Breakdown of the cartilage matrix is one of the hallmarks of osteoarthritis (OA). Cartilage destruction is predominantly mediated by cytokines and enzymes (1–4). During OA, a clear pericellular activation of chondrocytes is observed, which express large amounts of neoepitopes induced by metalloproteinases that drive pericellular breakdown of the matrix, eventually culminating in erosion (3). Pathologic changes that develop during OA are driven by at least 3 different tissues: cartilage, subchondral bone, and synovium (5, 6). Although OA has generally been described as a disease of the cartilage, the synovium is considered to contribute to the pathology during progression of the disease (7–11). A substantial group of OA patients (up to 50%) show activation of the synovium, not only during late phases, but also during early stages of the disease (8). In previous studies, we showed that activated synovial lining macrophages are crucial in regulating synovial inflammation and subsequent cartilage destruction during experimental OA. Selective elimination of synovial lining macrophages prior to induction of collagenase-induced OA diminished synovial activation and almost completely inhibited cartilage destruction (12).
Activated macrophages, which cover the inside of diarthrodial joints, produce a plethora of mediators, among them, cytokines such as interleukin-1β (IL-1β) (2). Although IL-1β has been considered important in mediating cartilage destruction (2, 13), its role in OA is still a matter of debate. The most prominent proteins released by activated macrophages are the calgranulins: myeloid-related protein 8 (MRP-8; also known as S100A8) and MRP-14 (also known as S100A9) (14, 15). These proteins belong to the group of damage-associated molecular patterns (DAMPs), which are crucial in innate immunity. Both proteins belong to the S100 family of calcium binding proteins, which comprises 24 members. They are expressed as homodimers and heterodimer assemblies. S100A8 is generally coexpressed with S100A9, and the heterodimer S100A8/A9 is translocated to membrane and cytoskeletal structures upon activation (16). In mice, S100A8 forms the active part, whereas S100A9 forms the regulating unit, which binds to S100A8 and thereby prevents its degradation. S100A8 stimulates macrophages via Toll-like receptor 4 (TLR-4) signaling (17, 18). When secreted, S100A8 exhibits proinflammatory functions, leading to activation of endothelial cells and phagocytes.
The homodimers S100A8 and S100A9 and the heterodimer S100A8/A9 accumulate in inflammatory fluids during arthritis (18, 19), and their levels correlate significantly with the severity of arthritis and were shown to predict a 10-year radiographic progression in RA patients (20). In previous studies, we found that S100A8/S100A9 is crucial in mediating cartilage destruction during experimental arthritis (21). S100A8 and S100A9 have recently also been described in experimental OA (22), and S100A8 appeared to be a potent stimulator of murine chondrocytes, thereby inducing a catabolic phenotype (23).
In the present study, we investigated whether these proteins are involved in synovial activation and cartilage destruction in experimental OA using 2 murine models that differ in the degree of synovial activation. In addition, we explored the clinical relevance of S100A8/A9 in samples of synovial biopsy tissues and sera from participants in the Cohort Hip and Cohort Knee (CHECK) early OA symptomatic cohort.
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In the present study, we found that S100A8 and S100A9 are pivotal proteins involved in mediating cartilage destruction during experimental collagenase-induced OA, in which synovial activation is important for the induction of joint pathology, but not in the destabilized medial meniscus model of OA, in which synovial activation is absent. Arthroscopic studies performed in the joints of OA patients suggest that localized proliferation and inflammatory changes in the synovium occur in ∼50% of OA patients (7, 8). Macrophages are the dominant cell types present in activated synovium of humans with OA, although B cells and T cells have also been previously described (11). As in human OA, macrophages were the predominant cell type in the inflamed synovium of mice with collagenase-induced OA, and no polymorphonuclear leukocytes were observed during the chronic phase of disease (between day 7 and day 42).
S100A8/S100A9 appeared to be important in sustaining synovial activation during collagenase-induced OA, since S100A9–/– mice, whose peripheral myeloid cells also lack the S100A8 protein (24), showed reduced synovial activation and cartilage destruction. Both the mRNA and protein levels of S100A8 and S100A9 remained high for extended periods (up to 21 days after injection) in the synovium of mice with collagenase-induced OA. S100A9 mRNA expression even increased between day 7 and day 21. This finding is consistent with the data from the human studies showing that biopsy samples from patients with early symptomatic OA as well as from patients with end-stage OA expressed high levels of S100A8 and S100A9 protein, suggesting a prolonged expression throughout the OA process. S100A8 and S100A9 have been shown to induce a specific inflammatory response in human microvascular endothelial cells, macrophages, osteoblasts, and chondroblasts (35). Synovial activated macrophages are the source of S100A8/S100A9 in collagenase-induced OA.
Apart from regulating synovial inflammation, macrophages have also been shown to mediate cartilage destruction. In previous studies, we found that removal of synovial macrophages prior to induction of experimental OA almost completely blocked metalloproteinase-induced cartilage destruction and osteophyte formation (12, 36). Synovial inflammation has been considered a factor that most likely contributes to dysregulation of chondrocyte function, favoring an imbalance between the catabolic and anabolic activities of the chondrocyte in remodeling the cartilage extracellular matrix (37). Earlier studies showed that intraarticular injection of S100A8 into murine knee joints strongly stimulate both cytokine and matrix metalloproteinase (MMP)/ADAMTS expression in the intima layer (23). Fibroblast-like type B cells lying within the intimal layer produce proteoglycans and at the same time are high producers of ADAMTS (38), which may explain the presence of large amounts of NITEGE neoepitopes within the intimal layer of mice with collagenase-induced OA. In contrast, stimulation of macrophages with S100A8 up-regulated only cytokines, but not MMP/ADAMTS, suggesting that the contribution of macrophages to cartilage destruction may be through the abundant release of catabolic cytokines and S100 species, thereby further stimulating fibroblast-like type B cells to produce ADAMTS.
IL-1β, IL-6, and TNFα are all capable of inducing MMPs, aggrecanases, and other catabolic factors (39). TNFα and IL-1β colocalized with MMPs 1, 3, 9, and 13 in regions of matrix depletion in OA cartilage (40). There is a strong relationship between the increased levels of catabolic enzymes and inflammatory mediators such as IL-1β. IL-1β was previously shown to be crucial in mediating cartilage destruction in experimental murine arthritis (41). The role of IL-1β in driving the pathologic changes of OA is, however, still a matter of debate (14). We found a 2-fold down-regulation of IL-1β in the synovium of the knee joint after a short initial phase of up-regulation. IL-1β and its effects are probably inhibited by cytokines such as IL-10 and IL-1Ra, which were highly expressed shortly after OA induction. IL-1β drives cartilage destruction by stimulating MMP production in chondrocytes. However, MMPs are secreted in a latent form and need an activation step in order to become activated. S100A8 protein may be involved in this by inducing MMP-activating factors, such as oxygen radicals (23).
Interestingly, when proinflammatory triggers such as SCW fragments were injected into a normal mouse knee joint, only a brief enhancement of S100A8 and S100A9 expression in the synovium was noted, indicating that the expression of both proteins is tightly controlled during acute inflammation. Expression of S100A8 and S100A9 in the synovium for prolonged periods during collagenase-induced OA implies that the expression becomes reactivated, which may be driven by cartilage degradation products released during OA. Interestingly, whereas S100A8 and S100A9 levels remained high up until day 21, expression of such cytokines as IL-1β,TNFα, and IL-6 were already strongly decreased at this time point.
Although the pathogenic triggers responsible for prolonged and specific up-regulation of S100A8/S100A9 in OA are not known, recent data point to a proinflammatory crosstalk between extracellular matrix metabolites and phagocyte-specific danger molecules. Elevated catabolic breakdown of the matrix by metalloproteinases leads to additional release of matrix products, such as biglycan, decorin, and aspirin (42). Biglycan has recently been shown to activate the inflammasome within macrophages (43) and may reactivate macrophages, forming a positive feedback loop with S100A8/S100A9. Interestingly, both molecules stimulate macrophages via TLR-4 (44).
Apart from having an effect on synovial inflammation, S100 proteins may have a direct effect on chondrocyte metabolism (23). Although homodimers and heterodimers of S100A8 and S100A9 are negatively charged, they may penetrate cartilage layers due to their small size (20 kd) (45). In a recent study of human chondrocytes isolated from OA cartilage that is reported elsewhere in this issue of Arthritis & Rheumatism (46), we found that not only S100A8, but also S100A9 was a potent stimulator of a catabolic phenotype, which is reflected by high levels of MMP-1, MMP-3, and MMP-9 release and inhibition of matrix molecules, type II collagen, and aggrecan. MMP-3 is a crucial enzyme involved in cartilage destruction during experimental arthritis (47), as well as during collagenase-induced OA (13).
The mechanism by which S100A8 activates chondrocytes is not known. Various receptors have been suggested to be involved in S100A8 signaling, such as TLR-4 (17, 48), RAGE (49), and N-glycans (50). In previous studies, it was shown that S100A8 signaling in macrophages occurs via TLR-4 (17). Using primary chondrocytes obtained at arthroplasty from patients with OA, we recently found that stimulation by S100A8 and S100A9 was significantly inhibited by the TLR-4 inhibitor TAK-242, but not by inhibitors of RAGE or N-glycans (46).
In synovial fluid samples from OA patients, abundant amounts of S100A8/S100A9 (up to 5–7 μg/ml) have been detected (51). Significantly elevated concentrations of S100, probably overflow from the joints, were also measured in the sera of patients in whom OA was suspected, as compared to sera from healthy controls. Interestingly, significantly higher levels were measured at baseline in OA patients who had developed clear cartilage destruction 2 years later. When a cutoff value of 600 ng/ml was used, above which the S100A8/A9 level was defined as increased, and only patients with severe OA progression (change in Kellgren/Lawrence score ≥3) were included in the analysis, a significantly higher number of patients with severe progression showed increased levels of S100A8/A9 (by chi-square test). The odds ratio was 7.5, indicating a highly increased risk of severe progression when S100A8/A9 levels are increased (results not shown). However, the numbers of strong progressors were very low (n = 8), and further research at later followup points are planned in order to determine the value of these proteins as markers of progression.
Synovial activation, which is clearly present in a subpopulation of OA patients, may explain the high levels of S100A8 and S100A9 found in synovial fluid, as well as in the blood, where they can easily be measured. Since S100A8 and S100A9 levels remain high for prolonged periods and since they are important stimulators of cartilage destruction, these proteins may be effective biomarkers for predicting progressive cartilage destruction in OA.
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All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. van Lent had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Van Lent, Blom, Schelbergen, Cats, Vogl, Roth, van den Berg.
Acquisition of data. Van Lent, Blom, Schelbergen, Slöetjes, Lafeber, Lems, Cats.
Analysis and interpretation of data. Van Lent, Blom, Schelbergen, Slöetjes, Cats, Vogl, Roth.