Figure 3: IFNγ renders Socs1^−/− BMDCs resistant to PGE2 but not to IL-10.

(a–e, g, h) Socs1^+/+ (black bars) and Socs1^−/− (grey bars) BMDCs (5×10⁵ cells per ml) induced from the bone marrow cells of Rag2^−/− and Socs1^−/−Rag2^−/− mice were stimulated for 12 h with indicated reagents, and the levels of IL-12p70 and TNFα in the culture supernatant were measured by ELISA. Error bars represent +s.d. Cells were stimulated in triplicate wells for each condition and s.d. was calculated from the values determined by ELISA. (a) Socs1^+/+ and Socs1^−/− BMDCs were stimulated with LPS (10 ng ml⁻¹) and murine IFNγ (10 ng ml⁻¹) in the presence of graded concentrations of recombinant murine IL-10. (b) Socs1^+/+ and Socs1^−/− BMDCs were co-cultured with 2.5×10⁵ cells per ml of CD4⁺CD25^high T cells in the presence of soluble anti-CD3 Ab (1 μg ml⁻¹). Cells were stimulated with LPS+IFNγ (10 ng ml⁻¹ each) with or without anti-IL-10-neutralizing Ab (αIL-10 Ab, 10 μg ml⁻¹). (c) Socs1^+/+ BMDCs were stimualted with LPS (10 ng ml⁻¹) in the presence or absence of graded concentrations of CME and αIL-10 Ab (10 μg ml⁻¹). (d) Socs1^+/+ and Socs1^−/− BMDCs were stimulated with LPS+IFNγ (10 ng ml⁻¹ each) in the presence of graded concentrations of CME (v/v, volume of CME/volume of solution (media+CME)). **P<0.01. (e) Socs1^+/+ BMDCs were stimulated with LPS (10 ng ml⁻¹) in the presence or absence of graded concentrations of CME. The CME filtrated through YM-3 ultrafiltration columns (Millipore), which remove molecules higher than 3,000 Da, retained a suppressive function. IL-10 was used as an experimental control. (f) Tissue extracts from the indicated organs were prepared as described in the Methods, and the levels of PGE2 were determined by ELISA. Error bars indicate +s.d. of triplicate ELISA measurements for each sample. (g) Socs1^+/+ BMDCs were stimulated with LPS (10 ng ml⁻¹) in the presence of PGE2 (1 nM) or graded concentrations of CME with or without PTGER4 antagonist (ONO-AE3-208; 100 nM). (h) Socs1^+/+ and Socs1^−/− BMDCs were stimulated with LPS+IFNγ (10 ng ml⁻¹ each) in the presence of graded concentrations of PGE2. **P<0.01. Data are representative of two (f) to three (a–e, g, h) independent experiments.