Glycyrrhizin enhances interleukin-12 production in peritoneal macrophages

Abstract

Interleukin-12 (IL-12) is a monocyte/macrophage-derived cytokine that plays a prominent role in the development of T helper type 1 (Th1) cell-mediated immune responses. Glycyrrhizin (GL), an aqueous extract of liquorice root, used as Chinese medicine, is known to have various immunomodulating activities. In this study, GL showed a dose-dependent priming effect on lipopolysaccharide (LPS)-induced IL-12 p40 and IL-12 p70 (heterodimer of p40 and p35) protein production by peritoneal macrophages (PM). The maximal effect was observed when GL was intraperitoneally administered 12 hr before the PM were harvested and stimulated in vitro with LPS. The increases in IL-12 p70 and p40 protein production were primarily due to up-regulated transcription of IL-12 p35 and p40 messenger RNAs (mRNAs), as demonstrated by RNase protection assay. The augmentation of IL-12 p40 mRNA expression induced by GL pretreatment was associated with increased NF-kappaB activation. Moreover, GL exhibited the same priming effect on IL-12 production in interferon-gamma knockout (IFN-gamma-/-) mice. The production of granulocyte-macrophage colony-stimulating factor (GM-CSF) was not induced at any time point after GL pretreatment. These findings demonstrated the ability of GL to enhance LPS-induced IL-12 production by peritoneal macrophages, and indicated that the priming effect of GL on IL-12 production was independent of both IFN-gamma and GM-CSF.

Figure 1

Effect of glycyrrhizin (GL) dose on LPS-induced IL-12 (p40 and p70) production by peritoneal macrophages (PM). C57BL/6 mice were injected i.p. with different concentrations of GL or saline and PEC were harvested 24 hr later. PEC (4 × 10⁶ cells/ml) were added to each well of a 24-well plate and incubated for 2 hr at 37°. Non-adherent cells were removed by washing vigorously three times with warm medium. PM were further incubated for 24 hr with LPS (10 µg/ml). IL-12 p40 (a) and p70 (b) in culture supernatants were measured by ELISA. Results are expressed as mean±SD of the mean values of 12 independent experiments.

Figure 2

Effect of glycyrrhizin (GL) dose on LPS-induced IFN-γ and IL-10 production. IFN-γ (a) and IL-10 (b) in the culture supernatants of PM cultures after in vivo pretreatment with different concentrations of GL were measured by ELISA. Results are expressed as mean±SD of the mean values of 12 independent experiments.

Figure 3

Time–courses of glycyrrhizin (GL) pretreatment on LPS-induced IL-12 production. C57BL/6 mice were injected i.p. with GL (60 mg/kg) or saline. PEC were harvested at the indicated time-points. Peritoneal macrophages (PM) were further incubated for 24 hr with LPS (10 µg/ml). IL-12 p40 (a) and p70 (b) in culture supernatants were measured by ELISA. Results are expressed as mean±SD of the mean values of eight independent experiments.

Figure 4

Time–courses of glycyrrhizin (GL) pretreatment on LPS-induced IFN-γ and IL-10 production. IFN-γ (a) and IL-10 (b) in the culture supernatants of PM after in vivo GL priming at different time-points were measured by ELISA. Results are expressed as mean±SD of the mean values of eight independent experiments.

Figure 5

Up-regulation of IL-12 p35 and p40 mRNA expression in glycyrrhizin (GL) -pretreated peritoneal macrophages (PM). C57BL/6 mice were injected i.p. with 60 mg/kg of GL or sterile saline and PEC were harvested 12 hr later. The PM were further stimulated with LPS (10 µg/ml) for 1 or 4 hr in vitro, and then IL-12 p35 and p40 mRNA were detected by RNase protection assay as described in the Materials and Methods. The experiment was performed three times with similar results.

Figure 6

Glycyrrhizin (GL) augments LPS-induced NF-κB-binding activities. (a) EMSA: C57BL/6 mice were pretreated with 60 mg/kg of GL or sterile saline and PEC were harvested 12 hr later. Peritoneal macrophages (PM) were further stimulated with LPS (10 µg/ml) for 30 min in vitro, and then NF-κB-binding activity was analysed by EMSA as described in the Materials and Methods. (b) Supershift EMSA: nuclear extracts were mixed with antibodies to p65 (RelA) or p50 NF-κB components or control antibody to GM-CSF. After a 30-min incubation, the probe was added and incubation was continued for an additional 20 min before electrophoresis. Supershift bands were indicated with an asterisk. Results are representative of three separate experiments.

Figure 7

Effect of glycyrrhizin (GL) on LPS-induced IL-12 production in IFN-γ^–/– knockout mice. IFN-γ^–/– knockout and IFN-γ^+/+ mice were injected i.p. with 60 mg/kg of GL for 24 hr before harvest of PEC. Peritoneal macrophages (PM) were further stimulated for 24 hr with LPS (10 µg/ml) in vitro. IL-12 p40 (a) and IL-12 p70 (b) in culture supernatants were measured by ELISA. Results are expressed as mean ±SD of the mean values of five independent experiments.