Cooperation of interleukin-17 and interferon-gamma on chemokine secretion in human fetal intestinal epithelial cells

Abstract

Interleukin (IL)-17 is a newly identified T cell-derived cytokine that can regulate the functions of a variety of cell types. In this study, we investigated the effects of IL-17 and interferon (IFN)-gamma on chemokine secretion in human fetal intestinal epithelial cells. IL-8 and monocyte chemoattractant protein (MCP)-1 secretion by the human fetal intestinal epithelial cell line, intestine-407, was evaluated by ELISA and Northern blot. The expression of IL-17 receptor (R) was analysed by a binding assay using [(125)I]-labelled IL-17. The activation of nuclear factor-kappa B (NF-kappa B), NF-IL6 and AP-1 was assessed by an electrophoretic gel mobility shift assay (EMSA). IL-17 induced a dose-dependent increase in IL-8 and MCP-1 secretion. The inducing effects of IL-17 on IL-8 and MCP-1 mRNA abundance reached a maximum as early as 3 h, and then gradually decreased. IL-17 and IFN-gamma synergistically increased IL-8 and MCP-1 secretion and mRNA abundance. IFN-gamma induced a weak increase in IL-17 R mRNA abundance, and incubation with IFN-gamma for 24 h enhanced [(125)I]-labelled IL-17-binding by 2.4-fold. IL-17 rapidly induced the phosphorylation and degradation of I kappa B alpha molecules, and the combination of IL-17 and IFN-gamma induced a marked increase in NF-kappa B DNA-binding activity as early as 1.5 h after the stimulation. Furthermore, this combination induced an increase in NF-IL-6 and AP-1 DNA-binding activity. In conclusion, it becomes clear that IL-17 is an inducer of IL-8 and MCP-1 secretion by human fetal intestinal epithelial cells. The combination of IL-17 with IFN-gamma synergistically enhanced chemokine secretion. These effects of IL-17 and IFN-gamma might play an important role in the inflammatory responses in the intestinal mucosa.

Fig. 1

Effects of IL-17 on IL-8 and MCP-1 secretion in intestine 407 cells. The cells were incubated for 48 h in the presence of increasing concentrations of IL-17. The amounts of IL-8 and MCP-1 were determined by ELISA. Values were expressed as mean±SD (n = 4).

Fig. 2

(a) Kinetics of IL-17-induced increase in IL-8 and MCP-1 mRNA abundance in intestine-407 cells. Intestine-407 cells were stimulated with IL-17 (500 ng/ml), and the abundance of IL-8 and MCP-1 mRNA was sequentially determined by Northern blotting. (b) Kinetics of IL-17-induced increase in IL-8 (b) and MCP-1. (c) Secretion in intestine-407 cells. Intestine-407 cells were stimulated with IL-17 (500 ng/ml), and the amounts of IL-8 and MCP-1 in supernatants were determined by ELISA. •, data of IL-17; ○, data of medium only. Values were expressed as mean±SD (n = 4).

Fig. 3

Effects of IFN-γ on IL-17-induced secretion of IL-8 (a) and MCP-1 (b). Intestine-407 cells were incubated for 48 h in the presence or absence of IL-17 (500 ng/ml), IFN-γ (200 U/ml), or IL-17 (500 ng/ml) plus IFN-γ (200 U/ml). The amounts of IL-8 and MCP-1 were determined by ELISA. Values were expressed as mean±SD (n = 4). Northern analysis of the effects of IL-17 and IFN-γ on IL-8 (c) and MCP-1 (d) mRNA abundance in intestine-407 cells. The cells were incubated for 3 h in the presence or absence of IL-17 (500 ng/ml), IFN-γ (200 U/ml), or IL-17 (500 ng/ml) plus IFN-γ (200 U/ml), and then Northern blotting was performed. The same membrane was used for the determination of IL-8 and MCP-1 mRNA expression.

Fig. 4

Kinetics of IL-17 receptor (R) mRNA abundance in intestine-407 cells. The cells were incubated in the presence of IL-17 (500 ng/ml), and IL-17 R mRNA abundance was sequentially determined by Northern blotting.

Fig. 5

Electrophoretic gel mobility shift assays (EMSA) for NF-κB (a), NF-IL6 (b) and AP-1 (c) DNA-binding activities. The cells were incubate with medium alone, IL-17 (500 ng/ml), IFN-γ (200 U/ml) or IL-17 (500 ng/ml) plus IFN-γ (200 U/ml) for 1·5 h, and then nuclear extracts were prepared. Lane 1, medium alone; lane 2, IL-17; lane 3, IFN-γ; lane 4, IL-17 plus IFN-γ; lane 5, IL-17 plus cold probe; lane 6, IL-17 plus antip50 antibody; lane 7, IL-17 plus antip65 antibody and (c) lane 1, medium alone; lane 2, IL-17; lane 3, IFN-γ; lane 4, IL-17 plus IFN-γ; lane 5, IL-17 plus cold probe. NS at the right side means non-specific band.

Fig. 6

Western blot analysis for IκBα degradation. The cells were stimulated with IL-17 (500 ng/ml), and then total IκBα molecules (lower lane) and phosphorylated IκBα molecules (upper lane) were sequentially analysed by Western blotting.