Studies On Mechanisms Of Multiple Cytokines Regulation Of HsBAFF Promoting B-cell Proliferation Via Erk1/2 And S6K1 Signaling

The present study, using cellular and molecular biology techniques and methods including cell culture, flow cytometry, Western blotting and RNA interference, etc., and employing Raji cell line as well as mouse spleen B lymphocytes and CD4~+T lymphocytes as experimental objects, studied the role of multiple cytokines in regulation of hsBAFF-dependent B-cell proliferation and survival, dissected the important action and significance of Erkl/2 and S6K1 signaling pathways in multiple cytokines’enhancement of hsBAFF contributing to B-cell proliferation and survival. Finally, proliferation effects and correlation of hsBAFF on mouse spleen B lymphocytes and CD4~+T lymphocytes were preliminarily discussed. The detailed results were summarized as follows:1 Multiple cytokines regulate hsBAFF-dependent B-cell proliferation and survivalRaji cells were stimulated with different concentrations of hsBAFF (0-0.25 ug/ml) for 48 h; or with different concentrations of IL-2, IL-4, IFN-γ or TNF-α (0-100 ng/ml) for 48 h, respectively; or with 0.25 μg/ml hsBAFF in the presence or absence of IL-2 (10 and 50 ng/ml), IL-4 (5 and 25 ng/ml), IFN-γ (10 and 100 ng/ml), TNF-α (10 and 50 ng/ml) for 48 h. After that, analysis for MTS, cell counting and flow cytometry was conducted, and live cells were counted by trypan blue exclusion. The results showed that hsBAFF promoted B-cell proliferation and survival dose-dependently. IL-2, IL-4, IFN-γ, TNF-α also promoted B-cell proliferation and survival in a concentration-dependent fashion, respectively. IL-2, IL-4, IFN-γ, TNF-α enhanced hsBAFF promoting B-cell proliferation and survival, respectively. The findings suggest that suitable IL-2, IL-4, IFN-γ or TNF-α reinforces hsBAFF-dependent B-cell proliferation and survival.2 Multiple cytokine strengthen hsBAFF-activated Erkl/2 and S6K1 pathways to improve B cells proliferation and survivalMouse spleen B lymphocytes and/or Raji cells were treated with 0-0.25 μg/ml hsBAFF for 12 h, or with/without 0.25 μg/ml hsBAFF for 0-24 h, or with/without 0.25 ug/ml hsBAFF in the presence or absence of IL-2 (10 and 50 ng/ml), IL-4 (5 and 25 ng/ml), IFN-γ (10 and 100 ng/ml). TNF-α (10 and 50 ng/ml) for 12 h or 48 h, or with/without 0.25 μg/ml hsBAFF in the presence or absence of single or multiple 50 ng/ml IL-2,25 ng/ml IL-4,100 ng/ml IFN-y and/or 50 ng/ml TNF-a for 12 h or 48 h. Cells were also treated with/without hsBAFF in the presence or absence of single or multiple IL-2, IL-4, IFN-γ and/or TNF-α for 12 h or 48 h following pretreatment with/without 5 μM U0126 for 1 h or 0.2 μg/ml rapamycin for 2 h. In addition, Raji cells, infected with shRNA Erkl/2, shRNA S6K1 and shRNA GFP, respectively, were treated with/without hsBAFF in the presence or absence of single or multiple IL-2, IL-4, IFN-γ and/or TNF-α for 12 h or 48 h. After that, cell proliferation was determined by MTS assay, live cells were counted by by trypan blue exclusion, and expressive changes of Erkl/2, S6K1 and S6 proteins were detected by Western blot analysis. The results showed that single or multiple IL-2, IL-4, IFN-γ and/or TNF-α powerfully reinforced hsBAFF promoting B-cell proliferation and survival through enhancing phosphorylation of Erkl/2 and S6K1/S6. Inhibition of Erkl/2 by μ0126 or downregulation of Erkl/2 by RNA interference significantly blocked phosphorylation of Erkl/2, as well as B-cell proliferation and survival promoted by combination single or multiple IL-2, IL-4, IFN-y and/or TNF-a with hsBAFF. Inhibition of S6K1 by rapamycin or downregulation of S6K1 by RNA interference also markedly reduced phosphorylation of S6K.1/S6, as well as B-cell proliferation and survival promoted by combination single or multiple IL-2, IL-4, IFN-y and/or TNF-a with hsBAFF. Our results suggest that single or multiple IL-2, IL-4, IFN-y and/or TNF-a strengthen hsBAFF-activated Erkl/2 and S6K1 pathways contributing to B-cell proliferation and survival.3 hsBAFF stimulates proliferation of mouse spleen B lymphocytes and CD4~+T lymphocytes and their correlationMouse spleen B lymphocytes and CD4~+T lymphocytes were treated with/without 0.25 μg/ml hsBAFF in the presence or absence of 4 μg/ml LPS or 20 μg/ml PHA for 48 h, respectively. Mixed mouse spleen B lymphocytes and CD4~+T lymphocytes were treated with/without 0.25 μg/ml hsBAFF in the presence or absence of 4μg/ml LPS or 20 μg/ml PHA for 48 h. Cultured supernatants of mouse spleen B lymphocytes and CD4~+T lymphocytes stimulated by 0.25 μg/ml hsBAFF were harvested, respectively, and then co-incubated with mouse spleen B lymphocytes in the presence or absence of 4 μg/ml LPS for 48 h. After that, cell proliferation and lymphocyte transformation rate (stimulation index, SI) were determined by MTS assay. The results showed that hsBAFF effectively promoted cell proliferation and transformation rate in mouse spleen B lymphocytes and CD4~+T lymphocytes. Meanwhile, cultured supernatants from hsBAFF-stimulated CD4~+T lymphocytes significantly promoted proliferation in mouse spleen B lymphocytes in response to hsBAFF. Our findings suggest that hsBAFF stimulates proliferation of B lymphocytes and CD4~+T lymphocytes; hsBAFF promotes proliferation of B lymphocytes by activating CD4~+T lymphocytes.