The Expression Of S1P1in B Lymphocyte At Different Differentiation Stage In Peyer’s Patches And Tfh Cell-secreted Cytokine Play A Role In The Upregulation Of Its Expression

Objective:To study the expression of S1P1in B lymphocyte at different differentiation stage(B220+IgA-B cells, B220+IgA+B cells, B220-IgA+plasma blast) and the effects of Tfhcell-secreted cytokine IL-4, IL-21, TGF-β1on S1P1’s expression of B lymphocyte.Methods:1. Peyer’s Patches were acquired from intestines of BALB/c mice whichwere sacrificedafter gavage in0.1%BSA every other day for two weeks and stained for B220, IgA, CD138and S1P1respectively by immunohistochemical methods.2. The expression of S1P1receptor were detected on membrane of B cells at differentstages (B220+IgA-B cells, B220+IgA+B cells, B220-IgA+plasma blast) using flowcytometry.3. PBMCs were isolated from spleens of BALB/c mice by Ficoll-Hypaque gradientcentrifugation and acquired B220+cells were sorted under sterile condition. Then Istimulated them with IL-4200ng/107cells, IL-21100ng/107cells and TGF-β15ng/107cells.Cells were harvested respectively after incubation for0hour,24hours,48hours,72hours,96hours and120hours. 4. Cells were harvested and divided into two parts, one part from IL-4、IL-21stimulatedgroup were used to study S1P1receptor on membrane of B220+cells from each groupusing flow cytometry. The other part from IL-4、IL-21、TGF-β1stimulated group weresubjected to extraction of total protein for western-blot of S1P1.Result:1. The immunohistochemical staining image showed a lot of B220+cells were locatedin the Germinal Center (GC) of Peyer’s patches, and IgA+cells are scattering in both darkband and light band, some of them gathered in light band. CD138+cells were scattered inthe whole GC. Meanwhile, more S1P1+cells were in light band than in dark band.2. Flow cytometry assay demonstrated the percentage of S1P1+cells was9.673±4.302in B220+IgA-B cells,36.39±9.193in B220+IgA+B cells,52.91±8.185in B220-IgA+plasma blasts.3. After the stimulation of IL-4, IL-21respectively for24hours,48hours,72hours,96hours. Flow cytometry assay shows that IL-4group’s S1P1+percent from0hour to72hours is3.02±1.232，6.23±3.600，5.75±3.216，11.86±3.614, which rises slowly, then itincreases significantly to38.36±5.558in96hours. Similarly, S1P1+percent of IL-21groupand IL-4and IL-21group increase to as high as46.77±7.893and43.77±3.316in96hours.However, S1P1+percent of control group just increases to15.16±4.327in96hours.4. Western-blot showed that the expressions of S1P1in B220+B cells were upregulatedby TGF-β1，IL-4and IL-21. But the increasing trend becomes flat after incubation withIL-4and IL-21for96hours and120hours.Conclusion:The above results demonstrate that B lymphocytes in Peyer’s Patches are becomingmature and migrate from the dark band to the light band of Germinal Center where they canbe activated by FDC and CD4+T lymphocyte and differentiated into plasmocytes. Alongwith the maturation of B cells, the expression of S1P1was upregulated in order to emigratefrom the lymphatic tissues. In addition, Tfh cell-secreted cytokines like TGF-β1，IL-4and IL-21can promote the expression of S1P1on B lymphocytes. However, after stimulationby IL-4and IL-21for over96hours, the expression trend to be flat, which predicts asaturated role of IL-4and IL-21in the expression of S1P1.