Figure 6

TNF-α and IL-17A expression in macrophages of wild-type and IL-4R KO mice. Wild-type or IL-4R KO mice were sensitized and challenged with OVA, and selected mice were inoculated with sham or RV. (A) Lungs were harvested and digested with collagenase IV. Cells were stimulated with cell stimulation cocktail for 5 h and stained with antibodies against macrophage surface markers, fixed, permeabilized and incubated with anti-TNF-α. CD11b + TNF-α + cells were analyzed in the CD45+ F4/80+ CD11c- fraction. A fluorescent minus one (FMO) control was utilized to confirm TNF-α signals. In this control, cells were incubated with all antibodies except anti-TNF-α. (B) The percentage of CD45+, F4/80+, CD11c-, CD11b+, TNF-α cells in the CD45+ F4/80+ CD11c- fraction (upper panel) and total CD45+ TNF-α + cells (lower panel) were calculated. (C) IL-17A producing macrophages were assessed by flow cytometry. Lung cells were stained with anti-IL-17A. CD45+, CD68+, F4/80+, CD11c- cells were analyzed for CD11b and IL-17A. An FMO control (all antibodies except anti-IL-17) was used to confirm IL-17 signals. (D) The percentage of CD45+, CD68+, F4/80+, CD11c-, CD11b+, IL-17A + cells in the CD45+ CD68+ fraction (upper panel) and total CD45+ IL-17A + cells (lower panel) were calculated. (E) Lung sections were stained with anti-IL-17A antibody. Immunohistochemistry shows DAB staining of round cells in the airway subepithelium. (F) Lungs were stained with AF555-conjugated anti-CD68 (red) and AF488-conjugated anti-IL-17A (green). Nuclei were stained with DAPI (blue). Immunofluorescence shows colocalization (yellow), indicating IL-17A production by CD68+ macrophages. (Mean ± SEM, n = 3-5 each group, *different from sham, P < 0.05, one-way ANOVA; †different from wild-type, P < 0.05, one-way ANOVA).