IL21 Dysregulation With Systemic Lupus Erythematosus

BackgroundWhen activatied B cells enter the light zone of germinal center (GC), they receive the selection provided by antigen-specific follicular T helper cell (Tfh) and follicular dentritic cell(fDC), which is one important mechanisms by which auto-reactive B cells are eliminated. In GC, B cells need to down-regulate CXCR4in order to migrate from dark zone to light zone. Tfhs in GC secrete IL21, an important regulative factor of B cell activity. IL21down-regulates CXCR4expression in B cells of rats. For B cells lacking specific BCR stimulation and appropriate T cell help, IL21inhibits their proliferation and/or initiates apoptosis, which may be an important mechanism to eliminate auto-reactive B cell. To see whether IL21regulates the expression of CXCR4and CXCR5on human B cells, and whether the abnormal expression of CXCR4and CXCR5on B cells of SLE(systemic lupus erythematosus) patients is related with IL21, causing B lymphocytes of SLE unable to enter light zone from dark zone of GC and auto-reactive B cells unable to be eliminated, we analyzed the change of CXCR4/5on SLE peripheral B lymphocytes following stimulation with IL21,CD40L and anti-IgM, and compared that with HC. To see whether the Akt is hyper-activated in B cells of SLE, and whether the negative-regulation of IL21toward Akt pathway is defective, and whether this causes the abnormal effect of IL21on CXCR4and the cells’ differentiation and survival, we compared how IL21acts differently to influence the pAkt, pStat3, and differentiation, survival of SLE and HC peripheral B lymphocytes, and we blocked the abnormally activated pAkt in SLE B lymphocytes with LY294002, a PI3K inhibitor, to see if the abnormal function of IL21was indeed caused by hyper-activation of Akt. To further research the possible mechanism that IL21abnormally regulates apoptosis in SLE, we detected the expression of two anti-apoptotic proteins mcll and flip which are regulated by Akt pathway under stimulation.MethodB lymphocytes from peripheral blood of SLE patients and health controls were cultured with the following stimulation:control, IL21, anti-IgM+CD40L, anti-IgM+CD40L+IL21. CXCR4and CXCR5were detected with flow cytometry at day3; intracellular pAkt and pStat3were detected at20minutes; B cell subpopulations were detected at day6; apoptosis was detected at day2. B lymphocytes of SLE are stimulated for another6days after being treated with LY294002for1hour, and then detected for changes of B cell subpopulations.Results1. CXCR4of HC B lymphocytes can be significantly down-regulated following stimulation of anti-IgM+CD40L or IL21(p<0.0001and p=0.0096, respectively) while that of SLE B lymphocytes can’t be effectively down-regulated (p=0.2575and p=0.2361, respectively)2. IL21can significantly up-regulate CXCR5and Stat3on HC and SLE peripheral B lymphocytes, with or without anti-IgM+CD40L.(CXCR5:for IL21group, HC p=0.0263, SLE p=0.0605; for anti-IgM+CD40LplusIL21group, HC p=0.0264, SLE p=0.0214. pStat3:for IL21group, HC p=0.0153, SLE p=0.0634; for anti-IgM+CD40LplusIL21group, HC p=0.0142, SLE p=0.0375). IL21can still up-regulate CXCR5after administrating LY294002, but can’t anymore after administrating AG490.3. Stimulation with IL21itself for20minutes can up-regulate pAkt in peripheral B cells of SLE, but has no significant effect on that of HC. Stimulation with anti-IgM+CD40L significantly up-regulated the pAkt in peripheral B cells of HC, and adding of IL21significantly down-regulated pAkt. Stimulation with anti-IgM+CD40L up-regulated pAkt in B cells of SLE up-regulated less significantly, and adding of IL21couldn’t down-regulate pAkt.4. After stimulation with anti-IgM+CD40L for3days, expression of mcll and flip in peripheral B lymphocytes of HC and SLE significantly increased, and the fold of increase in SLE is significantly greater than that in HC (mcll:p=0.0060, fold of increase, HC1.97±1.91, SLE4.71±2.63). Adding of IL21to anti-IgM+CD40L increased mcll in HC after3days (p=0.0242) while couldn’t change mcll in SLE significantly.5. After stimulation with IL21for6days, the percentate of IgD+CD38hi T1group、 CD27+CD38hi PC group and CD38+CD138+PC increased in peripheral B cell of SLE, and remained unchanged or decreased in HC. With pre-administration of LY294002to B lymphocytes of SLE, IL21couldn’t increase the percentage of these subgroups. 6. Stimulation with1121for2days induced the apoptosis of HC B cells, and inhibited the apoptosis of SLE B cells.ConclusionIn B lymphocytes of SLE, Akt is over-activated, and the regulation of IL21toward Akt pathway is abnormal. This may cause the defective down-regulation of CXCR4on B lymphocytes of SLE when stimulation with anti-IgM+CD40L or IL21, and the ability of IL21itself to promote the differentiation towards plasma cell and to inhibit apoptosis in peripheral B lymphocytes of SLE. The abnormal regulation of IL21toward differentiation and apoptosis can be iliminated after blocking the over-activity of Akt in B lymphocytes of SLE. The defective down-regulation of B cells of SLE may cause them unable to enter light zone from dark aone, thus escaping the selection and entering into peripheral; the abnormal effect of IL21on differentiation and apoptosis may turn B cells into plasma cells without T cell help, and may help B cells escape apoptosis. In all, the abnormal regulation of IL21of B cells in SLE may imply the mechanism by which auto-reactive B cells are generated in SLE.