Figure 3.
Reduced production of IL-6 in immunized GM-CSF−/− mice. WT and GM-CSF−/− mice were immunized s.c. with myhcα614-629/CFA as described in the Fig. 1 legend. (A) At the times indicated, CD11b+ and CD11c+ cells were isolated by magnetic sorting and cultured in the presence and absence of anti-CD40 for 20 h. IL-6 concentrations in the supernatant was measured by ELISA. (B) Expression of IL-6 relative to β-actin was assessed by quantitative PCR using cDNA generated from CD11b+ cells. (C) At day 7, CD11c+ cells were isolated by magnetic sorting and cultured in the presence and absence of anti-CD40 for 24 h (4 × 105 DC per well). IL-6, IL-23, and IL-12p40 levels in the supernatant were measured by ELISA. (D) Analysis of draining LN cells by flow cytometry at day 4 after immunization. Histograms show expression of CD80, CD86, CD40, and MHC class II gated on CD11c+ cells. Continuous and dotted lines represent cells of immunized WT and GM-CSF−/− mice, respectively. Shaded curves represent cells of naive mice. (E) BM-derived DC of WT and GM-CSF−/− mice were pulsed with 10 μg/ml myhcα614-629 and stimulated with 1 μg/ml LPS and 5 μg/ml anti-CD40 for 4 h before injection into WT and GM-CSF−/− mice at days 0, 2, and 4. At day 10, hearts were removed and myocarditis scored by histology. Horizontal lines indicate median. Error bars in A indicate SD of triplicate culture wells. Cells were isolated from pooled draining LN of three mice per time point. Error bars in C indicate SD of triplicate cultures of draining LN DC of four to five mice per time point. Data shown are representative of at least two experiments. *, P < 0.05.