Effects of tacrolimus on the secretion of chemokines CXCL9 and CXCL10 by γ-interferon-simulated HaCaT cells

Abstract

Objective To analyze effects of tacrolimus on the secretion of chemokines CXCL9 and CXCL10 by γ-interferon (IFN-γ) -simulated HaCaT cells, as well as phosphorylated Janus kinase 1 (p-JAK1) and phosphorylated signal transducer and activator of transcription 1 (p-STAT1) , and to explore the mechanism of tacrolimus in the treatment of vitiligo. Methods HaCaT cells were treated with 1, 10, 20, 40, 60, 80, 100, 120 mg/L tacrolimus solution separately for 4 hours, and methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate the cellular proliferative activity. HaCaT cells were divided into 4 groups: blank control group receiving no treatment, IFN-γ group treated with 500 U/ml IFN-γ for 12 or 48 hours, tacrolimus group treated with 20 mg/L tacrolimus for 4 hours, and tacrolimus + IFN-γ group treated with 20 mg/L tacrolimus for 4 hours followed by the treatment with 500 U/ml IFN-γ for 12 or 48 hours. Real-time fluorescence-based quantitative PCR was conducted to measure the mRNA expression of CXCL9 and CXCL10, Western blot analysis to determine the protein expression of CXCL9, CXCL10, p-JAK1, and p-STAT1, and enzyme-linked immunosorbent assay (ELISA) to detect the levels of CXCL9 and CXCL10 in the culture supernatants of HaCaT cells. Results Tacrolimus at the maximum concentration of 20 mg/L had no effect on the proliferation of HaCaT cells (P > 0.05) . After the pretreatment with 20 mg/L tacrolimus, the mRNA expression of CXCL9 and CXCL10 significantly decreased from 10 369.08 ± 7.99 and 290.02 ± 2.16 to 5 914.33 ± 4.59 and 114.96 ± 0.73, respectively, after the treatment with IFN-γ (both P < 0.01) , and the protein expression of CXCL9, CXCL10, p-JAK1, and p-STAT1 also significantly decreased from 8.47 ± 0.29, 7.87 ± 0.17, 4.20 ± 0.18 and 4.29 ± 0.11 to 7.36 ± 0.13, 7.36 ± 0.09, 2.60 ± 0.16 and 3.62 ± 0.19, respectively, after the treatment with IFN-γ (all P < 0.01) . Moreover, the levels of CXCL9 and CXCL10 in the culture supernatants of HaCaT cells significantly decreased in the IFN-γ group (1 213.36 ± 0.95, 1 722.41 ± 2.57, respectively) compared with the tacrolimus + IFN-γ group (426.45 ± 0.31, 554.12 ± 0.56, respectively, both P < 0.01) . Conclusion Tacrolimus can inhibit the secretion of CXCL9, CXCL10, p-JAK1 and p-STAT1 by HaCaT cells stimulated by IFN-γ. Key words: Vitiligo; Interferon-gamma; Chemokine CXCL9; Chemokine CXCL10; Janus kinases; STAT1 transcription factor; HaCaT cells; Tacrolimus