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Osteoarthritis, characterized by the breakdown of articular cartilage in synovial joints, has long been viewed as the result of “wear and tear”1. Although low-grade inflammation is detected in osteoarthritis, its role is unclear2–4. Here we identify a central role for the inflammatory complement system in the pathogenesis of osteoarthritis. Through proteomic and transcriptomic analyses of synovial fluids and membranes from individuals with osteoarthritis, we find that expression and activation of complement is abnormally high in human osteoarthritic joints. Using mice genetically deficient in C5, C6, or CD59a, we show that complement, and specifically the membrane attack complex (MAC)-mediated arm of complement, is critical to the development of arthritis in three different mouse models of osteoarthritis. Pharmacological modulation of complement in wild-type mice confirmed the results obtained with genetically deficient mice. Expression of inflammatory and degradative molecules was lower in chondrocytes from destabilized joints of C5-deficient mice than C5-sufficient mice, and MAC induced production of these molecules in cultured chondrocytes. Furthermore, MAC co-localized with matrix metalloprotease (MMP)-13 and with activated extracellular signal-regulated kinase (ERK) around chondrocytes in human osteoarthritic cartilage. Our findings indicate that dysregulation of complement in synovial joints plays a critical role in the pathogenesis of osteoarthritis.
The pathogenesis of osteoarthritis is unclear, and there are currently no treatments that prevent the development of osteoarthritis. Seeking to gain insight into osteoarthritis, we used mass spectrometry to identify proteins aberrantly expressed in synovial fluid—the fluid that bathes the synovial joints—of individuals with osteoarthritis. We discovered that proteins of the complement system are differentially expressed in osteoarthritic compared to healthy synovial fluids (Supplementary Table 1). Using less-sensitive proteomic techniques, we previously detected ten of these twelve differentially expressed complement proteins in osteoarthritic synovial fluids5. The complement system consists of three distinct pathways that converge at the formation of the C3 and C5 convertases, enzymes that mediate activation of the C5a anaphylatoxin and formation of MAC (comprising the complement effectors C5b-9) (Fig. 1a)6. Components of the classical (C1s and C4A) and alternative (factor B) pathways, the central components C3 and C5, and the C5, C7, and C9 components of MAC were all aberrantly expressed in synovial fluids from individuals with osteoarthritis (Fig. 1a and Supplementary Table 1).
Validating our proteomic results, ELISA analysis showed that levels of C3a (Fig. 1b) and C5b-9 (Fig. 1c) are significantly higher in synovial fluids from individuals with early-stage osteoarthritis than synovial fluids from healthy individuals. Thus, complement activation occurs in synovial joints early in the course of osteoarthritis and persists, albeit at a lower level, during the late phases of osteoarthritis (Fig. 1c). Moreover, immunohistochemical analysis revealed the presence of MAC in synovium (data not shown) and around chondrocytes in cartilage (Fig. 1d) from individuals with end-stage osteoarthritis, consistent with previous findings7–9.
To determine whether the synovium is a source of complement components, we analyzed the expression of genes encoding complement-related proteins (those identified in synovial fluid; Supplementary Table 1) in synovial membranes from individuals with osteoarthritis and from healthy individuals. Analysis by unsupervised hierarchical clustering revealed two major clusters: one containing all the expression profiles from individuals with osteoarthritis (both early- and end-stage), and one containing all the profiles from healthy individuals (Fig. 1e and Supplementary Fig. 1). Interestingly, expression of transcripts encoding the complement effectors C7, C4A, factor B, C9, and C5 was markedly higher, and expression of transcripts encoding the complement inhibitors clusterin, factor H, C4-binding protein, and C1 inhibitor was markedly lower, in osteoarthritic compared to healthy synovial membranes. Our results suggest that the synovial membrane may take on a pathogenic role in osteoarthritis by contributing to excessive complement activation.
To investigate the role of complement in the pathogenesis of osteoarthritis, we used a mouse model of osteoarthritis induced by medial meniscectomy10. In humans, tearing of the meniscus often requires meniscectomy, which is a risk factor for knee osteoarthritis11. Because the C5 effector lies at the nexus of the complement cascade (Fig. 1a), we surgically induced osteoarthritis in C5-deficient (C5−)12 and C5-sufficient (C5+) mice. Sixteen weeks after surgery, C5− mice exhibited substantially less cartilage loss, osteophyte formation, and synovitis than did C5+ mice (Fig. 2a,b and Supplementary Fig. 2d). By contrast, osteoarthritis in this model was not affected by genetic deficiency in C3 (data not shown). That C3−/− mice were not protected against osteoarthritis can be explained by the observation that compensatory mechanisms operate in C3−/− mice: coagulation factors compensate for the lack of C3, allowing C5 activation to proceed even in the absence of C313. Corroborating our findings in the C5− congenic mouse strain, treatment with a neutralizing monoclonal antibody to C514 attenuated osteoarthritis in wild-type mice (Fig. 2c). We also tested the effect on osteoarthritis of CR2-fH—a fusion protein that inhibits activation of C3 and C515. Administration of CR2-fH attenuated the development of osteoarthritis in wild-type mice (Fig. 2d).
We next determined whether the MAC-mediated effector arm of the complement cascade is important in osteoarthritis. We found that mice deficient in C6, an integral component of the MAC (see Fig. 1a), were protected against the development of osteoarthritis and synovitis induced by medial meniscectomy (Fig. 2e,f and Supplementary Fig. 2d). Conversely, mice deficient in CD59a, an inhibitor of MAC6 (see Fig. 1a), developed more severe osteoarthritis and synovitis than their wild-type littermates (Fig. 2g,h and Supplementary Fig. 2a,d).
Not only meniscectomy but also meniscal tearing per se can lead to the development of osteoarthritis in humans11. We therefore also examined the role of complement in the destabilization of the medial meniscus (DMM) model of osteoarthritis16–18. We found that deficiency in CD59a accentuated the osteoarthritic phenotype in mice subjected to DMM (Fig. 2i,j and Supplementary Fig. 2b–d). Deficiency in CD59a also accentuated the milder osteoarthritis that developed spontaneously in aged mice (Supplementary Fig. 3). These findings suggest that MAC-mediated complement activity plays a pathogenic role in osteoarthritis of disparate etiologies.
Resistance to the development of histological osteoarthritis in complement-deficient mice translated to functional benefit. Twelve weeks after medial meniscectomy, C5− mice maintained normal gait, whereas C5+ mice developed abnormal gait (Fig. 3a). Time-course studies revealed that neither C5+ nor C5− mice mice exhibited proteoglycan loss or cartilage degeneration two and four weeks after medial meniscectomy (Fig. 3b,c). This period of latency is similar to that observed in the DMM model16 and in humans who have undergone medial meniscectomy11. Eight and twelve weeks after surgery, however, C5+ mice exhibited significant proteoglycan and cartilage loss and synovitis, while C5− mice did not (Fig. 3b,c and Supplementary Fig. 4). The osteoarthritic phenotype was pronounced in these mice, most likely owing to their genetic background and age at the time of surgery, both factors that influence the severity of mouse osteoarthritis16.
Products of dysregulated cartilage remodeling and repair may contribute to joint inflammation in osteoarthritis19–22. We examined the ability of osteoarthritic cartilage or specific components of the extracellular matrix (ECM) of cartilage to activate complement in vitro. Pulverized osteoarthritic cartilage induced the formation of C5b-9, as did the ECM components fibromodulin and aggrecan but not the ECM components type II collagen and matrilin-3 (Fig. 4a,b and Supplementary Fig. 5). Fibromodulin, which can bind C1q and activate the complement cascade23, was present at higher levels in osteoarthritic compared to healthy synovial fluid (Fig. 4c). Other cartilage ECM components, such as cartilage oligomeric matrix protein, are also detected at abnormally high levels in osteoarthritic synovial fluid and can activate complement19,20. Together, these results suggest that release or exposure cartilage ECM components may contribute to the pathophysiology of osteoarthritis by activating complement.
Our in situ (Fig. 1d) and in vivo (Fig. 2e–j and Supplementary Figs. 2,3) findings indicate that MAC is important in mediating complement-related cartilage damage in osteoarthritis. But how might MAC damage cartilage? Extensive deposition of MAC induces cell lysis and necrotic cell death, whereas sublytic MAC can activate signaling pathways that drive the expression of proinflammatory and catabolic molecules24. Because many of the MAC-encircled chondrocytes in osteoarthritic cartilage appear morphologically intact (Figs. 1d, ,4h4h and Supplementary Fig. 6), we examined whether MAC induces the expression of proinflammatory and degradative enzymes in osteoarthritis. We first examined the expression of genes encoding such molecules in cultured chondrocytes coated with sublytic levels of MAC. Sublytic MAC increased the chondrocytes’ expression of multiple genes implicated in osteoarthritis: those encoding cartilage-degrading enzymes17,18,22 (MMPs, ADAMTSs); inflammatory cytokines25 (CCL2, CSF1, and CCL5); and cyclo-oxygenase 226 (Fig. 4d–f). Sublytic MAC also induced the expression of complement effectors (Fig. 4d); chondrocyte production of complement may thus synergize with complement derived from synovial membrane to amplify pathogenic complement signaling in osteoarthritis.
We next examined the effect of complement deficiency on the in vivo expression of these genes in destabilized mouse joints. Twenty weeks after DMM surgery, chondrocytes from the destabilized joints of C5-deficient mice, which are MAC deficient and protected against osteoarthritis (Figs. 2a,b and Fig. 3), expressed lower levels of these inflammatory and degradative mediators than did chondrocytes from the destabilized joints of C5-sufficient mice (Fig. 4g). mRNA levels of Jun and Fos, proinflammatory transcription factors whose expression is induced by MAC27,28, were also lower (Fig. 4g). Moreover, in human osteoarthritic cartilage, MAC co-localized with MMP-13 and with activated ERK-1/2 (Fig. 4h and Supplementary Fig. 6), a kinase that mediates resistance to MAC-mediated cell lysis29 and stimulates the expression of MMP-13 by inducing the expression of Fos30.
Here we show that the complement cascade is crucial to the pathogenesis of osteoarthritis. Cartilage ECM components released by or exposed in osteoarthritic cartilage may trigger the complement cascade. Additionally, dysregulation of gene expression in joint tissues may contribute to a local preponderance of complement effectors over inhibitors in osteoarthritis, permitting complement activation to proceed unchecked. Complement activation in turn results in the formation of MAC on chondrocytes, which either kills the cells or causes them to produce matrix-degrading enzymes, inflammatory mediators, and further complement effectors—all of which promote joint pathology.
Recent findings suggest that low-grade complement activation contributes to the development of other degenerative diseases, such as age-related macular degeneration31 and Alzheimer’s disease32. We propose that osteoarthritis can be added to this list of diseases. Our findings provide rationale for targeting the complement system as a disease-modifying therapy for osteoarthritis.
These studies were supported by a Rehabilitation Research and Development Merit Award from the Department of Veterans Affairs and US National Heart, Lung, and Blood Institute Proteomics Center contract N01 HV 28183 (W.H.R.); Northern California Chapter Arthritis Foundation Postdoctoral Fellowship Award (A.L.R.); New York Chapter Arthritis Foundation/Merck Osteoarthritis Research Fellowship Award, the Atlantic Philanthropies, American College of Rheumatology Research and Education Fund, and the Association of Specialty Professors (C.R.S.); and the Frankenthaler and Kohlberg Foundations (M.K.C).
Accession codes. Experimentally determined microarray data is available at GEO (accession code GSE32317).
AUTHOR CONTRIBUTIONSA.L.R. and W.H.R. initiated the investigation of complement in osteoarthritis, and Q.W. conducted key studies. Q.W., A.L.R., D.M. Larsen, H.H.W., and W.H.R. conducted the studies of osteoarthritis in mouse models. A.E., A.L.R., and M.S. performed the gait analysis studies. Q.W. and H.H.W. performed the in vitro MAC deposition experiments. C.M.L. and J.J.S. performed the immunohistochemical analyses of cartilage. J.F.C., G.B., S.Y.R., L.P., S.R.G., R.G., and D.M. Lee conducted or contributed to the proteomic analysis of osteoarthritic synovial fluid. S.Y.R. and D.M. Lee performed the ELISA analysis of osteoarthritic and healthy synovial fluids. C.R.S. and M.K.C. performed the gene-expression analysis of synovium, and G.B., R.G., and D.M. Lee contributed to the analysis of these datasets. A.L.R., C.M.L., J.J.S., and I.H. performed the in vitro complement activation and stimulation assays using samples provided by S.B.G. V.M.H., J.M.T. and N.B. provided the C5-specific antibody and the CR2-fH fusion protein; T.W-C the C6− and Cd59a−/− mice. V.M.H., T.M.L., and D.M. Lee provided scientific input. T.M.L., A.L.R., and W.H.R. wrote the manuscript, and Q.W., C.R.S, T.W-C., S.R.G., M.K.C., V.M.H., and D.M. Lee edited the manuscript. All authors analyzed the data and approved the final manuscript.
COMPETING FINANCIAL INTERESTS
D.M. Lee is currently employed by Novartis Pharma, AG. D.M. Lee and R.G. own equity in Synostics, Inc. J.M.T. and M.V.H. are consultants for Alexion Pharmaceuticals, Inc.