Induction of interleukin-12 production in mouse macrophages by berberine, a benzodioxoloquinolizine alkaloid, deviates CD4+ T cells from a Th2 to a Th1 response

Abstract

In this study we investigated whether berberine-mediated induction of interleukin-12 (IL-12) production in antigen-presenting cells could regulate a cytokine profile of antigen-primed CD4+ T helper (Th) cells. Pretreatment with berberine induced IL-12 production in both macrophages and dendritic cells, and significantly increased the levels of IL-12 production in lipopolysaccharide-stimulated macrophages and in CD40 ligand-stimulated dendritic cells. Importantly, berberine pretreatment of macrophages increased their ability to induce interferon-gamma (IFN-gamma) and reduced their ability to induce IL-4 in antigen-primed CD4+ T cells. Berberine did not influence the macrophage cell surface expression of the class II major histocompatibility complex molecule, the co-stimulatory molecules CD80 and CD86, and intracellular adhesion molecule-1. Addition of neutralizing anti-IL-12p40 monoclonal antibody to cultures of berberine-pretreated macrophages and CD4+ T cells restored IL-4 production in antigen-primed CD4+ T cells. The in vivo administration of berberine resulted in the enhanced induction of IL-12 production by macrophages when stimulated in vitro with lipopolysaccharide or heat-killed Listeria monocytogenes, leading to the inhibition of the Th type 2 cytokine profile (decreased IL-4 and increased IFN-gamma production) in antigen-primed CD4+ T cells. These findings may point to a possible therapeutic use of berberine or medicinal plants containing berberine in the Th type 2 cell-mediated immune diseases such as allergic diseases.

Figure 1

Pretreatment with berberine stimulates IL-12 production in mouse macrophages and dendritic cells. Mouse macrophages (a) or dendritic cells (b) were pretreated with berberine (0·1, 0·5, and 1·0 μg/ml) or untreated. After 6 hr, the cells were washed and stimulated in vitro with medium alone, or with either LPS (5 μg/ml) or CD40L–CHO cells for 48 hr. The culture supernatants were harvested, and the levels of IL-12 were evaluated by ELISA. The results are presented as the means ± SEM (n = 3). *P < 0·01, relative to groups which were not pretreated with berberine. **P < 0·05, relative to groups which were not pretreated with berberine.

Figure 2

Macrophages pretreated with berberine enhance IFN-γ and inhibit IL-4 production by antigen-primed CD4⁺ T cells. Macrophages (1 × 10⁵ cells/well) were pretreated with medium alone or 1·0 μg/ml berberine. After 6 hr, the cells were washed and incubated with KLH-primed CD4⁺ T cells (5 × 10⁵ cells/well) and KLH (10 μg/ml). 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 means ± SEM (n = 3). *P < 0·01, relative to an untreated group.

Figure 3

Cytofluorometric analysis of cell surface molecules on berberine-treated macrophages. Mouse macrophages were treated for 24 hr with medium alone or 1·0 μg/ml berberine and analysed for the expression of cell surface molecules by flow cytometry. Data are representative of three independent experiments.

Figure 4

Addition of neutralizing IL-12p40 mAb restores the decreased IL-4 production of T cells in cultures of berberine-pretreated macrophages and antigen-primed CD4⁺ T cells. KLH-primed CD4⁺ T cells were cultured with berberine (1·0 μg/ml)-pretreated macrophages in the presence of anti-IL-12p40 (10 μg/ml) and KLH (10 μg/ml). Culture supernatants were harvested 4 days later and assayed for IFN-γ (a) and IL-4 (b) by ELISA. The results are presented as the means ± SEM (n = 3). *P < 0·01, relative to each berberine-treated group in the absence of anti-IL-12p40.

Figure 5

Macrophages exposed to berberine in vivo increase levels of IL-12 production. Mice were injected in vivo with berberine (200 μg per mouse, i.p.). After 24 hr, macrophages were purified and stimulated with medium only, or with either LPS (5 μg/ml) or HKL (2 × 10⁶ bacteria per well). Culture supernatants were harvested 48 hr later and IL-12 levels were determined by ELISA. The results are presented as the means ± SEM (n = 3). *P < 0·05, relative to a saline-injected group.

Figure 6

Macrophages purified from mice treated in vivo with berberine regulate cytokine production in antigen-primed CD4⁺ T cells. DBA/2 mice were injected in vivo with either berberine (200 μg per mouse, i.p.) or saline. After 24 hr, macrophages were purified and incubated with KLH-primed CD4⁺ T cells and KLH (10 μg/ml). 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 means ± SEM (n = 3). *P < 0·05, relative to a saline-injected group.