Interleukin (IL)-4 is a major regulatory cytokine governing bioactive IL-12 production by mouse and human dendritic cells

Abstract

Interleukin (IL)-12 may be secreted as a bioactive T helper type 1 (Th1) cell-inducing heterodimer, as a monomer, or as an antagonistic homodimer. We analyzed the IL-12 produced by mouse splenic dendritic cells (DCs), human thymic DCs, and cultured human monocyte-derived DCs. IL-12 production required both a microbial or T cell-derived stimulus and an appropriate cytokine milieu. The different IL-12 forms were differentially regulated by the cytokines present rather than the stimulus used. IL-4 alone or together with granulocyte/macrophage colony-stimulating factor or interferon gamma effectively enhanced the production of the bioactive heterodimer and selectively reduced the antagonistic homodimer of IL-12. Therefore, IL-4, the major Th2-driving cytokine, provides a negative feedback causing DCs to produce the major Th1-inducing cytokine, bioactive IL-12.

Figure 1

IL-4 enhances IL-12 p70 production by mouse splenic DCs. Mouse splenic DCs were cultured with an IL-12 stimulus (LPS, anti-CD40 mAb, poly I:C, CpG, or SAC [10 μg/ml], or a mix of all stimuli together) in the presence of GM-CSF and IFN-γ with or without IL-4, for 18 h. The supernatants were assayed for IL-12 p70 content by ELISA. The result is typical of more than five experiments of this type.

Figure 2

Titration of the effect of IFN-γ on the production of IL-12 by mouse splenic DCs. Mouse splenic DCs were cultured with SAC (20 μg/ml) in the presence of titrated concentrations of IFN-γ, for 23 h. The supernatants were analyzed for IL-12 p70 and p40 by ELISA (A and B) and Western blotting (C). The error bars represent the variation within one typical experiment. The experiment has been repeated three times for the ELISA and two times for Western blots, with similar results.

Figure 3

Titration of the effect of GM-CSF on the production of IL-12 by mouse splenic DCs. Mouse splenic DCs were cultured with SAC (20 μg/ml) in the presence of IFN-γ and titrated concentrations of GM-CSF, for 23 h. The supernatants were analyzed for IL-12 p70 and p40 by ELISA (A and B) and Western blotting (C). The result is typical of three experiments with ELISA readouts and Western blotting.

Figure 5

The effects of combinations of IL-4, GM-CSF, and IFN-γ on the IL-12 production by mouse splenic DCs. Mouse splenic DCs were cultured with a mix of stimuli as used in Fig. 1 in the presence or the absence of IL-4, IFN-γ, or GM-CSF, for 23 h. The supernatants were analyzed for IL-12 p70 and p40 by ELISA (A and B) and Western blotting (C). The result is typical of more than five similar experiments with ELISA readouts and Western blotting.

Figure 4

Titration of the effect of IL-4 on the production of IL-12 by mouse splenic DCs. Mouse splenic DCs were cultured with a mix of stimuli as used in Fig. 1 (but with SAC used at only 5 μg/ml), in the presence of IFN-γ and GM-CSF and titrated concentrations of IL-4, for 18 h. A neutralizing mAb to IL-4 (10 μg/ml) was included for one set of conditions as indicated (gray bar). The supernatants were analyzed for IL-12 p70 and p40 by ELISA (A and B) and Western blotting (C). The result is typical of three similar experiments with ELISA readouts and Western blotting.

Figure 6

The effects of IL-4 on p70 expression are mediated by the regulation of p35 mRNA. Mouse splenic DCs were cultured with CpG alone or with the addition of IFN-γ, IL-4, or both, for 4 h. RNA was extracted from the cells and subjected to Northern transfer analysis. The relative amounts of radioactive p35-specific or p40-specific probes bound to the mRNA samples as quantitated using a PhosphorImager^® (Molecular Probes) are shown. A repeat experiment confirmed these findings.