Immunomodulatory Effect Of Koumine On T Cell-Dependent And-Independent Activation Of B Lymphocytes

Rheumatoid arthritis（RA）is a systemic and autoimmune disease mainly caused by inflammatory changes in synovial tissue.Its pathogenesis is complex and has not been fully elucidated.Early understanding of the pathogenic mechanism of RA was mainly focused on the immunodominant pattern of CD4⁺T cells,ie T helper cells（Th）.With the success of rituximab,which targeting B lymphocyte,in RA treatment,the important role of B lymphocytes in the pathogenesis of RA has gradually become highlighted.B cells participate in the regulation of immunity mainly through mediating humoral immunity.According to the participation of T cells,humoral immunity can be divided into T cell-dependent（TD）and non-dependent（TI）types.In the early researchs,the research group found that koumine,one of alkaloid monomer in Gelsemium elegans Benth.,showed a significant anti-RA effect and significantly inhibited the increase of serum anti-typeⅡcollagen IgG autoantibodies in typeⅡcollagen-induced arthritis（CIA）rats,suggesting that the koumine might have a regulatory effect on B cell-mediated humoral immunity,but the specific role and mechanism are not yet clear.This paper established TI and TD B-lymphocyte activation models in vitro and in vivo respectively to explore the immunomodulatory effects and functional characteristics of koumine on B lymphocytes,with a view to elucidating the anti-RA mechanism of koumine more comprehensively and provideing a scientific basis for its application in the treatment of RA.1 Inhibitory effects of koumine on activation,proliferation,differentiation and immunoglobulin secretion of T cell-dependent and independent primary B lymphocytesPrimary mouse B lymphocytes,as the research object,were obtained by immunomagnetic bead method.The TI and TD B cell activation models were induced by lipopolysaccharide（LPS）and anti-CD40 mAbs respectivcely,different concentrations of koumine（10、40、70μmol/L）were given simultaneously.Cell proliferation was detected by 5-bromodeoxy Uridine（Brd U）incorporation assay,the relative number of cells（cell surface CD69 and CD86 were used as early activation and activation markers and CD138 as differentiation markers）was detected by flow cytometry,the relative concentration of immunoglobulins in the cell culture supernatant was detected by the LEGENDplex ^TMassay to observe the effects of koumine on activation,proliferation,differentiation andimmunoglobulin secretion of T cell-dependent and-independent B lymphocytes in vitro.The results showed that the percentage of CD69⁺and CD86⁺cell in B lymphocytes induced by LPS or anti-CD40 mAbs was significantly increased,suggesting successful establishment of T cell-independent and-dependent B lymphocytes activation model of B lymphocytes,in which activation and differentiation effects of the TI model were weaker than those of the TD model,but differentiation and antibody secretion reactions were stronger than those of the TD model.In TI model induced by LPS,koumine had no signifigant effects on activation and proliferation of B lymphocytes,but had a significant concentration-dependent inhibition effect on differentiation of B lymphocytes and secretion of IgM,IgG2b and IgG3;While in TD model induced by anti-CD40 mAbs,koumine had a significant concentration-dependent inhibition effect on activation and proliferation of B lymphocytes and secretion of IgM in a concentration-dependent manner and had no significant effects on cell differentiation.2 Regulation effects of koumine on TI and TD humoral immune response2.1 Regulation effect of koumine on TI humoral immune responseC57BL/6J mice at 8-10 weeks were immunized with classic TI antigen NP-AECM-FICOLL for the establishment of TI humoral immune model,and the level of serum total antibody IgM,IgG3 and NP-specific antibodies NP-IgM and NP-IgG3 of the model were observed.The animals were divided into five groups with a control group,a humoral immune model group,and three koumine treatment model groups（0.4,2,and 10 mg/kg）.Mice were i.p.immunized with 100μg NP-AECM-FICOLL to establish TI humoral immune model.Immunized mice received koumine or vehicle by gastric gavage once a day for 7 consecutive days,from 1 h before injection on the experimental day（D0）and D1-6 days after injection.Mice were sacrificed and serum samples were collected 1 h after the completion of treatment and the antibody level was detected by ELISA.The results showed that in the TI immune response caused by NP-AECM-FICOLL,koumine had no significant effects on the total serum IgM and IgG3 of model mice,but had a significant dose-dependent inhibitory effect on the increase of NP-IgM and NP-IgG3.2.2 Regulation effects of koumine on TD humoral immune response2.2.1 Regulation effect of koumine on primary response of TD humoral immunityC57BL/6J mice at 8-10 weeks were immunized with classic TD antigen NP-CGG for the establishment of TD humoral immune model,and the level of serum total antibody IgM,IgG1 and NP-specific antibodies NP-IgM and NP-IgG1 of the model were observed.The animals were divided into five groups with a control group,a humoral immune model group,and three koumine treatment model groups（0.4,2,and10 mg/kg）.Mice were i.p.immunized with 100μg NP-CGG in alum to establish TD humoral immune model.Immunized mice received koumine or vehicle by gastric gavage once a day for 10 consecutive days,from 4 days after immunization.Mice were sacrificed and serum samples were collected 1 h after the completion of treatment and the antibody level was detected by ELISA.The results showed that total serum IgM level in mice treated with 2 mg/kg koumine and the IgG1 level in mice treated with 0.4mg/kg koumine were significantly lower than the model group;Koumine had a significant dose-dependent inhibitory effect on the increase of NP-IgG1.2.2.2 Regulation effect of koumine on secondary response of TD humoral immunityC57BL/6J mice at 8-10 weeks were immunized with NP-CGG for the establishment of TD humoral immune model.The animals were divided into five groups with a control group,a humoral immune model group,and three koumine treatment model groups（0.4,2,and 10 mg/kg）.C57BL/6J mice were i.p.immunized with 50μg NP-CGG in alum and boosted with 50μg NP-CGG in alum on D10 to establish TD humoral immune model.Immunized mice received koumine or vehicle by gastric gavage once a day for 10 consecutive days,from 4 days after first immunization.Mice were sacrificed and serum samples were collected 1 h after the completion of treatment and the antibody level was detected by ELISA,the proportion of GC B cells and isotype-switched GC B cells in the spleen of the mice was measured by flow cytometry.The results showed that koumine had no effects on the total serum IgM and IgG3 and NP-IgG3 level in model mice,but had a significant dose-dependent inhibitory effect on NP-IgM with no effects on the formation of the spleen germinal centers（GCs）and the survival and development of B cells.In summary,these results demonstrate that koumine has direct and indirect immunomodulatory effects on activated B lymphocytes.It can directly inhibit differentiation of B cells and autoantibody secretion under abnormal activation,and indirectly inhibit T cell-dependent activation and proliferation of B lymphocytes and then antibody secretion,which may be one of the important ways of anti-RA of koumine.Koumine has regulatory effects on both T cell-dependent and-independent humoral immunity,which normalizes the immune response mediated by abnormal activation of B-lymphocytes and reduce body damage caused by excessive secretion of autoantibodies with no significant effect on the survival,development of B lymphocytes and formation of GC.