Inhibition of interleukin-12 production by auranofin, an anti-rheumatic gold compound, deviates CD4(+) T cells from the Th1 to the Th2 pathway

Abstract

1. Interleukin-12 (IL-12) may play a central role in the development and progression of rheumatoid arthritis by driving the immune response towards T helper 1 (Th1) type responses characterized by high IFN-gamma and low IL-4 production. In this study we investigated the effect of auranofin (AF), an anti-rheumatic gold compound, on IL-12 production in mouse macrophages and dendritic cells, and studied whether AF-mediated inhibition of IL-12 production could regulate a cytokine profile of antigen (Ag)-primed CD4(+) Th cells. 2. Treatment with AF significantly inhibited IL-12 production in lipopolysaccharide (LPS)-stimulated macrophages and also in CD40L-stimulated dendritic cells. AF-pretreated macrophages reduced their ability to induce IFN-gamma and increased the ability to induce IL-4 in Ag-primed CD4(+) T cells. AF did not influence the cell surface expression of the class II MHC molecule and the costimulatory molecules CD80 and CD86. 3. Addition of recombinant IL-12 to cultures of AF-pretreated macrophages and CD4(+) T cells restored IFN-gamma production in Ag-primed CD4(+) T cells. 4. The in vivo administration of AF resulted in the inhibition of IL-12 production by macrophages stimulated in vitro with LPS or heat-killed Listeria monocytogenes (HKL), leading to the inhibition of Th1 cytokine profile (decreased IFN-gamma and increased IL-4 production) in Ag-primed CD4(+) T cells. 5. These findings may explain some known effects of AF including anti-rheumatic effects and the inhibition of encephalitogenicity, and point to a possible therapeutic use of AF in the Th1-mediated immune diseases such as autoimmune diseases.

Figure 1

Treatment with auranofin (AF) inhibits IL-12 production in mouse macrophages and dendritic cells. Mouse macrophages were stimulated with LPS (5 μg ml⁻¹) for 48 h in the absence or presence of varying concentrations of AF (A). Mouse DCs (1×10⁵ cells well⁻¹) were stimulated with 3×10⁴ cells well⁻¹ CD40L-CHO (B). Culture supernatants were harvested and cytokine levels were evaluated by ELISA. The results are presented as the mean±s.e.mean (n=3) of the percentage response of cytokine production of AF-treated cells compared with untreated control cells stimulated with LPS for macrophages or CD40L-CHO for DCs. Mean cytokine levels in the absence of AF were as follows: IL-12 p70, 650 pg ml⁻¹ for macrophages and 870 pg ml⁻¹ for DCs; IL-12 p40, 2.8 ng ml⁻¹ for macrophages and 3.9 ng ml⁻¹ for DCs; IL-10, 1.2 ng ml⁻¹ for macrophages. *P<0.05, relative to an untreated group.

Figure 2

Effect of AF on the expression of IL-12 gene at the mRNA level. Macrophages were stimulated with LPS (5 μg ml⁻¹) in the absence or presence of AF (0.1 and 1.0 μg ml⁻¹) for 6 h and total RNA was prepared from the cells. RT – PCR products for IL-12 p40 and β-actin (A), or for IL-6, IL-10, TNF-α, IL-12 p40 and β-actin (B) were analysed in 1.5% agarose gels.

Figure 3

Macrophages pretreated with AF inhibit IFN-γ and enhance IL-4 production by Ag-primed CD4⁺ T cells. Macrophages (1×10⁵ cells well⁻¹) were pretreated with medium alone or 1.0 μg ml⁻¹ AF. After 4 h, the cells were washed and incubated with KLH-primed CD4⁺ T cells (5×10⁵ cells well⁻¹) and KLH (10 μg ml⁻¹). Supernatants were harvested after 2 days for IL-12 (A) or after 4 days for IFN-γ (B) and IL-4 (C), and assayed by cytokine-specific ELISA. The results are presented as the mean±s.e.mean (n=3). *P<0.01, relative to an untreated group.

Figure 4

Cytofluorometric analysis of cell surface molecules on AF-treated macrophages. Mouse macrophages were treated for 24 h with medium alone or 1.0 μg ml⁻¹ AF and analysed for the expression of cell surface molecules by flow cytometry. Data are representative of three independent experiments.

Figure 5

Addition of recombinant IL-12 restores the decreased IFN-γ production of T cells in cultures of AF-pretreated macrophages and Ag-primed CD4⁺ T cells. KLH-primed CD4⁺ T cells were cultured with AF (1.0 μg ml⁻¹)-pretreated macrophages in the presence of rIL-12 (10 pg ml⁻¹) and KLH (10 μg ml⁻¹). Culture supernatants were harvested 4 days later and assayed for IFN-γ (A) and IL-4 (B) levels by ELISA. The results are presented as the mean±s.e.mean (n=3). *P<0.01, relative to each AF-treated group in the absence of rIL-12.

Figure 6

Macrophages exposed to AF in vivo decrease levels of IL-12 production. Mice were injected in vivo with AF (200 μg per mouse, i.p.). After 24 h, macrophages were purified and stimulated with either LPS (5 μg ml⁻¹) or HKL (2×10⁶ bacteria well⁻¹). Culture supernatants were harvested 48 h later and IL-12 levels were determined by ELISA. The results are presented as the mean±s.e.mean (n=4). *P<0.001, relative to a saline-injected group.

Figure 7

Macrophages purified from mice treated in vivo with AF regulate cytokine production in Ag-primed CD4⁺ T cells. DBA/2 mice were injected in vivo with either AF (200 μg per mouse, i.p.) or saline. After 24 h, macrophages were purified and incubated with KLH-primed CD4⁺ T cells and KLH (10 μg ml⁻¹). After 4 days, culture supernatants were harvested and assayed for IL-12 (A), IFN-γ (B) and IL-4 (C) levels by ELISA. The results are presented as the mean±s.e.mean (n=4). *P<0.001, relative to a saline-injected group.