Anti-tumour necrosis factor (TNF)-{alpha} therapy (etanercept) down-regulates serum matrix metalloproteinase (MMP)-3 and MMP-1 in rheumatoid arthritis

A. I. Catrina, J. Lampa, S. Ernestam1, E. af Klint, J. Bratt1, L. Klareskog and A.-K. Ulfgren

Department of Rheumatology, Karolinska Hospital and
1 Department of Rheumatology, Huddinge Hospital, Stockholm, Sweden


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives. Matrix metalloproteinases (MMPs) are cytokine-modulated enzymes that play an important role in the pathogenesis of rheumatoid arthritis (RA) by inducing bone resorption and cartilage destruction. This study evaluated the modulation of serum and synovial MMPs and their inhibitor, tissue inhibitor of matrix metalloproteinases (TIMP)-1, by therapy with soluble tumour necrosis factor (TNF) {alpha} receptor (etanercept).

Methods. Serum samples were collected from 60 RA patients at baseline and after 8 or 12 weeks of treatment. Paired synovial biopsies were obtained from 11 patients at two time points, before and after 8 weeks of treatment. We measured serum levels of MMP-1, MMP-3 and TIMP-1 by ELISA. Immunohistological analysis of synovial tissue was performed using monoclonal antibodies specific for MMP-1, MMP-3 and TIMP-1.

Results. Etanercept therapy significantly down-regulated serum levels of MMP-3 and MMP-1 in parallel with the reduction in inflammatory parameters (C-reactive protein concentration and erythrocyte sedimentation rate) in RA patients. Baseline pretreatment serum levels of MMP-3 correlated with changes in clinical disease activity during therapy. No consistent changes in serum level of TIMP-1 were observed, while ratios of MMP-1 and MMP-3 to TIMP-1 were down-regulated following etanercept treatment. Immunohistochemical analyses revealed great interindividual variability, with generally a high level of expression of MMP and low expression of TIMP. No significant change in the pattern or number of positive cells occurred during therapy.

Conclusions. In RA patients, etanercept therapy down-regulates serum levels of MMP-3 and MMP-1 and the ratio between MMPs and TIMP-1. This may be an important mechanism for the prevention of future development of joint damage.

KEY WORDS: Rheumatoid arthritis, Soluble tumour necrosis factor {alpha} receptor, Etanercept, Metalloproteinases, Tissue inhibitors of metalloproteinases.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Rheumatoid arthritis (RA) is an autoimmune inflammatory disease characterized by cartilage destruction and extracellular matrix degradation. Cytokines, such as tumour necrosis factor (TNF)-{alpha} and interleukin (IL)-1, play a major role in the pathogenesis of the disease, being able to induce bone resorption and cartilage destruction (for review see [1]). These two cytokines act synergistically to release matrix metalloproteinases (MMPs), a class of zinc-dependent peptidases participating in extracellular matrix degradation and remodelling. MMPs are synthesized and secreted as latent pro-enzymes and their activation is due to proteolysis of a pro-peptide domain at the N-terminus of the molecule.

At present there are at least 19 known human MMPs, which can be divided into four groups: the collagenases, the stromelysins, the gelatinases and the membrane-type MMPs (for review see [2]). Stromelysin 1 (MMP-3) and collagenase (MMP-1) are thought to be probably involved in the pathogenesis of RA [3]. MMP-3 levels are increased both in synovial fluid [4] and serum [5] in RA patients, with a highly significant correlation between matched samples [6], suggesting that serum MMP-3 is derived mainly from that synthesized in the synovium. MMP-1 (fibroblast collagenase) is detected in RA synovial fluid [7] and serum samples [8] at higher levels than in healthy controls. Tissue inhibitor of matrix metalloproteinase (TIMP)-1 is a specific inhibitor of MMPs. It is present in RA synovium to a lesser extent compared with MMPs, suggesting imbalance between the two components [9].

Treatment with TNF blocking agents, alone or in combination with methotrexate, has high clinical efficacy and delays joint destruction in RA (for review see [1]). Two TNF blocking agents are available for clinical use: infliximab (a chimeric monoclonal antibody) and etanercept (a recombinant TNF receptor–Fc fusion protein). Several mechanisms have been proposed to explain the clinical efficacy of infliximab, such as decreased cell recruitment at the site of inflammation [10], down-regulation of synovial cytokine expression [11] and reduction of MMP-1 and MMP-3 synthesis [12]. However, it is not known if etanercept therapy modulates the serum levels of MMPs and TIMP, and there are no reports on parallel studies of MMPs and TIMP in serum and synovial tissue during TNF blockade either for etanercept or for infliximab.


    Materials and methods
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 Abstract
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 Materials and methods
 Results
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Patients and samples
Sixty patients (50 females and 10 males, median age 53 yr, range 30–80 yr) meeting the American College of Rheumatology (ACR) criteria for RA [13] were recruited for this study. All patients had a history of failed therapy with at least one disease-modifying anti-rheumatic drug (DMARD) at trial entry. No patient received more than 10 mg prednisolone daily. All patients received a subcutaneous injection of 25 mg etanercept (Wyeth-Ayerst, Philadelphia, PA, USA) twice a week. Twenty-five patients were treated with etanercept alone. Thirty-two patients received etanercept in combination with methotrexate, one patient etanercept in combination with methotrexate and cyclosporin, one patient etanercept in combination with sulphasalazine, and one patient etanercept in combination with Reumacon (podophyllum) (Conpharm, Uppsala, Sweden), an anti-rheumatic agent [14] used on a licence basis in Sweden.

Serum samples were collected from all 60 patients before treatment and after 8 weeks of treatment (23 patients, group 1) or after 12 weeks of treatment (37 patients, group 2). Serum samples were stored at -20°C until assayed. Synovial biopsies were obtained at arthroscopy from 11 patients in group 1 before treatment and after 8 weeks of treatment.

Clinical and laboratory assessments
All patients were assessed for overall activity of the disease using the (disease activity score) DAS28 [15] before treatment and after 12 weeks of treatment. Changes in DAS28 score during therapy were calculated as the difference between the estimated value after 12 weeks of treatment and the baseline pretreatment value. The ACR response [16] to therapy was also recorded after 12 weeks of treatment for all 60 patients.

Laboratory parameters, namely C-reactive protein (CRP) concentration and erythrocyte sedimentation rate (ESR), were recorded before and after 12 weeks of treatment for all 60 patients.

Immunoassays
Serum levels of MMP-1, MMP-3 and TIMP-1 were measured by enzyme-linked immunosorbent assay (ELISA) (Amersham Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer's directions.

Serum samples were diluted (1:1 for MMP-1, between 1:1 and 1:8 for MMP-3 and between 1:10 and 1:20 for TIMP-1) and individual samples were assayed in duplicate. The MMP-1 kit recognized total human MMP-1, free MMP-1 and MMP-1 complexed with TIMP-1, but not MMP-1 complexed with {alpha}2-macroglobulin. The range of the assay was 6.25–100 ng/ml with sensitivity of 1.7 ng/ml. The MMP-3 assay recognized proMMP-3, active MMP-3 and MMP-3/TIMP complexes but not MMP-3 bound by {alpha}2-macroglobulin, in the range 3.75–120 ng/ml with sensitivity of 2.35 ng/ml. Total TIMP-1 (free TIMP-1 and TMP-1 complexed with MMPs) was recognized by ELISA in the range 3.13–50 ng/ml with sensitivity of 1.25 ng/ml. Ratios of MMP-3 to TIMP-1 and MMP-1 to TIMP-1 were calculated.

Tissue preparation and immunohistochemical analysis
Eleven patients from group 1 were subjected to knee arthroscopy before treatment and after 8 weeks of treatment. Biopsy specimens adjacent to the cartilage–pannus junction were snap-frozen in liquid nitrogen and then kept at -70°C until sectioned. Serial cryostat sections (7 µm) were fixed for 20 min with 2% (v/v) formaldehyde (Sigma Chemicals, St Louis, MO, USA) and stored at -70°C. The staining procedure has been published previously [17]. We used the following mouse monoclonal antibodies (all purchased from Oncogene, Cambridge, MA, USA): anti-MMP-1 IgG2a (41-1E5), anti-MMP-3 IgG1 (55-2A4) and anti-TIMP-1 IgG1 (7-6C1). Negative controls with matched IgG isotype and recombinant protein-blocked antibody were included for each marker [17].

Stained synovial biopsy sections were evaluated using computerized image analysis as described previously [18]. The areas of MMP and TIMP staining were expressed as percentages of the total area of counterstained tissue. Analysis of an entire tissue section typically involved 30–100 microscopic fields at a magnification of x250.

Statistical analysis
Differences between groups were analysed using the Mann–Whitney U-test and analysis for matched pairs was performed using Wilcoxon's signed rank test. Correlations between variables were assessed using the Spearman rank correlation test. P<0.05 was considered statistically significant.


    Results
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 Materials and methods
 Results
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 References
 
Clinical response
After 12 weeks of treatment, 47 of 60 patients (78.33%) fulfilled ACR20 criteria, while 13 of 60 patients (21.66%) showed no response to therapy. Clinical and laboratory parameters improved significantly after 12 weeks of treatment in all 60 patients, with reduction of the DAS28 score [from 6.01 to 4.02 (median values), P<0.0001], CRP level [from 34 mg/ml to 15 mg/ml (median values), P<0.0001] and ESR [from 42 to 27 mm/h (median values), P<0.0001].

Serum levels of MMPs and TIMPs
Table 1Go summarizes the results of ELISA analyses. Following treatment, the median serum level of MMP-3 decreased significantly in group 1 of patients from 68.1 to 46.5 ng/ml (P<0.001) and in group 2 of patients from 115.3 to 48.8 ng/ml (P<0.0001). The median serum level of MMP-1 decreased significantly in group 1 patients from 9.3 to 8.7 ng/ml (P<0.05) and in group 2 patients from 9.8 to 7.9 ng/ml (P=0.001). The median serum level of TIMP-1 was down-regulated following 8 weeks of treatment for patients in group 1 (from 433.2 to 371.6 ng/ml, P<0.05) but remained unchanged compared with the baseline value after 12 weeks for patients in group 2 (Table 1Go and Fig. 1Go). In group 1, the MMP-1:TIMP-1 and MMP-3:TIMP-1 ratios were not significantly changed after 8 weeks of treatment. In group 2, both the median MMP-1:TIMP-1 ratio and the median MMP-3:TIMP-1 ratio decreased significantly [from 0.025 to 0.020 (P<0.05) for MMP-1:TIMP-1 and from 0.27 to 0.12 (P<0.0001) for MMP-3:TIMP-1] (Table 1Go and Fig. 2Go).


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TABLE 1.  Serum levels of MMP-3, MMP-1 and TIMP-1 and their ratios before treatment (week 0) and after 8 weeks of treatment (group 1) or after 12 weeks of treatment (group 2)

 


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FIG. 1.  Changes in serum levels of MMP-3 (A), MMP-1 (B) and TIMP-1 (C) during etanercept treatment in group 1 (white boxes) and group 2 (grey boxes). Horizontal lines represent medians and whiskers the non-outlier values. *P<0.05; **P<0.01; ***P<0.001 (non-parametric Mann–Whitney U-test).

 


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FIG. 2.  Changes in MMP-1:TIMP-1 (A) and MMP-3:TIMP-1 (B) ratios during etanercept treatment in group 1 (white boxes) and group 2 (grey boxes). Horizontal lines represent medians and whiskers the non-outlier values. *P<0.05; ***P<0.001 (non-parametric Mann–Whitney U-test).

 
We did not observe any difference between cortisone-treated and non-treated patients or between patients treated with DMARDs and those not treated with DMARDs.

Correlations between serum levels of MMPs and TIMP-1 and disease parameters
In all 60 patients, the serum level of MMP-3 before treatment correlated with the baseline CRP level (P<0.001, r=0.42) and baseline ESR (P<0.01, r=0.38) (data not shown). Moreover, the baseline serum level of MMP-3 correlated with the change in DAS score after 12 weeks of treatment (P<0.05, r=-0.28) (data not shown).

Pretreatment serum level of TIMP-1 correlated with baseline CRP level (P<0.001, r=0.43) and ESR (P<0.01, r=0.34) (data not shown). Pretreatment serum level of MMP-1 did not correlate with any inflammatory parameters.

Immunohistochemical analysis
Table 2Go summarizes the results of immunohistochemical analysis of the biopsies obtained from 11 patients in group 1 at baseline and after 8 weeks of treatment. We observed great inter-individual variability even though most patients had a high level of MMP expression and a low level of TIMP expression (Fig. 3Go). Matrix metalloproteinases were present in all biopsies studied, both before and after treatment, while TIMP-1 was identified in 20 out of 22 biopsies investigated.


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TABLE 2.  Synovial expression of MMP-1, MMP-3 and TIMP-1 before treatment and after 8 weeks of etanercept treatment in 11 patients

 


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FIG. 3.  Photographs illustrating brown immunoperoxidase staining for MMPs and TIMP-1 in serial sections from cryopreserved synovial biopsies counterstained with haematoxylin. (A) MMP-1. (B) MMP-3. (C) TIMP-1. Original magnification x310. All sections are from the same patient before treatment.

 
MMP-3 was present mainly in the lining and endothelial cells. MMP-1 was expressed mainly in the sublining layer and around vessels and to a lesser extent in the lining layer. TIMP-1 showed a generally low level of expression, with positive signal in isolated areas.

No significant changes in the pattern or number of positive cells occurred after 8 weeks of treatment.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
MMPs have a major role in RA pathogenesis as mediators of cartilage and bone destruction, and this is the first report describing modulation of the MMP–TIMP system by soluble TNF-{alpha} receptors (etanercept) in RA patients.

We focused our study on MMP-3 and MMP-1, measuring their serum levels with ELISA kits that recognized both the active and inactive forms of MMPs. Serum levels of MMP-3 quantified by this method have been shown previously to correlate with radiological damage [1920] in RA patients. We found that etanercept therapy significantly decreased the serum level of MMP-3 as well as the ratio of MMP-3 to TIMP-1. These changes could explain the ability of anti-TNF therapy to prevent further development of joint damage. Serum levels of MMP-3 correlated highly with inflammatory markers (CRP and ESR), as shown previously [8, 21]. Moreover, our data indicate that baseline MMP-3 serum levels may predict the changes in DAS score during therapy.

We also observed a decrease in the serum level of MMP-1 during etanercept therapy, with reduction of the MMP-1:TIMP-1 ratio. It was shown previously that the serum level of MMP-1 correlated with the number of new erosions that developed in RA patients over 18 months of follow-up [22]. Thus, the decrease in the serum level of MMP-1 could partly explain the delay in radiological progression in patients with RA during etanercept treatment. The serum level of MMP-1 did not correlate with inflammatory parameters, suggesting that the effect of etanercept on the serum level of MMP-1 is independent of the down-regulation of inflammation.

The serum level of TIMP-1 before treatment correlated with both CRP and ESR. Interestingly, etanercept therapy did not influence the serum level of TIMP-1 consistently despite a general decrease in all inflammatory markers investigated. This finding suggests that TIMP-1 is influenced mainly by a pro- and/or anti-inflammatory mechanism other than one involving TNF.

Several reports have suggested that there might be an imbalance between MMP and TIMP expression in the RA synovium [3], allowing MMP activation and the subsequent destruction of joint components. A recent study reported that a high level of synovial expression of MMP-1 mRNA in early RA distinguished patients with more rapidly progressive erosive disease [23]. We did not observe any changes in the synovial expression of MMPs and TIMP-1 during etanercept therapy, however, even though biopsies were taken from areas adjacent to the cartilage pannus junction. Possible explanations for this finding are the low number of patients subjected to arthroscopy and/or lack of specificity between the recognition of active and inactive forms of MMPs.

In conclusion, we have demonstrated that therapy with soluble TNF-{alpha} receptors significantly decreases the serum levels of MMP-3 and MMP-1, with reductions in the ratios of MMPs to TIMP-1. The pretreatment serum level of MMP-3 may be considered a potential predictor of changes in clinical disease activity during treatment with etanercept.


    Acknowledgments
 
We thank Associate Professor R. A. Harris for linguistic advice. Marianne Engstrom provided excellent technical assistance. The study was supported by grants from the Swedish Medical Research Council, the Swedish Rheumatism Association, the insurance company AMF and from Freemasan Barnhuset Stockholm.


    Notes
 
Correspondence to: A. I. Catrina, Rheumatology Research Laboratory, CMM L8-004, Karolinska Hospital, S-17176, Stockholm, Sweden. Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Submitted 1 May 2001; Accepted 2 November 2001