Fig. 5.

GM-CSF produced by autoantigen-stimulated Ifng^−/−Il17a^−/− cells promotes eosinophil activation and migration (A) Bone-marrow derived eosinophils (bmEos) from WT B10.RIII mice were cultured with the supernatant of antigen (IRBP) re-stimulated splenocytes from EAU-induced WT or Ifng^−/−Il17a^−/− mice in the presence or absence of anti-GM-CSF antibody (10 μg/mL) or GM-CSF (100 ng/mL). After 2 days, the mean fluorescence intensity (MFI) of SiglecF and CD11b on bmEos was determined by FACS. Representative FACS plots with gates and histograms are shown on the left. (B) BmEos were placed into the upper chamber of a 96-well transwell chemotaxis assay plate with the supernatant of re-stimulated splenocytes in the lower chamber and incubated for 3 h. GM-CSF or eotaxin were used as positive controls. The total number of live cells that had migrated into the bottom chamber with the supernatant was counted and divided by the number of live cells that had migrated to medium alone to give the fold change over the medium control. Representative data from 2 independent experiments, each group contains 2 individual samples, and each sample was duplicated. Data shown as mean ± SEM. Significance was determined using unpaired t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.