Immuno Research) antibody were used as positive and negative controls, respectively (Fig. 1A). Histopathological Assessment of LPS-CIA For histopathology, the paws were removed and fixed in paraformaldehyde 4%, decalcified in TBD-2 Thermo-Shandon solution (Thermo Fisher Scientific) for 10 days, and embedded in paraffin. Sections were stained with H&E (Thermo Fisher Scientific) or safranin/fast green colorations (VWR International). Bone destruction, collagen integrity, and cell infiltration were evaluated by two blinded observers according to the following scales: 0–3 for bone destruction (0, normal; 1, slight degradation; 2, mild degradation; 3, complete destruction); 0–2 for collagen (0, normal; 1, mild loss; 2, complete loss); 0–2 for cell infiltration (0, normal; 1, mild infiltration; 2, severe infiltration). Immunohistochemistry Paws were cut into sections 5 mm thick. The deparaffinized and rehydrated sections were incubated with hydrochloric acid for antigen retrieval. After blocking endogenous peroxidase activity and non-specific binding using the ready to use Protein Block Serum free (Dako) slides were incubated with rabbit anti-S100A9, rabbit anti-S100A8 (10 mg/ml) or preimmune serum (1/1000) diluted in Tris-buffered saline overnight at 4uC. After washings, tissue sections were incubated with anti-rabbit IgG HRP diluted in Tris-buffered saline for 1 hour at RT. Detection was performed using the ready-to-use AEC solution (Dako). The slides were then counterstained with Mayer’s haematoxylin according to the manufacturer’s instructions (Dako). Western Blot Analysis Paws of sacrificed mice (5 days, at the top of inflammation) were cut at the ankle or wrist and frozen in dry ice. Fore and hind paws were separately homogenized using a polytron homogenizer in 1ml of RIPA buffer with a protease inhibitor cocktail. The homogenates were centrifuged at 1200 rpm for 5 min, and supernatants were then further centrifuged at 13,000 rpm for 15 min. Thirty microliters from a pool of 5 supernatants mixed in sample buffer were loaded on SDS-PAGE (15%). Proteins were detected using anti-7/4 Ab (R&D Systems), anti-Gr1 Ab (BD Biosciences), anti-IL-6 Ab (R&D Systems), or anti-TNFa Ab (PeproTech). ELISAs Murine blood was collected at 4 h, and 5 days after LPS injection, and centrifuged to obtain serum. Serum anti-collagen II antibody titers were measured using an anti-collagen detection kit according to the manufacturer’s instructions (Chondrex inc). S100A9 and calprotectin were quantified by a homemade ELISA kit. Briefly, plates were coated with 100 ml anti-S100A9 mAb (clone 2A5, 2 mg/ml) and left overnight at 4uC. After incubation, plates were washed three times and blocked with PBS/0.1% Tween-20/2% BSA for 1 h at room temperature (RT). Samples and standards were added and incubated for 1 h at RT. The plates were then washed and incubated with S100A8 polyclonal Ab (5 mg/ml) for 1 h at RT, for calprotectin detection, respec- tively. Following incubation, plates were washed and incubated with anti-rabbit IgG at a dilution of 1:5000 and revealed by using TMBS (Neogen Corporation), according to the manufacturer’s instructions. ELISA kits for murine IL-6 were obtained from PeproTech and used according to the manufacturer’s instructions within the range of 62–4000 pg/ml. Role of S100A9 in Arthritis Transmigration Assay Transwell chambers with a 6.5 mm diameter polycarbonate filter with a pore size of 3 mm (Corning) were used. HUVECs (ATCC) were grown to confluence on filters coated with 0.5% gelatine in PBS (Sigma-Aldrich). Confluence was confirmed before each experiment by measuring resistance using an ohmmeter. Neutrophil were obtained from heathly donnors and purified by centrifugation on Ficoll-Paque cushions (Wisent, inc) [19]. Fifty microliters of S100A9 or buffer (M199-5mM HEPES) and 50 ml of calcein-AM-labeled neutrophils were added to the upper well. IL- 8 (5 ng/ml in M199-5 mM HEPES) or buffer was added to the lower chamber and cells were allowed to migrate for 2 h at 37uC. After this incubation period, wells were gently shaken to dislodge the last transmigrated cells and the cells in the lower chambers were lysed by adding 150 ml Triton X-100 (5%) for 20 min in the dark and fluorescence was measured. Adhesion to Fibrinogen Immunoassay plates (96-well, Corning Inc., Corning, NY, USA) were coated overnight at 4uC with 50 ml of fibrinogen (2 mg/ml) in NaHCO3 0.1 M, pH 9.6. The plates were washed three times with 100 ml of HBSS-H (HBSS supplemented with 10 mM HEPES, pH 7.4) containing 1.3 mM Ca2+ and 0.8 mM Mg2+) before use. Fifty microliters of each stimulus solution (S100A9 at 80 mg/ml = 26, IL8 at 10 ng/ml = 26, alone or in combination) diluted in HBSS-H, were added to individual wells. Neutrophils were resuspended at 5 6 106 cells/ml in HBSS-H and labelled with 5 mM of the intracellular fluorescent dye calcein-AM (Calbiochem) for 20 min at 37uC. After washing, 50 ml of the cell suspension (5 6 106 cells/ml) was added to each well. Plates were centrifuged for 2 min at 250 6g and neutrophils were allowed to adhere for increasing periods of time at 37uC before being washed three times by immersion in cold PBS. Adherent cells were lysed by adding deionised distilled water, and fluorescence was measured at lex = 485 nm and lem = 530 nm using a fluorescence plate reader. Blocking Experiments Transmigration assays were performed as described in the material and methods section. Anti-S100A9, anti-CD11a, anti- CD11b, anti-CD18, or anti-ICAM-1 were mixed with neutrophils in the upper well during migration assays. Stimulation of Monocytes with S100A9 Proteins Monocytes from healthy donors were purified using the Human Enrichment Cocktail for RosetteSepH kit (StemCell Technologies) according to the manufacturer’s instructions. Cell suspensions were adjusted to a density of 1 6 106 cells per ml in RPMI containing 10% FBS and incubated in 48-well plates (Corning Life Sciences) with S100A9 (0.1–40 mg/ml). Culture supernatants were harvested 24 h later and tested for cytokine secretion. For some experiments, a time course (0–24 h) was also carried out. Cytokine arrays (R&D Systems) and ELISA for IL-6, TNFa, or MIP-1a (PeproTech) were performed according to the manufacturer’s instructions. Statistical Analyses Anti-collagen, S100A9, S100A8/A9 concentrations, collagen/ bone destruction, and cell infiltration were compared using one- way ANOVA followed by Tukey’s multiple comparison test of the means. A p-value ,0.05 was considered statistically significant. Secretion of murine or human cytokines was compared by t test. Protective efficacies of the different treatments were evaluated by PLOS ONE | www.plosone.org 3 September 2012 | Volume 7 | Issue 9 | e45478