Rheumatoid arthritis (RA) is characterized by inflammation and destruction of synovial joints. Fibroblast-like synoviocytes (FLS) harvested from synovial tissue of patients with RA can invade normal human cartilage in severe combined immunodeficient (SCID) mice and Matrigel basement membrane matrix in vitro. This study was undertaken to investigate the association of these in vitro characteristics with disease characteristics in patients with RA.
Synovial tissue samples from 72 RA and 49 osteoarthritis (OA) patients were obtained. Samples of different joints were collected from 7 patients with RA. The FLS invasiveness in Matrigel was studied, and the intraindividual and interindividual differences were compared. From the patients with FLS who exhibited the most extreme differences in in vitro ingrowth (most and least invasive FLS), radiographs of the hands and feet were collected and scored according to the Sharp/van der Heijde method to determine the relationship between in vitro invasion data and estimated yearly joint damage progression.
FLS from patients with RA were more invasive than FLS from patients with OA (P < 0.001). The mean intraindividual variation in FLS invasion was much less than the mean interindividual variation (mean ± SD 1,067 ± 926 and 3,845 ± 2,367 for intraindividual and interindividual variation, respectively; P = 0.035), which shows that the level of FLS invasion is a patient characteristic. The mean ± SEM Sharp score on radiographs of the hands or feet divided by the disease duration was 4.4 ± 1.1 units per year of disease duration in patients with the least invasive FLS (n = 9), which was much lower compared with the 21.8 ± 3.1 units per year of disease duration in patients with the most invasive FLS (n = 9) (P < 0.001).
The ex vivo invasive behavior of FLS from RA patients is associated with the rate of joint destruction and is a patient characteristic, given the much smaller intraindividual than interindividual FLS variation.
Rheumatoid arthritis (RA) is an autoimmune disease that predominantly targets the synovial joints and ultimately leads to joint destruction. The destructive process is suggested to be mediated, at least in part, by fibroblast-like synoviocytes (FLS) from the synovium because in a SCID mouse coimplementation model, it was shown that FLS from RA patients attach to and invade normal cartilage (1). Moreover, others have observed that in RA, FLS show characteristics of transformed cells, such as anchorage-independent growth (2), insensitivity to apoptosis, and increased proliferation.
Processes that are associated with the change in FLS from normal to aggressive behavior are phosphorylation of the STAT-3 protein and elevated levels of the prooncogene c-myc (3). It is not yet known whether these features are noncausal associations or a causal factor. FLS in culture express large amounts of proteinases that can degrade extracellular matrix components such as collagens. One family of proteinases expressed by FLS are the matrix metalloproteinases (MMPs). FLS express MMPs 1, 3, 9, and 10, and the expression of these MMPs correlates with invasion (4). Other proteinase families that are expressed by RA FLS are the cathepsins and ADAMs.
FLS from patients with RA express several oncogenes at higher levels than do FLS from normal controls. Oncogenes that are up-regulated in RA are c-myc (11), ras (5), and p53 (6, 7). These data have led to the suggestion that aspects of behavior of FLS in RA resemble those of malignant tissue. However, whether the transformed behavior is of relevance to the disease characteristics of RA and, if so, to which characteristics has not been studied.
In this study, we investigated the association between in vitro characteristics of FLS and disease characteristics of RA patients. We addressed whether the degree of FLS invasion was comparable in different joints of the same patient (i.e., is FLS invasiveness a characteristic that occurs in multiple joints of the same patient or is it a random process), and whether the degree of in vitro invasion correlated with the degree of radiologic destruction.
MATERIALS AND METHODS
Patients and synovium.
Synovial tissue was obtained from 121 patients (72 with RA and 49 with osteoarthritis [OA]) at joint replacement surgery or synovectomy. Sixty-nine percent of the patients with RA were women and the mean ± SD age was 60 ± 14 years. Samples were obtained from knees (53 patients), elbows (17 patients), shoulders (13 patients), hips (26 patients), ankles (7 patients), wrists (4 patients), and feet (1 patient). All patients with RA met the criteria of the American College of Rheumatology (formerly, the American Rheumatism Association) (8). Tissue was harvested by an orthopedic surgeon (RGN) and collected in sterile phosphate buffered saline (PBS). Connective tissue and fat were removed, and tissue was digested with type IA collagenase (1 mg/ml; Sigma, St. Louis, MO) for at least 2 hours at 37°C. Cells were separated from the tissue using a 200-μm filter (NPBI, Emmer-Compascuum, The Netherlands) and cultured in 75-cm2 culture flasks (Cellstar; Greiner, Alphen aan den Rijn, The Netherlands) with Iscove's modified Dulbecco's medium (IMDM; BioWhittaker, Verviers, Belgium) supplemented with Glutamax (Gibco, Paisley, UK), penicillin and streptomycin (Boehringer, Mannheim, Germany), and 10% fetal calf serum (FCS; Gibco) at 37°C in an atmosphere of 5% CO2.
When the cells had grown to confluence, they were detached with 0.25% trypsin and split in a 1:3 ratio. For invasive growth analysis, passage 1 or 2 FLS were used. Light microscopy and Giemsa staining indicated that >95% of cells were FLS. As a control for the possible contamination of macrophages in the cell source, we reasoned that after several passages, the macrophages would be gone. Thus, the invasiveness of FLS from several patients was tested for several passages. This revealed stable invasiveness for several passages. Moreover, fluorescence-activated cell sorting of a randomly chosen subset of the samples revealed the absence of CD14-positive cells.
In vitro invasion assay.
Invasiveness of FLS was measured as previously described (4). Briefly, transwells (6.5 mm diameter, 8.0 μm pore width; Costar, Cambridge, MA) were coated with paraffin to prevent meniscus formation. Transwells were then preincubated with 100 μl IMDM for 30 minutes at 37°C. Transwells were coated overnight with 100 μl of 0.375 μg/ml Matrigel basement membrane matrix (Becton Dickinson, Mountain View, CA) in IMDM under sterile conditions in a laminar flow cabinet. The next day, the Matrigel-coated wells were incubated with 100 μl IMDM for 1 hour at 37°C. Cells were harvested as described above, and after removal of the medium, 200 μl of 100,000 FLS/ml in IMDM was seeded in the inner compartment of the transwell system. In the outer compartment, 900 μl IMDM/10% FCS/10% human serum was pipetted, and the cells were incubated for 3 days at 37°C in an atmosphere of 5% CO2. After 3 days, the cells were fixed with 2% glutaraldehyde in PBS for 30 minutes at room temperature. After removal of the glutaraldehyde and subsequent washing with PBS, the cells were stained with a crystal violet solution for 30 minutes at room temperature. The cells were thoroughly washed with PBS, and those that did not invade the transwell membrane were removed, together with the matrix, by cleaning the inner wells of the transwell system with a cotton swab. The number of cells that had grown through the matrix and the transwell membrane were counted under a light microscope. All experiments were performed in duplicate.
Radiologic destruction and FLS characteristics.
To compare the association between the degree of FLS invasiveness and joint destruction, radiographs of the hands and feet of patients with the most invasive FLS (n = 9) and the least invasive FLS (n = 9) were scored according to the Sharp/van der Heijde method (9). The person who scored the radiographs (AHH) was unaware of the clinical data and the study question. The total erosion and joint space narrowing scores, as well as the total Sharp/van der Heijde scores, were divided by the disease duration from the date of diagnosis to determine the yearly progression of radiologic joint damage (10).
Results are expressed as the mean ± SD. Differences in invasiveness of FLS between patients, differences between intraindividual and interindividual variation, and the radiographic scores of the patients with the most and least invasive FLS were compared by the Mann-Whitney U test. P values less than 0.05 were considered significant.
FLS were isolated from the tissues and cultured. When the cells had grown to confluency, the cells were harvested and tested for invasiveness, as previously described (4). RA FLS were significantly more invasive than OA FLS in this study (mean ± SD 2,884 ± 2,326 and 4,573 ± 2,502 for OA and RA FLS, respectively; P < 0.001) (Figure 1). These results are consistent with those of previous studies (1, 4).
We then studied whether the invasiveness of FLS obtained at different times from different joints exhibited the same invasive characteristics. Two different samples were obtained from 2 different joints of 7 patients with RA. The mean differences in FLS invasiveness of different samples from an individual patient were significantly less than the mean differences between patients (mean ± SD 1,067 ± 926 and 3,845 ± 2,367 for intra- and interindividual variation, respectively; P = 0.035) (Figure 2). Thus, the variation within patients was smaller than the variation between patients. From the group of OA patients, 3 patients were operated upon twice. This group was too small for an analysis of the differences between intraindividual and interindividual variation.
Next, we addressed whether the in vitro invasiveness of FLS was correlated with radiologic joint destruction in RA. It has been shown that the estimated annual rate of destruction obtained by dividing the Sharp/van der Heijde score for radiographs of hands and feet by the disease duration correlates well with the destruction rate reported in observational studies using multiple measurements (10). We assessed the association between the invasiveness of FLS and radiologic joint destruction.
Sharp/van der Heijde scores from the 9 patients with the most invasive FLS and the 9 patients with the least invasive FLS were determined, and the estimated yearly destruction rates were compared (Figure 3). For patients with the most invasive FLS, the mean ± SEM total Sharp/van der Heijde score per year of disease duration was 21.8 ± 3.1, with a mean ± SEM erosion score of 13.3 ± 1.7 and a narrowing score of 8.5 ± 1.6. For patients with the least invasive FLS, the mean ± SEM total Sharp/van der Heijde score per year of disease duration was 4.4 ± 1.1, with an erosion score of 2.5 ± 0.7 and a narrowing score of 1.9 ± 0.5. No difference in disease duration was observed between patients with the most and the least invasive FLS (mean ± SEM 15.9 ± 11.5 versus 15.0 ± 7.3 years, respectively; P = 0.85). These results show a strong association between invasiveness of FLS and radiologically estimated yearly rate of joint destruction (P < 0.001).
Our findings show that in RA, the intraindividual variation in FLS invasiveness is much lower than the interindividual variation, indicating that the invasive behavior of FLS is a characteristic of individual RA patients. Furthermore, this study is the first to show that in vitro FLS invasiveness is associated with radiologic joint destruction. Patients with the least invasive FLS had significantly lower Sharp/van der Heijde scores per year of disease duration than did patients with the most invasive FLS. This suggests that the invasive behavior of FLS is relevant to the pathogenesis of RA.
The finding of a rather large variation in the rate of invasion of FLS between patients implies that the mechanism or processes underlying the invasive behavior of FLS differs between individuals. The mechanism that leads to the transformation of FLS is not fully understood.
Previous studies have shown a myriad of alterations in the behavior of FLS in RA. One very striking change in FLS is the expression of oncogenes (11). Oncogenes that are up-regulated in RA are c-myc (12, 13), ras (5), p53, and others. Inhibition of the Ras pathway reduced expression of MMP-1 and MMP-3. Inhibition of both the Ras and c-Myc pathways also reduced invasion into normal human cartilage in the SCID mouse coimplantation model (14). It has also been shown that down-regulation of p53 influences the proliferation and invasion of RA FLS (15, 16). Transformation of FLS is different in different individuals, suggesting that a genetic component plays a role.
This study shows that the transformed behavior of FLS in patients with RA is a patient characteristic and is strongly associated with clinical joint destruction. We used FLS from passages 1 and 2 in this study. Although no macrophages were detected in these samples by light microscopy and Giemsa staining, and no CD14-positive cells were detected in a randomly chosen subset of the samples, we cannot exclude unambiguously that macrophages contaminated the cells. However, the experiments in which FLS from different passages and from different donors were compared yielded similar levels of invasiveness, which is evidence against major artifacts from macrophages. Further research should elucidate the exact mechanism of transformation of FLS and the role of a genetic component.