Active synovial matrix metalloproteinase-2 is associated with radiographic erosions in patients with early synovitis

Abstract

Serum and synovial tissue expression of the matrix metalloproteinase (MMP)-2 and -9 and their molecular regulators, MMP-14 and TIMP-2 was examined in 28 patients with inflammatory early synovitis and 4 healthy volunteers and correlated with the presence of erosions in the patients. Immunohistological staining of MMP-2, MMP-14 and TIMP-2 localized to corresponding areas in the synovial lining layer and was almost absent in normal synovium. Patients with radiographic erosions had significantly higher levels of active MMP-2 than patients with no erosions, suggesting that activated MMP-2 levels in synovial tissue may be a marker for a more aggressive synovial lesion. In cancer the gelatinases [matrix metalloproteinase (MMP)-2 and MMP-9] have been shown to be associated with tissue invasion and metastatic disease. In patients with inflammatory arthritis the gelatinases are expressed in the synovial membrane, and have been implicated in synovial tissue invasion into adjacent cartilage and bone. It is hypothesized that an imbalance between the activators and inhibitors of the gelatinases results in higher levels of activity, enhanced local proteolysis, and bone erosion. To determine whether the expression and activity levels of MMP-2 and MMP-9, and their regulators MMP-14 and tissue inhibitor of metalloproteinase (TIMP), are associated with early erosion formation in patients with synovitis of recent onset. A subset of 66 patients was selected from a larger early synovitis cohort on the basis of tissue availability for the study of synovial tissue and serum gelatinase expression. Patients with peripheral joint synovitis of less than 1 years' duration were evaluated clinically and serologically on four visits over a period of 12 months. At the initial visit, patients underwent a synovial tissue biopsy of one swollen joint, and patients had radiographic evaluation of hands and feet initially and at 1year. Serum MMP-1, MMP-2, MMP-9, MMP-14, and TIMP-1 and TIMP-2 levels were determined, and synovial tissue was examined by immunohistology for the expression of MMP-2 and MMP-9, and their molecular regulators. Gelatinolytic activity for MMP-2 and MMP-9 was quantified using a sensitive, tissue-based gel zymography technique. Four healthy individuals underwent closed synovial biopsy and their synovial tissues were similarly analyzed. ). Tissue expression of MMP-2 and MMP-9, however, did not correlate with the serum levels of these enzymes. < 0.001). MMP-2 and MMP-9 are thought to play an important role in the evolution of joint erosions in patients with an inflammatory arthritis. Most studies have concentrated on the contribution of MMP-9 to the synovitis, because synovial fluid and serum MMP-9 levels are markedly increased in inflammatory arthropathies. Previously reported serum levels of MMP-9 have varied widely. In the present sample of patients with synovitis of recent onset, serum MMP-9 levels were elevated in only 21%. Moreover, these elevations were not specific for RA, the tissue expression of MMP-9 was focal, and the levels of MMP-9 activity were not well correlated with early erosions. Although serum MMP-2 levels were not of prognostic value, high synovial tissue levels of MMP-2 activity were significantly correlated with the presence of early erosions. This may reflect augmented activation of MMP-2 by the relatively high levels of MMP-14 and low levels of TIMP-2 seen in these tissues. We were able to localize the components of this trimolecular complex to the synovial lining layer in consecutive tissue sections, a finding that is consistent with their colocalization. In conclusion, we have provided evidence that active MMP-2 complexes are detectable in the inflamed RA synovium and may be involved in the development of early bony erosions. These results suggest that strategies to inhibit the activation of MMP-2 may have the potential for retarding or preventing early erosions in patients with inflammatory arthritis.

Introduction:

In cancer the gelatinases [matrix metalloproteinase (MMP)-2 and MMP-9] have been shown to be associated with tissue invasion and metastatic disease. In patients with inflammatory arthritis the gelatinases are expressed in the synovial membrane, and have been implicated in synovial tissue invasion into adjacent cartilage and bone. It is hypothesized that an imbalance between the activators and inhibitors of the gelatinases results in higher levels of activity, enhanced local proteolysis, and bone erosion.

Objectives:

To determine whether the expression and activity levels of MMP-2 and MMP-9, and their regulators MMP-14 and tissue inhibitor of metalloproteinase (TIMP), are associated with early erosion formation in patients with synovitis of recent onset.

Patients and method:

A subset of 66 patients was selected from a larger early synovitis cohort on the basis of tissue availability for the study of synovial tissue and serum gelatinase expression. Patients with peripheral joint synovitis of less than 1 years' duration were evaluated clinically and serologically on four visits over a period of 12 months. At the initial visit, patients underwent a synovial tissue biopsy of one swollen joint, and patients had radiographic evaluation of hands and feet initially and at 1year. Serum MMP-1, MMP-2, MMP-9, MMP-14, and TIMP-1 and TIMP-2 levels were determined, and synovial tissue was examined by immunohistology for the expression of MMP-2 and MMP-9, and their molecular regulators. Gelatinolytic activity for MMP-2 and MMP-9 was quantified using a sensitive, tissue-based gel zymography technique. Four healthy individuals underwent closed synovial biopsy and their synovial tissues were similarly analyzed.

Results:

). Tissue expression of MMP-2 and MMP-9, however, did not correlate with the serum levels of these enzymes.

< 0.001).

Discussion:

MMP-2 and MMP-9 are thought to play an important role in the evolution of joint erosions in patients with an inflammatory arthritis. Most studies have concentrated on the contribution of MMP-9 to the synovitis, because synovial fluid and serum MMP-9 levels are markedly increased in inflammatory arthropathies. Previously reported serum levels of MMP-9 have varied widely. In the present sample of patients with synovitis of recent onset, serum MMP-9 levels were elevated in only 21%. Moreover, these elevations were not specific for RA, the tissue expression of MMP-9 was focal, and the levels of MMP-9 activity were not well correlated with early erosions. Although serum MMP-2 levels were not of prognostic value, high synovial tissue levels of MMP-2 activity were significantly correlated with the presence of early erosions. This may reflect augmented activation of MMP-2 by the relatively high levels of MMP-14 and low levels of TIMP-2 seen in these tissues. We were able to localize the components of this trimolecular complex to the synovial lining layer in consecutive tissue sections, a finding that is consistent with their colocalization.

In conclusion, we have provided evidence that active MMP-2 complexes are detectable in the inflamed RA synovium and may be involved in the development of early bony erosions. These results suggest that strategies to inhibit the activation of MMP-2 may have the potential for retarding or preventing early erosions in patients with inflammatory arthritis.

Introduction

].

], and thus may also play a similar role in the invasion of synovial pannus.

]. In the present study we evaluated the levels of active MMP-2 and MMP-9 in small synovial biopsy samples obtained from patients with synovitis of recent onset. We also examined the synovial expression of the gelatinases by immunohistology, as well as their serum levels in order to determine whether these indices may predict erosions in RA patients.

Patient population

], and 14 could not be classified and were diagnosed with undifferentiated arthritis.

] from an inflamed joint, typically a knee. Tender and swollen joint counts were obtained from 66 joint areas; hips were excluded. Anterioposterior and lateral radiographs of the hands and feet were obtained at the initial visit and at 1-year follow-up visit. Radiographs were read by experienced radiologists, who were unaware of the patient's diagnosis or test results, and were scored for erosions. Erosions were defined as unequivocal loss of cortical and subcortical bone in at least two different joints of either the hands or feet. Of the 66 patients who underwent a clinical evaluation and analysis of serum MMP levels, 28 had synovial tissue biopsies with detectable lining layer and large enough samples to determine MMP and TIMP expression by gel zymography and immunohistochemistry.

= 4) were recruited under the same protocol and a synovial knee biopsy was performed once signed informed consent was obtained. These tissues were also assayed by gel zymography and stained immunohistochemically.

Antibodies and gels

was used as negative controlin the staining experiments. Enzyme-linked immunosorbent assay (ELISA) kits for serum MMP-1, TIMP-1 and TIMP-2, and the activity assays for MMP-2 and MMP-9 were purchased from Biotrak (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Precast zymography gels containing 10% Tris glycine with 0.1% gelatin as substrate were purchased from Novex (San Diego, CA, USA), and protein quantification kits were purchased from Bio-Rad Laboratories (Hercules, CA, USA).

Matrix metalloproteinase and tissue inhibitor of metalloproteinase enzyme-linked immunosorbent assay

ELISA assays for MMP-1, TIMP-1, and TIMP-2 were performed according to the manufacturer's instructions. Briefly, 100 μ l diluted serum samples were pipetted in duplicates into the appropriate ELISA microtiter wells and incubated for 2 h at 20-25 °C. Wells were washed and incubated with 100 μl of the antiserum for 2 h at 20-25°C, and incubated with 100 μl of the peroxidase conjugate for 1h. After washing 100 μl of 3,3',5,5' -tetramethylbenzi-dine/hydrogen peroxide substrate were pipetted into the wells and incubated for 30 min at room temperature. Absorbance at 630 nm was measured spectrophotometrically in an automated plate reader.

Serum matrix metalloproteinase activity assays

Active and total MMP-2 and MMP-9 were measured by activity assays. Assays were performed according to the manufacturer's instructions. Briefly, 100 μl diluted serum samples were plated in quadruplicate and refrigerated at 4 °C overnight. Plates were washed in an automated plate washer and 50 μl of a 1mol/l p-aminophenylmercuric acetate, which activates pro-MMP, was added only into duplicate wells in which total MMP activity was to be measured. After 2 h of incubation at 37 °C, 50 μl detection reagent containing a modified urokinase and S-2444 peptide substrate was added to all wells. Plates were read spectrophotometrically at time 0 and after a 2-h incubation at 37 ºC for 2 h at an absorbance of 410 nm. MMP activity was represented by the change of absorbance over time. These values were compared with a standard curve of serial dilutions of a known concentration of activated enzyme. Normal range of values was provided by the manufacturer.

Gel zymography

, and 0.02% Brijdetergent. The gels were subsequently stained with 0.5% Comassie blue (G-250), destained with 30% methanol and 10% acetic acid, and incubated in 30% methanol and 5% glycerol. They were dried between cellophane sheets. Areas of gelatinase activity appeared as nonstaining bands on the gels.

. Each value was normalized against the protein concentration, determined by a protein quantitation kit according to the manufacturer's instructions, and reported as gelatinase activity in nanograms per milligrams of protein. Gelatinolytic bands at 92, 72, and 62 kDA represent latent MMP-2, latent MMP-9, and active MMP-2 activity, respectively.

Immunohistochemistry

antibodies or no primary antibody were performed with each experiment. Slides were washed and incubated with goat antimouse biotinylated antibody at a 1 : 750 dilution followed by incubation with streptavidine horseradish peroxidase. Slides were developed with a substrate chromogen solution until a brown reaction product was observed, then counterstained with hematoxylin and coverslipped.

Histologic evaluation of the synovial tissue

Synovial tissues suitable for analysis were histologically evaluated for the presence and degree of inflammation. All tissues were analyzed in a blinded manner by two independent observers. Proliferation of the lining layer, inflammatory cellular infiltration of the sublining layer, lymphocytic aggregates, and stromal proliferation were scored semiquantitatively from 0 to 3 in five high-power fields (HPFs; 400 ×) for each category. Scores were added and divided by four to derive a tissue composite index. Tissue scores for MMP expression were obtained by counting the number of positive cells in the lining layer and sublining layer (one HPF underneath the lining layer) in five representative HPFs (400×) and expressed as positive cells/HPF.

Statistical methods

, analysis of variance, and Kruskal-Wallis tests.

Patient clinical features

. Patients with RA were older, had more tender and swollen joints, were rheumatoid factor positive, and were more likely to be on disease-modifying antirheumatic drug therapy than were non-RA patients. Of the patients with RA, 12 had erosions at multiple sites by 1 year, whereas none of the non-RA patients had developed definite erosions. Patient characteristics in the subset of 28 patients who underwent synovial tissue immunohistology and gel zymography did not differ significantly from the larger sample (data not shown).

Serum levels of the metalloproteinases and their inhibitors in patients with early synovitis

< 0.001). overall serum mmp-2 and mmp-9 levels did not correlate with tissue expression of these enzymes (data not shown).

Localization of MMP-2, MMP-9, MMP-14, and TIMP-2 expression in synovial tissue

. Of note, the antibodies used to detect MMP-2 recognized only the latent zymogen form. MMP-2 was widely expressed in the synovial lining layer and in areas of stromal proliferation in the sublining and stromal layer. MMP-9 expression was more focal and was observed sparsely in the lining layer and in the endothelium of single vessels in both RA and non-RA tissues. MMP-14 and TIMP-2 were detected primarily in the lining layer on consecutive tissue sections.

Comparisons of tissue levels of MMP-2, MMP-9, MMP-14, and TIMP-2

= 0.01). Activated and latent MMP-2 activities, as measured by gel zymography, tended to be higher overall in the RA patients than in the non-RA patients, but results were not statistically different.

Radiographic erosions are associated with higher MMP-2 activity in the synovial tissue

< 0.05). this trend was also observed for mmp-9, but did not reach statistical significance, probably due to the very wide standard deviation (4.0 ± 1.3 versus 28.6 ± 71.2 ng/mg in normal individuals and in the patients, respectively; > P = 0.07).

Discussion

]. Furthermore, these two MMPs differ in tissue distribution and transcriptional regulation. These observations suggest that MMP-2 and MMP-9 may contribute differentially to the pathophysiologic processes that lead to joint destruction.

].

].

]. Although the results of these experiments do not prove that the expression of these molecules occurs on the same cell, they are consistent with the view that the synovial lining layer is a site of MMP-2 activation.

Because of the blind nature of the biopsy technique used in the present study, we did not specifically sample areas of synovium directly adjacent to cartilage and bone. We did, however, ensure that all of the needle biopsy samples examined immunohistologically and zymographically had clear evidence of a well-defined synovial lining layer, were of adequate size, and were thus appropriate for comparative evaluation. The selection of biopsy material with detectable lining layer and sufficiently large size for analysis may have biased our results toward patients with more proliferative synovial lesions, and possibly more severe disease. This selection bias would have occurred in the RA and non-RA patients, however, and is therefore unlikely to account for the differences seen in MMP-2 activity between the patient groups. We compared the synovium of patients with early RA with that of patients with other forms of early synovitis, and with that of normal volunteers. The RA samples tended to have the highest expression levels of MMP-2 and MMP-14, but they exhibited low levels of TIMP-2 expression. More importantly, we showed that the patients with radiographic erosions, all of whom had RA, had the highest levels of active MMP-2 by gel zymography. We therefore propose that the augmented levels of activated MMP-2 detected in the synovia of patients with early erosive RA may relate, at least in part, to an imbalance between activation by MMP-14 and inhibition by TIMP-2.

Immunohistologically, MMP-9 expression was clearly higher in inflamed synovium than in normal synovium, where it was virtually undetectable. Furthermore, MMP-9 activity levels tended to be higher in the synovial samples of patients with radiographic erosions than those of patients without erosions, but the measured values varied widely in the specimens examined. The focal expression of MMP-9 in these synovial tissues, combined with the small number of samples examined, might have contributed to the wide tissue variations observed.

].

]. In contrast, this is not the case for MMP-1, because no blood cell carries a preformed secretory granule of this enzyme and this may explain why MMP-1 levels relate more closely to radiographic erosions. The measurement of serum MMP levels in the context of controlled therapy trials has not been well explored.

In the present study we provided evidence that active MMP-2 complexes are expressed in the synovium and may be involved in the development of bony erosions. Therapeutic strategies to inhibit a broad spectrum of MMPs, including MMP-2 and possibly MMP-9, may therefore present a more powerful approach to retard or prevent early erosions in patients with an inflammatory arthropathy.

Figures and Tables

Matrix metalloproteinase (MMP)-2 and MMP-9 gelatinolytic activity in patients with erosions, in patients with no erosions, and in normal control individuals.

(isotype control).

Patient description of demographic, clinical and treatment characteristics

Comparisons were made between groups. Values are presented either as means ± standard deviation or as total numbers (%).CRP,C-reactive protein; DMARD, disease-modifying antirheumatic drug; ESR, erythrocyte sedimentation rate; RA, rheumatoid arthritis; RF = Rheumatoid factor.

Determination of serum matrix metalloproteinase and tissue inhibitor of metalloproteinase concentrations

<0.01. crp, c-reactive protein; timp, tissue inhibitor of metalloproteinase.

Immunohistologic and zymographic expression of matrix metalloproteinases in synovial tissues from patients with recent onset ofArthritis

< 0.01, versus normal volunteers. mmp, matrix metalloproteinase; timp, tissue inhibitor of metalloproteinase.

Keywords

• early synovitis
• erosion
• metalloproteinase
• matrix metalloproteinase-2
• rheumatoid arthritis