ARTHRITIS & RHEUMATISM Vol. 64, No. 5, May 2012, pp 1477–1487 DOI 10.1002/art.33495 © 2012, American College of Rheumatology Alarmins S100A8 and S100A9 Elicit a Catabolic Effect in Human Osteoarthritic Chondrocytes That Is Dependent on Toll-like Receptor 4 Rik F. P. Schelbergen, 1 Arjen B. Blom, 1 Martijn H. J. van den Bosch, 1 Annet Slo ¨etjes, 1 Shahla Abdollahi-Roodsaz, 1 B. Wim Schreurs, 1 John S. Mort, 2 Thomas Vogl, 3 Johannes Roth, 3 Wim B. van den Berg, 1 and Peter L. E. M. van Lent 1 Objective. S100A8 and S100A9 are two Ca 2 binding proteins classified as damage-associated molec- ular patterns or alarmins that are found in high amounts in the synovial fluid of osteoarthritis (OA) patients. The purpose of this study was to inves- tigate whether S100A8 and/or S100A9 can interact with chondrocytes from OA patients to increase catabolic mediators. Methods. Using immunohistochemistry, we stained for S100A8 and S100A9 protein, matrix metal- loproteinases (MMPs), and a cartilage-breakdown epitope specific for MMPs (VDIPEN) in cartilage from OA donors. Isolated chondrocytes or explants from OA and non-OA donors were stimulated with S100A8 and/or S100A9. Messenger RNA and protein levels of MMPs, cytokines, and cartilage matrix molecules were determined with quantitative reverse transcription– polymerase chain reaction and Luminex techniques, respectively. For receptor blocking studies, specific in- hibitors for Toll-like receptor 4 (TLR-4), receptor for advanced glycation end products (RAGE), and carbox- ylated glycans were used. Results. In cartilage from OA patients, the ex- pression of S100A8 and S100A9 protein close to chon- drocytes was associated with proteoglycan depletion and expression of MMP-1, MMP-3, and VDIPEN. Stimula- tion of chondrocytes with S100A8 and S100A9 caused a strong up-regulation of catabolic markers (MMPs 1, 3, 9, and 13, interleukin-6 [IL-6], IL-8, and monocyte chemotactic protein 1) and down-regulation of anabolic markers (aggrecan and type II collagen), thereby favor- ing cartilage breakdown. Blocking TLR-4, but not car- boxylated glycans or RAGE, inhibited the S100 effect. The catabolic S100 effect was significantly more pro- nounced in chondrocytes from OA patients as compared to those from non-OA patients, possibly due to higher TLR-4 expression. Conclusion. S100A8 and S100A9 have a catabolic effect on human chondrocytes that is TLR-4 dependent. OA chondrocytes are more sensitive than normal chon- drocytes to S100 stimulation. During osteoarthritis (OA), the erosion of carti- lage is an important cause of pain and disability in the joint. Cartilage damage associated with OA is mainly caused by a shift in the balance between the resorptive (or catabolic) and synthetic (or anabolic) capacities of chondrocytes. Catabolic activities of OA chondrocytes are characterized by elevated release of cartilage- degrading enzymes, such as matrix metalloproteinases (MMPs), while anabolic activities of OA chondrocytes result in aggrecan and type II collagen production (1). Although OA is generally considered a disease of the cartilage, evidence is accumulating that synovial activation and proinflammatory mediators released after synovial activation might contribute to the induction of cartilage damage in OA (2–4). Arthroscopic studies suggest that synovial activation occurs in 50% of OA patients and that it is associated with more severe Supported by the Dutch Arthritis Foundation (grant 07-2- 301). 1 Rik F. P. Schelbergen, MSc, Arjen B. Blom, PhD, Martijn H. J. van den Bosch, MSc, Annet Slo ¨etjes, BSc, Shahla Abdollahi- Roodsaz, PhD, B. Wim Schreurs, MD, Wim B. van den Berg, PhD, Peter L. E. M. van Lent, PhD: Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; 2 John S. Mort, PhD: Shriners Hospital for Children, Montreal, Quebec, Canada; 3 Thomas Vogl, PhD, Johannes Roth, MD: University of Muenster, Muenster, Ger- many. Address correspondence to Peter L. E. M. van Lent, PhD, Rheumatology Research and Advanced Therapeutics, Department of Rheumatology, Radboud University Nijmegen Medical Centre, 272, Geert Grooteplein 28, PO Box 9101, 6500 HB Nijmegen, The Neth- erlands. E-mail: P.vanLent@reuma.umcn.nl. Submitted for publication May 16, 2011; accepted in revised form November 17, 2011. 1477