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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.
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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.
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Objective To explore the differential expression of Toll-like receptor 4(TLR4)mRNA in peripheral blood mononuclear cells(PBMCs)between the patients with ankylosing spondylitis(AS),rheumatoid arthritis(RA)and the healthy individuals as well as its clinical significance.Methods The expression level of TLR4 mRNA in PBMCs was determined from 60 AS patients,30 RA patients and 30 healthy controls by real-time fluorescence quantitative RT-PCR.Erythrocyte sedimentation rate(ESR)and plasma C-reactive protein(CRP)were detected automatically respectively by ESR automatic analyzer and specific protein electrophoresis analyzer.Results The expression level of TLR4 mRNA in PBMC was significantly higher in patients with AS or RA than in the controls,while no significant difference was observed between the two disease groups.Furthermore,both HLA-B27-positive and -negative AS patients showed higher TLR4 mRNA level than healthy controls,and HLA-B27-positive AS patients higher than RA patients.However,there was no significant difference between HLA-B27-positive,-negative AS and RA patients.In HLA-B27-positive AS patients,close correlations between TLR4 mRNA and ESR or CRP were observed,but no correlation observed in HLA-B27 negative patients.In RA patients,the level of TLR4 mRNA correlated with CRP.Conclusion The expression level of TLR4 mRNA is elevated in PBMC from either AS or RA patients.Although the upregulation of TLR4 mRNA is not associated with whether HLA-B27 is positive or not in AS patients,it is more significant in HLA-B27-positive AS than in RA.Furthermore,the correlations between the expression of TLR4 mRNA and ESR or CRP are influenced by HLA-B27 antigen in AS patients.In conclusion,the present results indicate that the abnormal expression of TLR4 might play a role in the development and progression of AS and RA.