Recombinant Human B Lymphocyte Stimulator Factor (of Hsblys) To Adjust The Effect And Mechanism Of B Lymphocyte Immune

The present study chose mice and splenic B lymphocytes (B cells) of mice, and employed modern biological techniques in order to systematically observe the effect of recombinant human soluble BLyS(hsBLyS) on the proliferation, survival, differentiation and intracellular Ca²⁺ signal mechanism of splenic B cells in vitro and in vivo. The regulation and mechanism of hsBLyS on the B-cell differentiation and immune system was discussed from physiology, immunology and cytology angles. The results were summarized as follows:1 Effect of hsBLyS on function activity of splenic B cells in vitroB cells were isolated from spleen suspensions with anti-CD19 FluoroBeads, then resuspended and diluted to 2× 10⁵ cells/ml. The freshly isolated B cells were cultured with hsBLyS and/or anti-IgM in different dose. After the culture, The absorbance was measured by the MTT cellular proliferation assay. The hsBLyS alone can stimulate B cell proliferation in a dose-dependent manner within 0.5-2 μ g/ml. The results of B cells costimulated with hsBLyS and anti-IgM close to those of untreated B cells, however the proliferation activity of B cells was dramatically enhanced by further stimulation with LPS. The stimulating index was markedly higher than that of untreated cells. hsBLyS can prolong the survival B cells in vitro. It was suggested that hsBLyS can induce B-cell proliferation and survival. The immune activity of B cells can be enhanced by hsBLyS costimulated with anti-IgM.2 Comparison on effect of hsBLyS monomer and trimer on splenic B cells in vitroB cells were isolated from spleen suspensions with anti-CD 19 FluoroBeads, then resuspended and diluted to 2 × 10⁵ cells/ml. The freshly isolated B cells were cultured with hsBLyS monomer or trimer in the absence or presence of anti-IgM in different dose. After the culture, The absorbance was measured by the MTT cellular proliferation assay. The proliferation activity of B cells cultured with hsBLyS trimer was higher or significantly higher than that of untreated B cells, however no obvious differences were observed between B cells cultured with hsBLyS monomer and untreated cells. To compare the changes of B-cell proliferation with hsBLyS monomer or trimer, we confirm that the trimeric molecule is the functional unit for hsBLyS.3. Effect of hsBLyS on differentiation of splenic B cells in vitroThere were two series for the experiment. (1) In the first series, Freshly isolated splenocytes in spleen suspensions were randomly divided into control group, hsBLyS group, anti-IgM group, hsBLyS+anti-IgM group with triplicate of each group, simultaneously incubated for 72 h with hsBLyS and/or anti-IgM. (2) In the second series, the groups were the same as the first series. The splenocytes were pretreated for 24 h in standard medium with hsBLyS and/or 1 anti-IgM, and further cultured for 48h in standard medium with 10μg/ml of LPS. After the cultures, the cells stained with monoclonal antibodies (mAbs) PE-conjugated anti-CD45R/B220 and FITC-conjugated anti-CD21 and analyzed in a fluorescence-activated cell sorter (FACS) Vantage SE flow cytometer with ModFit 2.0 software. The T1 B-cellpopulation account in the absence or presence hsBLyS was not significantly difference, however T2 B-cell population account treated by hsBLyS were markedly higher than that untreated by hsBLyS. After hsBLyS stimulated B cells, the account of Tl and T2 B cell was further enhanced by LPS. It seems that hsBLyS can induce development of immature B-cell and promote the differentiation from Tl to T2 B-cell.4 Effects of hsBLyS on function activities of splenic B cells and NK cells in miceForty-eight healthy ICR mice were chosen and randomly divided into a control group (group C) and five hsBLyS groups of different dosages which were diluted with PBS and injected in the abdominal cavity with 0.0lmg, 0.1 mg, 0.5mg, lmg and 2mg of hsBLyS per kg body weight, respectively, and group C with PBS of the same dosage in group 2mg/kg over eight days. The proliferation and the differentiation of splenic B cells, the activity of NK cells were analyzed on the 4th and 8th days. During the entire experimental period, the proliferation of splenic B cells was higher or significantly higher in animals administrated hsBLyS from 0.1-2mg/kg than that of control group. The account of splenic B cell was markedly higher in hsBLyS groups with 0.5 and 1 mg/kg than in control group on the 8th day, however, the account in other groups was lower than that in group C but no differences were observed. The account of Tl B-cell in each group was obvious differences with group C, except hsBLyS group with 0.1 mg/kg. The account of T2 B-cell in hsBLyS groups with 0.K 0.5 and 1 mg/kg was higher or markedly higher than that in group C, however, the result in hsBLyS group with 2mg/kg was lower than that in group C. The activity of splenic NK cell was higher or significantly higher in hsBLyS groups with 0.01 to 2 mg/kg than in control group on the 4th day. It suggested that suitable dosage of hsBLyS can greatly activate proliferation of splenic B cells in mice, promote its development, and upregulate NK cell activity.5. Discussion on intracellular Ca2+ signal mechanism of promoted role of hsBLyS on B lymphocyteFreshly isolated splenocytes in spleen suspensions from mice were cultured in the absence or presence of hsBLyS for 72 h(5%CC^ 37°C). After the cultures, the lymphocytes of spleen were collected and marked with monoclonal antibody B220, then loaded with fluorescence probe Fluo-3/AM. A laser scanning confocal microscope was utilized to measure fluorescence intensity of [Ca2+]i in B lymphocyte through fluorescence visualization. B lymphocytes cultured with hsBLyS increased significantly as compared with that of untreated cells. The [Ca2+]i fluorescence intensity in B lymphocytes cultured with hsBLyS maintained relative high level, however, with tendency to reduce in cells untreated with hsBLyS. The [Ca2+]j fluorescence pixels of hsBLyS-treated B cells was significantly higher than that of untreated cells. It was implied that the promoted role of hsBLyS on B cell hsBLyS may play an effective immune regulation through activating [Ca2+]j signal and maintaining its relative high level.