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

Radiographic erosions are characteristic of a number of chronic inflammatory arthropathies, and are associated with articular destruction leading to functional loss and disability [1,2]. The prevention or retardation of erosions is a major objective for early therapeutic interventions in patients with new onset arthritis [3]. Although the pathologic processes that underlie the development of erosions are incompletely understood, a considerable body of evidence has suggested that a pannus of cells that originate in chronically inflamed, proliferative synovial tissue becomes locally invasive, and enzymatically degrades the matrix of the articular cartilage and periarticular bone [4].

Although a number of enzymes have been shown to be involved in erosion formation, several members of the matrix metalloproteinase (MMP) family are thought to play a central role in the degradation of the extracellular matrix of articular bone and cartilage. Of the MMPs, the collagenases [5,6] and stromelysins [7] have been the best studied, and marked elevations in serum and synovial levels have been found in rheumatoid arthritis (RA) [8]. A number of studies have shown that the serum and synovial levels of both gelatinases MMP-2 [9,10] and MMP-9 [11,12,13] are similarly elevated in RA. Although both of these MMPs are expressed at the junction between synovial pannus and cartilage or bone [14], further evidence of their direct role in erosion formation is lacking. In addition members of this subfamily of MMPs are involved in the invasiveness of cancers [15,16], and thus may also play a similar role in the invasion of synovial pannus.

The regulation of MMP activity is complex, and occurs at multiple levels [17,18,19]. In most cases, MMPs are secreted as proenzymes and then undergo extracellular activation. Few studies have evaluated the levels of active MMPs in situ. Gel zymography has proved to be a sensitive and effective method of detecting the activity of gelatinases in small tissue samples [20]. 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.

Joint destruction is one of the hallmarks of erosive arthropathies and leads to significant morbidity and disability. Newer imaging studies have concluded that erosion formation can occur very early in the disease [25]. Detailed pathologic studies, although generally performed on destroyed joints obtained at the time of arthroplasty, have suggested that these erosions result from direct invasion of synovial tissue into the adjacent articular bone and cartilage. It has been postulated that the locally invasive cells that form the aggressive pannus have acquired a transformed phenotype, and analogies to invasive cancer cells have been made [26]. Several metalloproteinases that are capable of the proteolytic degradation of extracellular matrix are thought to play a major role in the dissolution of the bone and cartilage matrix, allowing the invasive cells to migrate into the stroma across cell boundaries. Although the relative contribution of each MMP to this process remains unclear, the gelatinases, particularly MMP-2, appear to play a prominent role in tissue invasion [16]. In patients with arthritis, most studies have concentrated on the contribution of MMP-9 to the underlying mechanism of joint destruction. In contrast to MMP-2, MMP-9 levels tend to be higher in inflammatory arthropathies than in traumatic, osteoarthritic, and tissue repair processes [11,12,27]. 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.

In the joint, MMP-9 is expressed in macrophages in the synovial lining layer, in vascular endothelial cells, leukocytes, chondrocytes, and osteoclasts. It is also expressed on cultured fibroblasts and on activated fibroblasts in vivo [11,28]. MMP-9 is inducible by inflammatory cytokines such as tumor necrosis factor-α and interleukin-1 [19,29], and the post-transcriptional regulation in vivo remains incompletely understood. MMP-9 is secreted in an inactive zymogen form and can be activated in vitro by the plasmin cascade and through other MMPs [30,31,32,33]. In its inactive form MMP-9 is complexed to TIMP-1 [34]. The regulation of MMPs by inflammatory cytokines, and the expression of MMP-9 in macrophages and vascular structures have previously suggested that MMP-9 may be associated with the synovial inflammation and neovascularization [35,36].

In contrast to MMP-9, MMP-2, in the inactive zymogen form, is constitutively expressed by many cell types, including fibroblasts and endothelial cells, and is involved in many aspects of normal tissue remodeling and angiogenesis [37,38]. Its unique regulatory requirements at the cell surface have suggested a special role for MMP-2 in tissue invasion. The transcriptional and post-translational activation of this enzyme is tightly regulated [16]. MMP-2 differs from several other members of the metalloproteinase family in that its transcription is not induced in response to the proinflammatory cytokines tumor necrosis factor-α and interleukin-1, and seems primarily regulated post-translationally. As is characteristic of most of the MMPs, MMP-2 is produced as a proenzyme, and requires activation by enzymatic cleavage. Evidence from several investigators has suggested that MMP-2 in its active form is bound in a trimolecular complex at the cell surface [39,40,41,42]. Pro-MMP-2 complexed with TIMP-2 binds to a TIMP-2 binding site on the membrane-bound MMP-14 receptor, and through a conformational change the catalytic domain becomes exposed forming the active enzyme complex. Upon dissociation from the MMP-14 receptor, TIMP-2 binds the catalytic site and suppresses the activity. Thus, the activity of MMP-2 is regulated locally by a delicate balance between MMP-14 activation and TIMP-2 inhibition. Studies have localized this trimolecular complex to the invadopodia of malignant cells at sites of tissue invasion into stroma [43,44] and serum levels and high tissue expression levels of active MMP-2 are predictors of disease severity and shortened survival in several malignancies [15,16].

In view of the analogies between erosion formation in arthritis and tumor invasion in cancer, we hypothesized that activated MMP-2 and possibly MMP-9 might play an important role in the early stages of invasion of synovial pannus into bone and cartilage. Synovial tissue specimens obtained from a cohort of patients with early synovitis were examined for the presence of MMP-9 and components of the MMP-2-associated trimolecular complex. A highly sensitive zymographic technique was used to evaluate tissue levels of active and latent MMP-2. Our immunohistologic data suggest that MMP-2, TIMP-2, and MMP-14 are all localized to the synovial lining layer. Previous reports have found colocalization of these molecules in areas of tissue remodeling, loosening of prosthetic joints [45,46,47], and in the cartilage-pannus junction [14]. 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.

Overall, the present observations suggest an association between the presence of active synovial gelatinases and the early development of erosive articular damage. Although the evidence is indirect, these data are consistent with the hypothesis that high levels of activated synovial gelatinases reflect augmented synovial invasiveness; these observations are similar to those made in the context of invasive cancers [16]. Although there continues to be controversy regarding the origins of the invasive cells at the junction of cartilage/bone and pannus, considerable evidence has accumulated that the lining cells in RA synovium exhibit a 'transformed' phenotype that is typical of invasive cells [26,48].

We attempted to evaluate the more readily available serum levels of active MMPs as predictors of joint erosions. Overall, only the serum levels of MMP-1 were significantly associated with erosions and with CRP levels. Persistent CRP elevation in RA patients has been shown to be associated with the progression of radiographic erosions [49], but a previous RA study failed to associate MMP-1 levels with erosions [50]. In general, serum levels of MMPs have tended to vary widely in the reported studies [9,11,12,51], and appear not to be of prognostic value. Furthermore, degranulation of platelets and neutrophils may contribute to the elevation of serum levels of MMP-2 and MMP-9 seen in the patients, and may be an explanation for the high baseline level in the serum [52,53]. 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.