comparing arthritis clinical scores using repeated measures ANOVA. Neutrophil migration was compared using one-way ANOVA followed by Dunett’s multiple comparison test. A p-value ,0.05 was considered statistically significant. Results S100A9 and Calprotectin are Rapidly Secreted during Arthritis Development Collagen-induced arthritis (CIA) is commonly used as a model of RA because it shares immunological and pathological features with human RA [21,22]. Here we chose the murine lipopolysac- charide (LPS)-CIA model to synchronize arthritis within experi- mental groups, as all animals develop arthritis within 24h following LPS injection. To ensure that all groups synthesize similar levels of anti-collagen II, the sera of 10 mice from each of the different groups were collected 4 h after the LPS boost to perform an enzyme-linked immunosorbent assay (ELISA). As expected, all groups showed similar concentrations of anti-collagen II ranging from 185 to 326 mg immunoglobulin G (IgG)/ml (Fig. 2A, not statistically significant between groups). These results confirmed that arthritis was similarly triggered in all groups, and that further observations may be attributed to specific treatments received by each group. S100A8 and S100A9 are known to be expressed in the synovium of RA patients [23]. We found expression of both Role of S100A9 in Arthritis proteins in paw of arthritic mouse in the CIA models (Fig. 2B). Because S100A9 and S100A8/A9 are early markers of inflam- mation in various chronic inflammatory diseases including RA [7,8,9,10], we used ELISA to quantify calprotectin in the serum of mice collected 4 h after LPS injection. The expression of calprotectin was strongly upregulated in the isotype control- treated and anti-TNFa-treated groups compared to na¨ıve mice (Fig. 2C). As expected, a strong decrease in the levels of S100A8/ A9 proteins was observed in mice treated with the anti-S100A9 antibody (2.161.0 mg/ml for S100A8/A9, ), compared to levels seen in mice treated with the isotype control Ab (and 7.561.5 mg/ ml). Anti-S100A9 Decreases Arthritis Severity, Bone/cartilage Destruction, and Cell Infiltration Arthritis severity in the murine LPS-CIA model was scored on a scale of 0 to 4 per paw, with a maximum possible score of 16 per animal, based on inflammation markers (redness and swelling). Mice treated with anti-S100A9 showed markedly decreased arthritis severity scores compared to the isotype control group. The first signs of inflammation such as redness or slight swelling were detected at approximately day 2 and disease activity was maximal on day 5 after LPS boost. Anti-S100A9 and anti-TNFa treatment significantly reduced the maximum arthritis score median, from 6.8 in the isotype control-treated group to 3 and 1.4 in groups treated with anti-S100A9 and anti-TNFa, respec- Figure 2. Effect of anti-S100A9, TNFa, or isotype control Abs on the LPS-CIA clinical score. (A) Anti-collagen II Abs were measured in mouse serum 4 h after LPS injection. The results are expressed as mg of IgG Ab/ml of serum. (B) S100A8 and S100A9 expression in arthritic paws. Paw tissue sections were stained with rabbit pre-immune serum, rabbit anti-S100A8, or rabbit anti-S100A9 polyclonal IgGs. B: bone, Js: joint space. (Magnification 1000X). (C) Four hours after LPS injection, sera were collected and ELISA S100A8/A9 were performed *p,0.05, one-way ANOVA test (n = 10 paws/group), Tukey’s Multiple Comparison Test (D) Clinical score as assessed by two blinded observers. Data are the mean scores calculated from at least 15 mice per group until day 19. doi:10.1371/journal.pone.0045478.g002 PLOS ONE | www.plosone.org 4 September 2012 | Volume 7 | Issue 9 | e45478