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

Fibroblast-like synoviocytes (FLS) play a prominent role in rheumatoid synovitis and degradation of the extracellular matrix through the production of inammatory 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.

Synovial uid 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 uid 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% CO 2 in cell culture asks. The culture medium was exchanged every three days until the cells were frozen. FLSs were used for experiments after ve passages.
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 sacri ced 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 ve passages. 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 ow 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-Llysine-treated coverslips. After cell xation, 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 xation 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 lopodia 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. and acetone for 20 minutes each. After dehydration, in ltration 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 semi ne sections were obtained from the surface of the blocks with the aid of glass razors and stained with toluidine-sodium borate blue. Ultrathin 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).

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 signi cant effects at p-values <0.05.

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 laments, which are lamentous 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 signi cant 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 uid and secrete hyaluronic acid and surfactant (proteins and lipids) [24]. Lamellar bodies at various stages of maturation were identi ed in the same cell 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, signi cant 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 in amed 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 in ammation 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][28][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 ndings of other studies [5,14,15,[29][30][31][32][33][34][35][36][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 in amed 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 insigni cant, 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 rst 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][47][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 bene ts these models bring, the models must be interpreted with care since they do not completely resemble human disease. Our results, despite demonstrating signi cant 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 in ammatory 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.