| Background Aplastic anemia(AA) is defined as an immune-mediated bone marrow failure syndrome, characterized by pancytopenia and hypocellular bone marrow. At present, the pathogenesis of AA is concerned chiefly with abnormal hematopoietic stem cells, hematopoietic microenvironment abnormality and disorder of immune system. In aplastic anemia, Th1 and CTL are activated while the number and IFN-gamma and TNF-alpha function of Tregs decline, which is identified with the biological effects of interleukin 27. The present study showed that the concentration of the IL-27 in bone marrow supernatant were higher than in normal control group. And the level of IL-27 in bone marrow supernatant is closely related to the severity of the patients. The more serious of the disease, the higher of the level of IL-27. This leads us to explore the effects of IL-27 on immune cells in aplastic anemia patients, especially the antigen presenting cells' effects on T cell response. Therefore, the aim of this study is to explore the influence of IL-27 on cell proliferation and apoptosis, cytokine secretion, cell factor m RNA level of antigen presenting cells, mainly including dendritic cells and monocytes in aplastic anemia patients. Moreover, we hope to clarify the role of IL-27 in the pathological mechanism of aplastic anemia patients.Objective The current research was to detect the effects of IL-27 on dendritic cells and monocytes in the patients with aplastic anemia.Methods(1) The peripheral blood mononuclear cells of AA were separated by Ficoll density gradient centrifugation in which cells could be isolated in different levels, and then dendritic cells and monocytes were separated by the Magnetic cell sorting(MACS) from peripheral blood mononuclear cells. The cell purity was tested by flow cytometry;(2) The proliferation activation of IL-27 on dendritic cells and monocytes was detected by CCK-8;(3) Annexinv-PI was marked on dendritic cells and monocytes and then tested by flow cytometry(FCM);(4) Costimulating molecules with immune fluorescent tags were marked on dendritic cells and monocytes, then detected by flow cytometry;(5) The effects of IL-27 on IL-12, IL-23 and IL-27 were measured by ELISA;(6) Real-time quantitative polymerase chain reaction(Real-time RT-PCR) was used to detect the m RNA of IL-12, IL-23 and IL-27 in dendritic cells and monocytes.Results(1)CCK-8 detects dendritic cells in patients with AA after cell culture, IL-27 can promote the dendritic cells proliferation(without IL-27 group OD value is 0.3009±0.0091, with 50ng/ml IL-27 group OD value is 0.3354±0.0077, P=0.012);(2) IL-27 can lessen the rate of apoptosis in patients' dendritic cells with AA(The apoptosis rate without IL-27 groups is 51.750±2.454% while the apoptosis rate with 50ng/ml IL-27 groups is 41.750±2.235%, P=0.006);(3) IL-27 can increase the costimulating molecules CD80, CD86 on the surface of dendritic cells in patients with aplastic anemia;(4) After 48 h stimulation of IL-27, the levels of cytokines IL-12, IL-23 and IL-27 secreted by dendritic cells were increased(the level of IL-12, IL-23 and IL-27 without IL-27 group respectively is 29.000±1.042 pg/ml, 60.850±3.589 pg/ml, 38.580±1.018 pg/ml, with 50ng/ml IL-27 group respectively is 36.950±0.869 pg/ml?79.370 ±3.441 pg/ml?44.710±2.138 pg/ml, P value is 0.000, 0.005, 0.003 respectively);(5) RT-PCR revealed the levels of IL-12p35?IL-12p40?IL-23p19 ?IL-23p40?IL-27p28?IL-27EBI3 relative quantitive m RNA expression were up-regulated in AA patients' dendritic cells when culturing cells with IL-27(IL-12p35?IL-12p40?IL-23p19 ?IL-23p40?IL-27p28?IL-27EBI3 relative quantitive m RNA expression without IL-27 group is 1.000±0.071?1.000±0.073, 1.000±0.050, 1.000±0.073, 1.000±0.069, 1.000±0.063, with 50ng/ml IL-27 group is 1.695±0.193, 1.585±0.254, 2.285±0.404, 1.585±0.254, 4.158±0.320, 1.647±0.224, P value is 0.009, 0.037, 0.024, 0.037, 0.000, 0.035 respectively);(6) CCK-8 detects monocytes in patients with AA after cell culture, IL-27 can not promote the monocytes proliferation(without IL-27 group OD value is 0.2238±0.0102, with 50ng/ml IL-27 group OD value is 0.2435±0.0116, P=0.218);(7) IL-27 can lessen the rate of apoptosis in patients' monocytes with AA(The apoptosis rate without IL-27 groups is 42.100±3.734% while the apoptosis rate with 50ng/ml IL-27 groups is 27.560±2.328%, P=0.002);(8) After 48 h stimulation of IL-27, the levels of cytokines IL-12, IL-23 and IL-27 secreted by monocytes were increased(the level of IL-12, IL-23 and IL-27 without IL-27 group respectively is 25.210±1.591pg/ml, 35.540±1.728 pg/ml, 26.690±1.133pg/ml, with 50ng/ml IL-27 group respectively is 31.660±1.032 pg/ml?43.620±2.353pg/ml?37.440±1.640 pg/ml, P value is 0.007, 0.020, 0.000 respectively);(9) RT-PCR revealed the levels of IL-12p35?IL-12p40?IL-23p19 ?IL-23p40?IL-27p28?IL-27EBI3 relative quantitive m RNA expression were up-regulated in AA patients' monocytes when culturing cells with 50ng/ml IL-27(IL-12p35?IL-12p40?IL-23p19 ?IL-23p40?IL-27p28?IL-27EBI3 relative quantitive m RNA expression without IL-27 group is 1.000±0.082, 1.000±0.075, 1.000±0.067, 1.000±0.075, 1.000±0.061, 1.000±0.069, with 50ng/ml IL-27 group is 1.546±0.164, 1.780±0.148, 1.598±0.151, 1.780±0.148, 4.114±0.307, 2.209±0.340, P value is 0.046?0.001?0.004?0.001?0.000?0.009 respectively).Conclusion IL-27 can promote proliferation and inhibt apoptosis of dendritic cells in peripheral blood of AA patients; IL-27 can upregulate the costimulating molecules on the surface of the dendritic cells; IL-27 promote the secretion and expression of cytokines IL-12, IL-23 and IL-27 by dendritic cells in peripheral blood of apastic anemia; IL-27 can not promote proliferation but inhibt apoptosis of monocytes in peripheral blood of AA patients; IL-27 promote the secretion and expression of cytokines IL-12, IL-23 and IL-27 by monocytes in peripheral blood of apastic anemia. All these datas show that IL-27 plays a driving role on dendritic cells and monocytes in the pathogenesis of AA. |
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