Michael A. DiMicco

Mechanical injury potentiates proteoglycan catabolism induced by interleukin‐6 with soluble interleukin‐6 receptor and tumor necrosis factor α in immature bovine and adult human articular cartilage

OBJECTIVE Traumatic joint injury can damage cartilage and release inflammatory cytokines from adjacent joint tissue. The present study was undertaken to study the combined effects of compression injury, tumor necrosis factor alpha (TNFalpha), and interleukin-6 (IL-6) and its soluble receptor (sIL-6R) on immature bovine and adult human knee and ankle cartilage, using an in vitro model, and to test the hypothesis that endogenous IL-6 plays a role in proteoglycan loss caused by a combination of injury and TNFalpha. METHODS Injured or uninjured cartilage disks were incubated with or without TNFalpha and/or IL-6/sIL-6R. Additional samples were preincubated with an IL-6-blocking antibody Fab fragment and subjected to injury and TNFalpha treatment. Treatment effects were assessed by histologic analysis, measurement of glycosaminoglycan (GAG) loss, Western blot to determine proteoglycan degradation, zymography, radiolabeling to determine chondrocyte biosynthesis, and Western blot and enzyme-linked immunosorbent assay to determine chondrocyte production of IL-6. RESULTS In bovine cartilage samples, injury combined with TNFalpha and IL-6/sIL-6R exposure caused the most severe GAG loss. Findings in human knee and ankle cartilage were strikingly similar to those in bovine samples, although in human ankle tissue, the GAG loss was less severe than that observed in human knee tissue. Without exogenous IL-6/sIL-6R, injury plus TNFalpha exposure up-regulated chondrocyte production of IL-6, but incubation with the IL-6-blocking Fab significantly reduced proteoglycan degradation. CONCLUSION Our findings indicate that mechanical injury potentiates the catabolic effects of TNFalpha and IL-6/sIL-6R in causing proteoglycan degradation in human and bovine cartilage. The temporal and spatial evolution of degradation suggests the importance of transport of biomolecules, which may be altered by overload injury. The catabolic effects of injury plus TNFalpha appeared partly due to endogenous IL-6, since GAG loss was partially abrogated by an IL-6-blocking Fab.

Co-culture of Mechanically Injured Cartilage with Joint Capsule Tissue Alters Chondrocyte Expression Patterns and Increases ADAMTS5 Production

We studied changes in chondrocyte gene expression, aggrecan degradation, and aggrecanase production and activity in normal and mechanically injured cartilage co-cultured with joint capsule tissue. Chondrocyte expression of 21 genes was measured at 1, 2, 4, 6, 12, and 24h after treatment; clustering analysis enabled identification of co-expression profiles. Aggrecan fragments retained in cartilage and released to medium and loss of cartilage sGAG were quantified. Increased expression of MMP-13 and ADAMTS4 clustered with effects of co-culture, while increased expression of ADAMTS5, MMP-3, TGF-beta, c-fos, c-jun clustered with cartilage injury. ADAMTS5 protein within cartilage (immunohistochemistry) increased following injury and with co-culture. Cartilage sGAG decreased over 16-days, most severely following injury plus co-culture. Cartilage aggrecan was cleaved at aggrecanase sites in the interglobular and C-terminal domains, resulting in loss of the G3 domain, especially after injury plus co-culture. Together, these results support the hypothesis that interactions between injured cartilage and other joint tissues are important in matrix catabolism after joint injury.

A53 MECHANICAL INJURY POTENTIATES THE COMBINED EFFECTS OF TNF-α AND IL-6/sIL-6R ON PROTEOGLYCAN CATABOLISM IN BOVINE CARTILAGE

+Sui, Y; **Song, X-Y; +**Lee, J H; *DiMicco, M; **Blake, S M; *Hung, H; **James, I; ** Lark, M W; +§¶*Grodzinsky, A J +Biological, §Mechanical, ¶Electrical Engineering, *Center for Biomedical Engineering, MIT, Cambridge, MA; **Centocor R&D, Inc, Radnor, PA ysui@mit.edu INTRODUCTION: Acute traumatic joint injury increases the risk of developing osteoarthritis (OA) [1]. The mechanisms by which injury causes chronic cartilage degradation in vivo are not fully understood, but elevated levels of injury-induced pro-inflammatory cytokines [2], including TNF-α and IL-6 [3], may play pivotal roles in the pathogenesis of OA. TNF-α causes a synergistic loss of PG from mechanically-injured cartilage in vitro [4], but the pathways regulating this synergy are unclear. The objectives of this study were to (1) examine the combined effect of TNF-α and IL-6/sIL6R on proteoglycan degradation in mechanically-injured cartilage, and (2) to determine the role of endogenous IL-6 in the cartilage catabolism induced by both TNF-α and mechanical injury. METHODS: Cartilage disks (3 mm diam., 1 mm thick) were harvested from the middle zone of the femoropatellar grooves of 1-2-week old calves, and equilibrated for 2 days in normal medium (DMEM + 1% ITS) prior to treatment. Cytokine and Mechanical Injury Treatments: Location-matched disks were either injuriously compressed (50% strain, 100%/second strain rate), cultured in medium with rhTNF-α (25 ng/ml), treated with rhIL-6 (50 ng/ml) plus soluble IL-6 receptor (sIL-6R, 250ng/ml), or treated with combinations of these three conditions (Fig.1). Culture was terminated after 6 days of treatment. In a separate experiment, half the cartilage disks (Fig. 3) were pre-equilibrated for 6 days with an IL-6 blocking Fab fragment (50 ug/ml, Centocor, J&J) prior to treatment; the remaining disks were incubated in normal medium during this period. Afterward, disks were either injuriously compressed, incubated with rhTNF-α (25 ng/ml), or treated with combined injury + TNF-α. Disks that were pre-treated with the IL-6 blocking Fab fragment continued to receive Fab fragments until the termination of the experiment. Aggrecan Western Blotting, GAG Content and Histology: Culture medium from each condition was collected on day 2, 4 and 6 after the initial injury and/or cytokine treatments. Concentrated medium was used to perform Western blot analysis using a monoclonal Ab specific to the G1NITEGE fragment of aggrecan (kindly provided by C. Flannery, Wyeth). DMMB dye was used to quantify sGAG released into the medium. Selected cartilage samples were fixed in gluteraldehyde with RHT, paraffin-embedded, sectioned, and stained with Toluidine Blue. Additional disks were radiolabeled during days 4-6 with 5 μCi/ml SO4 to assess proteoglycan synthesis. RESULTS: 3-way ANOVA analyses followed by post-hoc Tukey’s pairwise comparisons showed that TNF-α formed interactions with both IL-6/sIL-6R (p<0.001) and injurious compression (p<0.001) causing increased sGAG release (Fig. 1(i)) and decreased proteoglycan synthesis (data not shown). While IL-6/sIL-6R significantly augmented TNF-αinduced proteoglycan degradation (Fig.1(i)B,G), the largest amount of GAG loss was caused by the combination of injury+TNF-α + IL-6/sIL6R (Fig.1(i)D). Histology showed that GAG loss was not uniform across the disk cross-section, but instead was initiated at the disk periphery and progressed towards the disk center with time (Fig. 2). The most rapid, severe progression of GAG loss was observed in disks treated with the combination of injury + TNF-α + IL-6/sIL-6R (Fig. 2e,f). Analysis of conditioned medium for aggrecan fragments by Western blotting demonstrated that the most dramatic release of aggrecanase-generated cleavage products occurred in response to treatment with TNF-α + IL6/sIL-6R, both with and without injurious compression (Fig. 1(ii)B,D). The IL-6 blocking Fab fragment was effective in neutralizing exogenous rhIL-6 in the medium, and was not toxic to cells (data not shown). In separate studies, TNF-α + mechanical injury caused greater GAG loss than either treatment alone (Fig.3G,H,I). Importantly, the IL-6 blocking Fab fragment significantly reduced the combined catabolic effects of TNF-α + mechanical injury on GAG loss, with no exogenous IL-6 present (Fig.3D,I). DISCUSSION: We found that the combined treatment with TNF-α and IL-6/sIL-6R induced significantly more GAG loss than either cytokine alone did, consistent with previous studies of TNF-α/IL-6 treatment [3], and suggesting this catabolic response was associated with aggrecanase (but not MMP) activity. Additionally, we now report that the catabolic effect of TNF-α, and the combined effect of TNF-α + IL-6/sIL-6R are both highly potentiated by mechanical injury. The degradative effects of injury + TNF-α appear to be due, in part, to the action of endogenous IL-6, as sGAG loss was partly abrogated by the IL-6 blocking Fab fragment. This result is also consistent with the increased loss of sGAG upon addition of exogenous IL-6 to the combination of TNF-α and mechanical injury. Histology observations (Fig. 2) suggest that the kinetics of cartilage degradation is not merely a consequence of the activities of proteolytic enzymes, but it also depends strongly on the transport of cytokines, proteases, anti-IL-6 Fab and other cartilage biomolecules, which may be altered by overload injury. In conclusion, our study suggests that pro-inflammatory cytokines, whose productions are elevated by traumatic joint injury, can interact to potentiate cartilage catabolism. The mechanobiological (cell-mediated) responses to overload [5], as well as altered transport of cytokines and proteases in the damaged matrix, may both be affected by joint injury, making the damaged cartilage tissue more susceptible to further degradation by biochemical mediators. REFERENCES:[1] Gelber+, Ann Intern Med 133:3211, ‘00; [2] Irie+, Knee 10:93, ‘03; [3] Flannery+, Matrix Biol, 19:549, ‘00. [4] Patwari+, Arth Rheum, 48:1292, ‘03 [5] Lee+, Arth Rheum, 52:2386, ‘05. Acknowledgements: Supported by Centocor and NIH Grant AR45779