Rheumatoid arthritis (RA) results in destructive changes affecting joints, ligaments, and tendons. Much of the research into the molecular causes of RA has focused on the synovial-lined joints; the involvement of soft tissue, particularly the tendons and ligaments, has only been recognized relatively recently.
At least 50% of patients with RA have tendon involvement (1); the affected tendons are surrounded by synovial sheaths. Proliferative tenosynovitis may lead to tendon rupture, but even if this does not occur, persistent tenosynovitis leads to chronic scarring and adhesions, with reduced excursion and ultimately impaired function. Approximately 50% of patients have been found to have infiltration of the tendon by tenosynovium at the time of prophylactic tenosynovectomy (2). Invasion of the tendon by tenosynovium is associated with multiple tendon ruptures (3). Multiple ruptures lead to poor outcomes (4, 5), as does synovial invasion of the tendon (1). Once the tendon has ruptured, direct repair is impossible, and reconstruction depends on tendon transfer or tendon grafts.
While it is known that a combination of mechanical attrition, ischemia, and synovial proliferation and invasion causes tendons to rupture in RA, the exact mechanism by which the synovium brings about this destruction is unknown. Histologically, invasive tenosynovium has the characteristics of chronic inflammatory tissue. At the electron microscopic level, the affected tendons show an alteration in collagen structure, with an increased number of thin collagen fibrils and degenerative nodules known as Luse bodies (6). While there are reports regarding the histologic changes to the tendon in RA, surprisingly little is known regarding the involvement of proinflammatory cytokines or proteolytic enzymes in this process. It has, however, been demonstrated that tendon tissue cells can actively secrete matrix metalloproteinase 1 (MMP-1) and tissue inhibitor of metalloproteinases 1 (TIMP-1), suggesting that tendon is capable of producing a remodeling response (7).
More is known regarding cytokine and proteolytic enzyme activity in RA joint synovium. Destruction of cartilage and other components of connective tissue is thought to involve the actions of proteolytic enzymes, including the MMPs (8). MMPs are enzymes produced by activated macrophages, fibroblasts, and chondrocytes in response to proinflammatory cytokines such as interleukin-1 (IL-1) and tumor necrosis factor α (TNFα) (8).
The importance of TNFα in the pathogenesis of RA was emphasized by the success of studies on TNFα blockade (9). TNFα and IL-1 directly induce proteolytic enzyme synthesis and indirectly induce other cytokines that can perpetuate the inflammation (10). Another cytokine of interest in RA is IL-6. IL-6 is abundant in both serum and synovial fluid in patients with RA (11) and displays proinflammatory as well as protective properties.
MMPs, a family of zinc- and calcium-dependent endopeptidases, possess the ability to break down the extracellular matrix macromolecules associated with tissue destruction in various pathologic conditions. MMPs 1 and 13 are both capable of cleaving type I collagen, the primary constituent of tendon, as is MMP-2 in high concentrations (12). MMP-3 degrades other important components of the extracellular matrix; serum levels of MMP-3 have been shown to be elevated in patients with RA and reduced in response to anti-TNFα therapy (13). Because MMPs bind and degrade specific substrates, they can be potentially pathogenic if their regulatory mechanisms are not tightly controlled. Regulation is achieved at the levels of transcription and translation and by enzyme action and the production of TIMPs. TIMPs are specific inhibitors of MMPs that bind with them in a 1:1 noncovalent complex (8). RA patients show elevated levels of TIMP-1 in synovial fluid and serum, but it is the imbalance of the enzyme:inhibitor ratio, with up-regulation of MMPs, that allows continued matrix destruction (8).
In addition to the actions of the cytokines and proteolytic enzymes, it has been demonstrated that the inflammatory process in RA joint synovium is maintained by the continued development of new blood vessels, a process known as angiogenesis, in response to increasing metabolic demands of the tissue. Angiogenesis is a tightly governed process that depends on the balance of pro- and antiangiogenic stimuli. An important proangiogenic factor in the pathogenesis of RA is vascular endothelial growth factor (VEGF). RA synovial joint tissue has been shown to express VEGF predominantly in macrophages and lining cells, as well as in endothelial cells lining small blood vessels within the joint pannus (14).
PATIENTS AND METHODS
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- PATIENTS AND METHODS
Synovial specimens were obtained from 18 RA patients undergoing wrist extensor tenosynovectomy between July 1999 and July 2000. All patients gave signed consent, and ethical approval was obtained from the Riverside Research Ethics Committee (London). Patients met the American College of Rheumatology (ACR; formerly, the American Rheumatism Association) 1987 revised criteria for RA (15) and had clinically significant tenosynovitis at the time of surgery. The mean age of patients was 56 years (range 25–73 years), and the ratio of men to women was 1:8. The mean duration from disease onset to time of surgery was 15.5 years (range 2–40 years). The following 3 types of synovial specimens were obtained from each patient, as appropriate: synovium enveloping tendons (n = 17), synovium that had invaded tendons (n = 13), and synovium from the wrist joint (our control; n = 17). All patients continued to take their medication, including steroids, throughout the operative period.
Preparation of RA synovial membrane cells
Synovial membrane tissue was digested in RPMI 1640 (Sigma, Poole, UK) containing heat-inactivated 5% (volume/volume) fetal calf serum (FCS; Flow, High Wycombe, UK), 1 mg/ml type A collagenase (Boehringer, Mannheim, Germany), and 0.15 mg/ml DNase I (Sigma) for up to 2 hours at 37°C. Digested tissue was pipetted through a 200-μm2 nylon mesh into a sterile beaker. Cells were spun down, and trypan blue was used to check cell viability. Viable cells (106/well) were counted and cultured in RPMI 1640 containing 5% heat-inactivated FCS in 24-well culture plates (Falcon, Lincoln Park, NJ) at 37°C in 5% CO2 for 48 hours. Cell supernatants were harvested and stored at −70°C until required for analysis.
Enzyme-linked immunosorbent assays (ELISAs)
Cytokine, VEGF, MMP, and TIMP-1 production was measured in synovial cell culture supernatants by standard sandwich ELISA techniques using specific monoclonal and polyclonal antibodies.
Reagents used for the TNFα ELISA were provided by Dr. W. Buurman (University of Limburg, Maastricht, The Netherlands). The TNFα ELISA was performed as previously described (16). The range of the ELISA was 6.8–5,000 pg/ml, and its sensitivity was 40 pg/ml.
Reagents for the IL-6 ELISA, coat LN1-314-14 and detect LN1-14-110, were provided by Dr. F. Di Padova (Sandoz, Basel, Switzerland). The ELISA was carried out as previously described (17). The range of the ELISA was 13–10,000 pg/ml, and its sensitivity was 40 pg/ml.
Levels of VEGF in RA synovial membrane cell culture supernatants were measured by ELISA (R&D Systems, Abingdon, UK) in accordance with the manufacturer's instructions.
MMP and TIMP-1 immunoassays
MMPs 1, 2, 3, and 13 and TIMP-1 were assayed by ELISA (Amersham Life Science, Amersham Place, Buckinghamshire, UK), and all steps were performed according to the manufacturer's instructions. The assays for MMPs 1, 3, and 13 detect activated MMPs, pro–matrix metalloproteinases (proMMPs), and MMPs complexed to TIMPs. The assay for MMP-2 detects both the free form of proMMP-2 and proMMP-2 complexed to TIMP-2, but does not detect active MMP-2. Culture supernatants of synovial cells from individual patients were assayed in duplicate on the same plate.
Gelatin zymography was performed as previously described (18). Supernatant diluted 1:10 (10 μl) was loaded onto the gel alongside the positive controls of 10 ng activated recombinant MMP-1 expressed in Escherichia coli and 0.25 ng MMP-2 expressed in human uterine cervical fibroblasts (a gift from Prof. H. Nagase, Kennedy Institute of Rheumatology, London, UK). Molecular weight markers from Amersham Pharmacia Biotech (Amersham Place, Buckinghamshire, UK) were used.
Results were expressed as the mean ± SD of duplicate samples. Analysis was performed using the Prism software (GraphPad Software, San Diego, CA) for the PC. Comparison between synovial sample groups was performed using Student's paired 2-tailed t-tests for normally distributed data and the Wilcoxon signed rank test for nonparametric data (MMP-13 produced by encapsulating tenosynovium). P values less than or equal to 0.05 were considered significant.
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- PATIENTS AND METHODS
Both encapsulating (mean ± SD 716 ± 831.2 pg/ml; P = 0.82) and invasive (745.9 ± 1,101.3 pg/ml; P = 0.51) tenosynovium were found to produce TNFα spontaneously and in levels very similar to those produced by wrist joint synovium (748.3 ± 938.7 pg/ml), suggesting that this cytokine may be important in the process of tenosynovitis and tendon destruction, as it is in joint destruction. The downstream cytokine IL-6 was also detected in all RA synovial membrane cultures from the different sites, but was produced in much lower levels by encapsulating (497.3 ± 359.8 ng/ml; P = 0.03) and invasive (533 ± 517.8 ng/ml; P = 0.24) tenosynovium compared with wrist joint synovium (835.7 ± 537.9 ng/ml), suggesting that while production of TNFα may be uniform among synovial groups, differences in downstream cytokines may exist (Table 1).
Table 1. Levels of cytokines, vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMPs), and tissue inhibitor of metalloproteinases 1 (TIMP-1) produced by different synovial membrane cell culture groups as measured by enzyme-linked immunosorbent assay*
| ||Synovial sample||P†|
|Wrist joint synovium vs. encapsulating tenosynovium||Wrist joint synovium vs. invasive tenosynovium||Encapsulating tenosynovium vs. invasive tenosynovium|
|Wrist joint synovium||Encapsulating tenosynovium||Invasive tenosynovium|
|TNFα, pg/ml||748.3 ± 938.7 (14)||716 ± 831.2 (14)||745.9 ± 1,101.3 (10)||0.82||0.51||0.95|
|IL-6, ng/ml||835.7 ± 537.9 (12)||497.3 ± 359.8 (13)||533 ± 517.8 (10)||0.03‡||0.24||0.53|
|VEGF, ng/ml||8.9 ± 6.3 (14)||7.1 ± 6.5 (14)||8.1 ± 8.8 (10)||0.42||0.65||0.34|
|MMP-1, ng/ml||864.4 ± 1,097.2 (11)||214.2 ± 251.8 (11)||514.6 ± 762.8 (7)||0.12||0.26||0.28|
|MMP-2, ng/ml||493.6 ± 317.6 (14)||478.2 ± 462.1 (12)||734.7 ± 692.6 (10)||0.59||0.43||0.30|
|MMP-3, μg/ml||20.1 ± 15.5 (12)||17 ± 20.9 (14)||14.7 ± 11.8 (10)||0.06||0.27||0.59|
|MMP-13, ng/ml||14.2 ± 16.2 (14)||1.7 ± 2.9 (15)||4.5 ± 6.7 (10)||0.009‡§||0.25||0.46§|
|TIMP-1, ng/ml||1,138.6 ± 1,073 (12)||1,249.3 ± 1,065.6 (12)||1,336.8 ± 736.5 (8)||0.59||0.52||0.95|
While the levels of proinflammatory cytokines were similar in encapsulating and invasive tenosynovium, the levels of MMPs differed (Table 1 and Figure 1). Lowest levels of MMP-1 (214.2 ± 251.8 ng/ml) and MMP-13 (1.7 ± 2.9 ng/ml) were found in encapsulating tenosynovium; interestingly, however, invasive tenosynovium produced almost 2.5-fold more MMP-1 (514.6 ± 762.8 ng/ml; P = 0.28) and >2.5-fold more MMP-13 (4.5 ± 6.7 ng/ml; P = 0.46) than did synovium surrounding the tendon.
Figure 1. Levels of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases 1 (TIMP-1) in synovial sample groups. Synovial membrane cells from wrist joint synovium, encapsulating tenosynovium, and invasive tenosynovium were cultured for 48 hours. Supernatants were harvested, and levels of MMPs and TIMP-1 were determined by enzyme-linked immunosorbent assay. Bars represent mean values. Note logarithmic scale. Invasive tenosynovium produced ∼2.5-fold more MMP-1 and MMP-13 than encapsulating tenosynovium. Invasive tenosynovium also produced ∼1.5-fold more of the enzyme MMP-2 than both wrist joint synovium and encapsulating tenosynovium. Significantly lower levels of MMP-13 were produced by encapsulating tenosynovium compared with wrist joint synovium (P = 0.009). Levels of MMP-3 and TIMP-1 were similar in all synovial culture groups.
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MMP-2 levels were similar in wrist joint synovium (493.6 ± 317.6 ng/ml) and encapsulating tenosynovium (478.2 ± 462.1 ng/ml) (P = 0.59), but the highest levels of this enzyme were produced by invasive tenosynovium samples (734.7 ± 692.6 ng/ml). Unlike all the other MMPs measured, MMP-2 was produced in higher amounts by invasive tenosynovium compared with both of the other groups.
MMP-3 is an important enzyme in RA, degrading extracellular matrix components such as aggrecan, fibronectin, laminin, elastin, and gelatin. Interestingly, levels of MMP-3 were similar in all groups of synovia, indicating the enhanced proteolytic nature of tenosynovium in RA.
Our results showed slightly higher levels of the MMP inhibitor TIMP-1 in invasive tenosynovium (1,336.8 ± 736.5 ng/ml) compared with encapsulating tenosynovium (1,249.3 ± 1,065.6 ng/ml) (P = 0.95), a finding that would be expected considering the increased production of the majority of MMPs by this group. However, the increased production of MMPs 1, 2, and 13 by invasive compared with encapsulating tenosynovium suggests that the small increase in TIMP-1 production would result in an imbalance in the inhibitor:enzyme ratio, in favor of the enzyme, in the invasive tenosynovium group compared with the encapsulating tenosynovium group.
Zymography demonstrated enzyme activity against gelatin in all of the 3 sample groups in all 9 patients tested (a representative gel is shown in Figure 2). Indeed, many of the invasive tenosynovium samples showed both an increase in the activity of particular enzyme species (larger zones of digestion) and the appearance of additional bands (indicating the presence of additional proteolytic species) compared with the other types of synovium. While zymography demonstrates potential activity, it does not allow for accurate identification of the MMP responsible for a particular zone of digestion because many of the enzymes have similar molecular weights. Some of the bands may represent proforms of the enzymes that are latent in vivo but activated by the zymography process. The results do show, however, that RA tenosynovial cells, like wrist joint synovium, secrete activatable enzymes, and suggest that invasive tenosynovium produces a wider spectrum of enzymatic activity than does encapsulating tenosynovium.
Figure 2. Gelatin zymography for analysis of enzyme activity among the 3 synovial sample groups in 9 patients. The representative gel shown demonstrates enzyme activity in all 3 synovial supernatants from 1 patient, a trend that was apparent in all other patients tested. MMP-1 and MMP-2 positive controls are included. It must be noted, however, that zymography does not allow unequivocal identification of the enzyme responsible for each band of lysis based on the molecular weight of the band alone. ProMMP-2 = pro–matrix metalloproteinase 2 (see Figure 1 for other definitions).
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Angiogenesis is important in the development of the RA joint pannus, and mean ± SD levels of VEGF were very similar both in wrist joint synovium (8.9 ± 6.3 ng/ml) and in tenosynovium samples (7.1 ± 6.5 ng/ml in encapsulating tenosynovium, 8.1 ± 8.8 ng/ml in invasive tenosynovium). This finding suggests that angiogenesis may be an important process in tenosynovitis (Table 1).
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- PATIENTS AND METHODS
While there has been much research into the effects of cytokines, VEGF, and MMPs in synovial joint disease in RA, little is known of their effects on tendons, despite the fact that tendons are affected in many patients with RA. In our study, all synovial membrane cultures contained a complex, but pathophysiologically relevant, mixture of cells. While it was not surprising to find high levels of proinflammatory cytokines and proteolytic enzymes in wrist joint synovium, the observation that tenosynovium produced similar profiles, although at lower levels, shows that tenosynovium is capable of producing these destructive proteins.
In high concentrations, MMP-2 is capable of slowly degrading type I collagen (12), and, in our study, MMP-2 was produced in maximal amounts by invasive tenosynovium cultures, suggesting its importance in tendon destruction. Of further interest were the differences between these sample groups in levels of the enzymes MMP-1 and MMP-13. Both of these enzymes act against type I collagen, and mean levels of both enzymes were ∼2.5-fold higher in invasive tenosynovium than in encapsulating tenosynovium.
It is known that tenosynovial invasion of the tendon is associated with multiple tendon ruptures and a poorer prognosis (1, 3), but the precise mechanism of tendon invasion and degradation is unknown. Our results suggest that increased production of the enzymes MMP-1, MMP-2, and MMP-13 by invasive tenosynovium compared with that by synovium simply surrounding the tendon may be important.
Our results also showed similar levels of TIMP-1 in the different synovial sample groups, suggesting that the increase in metalloproteinase production in the invasive versus the encapsulating tenosynovial tissue is not compensated sufficiently by an increase in TIMP-1 production. This lends further weight to the argument that these MMPs may be implicated in tendon destruction.
It remains unknown why tenosynovium should become invasive, although it is unlikely to result simply from an increase in total cell number. Mean total cell numbers obtained from samples at the time of surgery were highest in wrist joint synovium and encapsulating tenosynovium and lowest in invasive tenosynovium (data not shown). Studies were performed on 2 patients to observe the cellular composition of the different types of synovium to explore the possibility that differences in MMP levels were due to differences in cell composition (Table 2). Flow cytometry using fluorochrome-conjugated antibodies (fluorescence-activated cell sorter analysis) was employed with markers for lymphocytes (CD45), monocytes (CD14), and T cells (CD3), as well as with markers of activation (HLA–DR). Although this study was very limited, it provided some idea of the cellular makeup of the samples.
Table 2. Cellular composition of samples (n = 2 patients) and cell activation as measured by flow cytometry*
| ||Wrist joint synovium||Encapsulating tenosynovium||Invasive tenosynovium|
Wrist synovium in both patients contained more fibroblasts, which provides a possible explanation for the higher levels of IL-6 found in wrist joint synovium compared with tenosynovium. Populations of lymphocytes and monocytes were higher in tenosynovium samples compared with wrist joint synovium samples, while levels of T cells were similar in all groups. Interestingly, tenosynovium had a higher proportion of activated cells compared with wrist joint synovium. While there were differences in cellular composition between wrist joint synovium and tenosynovium, the compositions of encapsulating and invasive tenosynovium were almost identical. This suggests that more complex mechanisms must be operating to explain the differences seen in MMP production demonstrated in these two groups.
Zymography demonstrated potential enzyme activity in the 3 different sample groups, with tenosynovium samples displaying at least as much enzyme activity as wrist joint synovium samples. While it is difficult to identify the MMP responsible for the band of digestion by molecular weight alone, since many MMPs have active forms of similar sizes, our results do suggest that enzymes with activation potential are produced by tenosynovium cultures. Inhibition of other classes of proteolytic enzymes showed that the enzyme degradation witnessed was the result of metalloproteinase activity (Jain A, et al: unpublished observations). Further work using Western blotting techniques should aid in accurately identifying the specific MMPs responsible for the zones of digestion demonstrated by zymography.
To proliferate, a cell mass needs an associated increase in vasculature to support its metabolic requirements, and this is facilitated by angiogenesis, an important process in RA. VEGF is the most endothelial cell–specific angiogenic factor characterized to date. VEGF was detectable in all synovium sample groups, and the levels were similar. This suggests that, as in joint disease, angiogenesis may be an important factor in allowing tenosynovial proliferation and invasion.
In conclusion, we have demonstrated that tenosynovium is capable of producing cytokines and proteolytic enzymes known to be important in the tissue destruction seen in RA. The proteolytic enzymes MMP-1, MMP-2, and MMP-13 are produced in higher amounts by invasive tenosynovium compared with encapsulating tenosynovium, suggesting a possible explanation for the worse prognosis and increased rupture rate seen in patients with invasive tendon disease.