, USA]) and kept at 37 °C and 5% CO2 in cell culture flasks. The culture medium was exchanged every three days until the cells were frozen. FLSs were used for experiments after five passages.

Sample collection and culture of mouse collagen-induced arthritis cells

Four (n = 4) male DBA1/J mice (8–12 weeks, average weight of 20 grams) were used to obtain CIA-FLSs. The animals were immunized with 50 µL of an emulsion containing an equal volume of type II bovine collagen (Chondrex; Washington, USA) and Freund's complete adjuvant (Merck, Saint Louis, USA) by intradermal injection at a distance of 1.5 cm from the base of the tail on day zero. The booster was administered on day 18. After ten days, the animals were sacrificed by cervical dislocation. The synovial tissue was removed from the joint and processed with a 1 mg/ml collagenase solution I (Merck, Saint Louis, USA) for one hour. After centrifugation, the pellet was resuspended in complete medium and cultured in the same way described for human samples until the cells were frozen. FLSs were used for experiments after five passages.

Immunophenotyping

RA-FLSs and CIA-FLSs were plated at 2 × 105 cells/well in 20 µl/well and diluted in 20 µl of flow cytometry buffer solution (PBS containing 1% fetal bovine serum and 0.01% azide). For human FLSs, anti-human CD14 PerCP-Cyanine 5.5 (ref. 45–0149, eBioscience), anti-human CD45 V450 (ref. 560368, BD Biosciences), and anti-human CD90 Phycoerythrin (PE) (ref. 561970, BD Biosciences) monoclonal antibodies and V450-, PE- and PerCP-labeled isotype control antibodies were used (all from BD Biosciences), and unlabeled control cells were included in all experiments. For mouse FLSs, anti-mouse CD45 V450 (ref. 19264, BD Biosciences) and anti-rat CD90.1 PE (ref. 554898, BD Biosciences) monoclonal antibodies, V450- and PE-labeled isotype control antibodies (all BD Biosciences) and unlabeled cells were used. Cells were labeled with antibodies for 20 minutes at 4 °C in the dark, washed, resuspended in 1 × PBS and analyzed on a FACSCanto II flow cytometer (BD Biosciences). A minimum of 100,000 events were acquired for each sample, and the acquisitions were processed using Diva software (BD Biosciences). FlowJo software (Tree Star) was used to analyze the data.

Electron microscopy

Morphological analysis was performed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). For SEM, RA-FLSs and CIA-FLSs were cultured without stimulation on poly-L-lysine-treated coverslips. After cell fixation, the media was aspirated, and the cells were washed with 1 × saline phosphate buffer (SPB). The cultures were then immersed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer. After 120 minutes, the fixation solution was aspirated and replaced with 0.1 M cacodylate buffer. The samples were washed in aldehyde three times for 10 minutes each in 0.05 M cacodylate buffer and immersed in 1% osmium tetraoxide (OsO4) solution in 0.05 M cacodylate buffer for one hour at room temperature. The samples were washed in distilled water and dehydrated with increasing concentrations of alcohol (30, 50, 70, 90 and 100%) and acetone for 10 minutes each. The dehydrated samples were dried until the acetone was completely removed. The samples were visualized by SEM (FEG - Quanta 200 FEI). Cell shape, the emission of cytoplasmic projections, and the presence of filopodia and lamellipodia were analyzed in 20 RA-FLSs and 20 CIA-FLSs.

For TEM analysis of unstimulated RA-FLS and CIA-FLS, the preparation included aspiration of DMEM followed by washes with SPB. The cultures were then fixed for 120 minutes with 2.5% glutaraldehyde in 0.1 M cacodylate buffer at 4 °C. The fixed samples were scraped off with a spatula, transferred to sterile 50 ml tubes and centrifuged at 3000 rpm for 5 minutes. The pellets were fixed in 2% osmium tetroxide (Sigma-Aldrich, Sao Paulo, Brazil) in 0.1 M cacodylate buffer for 2 hours at room temperature. After being washed three times with distilled water for 15 minutes each, the samples were incubated with 2% uranyl acetate (EMS) for 24 hours at room temperature in the dark. The samples were then washed with distilled water as described above and dehydrated in an increasing series of alcohol (35, 50, 70, 85, 95, and 100%) and acetone for 20 minutes each. After dehydration, infiltration was followed by incubation in a 1:2, 1:1 and 1:2 mixture of acetone-Epon resin (EMBed Resin 812, EMS) and pure resin for 3 hours. Subsequently, the cells were incubated in the same resin in BEEM capsules (Ted Pella, California, USA) and polymerized at 60 °C for 48 hours. After polymerization of the resin, 300 nm semifine sections were obtained from the surface of the blocks with the aid of glass razors and stained with toluidine-sodium borate blue. Ultrathin sections (~ 60 nm) were obtained on a Leica EM UC6 ultramicrotome (Leica Microsystems GmbH, Wetzlar, Germany) using a diamond razor. The sections were mounted on 200 mesh copper (Ted Pella), stained with lead citrate (Merck KGaA, Darmstadt, Germany) for 5 minutes and analyzed under a transmission electron microscope (Tecnai G2-12 Spirit Biotwin Thermo Fischer Scientific/FEI 2006, Eindhoven, the Netherlands) at 120 kV. Cells were visualized by MET (Tecnai G2-12 Spirit Biotwin FEI − 120 kV). To investigate the morphological characteristics of synoviocytes, images of 20 cells per group revealing the entire cellular profile, including the nucleus, and subcellular details were obtained at 4200-fold, 16500-fold and 20500-fold magnifications. The basic ultrastructural analyses focused on organelles involved with the classic cell synthesis and secretion pathway: the nucleus (N), rough endoplasmic reticulum (RER), Golgi apparatus (GA) and mitochondria (MT). The morphometric analyses were focused on organelles involved in cellular pinocytosis (pinocytosis (P) and pinocytic vesicles (PV)), lamellar bodies (LBs) and MT. The morphometric analysis was based on the relative quantity of each organelle per number of cells.

Stimuli and Treatments

RA-FLSs and CIA-FLSs were plated in 24-well plates (1.5 × 105 cells/well) in DMEM with 1% P/S/amphotericin B and 1% FBS for 24 hours at 37 °C in 5% CO2. The cells were then stimulated for 24 hours with 50 ng/ml TNF-α (ref.: human 300-01A e murine 315-01A, Peprotech) and 1 ng/ml IL-1β (ref.: human 200-01B e murine 211-11B, Peprotech). A dose-response curve was obtained to determine the appropriate concentration of each stimulus (TNF-α and IL-1β) (data not shown).

After stimulation, the supernatant was used to measure the production of IL-6 and MMP-3 by ELISA (R&D Systems) according to the manufacturer's protocols. For signaling pathway analysis, both cell types were transferred to six-well plates (1 × 106 cells/well) and stimulated with 50 ng/ml TNF-α and 1 ng/ml IL-1β for 30 minutes. After stimulation, the cells were lysed. The cell lysate buffer contained 25 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 10 mM sodium fluoride, 10% glycerin, 1 mM phenylmethylsulfonyl fluoride and 1 protease inhibitor cocktail set I (Calbiochem, California, USA). The following phosphorylated proteins in the cell lysate were analyzed: ERK1/2 (ref: 9101, Cell Signaling), p38 (ref: 4511, Cell Signaling), and JNK (ref: 9251, Cell Signaling), and the transcription factors NF-κB (ref: 3033, Cell Signaling) and β-actin (ref: ARG53987, Arigo) were analyzed.

Statistical analysis

The Mann-Whitney U test nonparametric analysis was used to compare MMP-3 and IL-6 concentrations between the TNF-α- and IL-1β-stimulated groups and to compare the amounts of pinocytosis, pinocytic vesicles, mitochondria and lamellar bodies between RA-FLSs and CIA-FLSs. GraphPad Prisma version 6.01 was used for data analysis and graph production. We considered evidence of significant effects at p-values ​​<0.05.

Results

Immunophenotyping

RA-FLSs and CIA-FLSs were positive for the FLS marker CD90 (Figs. 1A and B, and C). RA-FLSs were negative for the leukocyte marker CD45 and the monocyte/macrophage marker CD14 (Fig. 1A and B). In CIA-FLSs, controls for leukocyte markers (CD45) also demonstrated the presence of a pure FLS culture (< 1% CD45+) (Fig. 1D).

Electron microscopy

Scanning Electron Microscopy

RA-FLS and CIA-FLS cultures exhibited populations of fusiform FLSs with cytoplasmic expansions forming branches. Both cell types were anchored to the substrate with numerous cytoplasmic projections at the lamellipodia ends, and the protein structure of the actin cytoskeleton protruded at the mobile end of the cell [23]. Lamellipodia propelled entire cell structures through the substrate (Fig. 2).

The cytoplasmic projections that extended beyond the lamellipodia border on migrating cells were often anchored to neighboring cells (Fig. 2).

Transmission Electron Microscopy

Evaluation of RA-FLSs and CIA-FLSs by Transmission Electron Microscopy (TEM) revealed subcellular characteristics of intense cellular activity, represented by a large and euchromatic nucleus with a thin layer of heterochromatin, a prominent and well-developed rough endoplasmic reticulum and Golgi apparatus, and the presence of several mitochondria close to these organelles (Fig. 3). As shown in Fig. 4, we observed a high concentration of intermediate filaments, which are filamentous protein structures that make up the cytoskeleton of cells and assist in cell morphology. Ultrastructural differences were observed between RA-FLSs and CIA-FLSs in the degree of pinocytosis (number of plasma membrane invaginations) and the number of pinocytotic vesicles, which were related to the process of cellular pinocytosis. Both structures were more frequently seen in RA-FLSs than in CIA-FLSs (p < 0.05) (Fig. 4). No significant difference was observed in the number of mitochondria.

Lamellar bodies were observed in RA-FLS and CIA-FLS cultures (Fig. 3). Lamellar bodies consist of cavitary structures with circular walls in unique juxtaposition to FLSs in the synovial environment; these structures are secreted by exocytosis in the synovial fluid and secrete hyaluronic acid and surfactant (proteins and lipids) [24]. Lamellar bodies at various stages of maturation were identified in the same cell (Fig. 3C). No significant difference was observed in the number of lamellar bodies between RA-FLSs and CIA-FLSs (data not shown). Adjacent FLSs were observed to be in close contact through their cytoplasmic projections, corresponding to the electron-dense regions at the communication/adherent junction sites.

Functional analysis

Figure 5 shows the activation of MAPK and p-NF-κB signaling in RA-FLSs and CIA-FLSs after stimulation with TNF-α and IL-1β. In RA-FLSs and CIA-FLSs, TNF-α activated p-ERK1/2 (2.25-fold and 2.44-fold, respectively), p-NF-κB (3.91-fold and 1.18-fold, respectively) and p-P38 (1.48-fold and 4.89-fold, respectively), and TNF-α activate p-JNK (13.74-fold) only in RA-FLSs. On the other hand, IL-1β activated p-P38 (56.06-fold and 1.31-fold, respectively) and p-NFκB (35.96-fold and 1.07-fold, respectively) in RA-FLSs and CIA-FLSs and only activated p-ERK1/2 and p-JNK in RA-FLSs (11.96-fold and 142.31-fold, respectively) (Fig. 5).

Similarly, MMP-3 and IL-6 (Fig. 6) were constitutively expressed by RA-FLSs and CIA-FLSs even when the cells were unstimulated. In RA-FLSs, significant increases in IL-6 and MMP-3 production were observed after stimulation with TNF-α and IL-1β (p = 0.0267; p = 0.0405; p = 0.0405 and p = 0.0075, respectively) (Fig. 6A and 6B)). However, in CIA-FLSs, there was an increase in IL-6 production only after TNF-α stimulation (p = 0.0026). No difference in MMP-3 production after TNF-α stimulation or IL-6 or MMP-3 after IL-1β stimulation (Figs. 6C and 6D) was detected.

Discussion

Many in vitro studies use cells from experimental models to study the etiopathogenesis of RA [25]. FLSs from experimental models are frequently used in the search of therapeutic targets and to study the effects of new drugs, and it is well established that FLSs play important roles in cartilage and bone destruction in the joints of patients with RA [2]. Previous studies have shown that histopathological and pathological characteristics in the inflamed tissues of mice with CIA and patients with RA are similar [26]. However, the present study showed differences in the production of important mediators of inflammation in RA, and different signaling pathways were activated between human- and mouse-derived FLSs in vitro, which may be explained by their ultrastructural differences.

In accordance with our data, upregulation of IL-6 and MMP-3 after in vitro stimulation with TNF-α and IL-1β in RA-FLS has been previously demonstrated [20, 27–29]. This stimulation was mediated by activation of the MAPK pathways (ERK, p38 and JNK), as well as the transcription factor NF-κB, and was supported by the findings of other studies [5, 14, 15, 29–37]. Unlike RA-FLSs, in CIA-FLS, after IL-1β stimulation, there was a lack of activation of the MAPK pathways and NF-κB and, consequently, the absence of an increase in the production of IL-6 and MMP-3. Although IL-1β is present on the inflamed synovium in CIA [38, 39], few studies have demonstrated the expression of the IL-1β receptor (IL1r1) in CIA-FLSs [37]. The low expression of the IL-1β receptor in CIA-FLSs could explain the lack of activation of downstream signaling pathways and the lack of an increase in MMP-3 and IL-6 production [40]. However, IL-6 production has been observed after IL-1β stimulation in CIA-FLSs [40], but the researchers did not analyze the activation of the signaling pathways that was reported in our work. Our results suggest that the low activation and nonactivation of signaling pathways (p-ERK, p-p38, p-JNK and NF-κB transcription factor) was consistent with the lack of production of MMP-3 and IL-6 [41].

Furthermore, TNF-α plays a major role in the pathogenesis of RA, as well as in CIA [2, 39, 42, 43]. Both groups of cells responded to TNF-α stimulation by activation of the p-ERK, p-p38 and p-NF-κB signaling pathways. These activations mediate an increase in IL-6 production, but only JNK was not phosphorylated in CIA-FLSs. Despite studies showing the activation of this pathway in CIA-FLSs [38], we demonstrated that MMP-3 production was insignificant, which was consistent with the lack of activation of p-JNK [33, 36]. It is possible that MMP-3 does not play as important a role in CIA as it does in RA patients [6, 44, 45].

In addition to differences in the responses between RA-FLSs and CIA-FLSs, we found important morphological differences between these cells. Our data demonstrate for the first time a higher degree of pinocytosis and more pinocytotic vesicles in human cells than in mouse cells. Pinocytosis, a type of endocytosis, plays a critical role in cell transport, endocytosis, signal transduction, and cell proliferation [46–48]. When endocytosis occurs, the cell extends and folds around the extracellular material, forming a pocket and then creating pinocytic vesicles that are absorbed [49, 50]. Therefore, these morphological differences could indicate increased functional activity in RA-FLSs.

Despite these differences, many similarities between RA-FLS and CIA-FLS morphology were observed, including the presence of lamellar bodies, fusiform shape, the presence of lamellipodia, large euchromatic nucleus, prominent and well-developed rough endoplasmic reticulum and Golgi apparatus, and the presence of several mitochondria nearby to these organelles [24].

Experimental models play an important role in the study of disease mechanisms and the search for possible treatments. However, despite the benefits these models bring, the models must be interpreted with care since they do not completely resemble human disease. Our results, despite demonstrating significant differences in vitro between human FLSs and FLS models, do not diminish the relevance of experimental models in studies.

Conclusion

In conclusion, our in vitro results show differences between the ultrastructural morphology and functional responses of RA-FLSs and CIA-FLSs regarding inflammatory cytokines involved in the pathophysiology of RA. The differences found in our study suggest a possible limitation of the use of CIA-FLSs for in vitro studies related to etiopathogenesis and new therapeutic targets in RA.

Abbreviations

FLS: Fibroblasts like synoviocytes; RA: Rheumatoid Arthritis; CIA: Collagen-Induced Arthritis; IL: Interleukin; MMP: Metalloproteinase; TNF: Tumor Necrosis Factor; ACR: American College of Rheumatology; RPM: Revolutions Per Minute; PE: Phycoerythrin; SEM: Scanning Electron Microscopy; TEM: Transmission Electron Microscopy; SPB: Saline Phosphate Buffer; EMS: Uranyl Acetate; N: Nucleus; RER: Rough Endoplasmic Reticulum; GA: Golgi Apparatus; MT: Mitochondria; P: Pinocytosis; PV: Pinocytic Vesicles; LBs: Lamellar Bodies

Declarations

Ethics approval

The Ethics Committee approved the protocol (CAAE Human Research Ethics Committee: 08387918.6.0000.5149). Informed consent was obtained from all participants in the study. All procedures using mice were in accordance with the National Institutes of Health Guide for the Care and Use of Animals. The Animal Use Ethics Committee (Protocol 293/2018) approved the animal study.

Consent for publication

Not applicable

Availability of data and materials

The datasets during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests: none declared.

Funding

This study received support from Fundo de Apoio à Pesquisa e Ensino - Sociedade Brasileira de Reumatologia (FAPE-SBR), Fundação de Amparo à Pesquisa de Minas Gerais (FAPEMIG), and Conselho Nacional de Desenvolvimento Tecnológico (CNPq).

Authors’ contributions

MVMA, AMK, CRLM conceived and designed the experiments. CRLM performed the experiments and analyzed data. CRLM, FFD, PGO, GGR contributed to acquisition and interpretation of the data. Contributed reagents/materials/analysis tools: MVMA, AMK, RMX. All authors read and approved the final manuscript.

Acknowledgements

This study received support from Fundo de Apoio à Pesquisa e Ensino - Sociedade Brasileira de Reumatologia (FAPE-SBR), Fundação de Amparo à Pesquisa de Minas Gerais (FAPEMIG), and Conselho Nacional de Desenvolvimento Tecnológico (CNPq).

Authors' information

1 Laboratory of Scientific Research - Professor Lineu Freire-Maia, Faculty of Medicine, Universidade Federal de Minas Gerais. 2 Post Graduate Program in Sciences Applied to Adult Health Care, Federal University of Minas Gerais, Belo Horizonte, Brazil. 3 Laboratory of Scientific Research, Institute of Biological Sciences, Universidade Federal de Minas Gerais. 4 Hospital das Clínicas, Universidade Federal de Minas Gerais. 5 Assistant Project Scientist at Medicine Department, University of California of San Diego. 6 Internal Medicine Department, Faculty of Medicine, Universidade Federal do Rio Grande do Sul.

References

  1. McInnes IB, Schett G. The pathogenesis of rheumatoid arthritis. The New England journal of medicine. 2011;365(23):2205-19.
  2. Tu J, Hong W, Zhang P, Wang X, Korner H, Wei W. Ontology and Function of Fibroblast-Like and Macrophage-Like Synoviocytes: How Do They Talk to Each Other and Can They Be Targeted for Rheumatoid Arthritis Therapy? Frontiers in immunology. 2018;9:1467.
  3. Croft AP, Naylor AJ, Marshall JL, Hardie DL, Zimmermann B, Turner J, et al. Rheumatoid synovial fibroblasts differentiate into distinct subsets in the presence of cytokines and cartilage. Arthritis research & therapy. 2016;18(1):270.
  4. Ekwall AK, Eisler T, Anderberg C, Jin C, Karlsson N, Brisslert M, Bokarewa MI. The tumour-associated glycoprotein podoplanin is expressed in fibroblast-like synoviocytes of the hyperplastic synovial lining layer in rheumatoid arthritis. Arthritis research & therapy. 2011;13(2):R40.
  5. Hammaker DR, Boyle DL, Chabaud-Riou M, Firestein GS. Regulation of c-Jun N-terminal kinase by MEKK-2 and mitogen-activated protein kinase kinase kinases in rheumatoid arthritis. Journal of immunology. 2004;172(3):1612-8.
  6. Sugiyama E. Role of matrix metalloproteinase-3 in joint destruction in rheumatoid arthritis. Clinical calcium. 2007;17(4):528-34.
  7. Jiao Z, Wang W, Ma J, Wang S, Su Z, Xu H. Notch signaling mediates TNF-alpha-induced IL-6 production in cultured fibroblast-like synoviocytes from rheumatoid arthritis. Clinical & developmental immunology. 2012;2012:350209.
  8. Migita K, Iwanaga N, Izumi Y, Kawahara C, Kumagai K, Nakamura T, et al. TNF-alpha-induced miR-155 regulates IL-6 signaling in rheumatoid synovial fibroblasts. BMC research notes. 2017;10(1):403.
  9. Mor A, Abramson SB, Pillinger MH. The fibroblast-like synovial cell in rheumatoid arthritis: a key player in inflammation and joint destruction. Clinical immunology. 2005;115(2):118-28.
  10. Tran CN, Lundy SK, White PT, Endres JL, Motyl CD, Gupta R, et al. Molecular interactions between T cells and fibroblast-like synoviocytes: role of membrane tumor necrosis factor-alpha on cytokine-activated T cells. The American journal of pathology. 2007;171(5):1588-98.
  11. Bucala R, Ritchlin C, Winchester R, Cerami A. Constitutive production of inflammatory and mitogenic cytokines by rheumatoid synovial fibroblasts. The Journal of experimental medicine. 1991;173(3):569-74.
  12. Neumann E, Lefevre S, Zimmermann B, Gay S, Muller-Ladner U. Rheumatoid arthritis progression mediated by activated synovial fibroblasts. Trends in molecular medicine. 2010;16(10):458-68.
  13. Perlman H, Bradley K, Liu H, Cole S, Shamiyeh E, Smith RC, et al. IL-6 and matrix metalloproteinase-1 are regulated by the cyclin-dependent kinase inhibitor p21 in synovial fibroblasts. Journal of immunology. 2003;170(2):838-45. doi: 10.4049/jimmunol.170.2.838.
  14. Sweeney SE, Firestein GS. Mitogen activated protein kinase inhibitors: where are we now and where are we going? Annals of the rheumatic diseases. 2006;65 Suppl 3:iii83-8.
  15. Thalhamer T, McGrath MA, Harnett MM. MAPKs and their relevance to arthritis and inflammation. Rheumatology. 2008;47(4):409-14.
  16. Rosengren S, Corr M, Boyle DL. Platelet-derived growth factor and transforming growth factor beta synergistically potentiate inflammatory mediator synthesis by fibroblast-like synoviocytes. Arthritis research & therapy. 2010;12(2):R65.
  17. Courtenay JS, Dallman MJ, Dayan AD, Martin A, Mosedale B. Immunisation against heterologous type II collagen induces arthritis in mice. Nature. 1980;283(5748):666-8.
  18. Holmdahl R, Andersson M, Goldschmidt TJ, Gustafsson K, Jansson L, Mo JA. Type II collagen autoimmunity in animals and provocations leading to arthritis. Immunological reviews. 1990;118:193-232. PubMed PMID: 2079326.
  19. Inoue T, Kohno M, Nagahara H, Murakami K, Sagawa T, Kasahara A, et al. Upregulation of sphingosine-1-phosphate receptor 3 on fibroblast-like synoviocytes is associated with the development of collagen-induced arthritis via increased interleukin-6 production. PloS one. 2019;14(6):e0218090.
  20. Lee A, Choi SJ, Park K, Park JW, Kim K, Choi K, et al. Detection of active matrix metalloproteinase-3 in serum and fibroblast-like synoviocytes of collagen-induced arthritis mice. Bioconjugate chemistry. 2013;24(6):1068-74. doi: 10.1021/bc4001273.
  21. Zhai T, Gao C, Huo R, Sheng H, Sun S, Xie J, et al. Cyr61 participates in the pathogenesis of rheumatoid arthritis via promoting MMP-3 expression by fibroblast-like synoviocytes. Modern rheumatology. 2017;27(3):466-75.
  22. Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis and rheumatism. 1988;31(3):315-24.
  23. Vuorio E, Takala I, Pulkki K, Einola S. Effects of sodium aurothiomalate on hyaluronic acid synthesis in normal and rheumatoid synovial fibroblast cultures. Scandinavian journal of rheumatology. 1979;8(3):173-6.
  24. Vandenabeele F, De Bari C, Moreels M, Lambrichts I, Dell'Accio F, Lippens PL, et al. Morphological and immunocytochemical characterization of cultured fibroblast-like cells derived from adult human synovial membrane. Archives of histology and cytology. 2003;66(2):145-53.
  25. Hu Y, Cheng W, Cai W, Yue Y, Li J, Zhang P. Advances in research on animal models of rheumatoid arthritis. Clinical rheumatology. 2013;32(2):161-5.
  26. Jia Q, Wang T, Wang X, Xu H, Liu Y, Wang Y, et al. Astragalin Suppresses Inflammatory Responses and Bone Destruction in Mice with Collagen-Induced Arthritis and in Human Fibroblast-Like Synoviocytes. Frontiers in pharmacology. 2019;10:94.
  27. Bartok B, Firestein GS. Fibroblast-like synoviocytes: key effector cells in rheumatoid arthritis. Immunological reviews. 2010;233(1):233-55.
  28. Baumann H, Kushner I. Production of interleukin-6 by synovial fibroblasts in rheumatoid arthritis. The American journal of pathology. 1998;152(3):641-4.
  29. El-Kady IM, El-Masry SA. Pro-inflammatory and anti-inflammatory cytokines profile in rheumatoid arthritis patients. The Egyptian journal of immunology. 2008;15(1):109-14.
  30. Bottini N, Firestein GS. Duality of fibroblast-like synoviocytes in RA: passive responders and imprinted aggressors. Nature reviews Rheumatology. 2013;9(1):24-33.
  31. Firestein GS, Manning AM. Signal transduction and transcription factors in rheumatic disease. Arthritis and rheumatism. 1999;42(4):609-21.
  32. Gortz B, Hayer S, Tuerck B, Zwerina J, Smolen JS, Schett G. Tumour necrosis factor activates the mitogen-activated protein kinases p38alpha and ERK in the synovial membrane in vivo. Arthritis research & therapy. 2005;7(5):R1140-7.
  33. Han Z, Boyle DL, Chang L, Bennett B, Karin M, Yang L, et al. c-Jun N-terminal kinase is required for metalloproteinase expression and joint destruction in inflammatory arthritis. The Journal of clinical investigation. 2001;108(1):73-81.
  34. Kaneko M, Tomita T, Nakase T, Ohsawa Y, Seki H, Takeuchi E, et al. Expression of proteinases and inflammatory cytokines in subchondral bone regions in the destructive joint of rheumatoid arthritis. Rheumatology. 2001;40(3):247-55.
  35. Lu H, Sun T, Yao L, Zhang Y. Role of protein tyrosine kinase in IL-1 beta induced activation of mitogen-activated protein kinase in fibroblast-like synoviocytes of rheumatoid arthritis. Chinese medical journal. 2000;113(10):872-6.
  36. Simmonds RE, Foxwell BM. Signalling, inflammation and arthritis: NF-kappaB and its relevance to arthritis and inflammation. Rheumatology. 2008;47(5):584-90.
  37. Van Holten J, Reedquist K, Sattonet-Roche P, Smeets TJ, Plater-Zyberk C, Vervoordeldonk MJ, et al. Treatment with recombinant interferon-beta reduces inflammation and slows cartilage destruction in the collagen-induced arthritis model of rheumatoid arthritis. Arthritis research & therapy. 2004;6(3):R239-49.
  38. Lee J, Hong EC, Jeong H, Hwang JW, Kim H, Bae EK, et al. A novel histone deacetylase 6-selective inhibitor suppresses synovial inflammation and joint destruction in a collagen antibody-induced arthritis mouse model. International journal of rheumatic diseases. 2015;18(5):514-23.
  39. Liu Z, Zhou L, Ma X, Sun S, Qiu H, Li H, et al. Inhibitory effects of tubeimoside I on synoviocytes and collagen-induced arthritis in rats. Journal of cellular physiology. 2018;233(11):8740-53.
  40. Wang X, Xia S, Fu B. RNAseq analysis of synovial fibroblasts in human rheumatoid arthritis. Molecular medicine reports. 2014;10(1):241-7.
  41. Shimizu K, Nakajima A, Sudo K, Liu Y, Mizoroki A, Ikarashi T, et al. IL-1 receptor type 2 suppresses collagen-induced arthritis by inhibiting IL-1 signal on macrophages. Journal of immunology. 2015;194(7):3156-68.
  42. Joosten LA, Helsen MM, van de Loo FA, van den Berg WB. Anticytokine treatment of established type II collagen-induced arthritis in DBA/1 mice. A comparative study using anti-TNF alpha, anti-IL-1 alpha/beta, and IL-1Ra. Arthritis and rheumatism. 1996;39(5):797-809.
  43. Weinblatt ME, Kremer JM, Bankhurst AD, Bulpitt KJ, Fleischmann RM, Fox RI, et al. A trial of etanercept, a recombinant tumor necrosis factor receptor:Fc fusion protein, in patients with rheumatoid arthritis receiving methotrexate. The New England journal of medicine. 1999;340(4):253-9.
  44. Hammaker D, Sweeney S, Firestein GS. Signal transduction networks in rheumatoid arthritis. Annals of the rheumatic diseases. 2003;62 Suppl 2:ii86-9.
  45. Woolley DE, Evanson JM. Collagenase and its natural inhibitors in relation to the rheumatoid joint. Connective tissue research. 1977;5(1):31-5.
  46. Panariti A, Miserocchi G, Rivolta I. The effect of nanoparticle uptake on cellular behavior: disrupting or enabling functions? Nanotechnology, science and applications. 2012;5:87-100.
  47. Sinha B, Koster D, Ruez R, Gonnord P, Bastiani M, Abankwa D, et al. Cells respond to mechanical stress by rapid disassembly of caveolae. Cell. 2011;144(3):402-13.
  48. Soldati T, Schliwa M. Powering membrane traffic in endocytosis and recycling. Nature reviews Molecular cell biology. 2006;7(12):897-908.
  49. Singer, II. Microfilament bundles and the control of pinocytotic vesicle distribution at the surfaces of normal and transformed fibroblasts. Experimental cell research. 1979;122(2):251-64.
  50. Stillwell W. An Introduction to Biological Membranes. 2 edition ed2016. 590 p.