Rheumatology

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Rheumatology 2001; 40: 988-994
© 2001 British Society for Rheumatology

Original Papers

Expression of chemokines and matrix metalloproteinases in early rheumatoid arthritis

A. Katrib^1,2, P. P. Tak³, J. V. Bertouch¹, C. Cuello², H. P. McNeil^1,2, T. J. M. Smeets³, M. C. Kraan³ and P. P. Youssef^2,4,

¹ Rheumatology Unit, Prince of Wales Hospital,
² Inflammation Research Unit, School of Pathology, University of New South Wales, Sydney, Australia,
³ Division of Clinical Immunology and Rheumatology, Academic Medical Centre, Amsterdam, The Netherlands and
⁴ The Combined Centre for the Rheumatic Diseases, Rachel Forster Hospital, Sydney, Australia

	Abstract

Top Abstract Introduction Materials and methods Results Discussion References

 
Objective. To compare macrophage infiltration and expression of chemokines and matrix metalloproteinases (MMPs) in synovial tissue between patients with early and long-standing rheumatoid arthritis (RA).

Methods. Knee synovial biopsies were taken from 22 patients with early (<1 yr) and 22 patients with long-standing (>5 yr) RA and immunostained with antibodies specific for CD68; macrophage inflammatory protein (MIP)-1 and monocyte chemoattractant protein (MCP)-1; MMP-1 and -3 and the tissue inhibitors of metalloproteinases (TIMP)-l and -2. Immunostaining was quantified using a colour video image analysis system.

Results. CD68+ macrophage infiltration and the expression of MIP-1, MCP-1, MMP-1, MMP-3, TIMP-1, and TIMP-2 were observed in synovial tissue of patients with early RA. In long-standing RA, there was a further increase in CD68+ macrophage infiltration and MIP-1 expression in the synovial lining layer. CD68 expression correlated with MIP-1 (R=0.39, P=0.01), but not with MCP-1 expression.

Conclusion. Macrophage accumulation, and the expression of chemokines and MMPs in synovial tissue occur in early RA. Targeting chemokines which play a role in the migration of macrophages into the joints may be of therapeutic benefit in RA patients.

KEY WORDS: Rheumatoid arthritis, Chemokines, Matrix metalloproteinases, Tissue inhibitors of matrix metalloproteinases.

	Introduction

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Rheumatoid arthritis (RA) is characterized by chronic synovial inflammation and progressive cartilage and bone destruction resulting in significant morbidity and early mortality despite treatment with conventional medication [1]. A number of studies have observed that joint erosions commonly occur early in disease (within the first 2 yr) and that erosions accumulate over time [2, 3]. Therefore, much research effort is currently focused on ‘early’ RA with the potential for targeting treatment prior to the development of irreversible disability [4].

One of the most significant positive correlates with local disease activity [5] and with the development of joint erosion [6, 7] in RA is the number of macrophages in the synovial membrane.

Macrophage accumulation in the RA synovial membrane is predominantly due to infiltration from the bloodstream rather than local cell division [8] and is likely to be mediated, in part, by the expression of selective chemotactic factors. Studies have identified two members of the ß chemokine family, monocyte chemoattractant protein-l (MCP-1) and macrophage inflammatory protein-l alpha (MIP-1), as likely ß chemokines responsible for mononuclear cell accumulation into RA synovium [9, 10]. Both molecules are significantly increased in RA serum, synovial fluid, and synovial tissue compared with osteoarthritis (OA) and other arthritides [9–11]. In RA, the levels of MCP-1 and MIP-1 are higher in synovial fluid compared with serum, reflecting local production [9, 10]. We are unaware of any published studies comparing the expression of these chemokines in early with long-standing RA.

A major factor in RA disease pathogenesis is irreparable degradation of the extracellular matrix by the enzymatic action of proteases, most importantly the matrix metalloproteinases (MMPs) [12–16]. There are natural inhibitors specific for MMPs produced locally by chondrocytes and fibroblast-like synoviocytes known as tissue inhibitors of metalloproteinases (TIMPs). Joint destruction in RA is probably due to a local imbalance between activated MMP and TIMP [17–19]. Several MMPs and TIMPs have been demonstrated in RA serum, synovial fluid, and synovial tissue. The best studied are MMP-1 and MMP-3 which have been found in higher levels in RA than in OA and normal controls [20]. In RA, serum levels of MMP-1 and MMP-3 tend to correlate with generalized clinical disease activity [20], whereas synovial fluid MMP-1, MMP-3 and TIMP-1 activity correlate with local joint inflammation [21].

In this study we determined the expression of MCP-l, MIP-1, MMP-1 and -3, and TIMP-1 and -2 in early RA (defined as <1 yr duration) and compared this with long-standing RA (defined as >5 yr duration) and correlated the expression of these mediators with macrophage infiltration in the synovium.

	Materials and methods

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Patients
Twenty-two patients with early RA (<1 yr duration, as measured from the first clinical signs of arthritis) and 22 patients with long-standing RA (>5 yr duration) were entered into the study. All patients had knee arthritis and fulfilled the 1987 American College of Rheumatology criteria for RA [22] at follow-up of at least 1 yr. None of the patients was taking glucocorticoids or cytotoxic disease-modifying anti-rheumatic drugs (DMARDs) such as methotrexate at the time the biopsies were performed. However, DMARDs such as hydroxychloroquine and sulphasalazine were allowed. All patients gave their informed consent and the study protocol was approved by the Medical Ethics Committee in The Netherlands.

One observer (PPT) performed a clinical assessment on the day of synovial biopsy collection. Data comprised the Ritchie articular index [23] and the number of swollen joints [24] (total 20 with groups of joints, e.g. metacarpophalangeal joints, considered as one joint). Laboratory assessments included C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR). Radiological assessments of hands, feet and affected joints were also performed for diagnostic purposes.

Reagents
Sodium chloride and hydrogen peroxide were purchased from Merck (Victoria, Australia); methanol from Ajax Chemicals (Sydney, Australia); diaminobenzidine (DAB) from Sigma (Sydney, Australia); bovine serum albumin (BSA) and proteinase K from Boehringer Mannheim (Mannheim, Germany); normal goat serum, biotinylated goat anti-mouse secondary antibody and the avidin–biotin–horseradish peroxidase complex from Vector Australian Laboratory Services (Sydney, Australia); methyl green from BDH Chemicals (Sydney, Australia).

Monoclonal antibodies
All antibodies used were murine monoclonals. Anti-MMP-1 (IgG_2a), anti-MMP-3 (IgG₁), anti-TIMP-1 (IgG₁), and anti-TIMP-2 (IgG₁) antibodies were purchased from ICN Biomedicals (Sydney, Australia) Anti-MIP-1 (IgG_2a) and anti-MCP-1 (IgG_2a) were purchased from R&D Systems Inc. (Minneapolis, USA). Anti-CD68 monoclonal antibody (IgG₃), irrelevant isotype-specific negative controls, and mouse IgG were purchased from Dako (Sydney, Australia).

Synovial tissue
An average of 15 biopsy specimens of synovial tissue were obtained with a Parker-Pearson needle from each patient. An average of four pieces of tissue were fixed in formalin and paraffin embedded for histological analysis with haematoxylin and eosin.

Immunohistochemistry
Serial sections were stained with the following monoclonal antibodies: anti-CD68 (EBM11), anti-MCP-1, anti-MIP-1, anti-MMP-1, anti-MMP-3, anti-TIMP-1, anti-TIMP-2 and with the relevant isotype-specific negative controls.

Formalin-fixed adjacent tissue sections (4 µm thickness) were digested and quenched for endogenous peroxidase as previously described [25]. For MIP-1, MCP-1 and CD68 staining, the slides were incubated in 25 µg/ml proteinase K for 20 min at 37°C. The slides were then washed with Tris-buffered saline (TBS) pH 7.6 (10x TBS: 250 mM Tris Base, 250 mM Tris HCl, 8.5% NaCl) and incubated with 20% goat serum for 20 min 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 4°C. Primary antibodies were diluted in 2% BSA/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 avidin–biotin–horseradish peroxidase complex was added for 60 min. Following further washes, the sections were incubated with the chromogen DAB for 10 min and counterstained with methyl green before being coverslipped. For each antibody, sections from all patients were processed in the same run. Negative controls were performed using irrelevant mouse isotype-specific controls, mouse IgG alone, normal goat serum alone or by omitting the secondary antibody. Specificity of staining was verified by adsorption of positive staining by preincubation with recombinant protein.

Quantification of immunostaining
The immunostained sections were examined by computer-assisted colour video image analysis, as previously described [26, 27]. 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 DAB staining, which is a measure of the average concentration of protein on the positively stained cells) were made by a masked observer (AK) who was unaware of the order of biopsies from any one patient. The reproducibility of measurements was within 10% (data not shown). Differences were mostly due to variability in field selection.

Statistical analysis
Results are given as mean±S.E.M. (standard error of the mean). Correlations were determined using the Spearman correlation coefficient. Comparisons were made using the non-parametric Mann–Whitney U-test. Differences were considered to be significant at P<0.05.

	Results

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Clinical features
The clinical and demographic characteristics of the patients are presented in Table 1. The two groups were well matched for age, sex, biochemical markers of disease activity (ESR and CRP) and baseline serum rheumatoid factor (RF). Of the 22 patients with early RA, erosions were present in five patients (23%) at the time of the biopsy and in 20 patients at follow-up at 12 months (91%), whereas 19 of the 22 patients (86%) with long-standing RA had erosions. The mean±S.E.M. disease duration in the early RA group was 6.5±0.6 months (range 2–12). The disease duration was <6 months in 12 of the 22 early RA patients; in two of these 22 patients, the disease duration was <3 months.

View this table: [in this window] [in a new window]   TABLE 1. Clinical and demographic characteristics of patients [mean (S.E.M.)]

 

Macrophage infiltration and MIP-1 expression are increased in long-standing RA
CD68, MIP-1 and MCP-1 expression (Fig. 1) was observed in all subjects independent of disease duration. In long-standing RA, CD68 and MIP-1 expression was on average increased in the synovial lining layer and sublining layers when compared with early disease (Fig. 2). There was no significant difference in MCP-1 expression between the two groups either in the lining or sublining layers (Fig. 2). There were no significant differences in MOD between the two groups (data not shown).

View larger version (120K): [in this window] [in a new window]   FIG. 1. High-power photomicrographs of knee synovial membrane immunostained with (A) an isotype-specific negative control and specific monoclonal antibodies against (B) CD68, (C) MCP-1, (D) MIP-1, (E) MMP-1, (F) MMP-3, (G) TIMP-1, or (H) TIMP-2. Original magnification x600.

 

View larger version (17K): [in this window] [in a new window]   FIG. 2. The expression of (a) CD68, (b) MIP-1 and (c) MCP-1 in early RA of less than 1 yr duration compared with long-standing RA of greater than 5 yr duration, in both the synovial lining layer and sublining layer. *P<0.05, P<0.01. RA <1 yr; RA >5 yr.

 
There was a positive correlation between CD68 and MIP-1 expression in the lining layer (r=0.39, P=0.01, Fig. 3). There was also a positive correlation in the sublining layer, although this was not significant (r=0.27, P=0.08, Fig. 3). CD68 expression did not correlate with MCP-1 expression in either the lining layer or the sublining layer (Fig. 3).

View larger version (32K): [in this window] [in a new window]   FIG. 3. Positive correlation between CD68+ macrophage infiltration and MIP-1 expression which is significant in the synovial lining layer (a) and approaches statistical significance in the sublining layer (b). CD68+ expression did not correlate with MCP-1 expression in either the lining layer (c) or sublining layer (d).

 

MMP and TIMP expression in early and long-standing RA
MMP-1, MMP-3, TlMP-1 and TIMP-2 expression (Fig. 1) was observed in the majority of subjects independent of disease duration. There were no significant differences in MMP-1 or MMP-3 expression in early compared with long-standing disease in the lining and sublining layers (Fig. 4). Similarly, there were no significant differences in TIMP-1 or TIMP-2 expression in the lining and sublining layers (Fig. 4). There were no significant differences in MOD between the two groups for either MMPs or TIMPs (data not shown).

View larger version (26K): [in this window] [in a new window]   FIG. 4. Expression of (a) MMP-1, (b) MMP-3, (c) TIMP-1 and (d) TIMP-2 in the synovial lining and sublining layers in RA of less than 1 yr duration compared with RA greater than 5 yr duration. RA <1 yr; RA <5 yr.

 

	Discussion

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In this study, macrophage infiltration in the synovial membrane was observed in very early RA (<6 months duration), a finding consistent with previous studies from our group and others [5, 28–30]. A novel observation was the expression of MCP-1 and MIP-1 in patients with very early disease (12 of our patients had disease duration <6 months). Leucocyte trafficking into sites of inflammation is a multistep phenomenon involving various mediators, including chemokines and cell adhesion molecules (CAMs) [31]. Clearly, the macrophage infiltration observed in very early disease may at least partially be mediated by the chemotactic effects of MCP-1 and MIP-1, and by the CAMs which our group has previously shown to be expressed in early disease [5, 28]. Apart from their chemotactic function, chemokines also activate CAMs [32] and thus play a dual role in leucocyte trafficking.

Macrophage infiltration of the synovial lining and the synovial sublining layers was on average greater in long-standing RA compared with early disease and there were modest correlations with increased expression of MIP-1, a macrophage chemoattractant. Previous studies have also shown a tendency towards more prominent lining layer hyperplasia and infiltration by macrophages in long-standing disease, but the differences in the semiquantitative scores did not reach statistical significance [5, 28]. We have previously shown that, as expected, digital image analysis and conventional counting of cells allow the detection of small differences, whereas semiquantitative analysis is less sensitive to change [33, 34].

We did not observe a significant change in MCP-1 expression or a correlation of macrophage infiltration with MCP-1 expression even though a number of in vitro and animal studies have suggested a role for both MIP-1 and MCP-1 in RA. Both MCP-1 and MCP-1 are found in RA serum and synovial fluid at significantly higher levels than OA and other arthritides [9–11]. They are produced by a range of RA synovial membrane cell types in culture, including synovial macrophages and fibroblasts, although macrophages appear to be the major source [9, 11]. The administration of neutralizing anti-MIP-1 and anti-MCP-1 reduces inflammation in animal models of arthritis in mice [11, 35, 36]. There are two pieces of evidence which favour the role of MIP-1 as a mononuclear chemoattractant in RA. First, the mononuclear infiltrate in the RA synovium predominantly expresses the CC chemokine receptor-5 (CCR5), the receptor for MIP-1 [37] and not the CC chemokine receptor-2 (CCR2), the receptor for MCP-1. Second, an association between a functional CCR5 and the development of RA has been observed [38]. Other chemokines which bind to CCR5 include MIP-1ß and RANTES (regulated on activation normal T cell expressed and secreted) which we did not study. This study lends some support to the notion that modulation of MIP-1 or CCR5 expression may alter the progression of RA. The reasons for the possible discordant up-regulation of MIP-1 and MCP-1 remain unexplained. These chemokines appear to be produced by the same cell types [9, 10]. Also interleukin-1 (IL-l) and tumour necrosis factor alpha (TNF) increase the production of both chemokines by RA synovial fibroblasts and macrophages [9, 10]. Our previous studies comparing early with long-standing RA have not found differences in TNF and IL-lß expression [5].

In this study we observed the expression of MMP-1 and MMP-3 in very early disease. This is not surprising in view of clinical studies which have observed joint erosions in very early disease [2, 3]. There were no significant differences in MMP-1, MMP-3, TIMP-1 or TIMP-2 expression between early and long-standing disease.

One limitation of this study was that we did not analyse synovium from areas adjacent to the cartilage–pannus junction (CPJ). The CPJ has been shown to be a site of preferential aggregation of macrophages [39], in which TNF and IL-lß are expressed [40–42]. Thus, it would seem feasible to extrapolate that the CPJ should be the site of increased ß chemokine and MMP expression in RA. Studies of the CPJ are currently underway. The patients in the early arthritis group generally had late-onset disease and therefore these results cannot necessarily be extrapolated to patients whose disease onset is at a younger age.

In summary, this study suggests that the chemokine MIP-1 plays an important role in the pathogenesis of RA by recruiting monocytes. MIP-1 is expressed in the RA synovium in very early disease, accumulates over time and correlates with increased macrophage infiltration in long-standing disease. The expression of MMP-1 and -3 is present in early disease and is not altered by disease duration. These results support the current treatment paradigm of intervening very early in disease and would support the targeting of chemokines and MMPs as a therapeutic option.

	Acknowledgments

 
P. Youssef is supported by a post-doctoral research grant from the University of New South Wales. This study was also supported by the Arthritis Foundation of Australia, the Royal Australasian College of Physicians and a grant from the Government Employees Research Fund.

	Notes

 
Correspondence to: P. P. Youssef, Inflammation Research Unit, School of Pathology, University of New South Wales, Sydney, 2052, Australia.

	References

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Submitted 20 April 2000; Accepted 2 April 2001

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