RESUMO
Objective To explore the effect of several cytokines, including interferon-γ, interleukin-10 and interlekin-4, on promoter activity of human BAFF (B-cell activating factor belonging to tumor necrosis factor family) gene. Methods A construct of phBAFF 1.02 containing sequence form -1349 bp to -329 bp of human BAFF gene, linking with chloramphenicol acetyltransferase (CAT) as reporter gene, was transiently transfected into human HL-60 cells, a kind of myeloid tumor cell lines. The cells were subsequently treated with IFN-γ, IL-10 and IL-4, and the CAT activity was assessed 24 hours after stimulation with each cytokines. Results IFN-γ of 5 ng/ml, IL-10 of 100 ng/ml could increase the CAT activity of phBAFF 1.02 to 4.18 and 2.13 folds respectively compared to the control. IL-4 at 100 ng/ml had no effect on promoter activity of human BAFF gene. Combination of IFN-γ, IL-10 and IL-4 could increase the CAT activity of phBAFF 1.02 to 3.41 and 1.58 folds respectively compared with controls. Conclusion IFN-γ and IL-10 can increase the promoter activity of human BAFF gene. IL-4 treatment can not affect the CAT activity driven by BAFF promoter. However, IL-4 can decrease the upregulating effect of IFN-γ and IL-10 on phBAFF1.02. These provide essential evidence for future study on the interaction mechanism of cytokines and BAFF in autoimmune diseases.
RESUMO
Objective To explore the effect of IFN-γ, IL-10 and IL-4 on B cell activating factor (BAFF) expression in human HL-60 cells, a kind of myeloid tumor cell lines, and its possible regulation mechanism. Methods Cultured human HL-60 cells were treated with IFN-γ, IL-10 and IL-4 for 1-3 days. The expression of membrane-bound BAFF on HL-60 cells was examined by flow cytometry, the amount of soluble BAFF was detected by ELISA assay, and the level of BAFF mRNA was tested by real-time PCR method. A functional 1021 bp fragment of the 5'-tlanking region of the human BAFF gene (-1349 to -329 bp) was cloned and investigated with serial 5'-deletion. The 5'-deleted promoters were recombinated with chloramphenicol acetyltransferase (CAT) as reporter gene. These five recombinant plasmids were transiently transfected to HL-60 cells with liposomal transfectian method. Promoters activities were determined by CAT reporter gene assay(CAT-ELISA) in those transfected cells treated with different cytokines. Results The results showed that the expression of membrane-bound BAFF, soluble BAFF and BAFF mRNA in human HL-60 cells were significantly elevated (P ＜ 0. 05) after incubated with IFN-γ and IL-10. In addition, IFN-γ and IL-10 showed significantly (P ＜ 0. 05) increased effects on promoter activity in human BAFF gane. And the cytokines-responsive sequences were located between -929 and -719 bp of the BAFF promoter region. Conclusion The enhancement of IFN-γ and IL-10 on BAFF expression and synthesis were regnla-ted by promoter activation. Our in vitro studies also raise the possibility to investigate the mechanisms regula-ting BAFF expression in other tumor cells of myeloid origin under pathological circumstances.