Caffeic acid phenethyl ester inhibits nuclear factor-kappaB and protein kinase B signalling pathways and induces caspase-3 expression in primary human CD4+ T cells

Abstract

Caffeic acid phenethyl ester (CAPE), an active component in propolis, is known to have anti-tumour, anti-inflammatory and anti-oxidant properties. In this study, the effects of CAPE on the functions of primary human CD4+ T cells were evaluated in vitro. CAPE significantly suppressed interferon (IFN)-gamma and interleukin (IL)-5 production and proliferation of CD4+ T cells stimulated by soluble anti-CD3 and anti-CD28 monoclonal antibodies in both healthy subjects and asthmatic patients. CAPE inhibited nuclear factor (NF)-kappaB activation and protein kinase B (Akt) phosphorylation, but not p38 mitogen-activated protein kinase (MAPK) phosphorylation in T cells. CAPE also induced active caspase-3 expression in CD4+ T cells; CCR4+CD4+ T cells were more sensitive to CAPE induction than CXCR3+CD4+ T cells. Together, these results indicate that CAPE inhibits cytokine production and proliferation of T cells, which might be related to the NF-kappaB and Akt signalling pathways, and that CCR4+CD4+ T cells are more sensitive to CAPE inhibition. This study provides a new insight into the mechanisms of CAPE for immune regulation and a rationale for the use of propolis for the treatment of allergic disorders.

Fig. 2

Caffeic acid phenethyl ester (CAPE) inhibits proliferation of human CD4⁺ T cells. CD4⁺ T cells (10⁶ cells/ml) were cultured for 48 h in the absence or presence of CAPE (10 µM) with soluble anti-CD3 and anti-CD28 monoclonal antibodies (2 µg/ml) stimulation. Tritiated thymidine [³H] incorporation for six healthy subjects and four asthmatic patients was measured by liquid scintillation counter and expressed as mean counts per minute (cpm). *P < 0·05; **P < 0·01.

Fig. 1

Caffeic acid phenethyl ester (CAPE) inhibits interferon (IFN)-γ and interleukin (IL)-5 production by human CD4⁺ T cells. CD4⁺ T cells (10⁶ cells/ml) were cultured for 48 h in the absence or presence of CAPE (10 µM) with soluble anti-CD3 and anti-CD28 monoclonal antibodies (2 µg/ml) stimulation. Cytokine levels in the culture supernatants for six healthy subjects and four asthmatic patients were measured by enzyme-linked immunosorbent assay. *P < 0·05; **P < 0·01; ***P < 0·001.

Fig. 3

Caffeic acid phenethyl ester (CAPE) inhibits the phosphorylation of inhibitor of κB (IκB) kinase (IKK)α/IKKβ and IκBα. (a) Human CD4⁺ T cells from healthy subjects were pretreated with CAPE at the indicated concentrations for 2 h, and then stimulated with phorbol myristate acetate (PMA) (50 ng/ml) and ionomycin (500 ng/ml). Cell lysates were collected for the levels of phosphorylation of the IKK complex after 10 min or phosphorylation of IκBα after 15 min, which were assayed by Western blotting with anti-phosphorylated IKKα (ser180)/IKKβ (ser181) and anti-phosphorylated IκBα polyclonal antibodies. (b) Human CD4⁺ T cells from mite-sensitized asthmatic patients were pretreated with CAPE at the indicated concentrations for 2 h, and then stimulated with soluble anti-CD3 and anti-CD28 monoclonal antibodies (2 µg/ml). Cell lysates were collected for the levels of phosphorylation of the IKK complex after 30 min or phosphorylation and degradation of IκBα after 40 min. Anti-α-tubulin mAb was used as an internal control. The quantification results were normalized with the first lane set as relative expression level of 1. Representative results of one of at least three independent experiments are shown.

Fig. 4

Caffeic acid phenethyl ester (CAPE) inhibits nuclear factor (NF)-κB activation. Human CD4⁺ T cells from healthy subjects were pretreated with CAPE at the indicated concentrations for 2 h, and then stimulated with phorbol myristate acetate (PMA) (50 ng/ml) and ionomycin (500 ng/ml) for 45 min. Nuclear fractions were analysed for NF-κB binding activity by electrophoretic mobility shift assay. To assess the specificity of the binding, 100-fold excess of cold NF-κB probe or a mutant probe was added to the PMA and ionomycin conditions. Representative results of one of at least three independent experiments are shown.

Fig. 5

Caffeic acid phenethyl ester (CAPE) inhibits the phosphorylation of protein kinase B (Akt) but not p38 mitogen-activated protein kinase (MAPK). (a) Human CD4⁺ T cells from healthy subjects were pretreated with CAPE at the indicated concentration for 2 h, and then stimulated with phorbol myristate acetate (PMA) (50 ng/ml) and ionomycin (500 ng/ml) for 15 min. Human CD4⁺ T cells from mite-sensitized asthmatic patients were pretreated with CAPE at the indicated concentrations for 2 h, and then stimulated with soluble anti-CD3 and anti-CD28 monoclonal antibodies (2 µg/ml) for 40 min. Cell lysates were collected and the levels of phosphorylation of p38 were assayed by Western blotting with anti-phosphorylated p38 polyclonal antibody; anti-total p38 polyclonal antibody was used as an internal control. (b) Human CD4⁺ T cells from healthy subjects were pretreated with CAPE at the indicated concentrations for 2 h, and then stimulated with soluble anti-CD3 and anti-CD28 mAbs (2 µg/ml) for 10 min. Cell lysates were collected and the levels of phosphorylation of Akt were assayed by Western blotting with anti-phosphorylated Akt (Ser473) polyclonal antibody; anti-total Akt polyclonal antibody was used for an internal control. The quantification results were normalized with the first lane set as relative expression level of 1. Representative results of one of at least three independent experiments are shown.

Fig. 6

Caffeic acid phenethyl ester (CAPE) induces active caspase-3 expression in human CD4⁺ T cells. Peripheral blood mononuclear cells (PBMCs) (10⁶ cells/ml) were cultured for 12 h at the indicated concentrations of CAPE (0–100 µM). Cells were stained with monoclonal antibodies for CD4, CCR4, CXCR3 and active caspase-3 and then analysed by flow cytometry. Results are means ± standard error of the mean of six separate experiments. *P < 0·05; **^{,^^}P < 0·01; ***^{,^^^}P < 0·001 compared to controls.

Fig. 7

The schematic model of inhibitory effects of CAPE. CAPE inhibits κB (IκB) kinase (IKKα)/IKKβ phosphorylation, then IκBα phosphorylation, degradation and further nuclear factor (NF)-κB activation and nuclear translocation, which enhances the transcription of inflammatory cytokines, chemokines, growth factor, adhesion molecules and inhibitors of apoptosis. CAPE also inhibits protein kinase B (Akt) phosphorylation, which promotes cell growth and survival. The inhibitory effect of CAPE results in the inhibition of cytokine production and proliferation in primary human CD4⁺ T cells.