Department of Rheumatology and Internal Diseases,
1 Department of Orthopaedic Surgery and
2 Department of Pathological Anatomy, Medical University of Bialystok, Bialystok, Poland
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Abstract |
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Methods. Tissue and serum samples were obtained from 37 patients with clinically active RA and 30 with osteoarthritis (OA). Morphological analysis allowed the division of RA synovial specimens into two distinct types. In 22 samples only diffuse infiltrates of mononuclear cells without further microanatomical organization were found. In 15 specimens we observed lymphocytic conglomerates with germinal centre-like structures. Serum concentrations of interstitial collagenase (MMP-1), stromelysin-1 (MMP-3), gelatinase B (MMP-9), TIMP-1 and TIMP-2 were measured with an ELISA technique.
Results. Unique serum profiles of MMPs and TIMPs were identified in each of the two histological types of RA synovitis. The serum concentrations of MMP-1, MMP-3 and MMP-9 were higher in RA patients than in OA patients used as a control group (P<0.001 for all comparisons). These three MMPs dominated in the serum of RA patients with follicular synovitis compared with those with diffuse synovitis (P<0.05, P<0.01 and P<0.001 respectively). The analysis of the serum concentrations of TIMP-1 and TIMP-2 showed that their levels were also elevated in RA patients compared with OA patients (P<0.001 and P<0.01 respectively). Only TIMP-1 was found in a significantly higher concentration in the serum of RA patients with follicular synovitis than in those with diffuse synovitis (P<0.05). The serum concentrations of MMPs and TIMP-1 clearly identified patients with two different histological types of rheumatoid synovitis and with OA. Additionally, the analysis of clinical data showed that the rheumatoid disease in patients with follicular synovitis seemed to be more active than in those with diffuse synovitis.
Conclusion. The morphological appearance of rheumatoid synovitis and the serum MMP and TIMP-1 profile were correlated with the clinical activity of the disease, confirming the heterogeneity of RA. These associations also suggest that patients with different histological forms of RA might require different treatment regimens.
KEY WORDS: Matrix metalloproteinases, MMPs, Tissue inhibitors of metalloproteinases, TIMPs, Rheumatoid arthritis, Histology, Heterogeneity.
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Introduction |
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Several investigators have shown genetic, histological, biological and clinical heterogeneity of RA [1014]. However, the associations between the morphological components of RA synovium and biological manifestation remain unclear. There are no histological findings that are typical of RA and distinguish it from other inflammatory arthropathies [3]. The only exception is the formation of TB cell follicles, which sometimes form germinal-like centres [1416]. It was also suggested that patients with such focal lymphoid aggregates represented a greater degree of immunological activation and greater potential for articular destruction [14]. Microscopic evaluation of rheumatoid synovium may demonstrate the existence of at least three histological types [17]. Lymphocytic clusters were found in a subgroup of RA patients. Some of these clusters resembled germinal centre-like structures with a central accumulation of B cells surrounded by T cells. A few RA patients had necrobiotic granulomas with a fibrinoid, necrotic centre lined with a collar of histiocytes, rarely with giant cells. The coexistence of follicular synovitis and granulomatous necrobiosis was not observed in the same samples. The remaining patients had only a diffuse infiltrate of mononuclear cells with no further morphological organization [17].
Each of the histological forms of rheumatoid synovitis might be characterized by a specific pattern of cytokine mRNA expression in the synovium [17]. The purpose of this study was to explore whether serum concentrations of MMPs and/or their inhibitors were also associated with the histological appearance of the disease.
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Materials and methods |
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Clinical and laboratory evaluation
The analysis included the duration of morning stiffness, the number of tender joints (Ritchie index) [19], the number of swollen joints, the erythrocyte sedimentation rate (ESR) and the rheumatoid factor level.
Histopathological assessment
Synovial tissue specimens were stained with haematoxylin and eosin. The density of the cell infiltrate and the topographical organization of mononuclear cells were analysed and classified as described previously [17]. Samples were assessed by one pathologist (BC), who was blind to the clinical and laboratory data.
Serum sample preparation
Blood specimens were clotted for 30 min and then centrifuged for 10 min at 1000 g. Serum aliquots were frozen at -80 °C immediately after sample collection.
Measurement of serum MMPs and TIMPs
Serum concentrations of MMP-1, MMP-3, MMP-9, TIMP-1 and TIMP-2 were measured with a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) (Biotrak; Amersham Pharmacia Biotech, Little Chalfont, UK) according to the manufacturer's instructions. The sensitivities were 1.7 ng/ml for MMP-1, 2.35 ng/ml for MMP-3, 0.6 ng/ml for MMP-9, 1.25 ng/ml TIMP-1 and 3.0 ng/ml for TIMP-2.
Statistical analysis
Normally distributed clinical data were analysed with the unpaired Student's t-test. The MannWhitney U-test was employed to analyse differences between non-normally distributed data on MMPs and TIMPs. Probabilities of differences in frequency distributions were assessed with Fisher's exact test. The analysis of correlations between variables was based on Spearman's rank test. P<0.05 was considered statistically significant.
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Results |
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Clinical and radiological findings
No significant differences in sex ratio, age or disease duration were observed between patients with the two morphological types of RA and those with OA. The ESR was higher in the RA than in the OA group (P<0.001) and higher in patients with follicular synovitis than in those with diffuse infiltration (P<0.05) (Table 1). The number of swollen joints was higher in patients with the follicular type of RA than in those with the diffuse type (P<0.01). Radiologically more advanced joint destruction (stage III or IV according to Steinbrocker's criteria) was more frequent in the group of patients with follicular synovitis than in the group with diffuse infiltration (P<0.05) (Table 1
). No other clinical parameters of disease activity differed significantly between the histological variants of RA.
All patients were receiving non-steroidal anti-inflammatory drugs. Sulphasalazine was used in the therapy of five (22.7%) and two (13.3%) RA patients with the diffuse and follicular type of synovitis respectively during the last 3 months before surgery (difference not significant) (Table 1). Seven (31.8%) RA patients with diffuse synovitis were being treated with methotrexate compared with 12 (80.0%) patients with the follicular histological form of the disease (P<0.01). Four (18.2%) and five (33.3%) RA patients with the diffuse and follicular type of synovitis respectively were receiving oral steroids (difference not significant). No patients received steroid injections in last 3 months before surgery.
Serum MMP concentrations
A two-site ELISA sandwich technique was used to measure the serum concentrations of MMPs. Our main interest was whether the levels of MMPs differed between patients with distinct histological types of synovitis. As shown in Fig. 2, the amounts of MMP-1 in serum were higher in patients with RA and the diffuse and follicular types of synovitis than in OA patients (P<0.001, P<0.01 and P<0.001 respectively). MMP-1 reached the highest concentration in patients with the follicular type of synovitis, differentiating them from those with the diffuse histological type of the disease (P<0.05).
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Serum TIMP concentrations
Serum concentrations of TIMPs were also measured with a two-site ELISA sandwich method. Like the MMP levels, the serum levels of TIMP-1 were higher in RA patients and in patients with the diffuse and follicular morphological forms of rheumatoid synovitis than in OA patients (P<0.001 for all comparisons) (Fig. 5). TIMP-1 reached the highest concentration in patients with the follicular type of synovitis and clearly differentiated these patients from patients with diffuse infiltrates (P<0.05).
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Correlations between clinical data and serum MMP and TIMP levels
Table 2 shows the correlations among clinical parameters of disease activity and between these variables and serum MMP and TIMP concentrations in RA patients.
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Concentration ratios of MMPs to TIMPs
As shown in Table 3, the calculated concentration ratios of MMP-1 to TIMP-1 or TIMP-2 did not differ significantly between OA and RA patients with the two histological patterns of synovitis. The concentration ratios of MMP-3, MMP-9 and total measured MMPs to TIMP-1, TIMP-2 and total measured TIMPs were higher in RA patients than in OA patients (P<0.001 for all comparisons), but were especially elevated among patients with the follicular type of rheumatoid synovitis. However, significant differences between the two histological types of RA were observed only for the ratios MMP-9/TIMP-1, MMP-9/TIMP-2 and total MMPs/total TIMPs (P<0.05, P<0.01 and P<0.05 respectively).
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Discussion |
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In the present study, we were able to distinguish only two of the morphological forms of rheumatoid synovitis described in the report referred to above [17]. Almost two-thirds of the RA synovium tissues were characterized by diffuse mononuclear cell infiltrates with no further microanatomical organization. Follicular clusters of lymphocytes with germinal centre-like structures were found in the remaining specimens. We did not find necrobiotic granulomas. However, in the previous study [17] these were very rare.
MMPs and TIMPs play an important role in the remodelling of the articular tissues [46, 9]. MMPs are responsible for the proteolytic degradation of extracellular matrix components. They are produced by several types of cell, such as chondrocytes, fibroblasts and macrophages [57, 20]. It was demonstrated that their synthesis is stimulated by cytokines such as IL-1 and TNF- [5, 6, 21]. The action of MMPs is regulated by TIMPs, the production of which is stimulated by cytokines such as transforming growth factor ß, IL-6 and IL-11 [5, 6, 22]. The aim of the present study was to explore whether the serum concentrations of MMPs and their inhibitors are associated with the histological appearance of the disease. Our main interest was whether the concentrations of MMPs and/or TIMPs might identify patients with distinct morphological types of rheumatoid synovitis.
Interstitial collagenase (MMP-1), produced mainly by synovial fibroblasts [20, 23], plays an important role in the degradation of articular cartilage and synovium [5, 6, 9]. Previous investigators have shown that, in RA, MMP-1 is present not only in synovium [20, 24, 25] but also in synovial fluid [25] and serum [23, 25]. However, only some of the previous work has shown that its concentration in RA synovial fluid [26] or serum [27, 28] differs significantly from concentrations in controls. That MMP-1 is expressed not only in patients with established erosive RA but also in patients with early arthritis implies that this protease may serve as a marker of the very early stage of the disease [29]. Our study confirms that the level of MMP-1 in serum is elevated in RA patients. Furthermore, we found that MMP-1 reached its highest concentration in patients with the follicular type of synovitis, differentiating them from those with diffuse synovitis.
We also demonstrated that the serum concentration of this protease correlated with MMP-3, MMP-9 and clinical markers of disease activity, such as ESR and the duration of morning stiffness. Among the group of 15 patients with the follicular type of the disease and with more advanced joint destruction, we also found significant correlations between the serum level of MMP-1 and the Ritchie index (r=0.573, P=0.0252) and the number of swollen joints (r=0.555, P=0.0312) (data not shown). In the case of patients with the diffuse type of synovitis and for all 37 RA patients, these correlations were not significant (data not shown). Therefore, MMP-1 may be more a marker of joint damage than of disease activity.
Stromelysin-1 (MMP-3) is known to degrade components of the extracellular matrix, including proteoglycans, gelatin, fibronectin, laminin and various types of collagens [5, 6, 9, 23]. It is also an activator of pro-MMPs, such as MMP-1 and MMP-9. This protease is produced mainly by fibroblasts [20]. Several studies have revealed that MMP-3 in RA is expressed in large amounts in synovium [20, 24], synovial fluid [26] and serum [23, 27, 28, 3032]. It was also shown that the concentration of MMP-3 in RA synovial fluid correlated with that in serum [3234]. Therefore, the serum concentration of MMP-3 may be a useful marker of inflammation activity in the joints of patients with RA. In our study, the serum level of MMP-3, like that of MMP-1, was also greater in RA than in OA patients. In particular, MMP-3 was the dominant metalloproteinase in follicular synovitis and could differentiate these patients clearly from those with only diffuse infiltrates. It was shown that the serum MMP-3 concentration correlated with the ESR and swollen joint count [30, 35, 36]. We demonstrated that the serum concentration of MMP-3 is also associated with the duration of morning stiffness, the Ritchie index and MMP-1 and MMP-9 concentrations. Others have also shown a correlation of MMP-1 with MMP-3 in RA synovial fluid [26]. These results suggest that the follicular histological form of RA can be considered as the more active form of the disease, and that the MMP-3 concentration could be a useful marker for identifying this form of RA. The levels of MMP-3 in the serum of RA patients, even during the early stages of the disease, were higher than those in controls, and appeared to increase as the disease progressed [32, 37, 38]. This suggests that the assessment of serum MMP-3 may be an important tool in the diagnosis of early onset of RA. Suppression of MMP-3 production might be an effective therapeutic approach for patients with early RA.
The serum concentration of gelatinase B (MMP-9), produced mainly by granulocytes [7, 39], was elevated in RA in comparison with OA patients. Other studies showed that MMP-9 was abundant in the RA synovium [7, 40] and found that its levels were increased in RA synovial fluid [7, 26, 40] and serum [7, 40] compared with controls. In our study the highest serum concentration of MMP-9 correlated with the presence of the follicular form of synovitis, in which the concentration was greater than in patients with the diffuse type of disease. This MMP discriminated clearly between these two variants of rheumatoid synovitis. Correlation of MMP-9 with disease activity markers such as ESR and the number of swollen joints seems to confirm the suggestion that RA is more severe in patients with the follicular type of synovitis.
The activity of MMPs is regulated by proenzyme production and/or by the activation of their endogenous inhibitors. There are at least two TIMPs. TIMP-1, the most widely distributed one, suppresses the activity of all MMPs [5, 6]. The presence of TIMP-1 in synovium [5, 6] and in increased amounts in RA synovial fluid [26, 32] or serum [23, 27, 32] has been shown in other studies. On the other hand, some investigators detected very similar levels of TIMP-1 in sera of RA and OA patients [28, 30]. In the present work we showed that the serum levels of TIMP-1, like those of the MMPs we studied, were elevated in RA patients compared with OA patients. Furthermore, TIMP-1 reached its highest concentration in patients with the follicular type of synovitis. Other studies showed that TIMP-1 correlated with ESR [30]. In our study it also correlated with the Ritchie index and the number of swollen joints. Up-regulation of TIMP-1 synthesis, especially in patients with the follicular form of rheumatoid synovitis, might be responsible for the suppression of synovium and cartilage destruction in patients with severe activity of the disease.
Like concentrations of TIMP-1, serum concentrations of TIMP-2 were also higher in patients with RA and with follicular synovitis than in OA patients. However, the TIMP-2 level did not differ significantly between the two histological patterns of RA. Others have also shown a higher serum concentration of TIMP-2 in RA than in controls [27].
Some studies suggest that the articular destruction in RA may be caused by the alteration in the balance between MMPs and TIMPs in favour of MMPs [7, 26, 33]. In our study, the calculated concentration ratios of MMP-3, MMP-9 and the ratios of total measured MMPs to TIMP-1, TIMP-2 and total measured TIMPs were higher in RA patients than in OA patients. They were especially increased in patients with follicular synovitis. However, significant differences between the morphological forms of RA were observed only for the ratios MMP-9/TIMP-1, MMP-9/TIMP-2 and total MMPs/total TIMPs. The analysis of ratios of individual MMPs or total measured MMPs to individual TIMPs or total measured TIMPs suggests that the relative production of TIMPs is decreased in RA compared with OA, especially in patients with the follicular type of synovitis. These data should be considered cautiously because we did not measure other proteinases that may be involved in the process of articular tissue destruction, or other inhibitors.
Radiological analysis demonstrated that more advanced joint destruction was more frequent among patients with follicular synovitis, characterized by higher levels of MMPs and TIMP-1 measured by us. Other investigators have also observed correlations of serum concentrations of MMP-3 and TIMP-1 with disease progression [32].
The analysis of the treatment regimens revealed that methotrexate (MTX) was more often used by patients with follicular synovitis than by those with diffuse synovitis. There is evidence that MTX may decrease the density of the mononuclear cell infiltration in RA synovium [4142]. It was suggested that MTX has an antiangiogenic effect and may reduce joint inflammation through the suppression of the growth of small blood vessels in the synovium, which is related to mononuclear cell migration and the proliferation of synovial tissue [4345]. However, there is also evidence that MTX does not influence cell density significantly in rheumatoid synovium [46, 47]. Moreover, it has also been shown that the density of the mononuclear cell infiltrate is higher in RA patients treated with MTX [48]. We are unaware of any data suggesting an effect of MTX on the formation of follicular structures in rheumatoid synovium.
Methotrexate has also been shown to reduce MMP-1 and MMP-3 production [46, 47, 49] and increase TIMP-1 production [50]. However, some studies suggest that MTX does not have a significant influence on MMP-3 [46] or TIMP-1 production [46, 49]. In our study we found that measured serum concentrations of MMPs and TIMPs and MMP/TIMP ratios were higher in patients with the follicular type of synovitis than in those with diffuse synovitis. This was observed despite more frequent MTX treatment in the group of patients with the follicular form than in those with the diffuse histological form of RA. Therefore, we surmise that the more frequent use of MTX in our patients with follicular than in the group with diffuse synovitis simply suggests greater activity of the disease in the former group of patients.
There were no other significant differences in treatment between the two RA groups that could have influenced the histological appearance of rheumatoid synovitis or the production of MMPs or TIMPs. No patients had received steroid injections in the 3 months before surgery.
Our finding that the distinct morphological appearances of synovitis are associated with different levels of serum MMPs and TIMP-1 confirms the heterogeneity of RA. We also observed correlations of serum MMP and TIMP concentrations with laboratory and clinical parameters of disease activity. Therefore, not only are MMP-1, MMP-3, MMP-9 and TIMP-1 good indicators of disease activity in RA but they might also predict the histological manifestation of the disease. The distinct histological, metalloproteinase and TIMP-1 patterns observed in RA patients in our study suggest the possibility of different responses to therapy regimens. However, a longitudinal study of patients at various stages in the disease process is clearly indicated.
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Acknowledgments |
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Notes |
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References |
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