Since the production of anti-CII IgG1 and IgG3 represent T cellCdependent and Cindependent responses, respectively (Stevens et al

Since the production of anti-CII IgG1 and IgG3 represent T cellCdependent and Cindependent responses, respectively (Stevens et al., 1988), the therapeutic effect of IQ-1S on CIA could be associated with direct inhibition of B cell activity. treatment. By contrast, the inactive ketone derivative 11and isomers) and Rabeximod (9-chloro-2,3-dimethyl-6-(Expression. This study was approved by the Institutional Review Table of University or college of California, San Diego School of Medicine (La Jolla, CA), and informed consent was obtained from all participants. Synovial tissue was obtained from patients with RA at the time of total joint replacement, as previously explained (Alvaro-Gracia et al., 1990). The diagnosis of RA conformed to American College of Rheumatology 1987 revised criteria (Arnett et al., 1988). The synovium was minced and incubated with 0.5 mg/ml collagenase type VIII (Sigma-Aldrich) in serum-free RPMI 1640 (Life Technologies, Grand Island, NY) for 2 hours at 37C, filtered, extensively washed, and cultured in Dulbeccos modified Eagles medium (DMEM) (Life Technologies) supplemented with 10% fetal bovine serum (FBS) (Gemini Bio Products, Calabasas, CA), penicillin, streptomycin, gentamicin, and glutamine in a humidified 5% CO2 atmosphere. Cells were allowed to adhere overnight, nonadherent cells were removed, and adherent fibroblast-like synoviocyte (FLSs) were split at 1:3 when 70%C80% confluent. FLSs were used from passages 3 through 9, during which time they are a homogeneous SPL-707 populace of cells (<1% CD11b positive, <1% phagocytic, and <1% FcmRNA analysis, FLSs were plated in six-well plates and cultured until 80% confluence, and they were subsequently serum starved (0.1% FBS/DMEM) for 24 hours. The cells were treated with IQ-1S (4, 10, and 25 activation (2 ng/ml) for 6 hours. The mRNA was isolated and reverse transcribed to obtain cDNA. Quantitative real-time polymerase chain reaction was performed using primer probe units for (Chondrex) was injected s.c. in the tail (Kochetkova et al., 2010, 2014). Using this method, nearly 100% of mice consistently showed clinical symptoms by day 25. IQ-1S (JNK inhibitor), IQ-18 (analog of IQ-1S, inactive for JNK), or sterile saline answer were injected intraperitoneally daily beginning at days ?1, 7, 14, or 25 relative to the CII challenge, as indicated, and continued until day SPL-707 31 or 38 after the CII challenge. Mice were scored using a level of 0C3 for each limb for any maximal total score of SPL-707 12, as previously explained (Kochetkova et al., 2010): 0, no indicators of inflammation; 1, mild redness or swelling of single digits; 2, significant swelling of ankle or wrist with erythema; and 3, severe swelling and erythema of multiple joints. Histopathology. Forty days after the CII challenge, animals were PRKD2 euthanized, and their limbs were fixed in 10% neutral buffered formalin and decalcified in 5% formic acid for 3C6 days. The joints were embedded in paraffin and cut at 8-for 10 minutes, and supernatants were filter sterilized (0.2 were measured in culture supernatants and homogenized paw tissues using ELISA packages (BD Biosciences, San Jose, CA) for mouse cytokines/chemokines. Circulation Cytometry. Upon termination of the disease course, LN cells were stained with fluorochrome-labeled anti-CD25 (BD Pharmingen, Franklin Lakes, NJ) and anti-CD4 monoclonal antibodies (eBioscience, San Diego, CA). For analysis of forkhead box p3 (Foxp3) intracellular expression, cells were further fixed in 2% paraformaldehyde, permeabilized with ice-cold methanol, and stained with fluorochrome-labeled anti-Foxp3 monoclonal Ab (eBioscience) or isotype control. Fluorescence was acquired on an LSR II circulation cytometer (BD Biosciences, San Diego, CA) with BD FACSDiva software. All samples were analyzed using FlowJo software (Tree Star, Ashland, OR). Statistical Analysis. The nonparametric MannCWhitney test was utilized for statistical analysis of CIA clinical scores, histology scores, and cartilage destruction. FLS data were analyzed by two-way analysis of variance with Tukeys multiple comparison test, and differences were considered statistically significant if < 0.05. The test and one-way analysis of variance were utilized for analysis of ELISA results and circulation cytometry data. Results were considered statistically significant if < 0.05. Results Characterization of IQ-1S.