Rheumatoid arthritis (RA) is characterized by a loss of joint function resulting from proteolytic degradation of articular cartilage. Chondrocytes synthesize and maintain the extracellular matrix (ECM) of cartilage, which is composed primarily of proteoglycan (aggrecan) and collagen. These structural components provide resistance to compressive forces and give the tissue its tensile strength. Cartilage degradation is mediated predominantly by the matrix metalloproteinases (MMPs), a family of potent enzymes that, collectively, can degrade all ECM components and that have been strongly implicated in arthritic joints (1). Aggrecanolysis is considered to be mediated by the ADAMTS proteinases (2), although this ECM component can be replaced relatively rapidly once the stimulus, such as interleukin-1 (IL-1), has been removed (3). In contrast, collagen is much less readily released, but when degradation does occur, tissue integrity is irreversibly lost (4). The collagenolytic MMPs (MMPs 1, 8, and 13) have all been implicated in pathologic collagenolysis (1) and require activation of their latent proforms via proteolytic removal of the propeptide, which can be MMP-mediated (5). Indeed, this activation has been shown to be a key step in cartilage collagenolysis (6).
We have previously shown that the combination of IL-1 and oncostatin M (OSM), cytokines known to be elevated in RA synovial fluid (7, 8), promotes the synergistic loss of collagen (and proteoglycan) from cartilage in vitro (7). Furthermore, we have also demonstrated that this combination induces a marked inflammatory arthritis in vivo, which is characterized by pronounced synovial hyperplasia, increased inflammatory infiltrate, marked cartilage and bone erosions, and elevated MMP expression (9, 10). In IL-1 plus OSM–treated human chondrocytes, the most striking observation is a pronounced induction of MMP-1 (10, 11), as well as other MMPs, ADAM proteinases, and ADAMTS proteinases (11). Traditionally, inflammation and destruction of bone and cartilage have been linked, although this may not be the case (12). These processes are complex and multifactorial. Studies to date have been restricted to a relatively small subset of those metalloproteinases considered to be important in cartilage ECM degradation, rather than focusing on the diversity of proteinases and other molecules that can be expressed by chondrocytes. Indeed, cytokine-induced cartilage catabolism is multifarious, and sequential proteolysis of ECM components can occur. It is known, for example, that aggrecanolysis is an early event, whereas collagenolysis takes place much later in the disease course (7, 13). Interestingly, aggrecan has been suggested to assist in maintenance of the ECM by protecting collagen fibrils from collagenolysis (14).
Most studies have focused on the genes that help mediate the destructive response following a procatabolic stimulus, such as that provided by IL-1 plus OSM, and it is apparent that a number of genes are likely to be regulated coordinately. Little, however, is known about the repair responses that chondrocytes may initiate following such stimuli. Herein we report the findings of microarray analyses of IL-1 plus OSM–treated chondrocytes. Our results provide new insight into the mechanisms by which this potent cytokine combination can affect the breakdown of cartilage as well as the repair responses initiated by chondrocytes.
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- MATERIALS AND METHODS
Although considerable data exist on the nature of the genes that contribute to pathologic cartilage destruction, such as MMPs (see ref.1 and references therein), less data are available on the repair responses that are invoked following a proinflammatory stimulus. The aim of the current study was to identify genes up-regulated by IL-1 plus OSM that may contribute to such a repair mechanism. We have shown that the combination of IL-1 and OSM up-regulates many of the MMPs known to play a key role in cartilage degradation (1), as well as some for which a defined role has yet to be demonstrated, including MMPs 10 and 12.
The data show that MMP-10 is synergistically up-regulated in chondrocytes by IL-1 plus OSM. Other studies have shown that MMP-10 can degrade aggrecan, link protein, and fibronectin, and that it activates proMMP-1 and proMMP-8 (1). MMP-12 (macrophage elastase) is also present in RA synovium (24) and can degrade elastin, fibronectin, and laminin in addition to cleaving and activating pro–tumor necrosis factor α (TNFα), proMMP-2, and proMMP-3 (25). MMP-12 also cleaves urokinase-type plasminogen activator receptor (uPAR), resulting in its inhibition (26). This cell-surface receptor localizes and enhances uPA activity, which converts plasminogen to plasmin; this has been implicated in procollagenase activation and cartilage collagenolysis (6). The array data also identified other genes that are likely to be involved in cartilage degradation, such as C1r. Expression of complement component in chondrocytes has been previously reported (27), and C1r activates C1s, which can degrade type II collagen and decorin (28). Serum amyloid A2 (SAA2) was also induced. SAA expression has been reported in RA synovial tissue (29), and SAA proteins induce MMP production in synovial fibroblasts (29), thereby enhancing ECM breakdown.
Various cytokines, chemokines, and their receptors involved in inflammatory processes were up-regulated. These include MCPs 1 and 3, IL-6, leukemia inhibitory factor (LIF), and the OSM-specific receptor OSMβR. IL-6, LIF, and OSM all belong to the glycoprotein-130–binding cytokine family (30). IL-6 and OSM are produced in RA synovium and can act synergistically with IL-1 and TNFα in the presence of their soluble receptors (7, 31) to promote cartilage breakdown (6, 9, 10, 21). An increase in the expression of OSMβR may lead to prolonged activation of OSM-mediated signaling pathways (30), and one component that facilitates such signaling, Jak-2 kinase, was also notably induced. Combined with the marked induction of IL-1β, this could result in an exacerbation of IL-1 plus OSM–induced effects within cartilage by the resident chondrocytes. This indicates that cartilage may be a much more active player in RA pathogenesis than previously thought.
The chemokines IL-8 and ENA-78 were synergistically up-regulated. We confirmed that KC, a murine equivalent to IL-8 (23), was up-regulated by IL-1 plus OSM in a murine model of arthritis. KC expression was primarily localized to the articular surfaces of the cartilage, concomitant with its role as a chemoattractant inducing the migration of neutrophils from the synovium toward the cartilage. IL-8 and ENA-78 are potent inducers of angiogenesis (32), which is a marked feature in our arthritis model (9, 10). IL-8 also contributes to the pathologic changes observed in arthritis through p38 MAPK pathway activation (33), which can lead to hypertrophic differentiation, alteration in collagen subtype expression, and cartilage calcification. PBEF was also up-regulated, and this cytokine perpetuates inflammation since it stimulates IL-6 and IL-8 expression (34). Cartilage may therefore inadvertently contribute to joint inflammation, since the inflammatory process is presumably initiated as a repair response to the original procatabolic stimulus.
Stimulation with IL-1 plus OSM significantly induced the calcium binding proteins S100 A8 and S100 A9. These proteins have been localized to RA synovial tissue, in particular, the synovium–pannus junction (35). S100 A8 and S100 A9 activate endothelium, promoting further recruitment of inflammatory cells into the synovium (36) and thus perpetuating inflammation. PTX-3 expression has been reported in RA synovium (37), but this study is the first to demonstrate PTX-3 expression by chondrocytes. Several functions have been attributed to PTX-3, including C1q binding, complement activation (38), and inhibition of angiogenesis through its interaction with fibroblast growth factor 2 (39).
A variety of other genes that represent a repair response mechanism were also up-regulated by IL-1 plus OSM. The serine protease inhibitors antileukopeptidase and squamous cell carcinoma antigen were induced. Antileukopeptidase prevents cartilage and bone erosion in anti–type II collagen antibody–induced arthritis (40). Activin A is a member of the transforming growth factor β (TGFβ) superfamily, mediating its affects through Smad transcription factors. The Array data, as well as immunolocalization, showed that activin A is significantly up-regulated by IL-1 plus OSM in chondrocytes. Previous studies have shown that activin A is expressed in RA synovial tissue and can induce the proliferation of fibroblast-like synoviocytes in culture (41). In osteoarthritic cartilage, activin A exhibits anabolic properties, inducing expression of tissue inhibitor of metalloproteinases 1 and increasing expression of type II collagen and proteoglycan synthesis in chondrocytes (42, 43). Our findings also indicate that activin A expression appears to be prolonged, since its expression was relatively unaltered regardless of the adenovirus titer used (data not shown). In cartilage, activin A expression may be a repair response, since it appears to have a protective role by preventing MMP-mediated cartilage degradation and promoting ECM-component synthesis. However, we have shown that, unlike TGFβ (44), activin A fails to prevent IL-1 plus OSM–mediated cartilage collagenolysis in an in vitro model of cartilage breakdown (Hartland S and Rowan AD: unpublished results).
Another markedly induced gene following IL-1 plus OSM stimulation was YKL-40 (chitinase-3–like protein 1), a protein that is reported to be present in degenerate articular cartilage and in inflamed, hyperplastic synovium (45, 46). Recent studies have shown that purified YKL-40 promotes connective tissue growth, provides a signal through kinase-mediated signaling pathways (47), and inhibits fibroblast responses to IL-1 through its effects on these pathways, resulting in a reduction in MMP-1, MMP-3, and IL-8 production (48). These data support the concept of a protective repair response elicited by chondrocyte-derived YKL-40. Indeed, it is known that 33% of conditioned medium from stimulated chondrocytes can exhibit YKL-40 (49), and our observations support this concept (Catterall JB, et al: unpublished results). Expression of the structural components decorin and fibronectin was increased by IL-1 plus OSM, again indicating a repair response aimed at synthesizing new ECM components. Another protein that may have a protective role is superoxide dismutase, which is an antioxidant that removes superoxide anions that have been implicated in hyaluronic acid and cartilage ECM damage. The role of this protein is supported by evidence showing that a genetic deficiency in superoxide dismutase results in an enhancement of collagen-induced arthritis in mice (50).
We have provided further evidence of marked induction of MMPs in chondrocytes following stimulation with IL-1 plus OSM, confirming their undoubted importance in the degradation of the cartilage ECM. We have also provided evidence that chondrocytes are capable of expressing a variety of factors following stimulation, some of which are protective. It would appear that these reparative mechanisms, initiated by chondrocytes, are ultimately overwhelmed by the continued inflammatory stimuli that predominate in cartilage catabolism. Our data suggest that blockade of such proinflammatory stimuli may inadvertently suppress potential repair mechanisms as well as catabolic processes, and such interventions therefore need to be fully evaluated. Moreover, our data indicate that cartilage may be an active player in the disease process, especially in RA, which is often viewed as a synovium-driven disease. Thus, microarray analyses have highlighted many genes that may play a role in prevention of cartilage breakdown and mechanisms of the repair response. These genes could be exploited for therapeutic intervention in the future.