IL-17 and Synoviocyte Survival averaged and expressed as the number of TUNEL positive cells/mm2, mean 6 SEM, * P,0.05 compared to WT mice. Synovial PCNA staining in knee joint sections from WT or IL-17R 2/2 mice with chronic streptococcal cell wall-induced arthritis (D). A representative image of PCNA staining in synovial sections (magnification 6200). The number of PCNA-positive cells was quantified in 10 high power fields, averaged and 2 expressed as the number of PCNA positive cells/mm , mean 6 SEM, * P,0.05 compared to WT mice. doi:10.1371/journal.pone.0013416.g007 Germany), p-65 (Search-LC GmbH, Heidelberg, Germany), b-actin (accession number: X00351, 59-TGTCCCTGTATGCCTCTGGT- 39 and 59-GATGTCACGCACGATTTCC-39) or GAPDH (Search- LC GmbH, Heidelberg, Germany) were used and genes measured by quantitative real-time RT-PCR (RT-PCR). Chronic SCW-induced arthritis model The mice and the arthritis model have been previously described in detail [30]. All in vivo mouse studies complied with national legislation and were approved by local authorities of the Care of Use of Animals of the University of Nijmegen, the Netherlands. Animals were housed under conventional conditions, water and standard laboratory chow were provided ad libitum. Specific pathogen–free C57BL/6 mice (males and females, ages 8– 10 weeks; National Cancer Institute, Frederick, MD) were used as WT controls for the IL-17R–/– mice. The IL-17R–/– mice were obtained from Dr. J. J. Peschon (Amgen, Seattle, WA). Arthritis was induced in WT and IL-17R–/– mice by the intraarticular injection of 25 mg of streptococcal cell wall (SCW) fragments in 6 ml of phosphate buffered saline into the knee joints. Chronic arthritis was induced by 5 repeated injections of 25 mg of SCW administered intraarticularly at 1-week intervals. On day 42, mice were killed by cervical dislocation and knee joints were isolated for histological analysis. The arthritis severity and inflammatory infiltrates were scored as previously described . Detection of synoviolin expressing cells Mouse joints were fixed in 10% formalin, decalcified in formic acid and embedded in paraffin. Briefly for single staining, synoviolin expression was measured by immunohistochemistry using 12.5 mg/ml of a polyclonal rabbit antibody against synoviolin (Abgent). In negative control sections, irrelevant antibody (goat serum) was applied., and counterstained with haematoxylin [46,47]. Cells positive for synoviolin were quantified by counting positive cells in ten consecutive high power fields (x 200). The number of positive cells per high power field was averaged and the results expressed as the number of synoviolin positive cells per mm2 using HistolabTM microvision version 5.9.2 References 1. Nakamura H, Tanaka H, Yoshino S (2004) Long-term results of multiple synovectomy for patients with refractory rheumatoid arthritis. Effects on disease activity and radiological progression. Clin Exp Rheumatol 22: 151–157. 2. Firestein G (2003) Evolving concepts of rheumatoid arthritis. Nature 423: 2003. 3. Toh M, Marotte H, Blond J, Jhumka U, Eljafari A, et al. (2006) Synoviolin is overexpressed in peripheral blood and synoviocytes in rheumatoid arthritis and remains elevated in nonresponders to infliximab. Arthritis Rheum 54: 2109–2118. 4. Amano T, Yamasaki S, Yagishita N, Tsuchimochi K, Shin H, et al. (2003) Synoviolin/Hrd1, an E3 ubiquitin ligase, as a novel pathogenic factor for arthropathy. Genes and Dev 17: 2436–2449. 5. Gao B, Calhoun K, Fang D (2006) The proinflammatory cytokines IL-1beta and TNF-alpha induce the expression of Synoviolin, an E3 ubiquitin ligase, in mouse synovial fibroblasts via the Erk1/2-ETS1 pathway. Arthritis Res Ther 8: R172. 6. Yamasaki S, Yagishita N, Sasaki T, Nakazawa M, Kato Y, et al. (2006) Cytoplasmic destruction of p53 by the endoplasmic reticulum-resident ubiquitin ligase ‘synoviolin’. EMBO J 26: 113–122. 7. Bettelli E, Carrier Y, Gao W, Korn T, Strom TB, et al. (2006) Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 441: 235–238. [48]. For double staining, sections were incubated with a polyclonal rabbit antibody against synoviolin (Abgent) followed by biotinylated anti-rabbit immunoglobulins, and streptavidin- peroxidase (DAKO, Glostrup, Denmark). Peroxidase was devel- oped by 3,39 diaminobenzidine chromogen solution (DAB) (DAKO, Glostrup, Denmark). Goat polyclonal anti-mouse CD20 (4 mg/ml, Santa Cruz Biotechnology, Europe) and goat polyclonal anti-mouse IL-17 antibodies (10 mg/ml, R&D Systems Europe, London, UK) were followed by biotinylated anti-goat immunoglobulins (DAKO, Glostrup, Denmark) and streptavidin- alkaline phosphatase (DAKO, Glostrup, Denmark). Alkaline phosphatase was revealed using Vector Blue as chromogen (blue color; Vector Labs, Burlingame, CA, USA). In negative control sections, irrelevant antibody (rabbit or goat serum) was applied. In situ apoptosis and proliferation in chronic SCW arthritis TUNEL staining was performed using an in situ death detection kit (Roche, Mannheim, Germany) according to the manufacturer’s instructions. Synovial proliferation was determined by immuno- staining using a specific Mab to the proliferation marker PCNA (Santa Cruz Biotechnology). Tunnel or PCNA-positive cells in murine synovial tissue samples were counted in a blinded manner in ten high-power fields per section, an average score was obtained and results expressed as the number of Tunnel or PCNA-positive cells mm2 using HistolabTM microvision version 5.9.2. Statistical analysis Results are expressed as the mean 6 SEM. Analysis was performed using the Mann Whitney U-test and P values ,0.05 considered statistically significant. 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