1 Rheumatology Unit, Prince of Wales Hospital, Sydney,
2 Inflammation Research Unit, School of Pathology, University of New South Wales, Sydney,
3 Department of Clinical Immunology and Rheumatology Unit, Flinders Medical Centre, Flinders Drive, Bedford Park, Adelaide,
4 Department of Medicine, Flinders University, Bedford Park, Adelaide and the Repatriation General Hospital, Adelaide, South Australia, Australia
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Abstract |
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Methods. Sequential arthroscopic biopsies of the knee were taken before and 24 h after pulse therapy (11 patients), at disease relapse (three patients) and after retreatment with pulse therapy (one patient). Immunoperoxidase staining for MMP-1 (interstitial collagenase), MMP-3 (stromelysin-1) and TIMP-1 was performed and the immunoreactive staining quantified by colour video image analysis.
Results. In the synovial lining layer, MMP-1 and TIMP-1 immunostaining was reduced by a mean of 47% (P = 0.02) and 72% (P = 0.05), respectively, 24 h after pulse methylprednisolone therapy. In the synovial sublining layer, MMP-1 was reduced by a mean of 51% (P = 0.08) and TIMP-1 by a mean of 73% (P = 0.02) 24 h after pulse methylprednisolone therapy. There was no change in MMP-3 staining in the synovial lining or sublining layer.
Conclusions. High-dose pulse methylprednisolone therapy is associated with a rapid (within 24 h) and substantial decrease in the expression of MMP-1 and TIMP-1 but not MMP-3 in the synovial membrane in RA.
KEY WORDS: Rheumatoid arthritis, Matrix metalloproteinases, TIMP, Corticosteroids.
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Introduction |
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Therapeutic intervention in RA is aimed at reducing inflammation and joint destruction. We have demonstrated previously that high-dose intravenous pulse methylprednisolone causes a marked improvement in clinical parameters of inflammation, a reduction in inflammatory cells in synovial fluid and a reduction in pro-inflammatory cytokines and cell adhesion molecules in the synovial membrane [3, 4]. The effects of pulse therapy with methylprednisolone on MMP and TIMP expression in the synovial membrane have not been studied previously. The one in vivo study of the effects of glucocorticoids on MMP and TIMP expression in RA synovium demonstrated a reduction in MMP-1 and TIMP-1 mRNA expression in the synovial lining of three patients treated with intra-articular glucocorticoids [2], but there was no documentation of protein expression. The present study was initiated to determine the effects of pulse therapy with methylprednisolone on the expression of MMP-1, MMP-3 and TIMP-1 in the synovial membrane of patients with active RA.
This project was approved by the institutional ethics committee of the Repatriation General Hospital, Adelaide, and informed consent was obtained from each patient.
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Patients and methods |
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Monoclonal antibodies
All antibodies used were murine monoclonals. Anti-MMP-1 (IgG2a), anti-MMP-3 (IgG1) and anti-TIMP-1 antibodies (IgG2b) were purchased from ICN Biomedicals (Sydney, Australia). Anti-CD68 mAb (IgG3) and mouse IgG were purchased from Dako (Sydney, Australia).
Patients
Eleven patients (eight males and three females) who fulfilled the 1987 American College of Rheumatology criteria for classical or definite RA [5] and who had at least one inflamed knee joint with an effusion were studied. Seven of these patients were part of a previous study [3]. The mean ± S.D. age was 69.5 ± 11.2 yr and disease duration 4.8 ± 5.2 yr. Five patients had a disease duration of 1 yr. Six patients were seropositive for IgM rheumatoid factor, with a mean ± S.D. of 572 ± 787 IU/ml (measured by rate nephelometry; normally <40 IU/ml). None of the patients had received glucocorticoids within the previous 3 months. Five patients were receiving weekly i.m. injections of gold, one in combination with methotrexate and another in combination with sulphasalazine. Three patients were receiving only non-steroidal anti-inflammatory drugs, the dose of which was not altered during the period of study. Three patients were not receiving any anti-rheumatic therapy at the commencement of this study. Four patients had proven erosive disease on X-rays of the hands or feet.
Treatment
All patients received 1000 mg methylprednisolone i.v. as the sodium hemisuccinate salt, as previously described [3, 4].
Patient assessment
Clinical assessment was performed using visual analogue scales (10 cm horizontal scales anchored at both ends) for pain, generalized stiffness and well-being, and by a count of tender joints. Serum C-reactive protein (CRP) was used as a laboratory assessment of inflammation.
Arthroscopic biopsies
Synovial membrane samples were obtained from the knee before and 24 h after pulse methylprednisolone (11 patients) using needle arthroscopic techniques and standard approaches as described previously [6]. A biopsy was obtained at disease relapse in two patients, and in one of these patients a biopsy was obtained after repeated treatment. Where possible, repeat biopsies were taken adjacent to previous biopsy sites.
Tissue processing and immunoperoxidase staining
Formalin-fixed adjacent tissue sections (4 µm thick) were digested and quenched for endogenous peroxidase as previously described [7]. Slides were incubated in 25 µg/ml proteinase K for 20 min at 37°C. Slides were then incubated with 20% goat serum for 2 h at room temperature to block non-specific binding sites, followed by incubation with optimized dilutions of the primary antibody overnight in a humidified chamber at room temperature. All dilutions of the primary antibody were in 2% bovine serum albumin/Tris-buffered saline (TBS). After further washes with TBS, the sections were incubated with biotinylated goat anti-mouse secondary antibody for 20 min at room temperature. The sections were again washed and an avidinbiotinhorseradish peroxidase complex was added for 60 min. After further washes, the sections were incubated with the chromogen AEC for 5 min and counterstained with haematoxylin before being mounted in crystal mount. All sections from the same patient were processed in the same run. Negative controls were performed using mouse IgG or normal goat serum alone, or by omitting the secondary antibody. Specificity of positive staining of the MMP-1 antibody was verified by adsorption of positive staining by preincubation with recombinant MMP-1. The specificities of the anti MMP-1, MMP-3 and TIMP-1 antibodies were determined using Western blots of supernatants derived from tumour necrosis factor (TNF-
)- and interleukin 1ß (IL-lß)-stimulated RA synovial fibroblasts (data not shown). A single immunoreactive band was observed with each antibody, corresponding to the correct molecular weight.
Quantification of immunostaining
The immunostained sections were examined by computer-assisted colour video image analysis as described previously [3, 4]. Measurements of the integrated optical density (IOD; proportional to the total amount of protein staining) and the mean optical density (MOD; equal to IOD divided by the area of AEC staining, which is a measure of the average concentration of protein in the positively stained cells) were made by a masked observer (P.W.) who was unaware of the order of biopsies from any one patient. The reproducibility of each measurement was within 10% (data not shown). This was mostly due to variability in field selection.
All sections from the same patient were analysed in the same sitting.
Statistical analysis
Results are given as mean ± S.E.M. Correlations were determined using the Spearman correlation coefficient. Comparisons were made using paired and unpaired t-tests as appropriate. Differences were considered to be significant at P < 0.05.
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Results |
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Pulse methylprednisolone therapy decreases TIMP-1 and MMP-1 but not MMP-3 expression in the synovial membrane.
Sequential arthroscopic knee biopsies were immunostained for MMP-1, MMP-3 and TIMP-1, and computer-assisted colour video image analysis was used to measure the MOD and the IOD. Due to the availability of tissue, MMP-1 staining was performed in all 11 patients, TIMP-1 staining in nine patients and MMP-3 staining in eight patients.
MMP-1, MMP-3 and TIMP-1 immunostaining was observed on the majority of synovial lining layer cells and on infiltrating sublining macrophages (Fig. 1). Vascular immunoreactivity was not observed.
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Pulse methylprednisolone therapy did not cause a statistically significant decrease in CD68 expression in the synovial lining or sublining, but this variable did show a trend to a decrease, which may not have been statistically significant because of the low number of patients (type II error). In the synovial lining, the pretreatment CD68 IOD was 6015 ± 1819 and the pretreatment MOD 0.52 ± 0.16; the post-treatment IOD was 4645 ± 1400 and the post-treatment MOD 0.51 ± 0.15. In the synovial sublining, the pretreatment CD68 IOD was 4253 ± 1282 and the pretreatment MOD 0.50 ± 0.15; the post-treatment IOD was 2543 ± 767 and the post-treatment MOD 0.51 ± 0.15.
Two patients had sequential arthroscopic synovial biopsies before and 24 h after pulse methylprednisolone therapy and at disease relapse, and in one patient a biopsy was taken after retreatment with pulse methylprednisolone (Fig. 2b). In both patients there was a decrease in MMP-1 by 24 h after pulse methylprednisolone therapy, which increased at relapse but not to the original levels and was further reduced with retreatment. In both patients there was an increase in MMP-3 levels with relapse. In only one of the patients was there an increase in TIMP-1 at relapse.
There was no significant difference in MMP-1, MMP-3 or TIMP-1 in patients with early disease (1 yr) compared with patients with disease of >1 yr duration (data not shown). MMP-1, MMP-3 and TIMP-1 expression was observed in all five patients with disease duration of
12 months. Similarly, the four patients with erosive disease did not differ significantly in MMP-1, TIMP-1 and MMP-3 expression from those without erosive disease (data not shown).
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Discussion |
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In this study, it was observed that glucocorticoids reduce the expression of both MMP-1 and TIMP-1. Although previous in vitro studies have demonstrated that glucocorticoids reduce MMP-1 but do not alter TIMP-1 production by human fibroblasts [11], our findings are consistent with a previous in vivo study of three patients receiving intra-articular glucocorticoids, in which a reduction was observed in both MMP-1 and TIMP-1 mRNA in the synovial membrane [2]. Glucocorticoid inhibition of MMP-1 and TIMP-1 may be mediated directly by transcriptional and post-transcriptional effects or indirectly by altering factors which regulate MMP-1 and TIMP-1 synthesis. For example, the glucocorticoidreceptor complex interferes with transcriptional activation at AP-1 sites [12], which are up-regulated during cytokine-induced MMP-1 and TIMP-1 expression [13, 14]. Also, glucocorticoid-induced reduction of MMP-1 mRNA stability may be mediated by an interaction with any of the three AU-rich sequences in the 3'-untranslated region of the MMP-1 gene [15, 16]. Finally, pro-inflammatory mediators such as TNF- up-regulate MMP-1 expression [17], and we have demonstrated previously that TNF-
expression in the synovial membrane is markedly reduced by pulse methylprednisolone therapy [3]. Unlike the production of MMP-1, that of TIMP-1 by RA synovial fibroblasts does not appear to be controlled by TNF [17] but is induced by IL-6 [18] and the IL-6 family of cytokines, especially oncostatin M [19]. Although we did not study the expression of IL-6 directly, the rapid reduction in serum CRP suggests an associated, similar reduction in serum IL-6, as this is the major cytokine controlling hepatic CRP synthesis [20]. We did not study the effects of pulse methylprednisolone therapy on oncostatin M, although this would be of interest.
It is interesting that pulse methylprednisolone therapy reduced the expression of MMP-1 and TIMP-1 but not that of MMP-3, even though, like MMP-1 and TIMP-1, AP-1 is activated in the induction of MMP-3 by pro-inflammatory mediators [21] and the MMP-3 gene has one AU-rich sequence in its 3'-untranslated region [15]. It has been demonstrated that MMP-1 and MMP-3 expression may be discoordinately regulated. For example, IL-1 is a more potent inducer of MMP-3 production by synovial fibroblasts than TNF-, but they act synergistically to induce MMP-1 [17] and we have previously demonstrated that pulse methylprednisolone therapy reduces TNF-
but not IL-1ß expression in RA synovium. Also, adenosine receptor agonists reduce MMP-1 but not MMP-3 production by cultured synoviocytes [22]. We have not studied the effects of pulse methylprednisolone therapy on intracellular adenosine and are unaware of any such studies. It has become increasingly recognized that high doses of glucocorticoids mediate non-genomic physicochemical actions which are not well understood [23]. It is possible that such effects may have mediated the differential effects of pulse methylprednisolone therapy on MMPs and TIMPs observed in this study.
A novel and significant observation of this study was that MMP-1, MMP-3 and TIMP-1 expression was present in synovial membrane of all five patients with RA of duration 1 yr, including one patient biopsied 1 month and another patient biopsied 2 months after symptom onset. This is not surprising in view of the common clinical observation of joint erosion early in the disease [24].
One limitation of this study was that we did not study synovium from areas adjacent to the cartilagepannus junction. We have observed that MMP-1 expression is increased at this site in RA (manuscript in preparation) and it is likely that the effect of pulse methylprednisolone therapy at this site determines whether joint erosion occurs. Also, we chose to study only a limited number of very important mediators of joint damage. Clearly, there are many other mediators of cartilage damage that have been described in the rheumatoid joint, and future studies of these may provide further insights into the mechanisms of action of glucocorticoids.
In summary, in this study it was observed that pulse methylprednisolone therapy reduced the expression of MMP-1 and TIMP-1 but not that of MMP-3, suggesting a possible mechanism by which glucocorticoids slow down but do not completely prevent joint erosions in RA.
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Acknowledgments |
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Notes |
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References |
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