Therapy studies of F8-IL10 in the CIA mouse model. (a) Combination with methotrexate. Arthritic mice were given injections with saline (black squares), methotrexate 100 μg intraperitoneally (open circles), F8-IL10 200 μg subcutaneously (black triangles), F8-IL10 200 μg intravenously (black circles), or a combination of F8-IL10 200 μg intravenously and methotrexate (MTX) 100 μg intraperitoneally (crosses). Injections were started at day 1 after arthritis onset and then repeated every third day for three injections per animal, as indicated by the arrows. The arthritic score was evaluated daily and expressed as the mean ± standard error of the mean (SEM) of eight mice per group. * 1 P < 0.05 versus saline; * 2 P < 0.05 versus F8-IL10 intravenously. (b) Comparison of targeted versus systemic application of IL10. Arthritic mice were injected subcutanously with saline (black squares), HyHel10-IL10 200 μg (open circles), F8-TNFRII (crosses), or F8-IL10 200 μg (black circles) every third day for three injections, as indicated by arrows. Arthritic score is expressed as the mean ± SEM of six to seven mice per group. * P < 0.05 versus saline. (c) Ex vivo immunohistochemical detection of F8-IL10 and HyHel10-IL10 in arthritis paws. Analysis of the arthritis paws at the end of therapy (day 12 for F8-IL10 and day 10 for HyHel10-IL10) showed that F8-IL10 is still detectable by immunohistochemistry using an anti-human-IL10-antibody. (d) Analysis of plasma cytokines levels at the end of therapy. F8-IL10-treated mice showed significantly decreased IL6 levels compared with the saline group. Furthermore, IL1b serum levels of F8-IL10-treated mice were below the lower limit of detection. * P < 0.05 versus saline. (e) Anti type-II collagen antibodies. Titers of bovine type II collagen-specific total IgG, IgG1 and IgG2a antibodies were determined by ELISA. A clear reduction of total IgG and IgG2a, but not IgG1, antibody levels was observed in F8-IL10-treated mice. * P < 0.05 versus saline.