Interleukin-4 and interleukin-13 enhance CCL26 production in a human keratinocyte cell line, HaCaT cells

Abstract

Eotaxin-2/CCL24 and eotaxin-3/CCL26 are CC chemokines and their receptor, CC chemokine receptor 3 is preferentially expressed on eosinophils. It was reported that vascular endothelial cells and dermal fibroblasts produced CCL26. However, the regulation of CCL24 and CCL26 production in keratinocytes has not been well documented. We investigated the expression and production of CCL24 and CCL26 in the human keratinocyte cell line, HaCaT cells. Reverse transcription and polymerase chain reaction was performed using these cells and Enzyme-linked immunosorbent assay was carried out using supernatant of these cells. The production of CCL24 in HaCaT cells was slightly enhanced by IL-4 and that of CCL26 was strongly enhanced by IL-4 and IL-13. Furthermore, TNF-alpha generated a synergistic effect on IL-4 enhanced CCL26 production. Dexamethasone, IFN-gamma and the p38 mitogen-activated protein kinase inhibitor SB202190 inhibited IL-4 enhanced CCL26 production. IL-4 enhanced production of CCL26 was inhibited by leflunomide and JAK inhibitor 1, but not by JAK3 inhibitor, which indicates that it is mediated by JAK1-STAT6-dependent pathway. This result also strongly suggests the involvement of the type 2 IL-4 receptor in IL-4 enhanced production of CCL26. These results suggest that keratinocytes are involved in the migration of CC chemokine receptor 3 positive cells such as eosinophils in a Th2-dominant situation like atopic dermatitis.

Fig. 1

CCL24 and CCL26 production in HaCaT cells were determined by ELISA. (a) CCL24 production was determined when they were stimulated by various stimuli. (b) CCL26 production was determined when they were stimulated by IL-4 and antihuman IL-4 antibody. (c) CCL26 production was determined when they were stimulated by TNF-α, IL-4 and IL-13. The error bars indicate standard error. ‘No’ means no stimulation. *P < 0·05 for the stimulus effect; **P < 0·05 for the inhibitory effect. ***P < 0·05 for the synergistic effect of TNF-α and IL-4.

Fig. 2

(a) CCL26 mRNA expression in HaCaT cells was determined by RT-PCR when they were stimulated with TNF-α, IL-4, IL-13 and antihuman IL-4 antibody. These results were parallel to those of ELISA. (b) CCL26 mRNA and G3PDH mRNA expression in IL-4-stimulated HaCaT cells were determined when the reverse transcriptase enzyme was omitted. RT means reverse transcriptase.

Fig. 3

CCL26 production in HaCaT cells was determined by ELISA. (a) Dexamethasone and IFN-γ significantly inhibited IL-4-enhanced CCL26 production. (b) SB202190, but not other reagents, significantly inhibited the IL-4 enhanced CCL26 production. These inhibitors, at the indicated concentrations, were effective in suppressing other cytokines in HaCaT cells. The error bars indicate standard error. **P < 0·05 for the inhibitory effect.

Fig. 4

CCL26 production in HaCaT cells was determined by ELISA when they were stimulated with IL-4, leflunomide, JAK inhibitor 1 or JAK3 inhibitor 1. Leflunomide and JAK inhibitor 1, but not JAK3 inhibitor 1, significantly inhibited the IL-4-enhanced CCL26 production from HaCaT cells. The error bars indicate standard error. **P < 0·05 for the inhibitory effect.

Fig. 5

CCL26 mRNA expression in HaCaT cells was determined by RT-PCR when they were stimulated with IL-4, leflunomide, JAK inhibitor 1 and JAK3 inhibitor 1. Leflunomide and JAK inhibitor 1, but not JAK3 inhibitor 1, inhibited IL-4-enhanced CCL26 mRNA expression dose-dependently. When the reverse transcriptase enzyme was omitted, neither CCL26 mRNA nor G3PDH mRNA was detected.

Fig. 6

Expression of components of IL-4 receptors in HaCaT cells was determined by RT-PCR. HaCaT cells weakly expressed mRNA of IL-2Rγ and strongly expressed IL-4Rα and IL-13Rα₁. When the reverse transcriptase enzyme was omitted, G3PDH mRNA was not detected. RT means reverse transcriptase.