Apoptosis,Proliferation,Metabolism And Clinical Significance Of CD19~+B Cells In AIHA/Evans Syndrome Patients

Autoimmune hemolytic anemia(AIHA)/Evans syndrome is caused by the body’s immune function disorder produces its own red blood cells and/or anti-platelet antibodies of autoimmune disease.Our previous study found that the number of CD5~+B lymphocytes was positively correlated with the severity of AIHA/Evans syndrome.It has been investigated that down-regulating the activity of B lymphocytes by inducing the apoptosis of CD5~+B lymphocytes can improve the clinical symptoms of patients with immune hemolytic anemia.Whether CD5~+B lymphocyte apoptosis is similar to CD5~-B lymphocytes in patients with AIHA/Evans syndrome.In this study,the expression of bcl-2 and bcl-xl,the activity of caspase-3,and the proportion of CD19~+B lymphocyte apoptosis cells in peripheral blood of AIHA/Evans syndrome patients and healthy controls were determined.The proliferation function and metabolic of CD19~+B lymphocytes in AIHA/Evans syndrome patients were detected,and the correlation between the indexes and clinical indexes were analyzed.So this study is divided into three aspects of CD19~+B lymphocytes apoptosis,proliferation and metabolism.Part 1 Apoptosis of CD19~+B lymphocytes and its clinical significance in patients with AIHA/Evans syndromeObjective:The expression level of bcl-2 and bcl-xl in CD19~+B lymphocytes and the apoptosis of CD19~+B lymphocytes in peripheral blood of patients with AIHA/Evans syndrome were detected to understand the apoptosis level of CD19~+B in peripheral blood of patients with AIHA/Evans syndrome.Methods:The subjects were patients with AIHA/Evans syndrome(hemolytic group:25 cases;remission group:15 cases)and 14 healthy controls.The expression of bcl-2and bcl-xl in CD19~+B lymphocytes and the number of apoptosis of CD19~+B lymphocytes in peripheral blood were detected by FCM.CD19~+B lymphocytes were sorted by magnetic activated cell sorting(MACS).The levels of bcl-2 and bcl-xl mRNA in CD19~+B lymphocytes were detected by RT-PCR.The activity of caspase-3in CD19~+B lymphocytes was detected by a microplate assay,and the correlation between the detection indexes and clinical indexes was analyzed.Results:1.The CD19~+CD5~+bcl-2~+/CD19~+CD5~+lymphocyte ratio in hemolytic patients[(17.40±25.02)%]was lower than that in remission patients[(35.74±35.79)%],but higher than that in healthy control group[(16.48±30.31)%].There were no significant difference among the three groups(P>0.05).The CD19~+CD5~+bcl-2~+/CD19~+lymphocyte ratio in hemolytic group(0.73±1.62)%],remission group[(1.41±2.14)%]and healthy group[(1.39±2.74)%]showed no statistical difference(P>0.05).The CD19~+CD5~+bcl-2~+/CD19~+lymphocyte ratio in AIHA/Evans syndrome patients[(0.98±1.84)%]was lower than that in healthy controls[(1.39±2.74)%],the difference was statistically significant(P<0.01).The ratio of CD19~+CD5~+bcl-2~+/CD19~+in hemolytic group was positively correlated with IgE(P=0.0046).The ratio of CD19~+CD5~+bcl-2~+/CD19~+CD5~+in remission group was positively correlated with TBIL,DBIL,IBIL,Ret%and absolute value of eosinophil(P<0.05).2.The ratio of CD19~+CD5~-bcl-2~+/CD19~+CD5~-in hemolytic group[(16.71±22.69)%]and remission group[(28.65±19.49)%]and healthy controls[(21.36±25.81)%]do not differ significantly.The ratio of CD19~+CD5~-bcl-2~+/CD19~+in hemolytic group[(8.33±20.54)%]was lower than that in remission group[(14.58±14.75)%]and healthy control group[(10.93±20.17)%].There was not statistically significant among the three groups(P>0.05).CD19~+CD5~-bcl-2~+/CD19~+cells in hemolytic group was positively correlated with DBIL(P=0.0447),and negatively correlated with RBC(P=0.0478).CD19~+CD5~-bcl-2~+/CD19~+CD5~-cells in hemolytic group was negatively correlated with Hb(P=0.0230).The ratio of CD19~+CD5~-bcl-2~+/CD19~+in remission group was positively correlated with TBIL,DBIL,IBIL,Ret%,absolute Ret value(P<0.05)and negatively correlated with C4,Hb(P<0.05).3.The expression level of bcl-2 mRNA in CD19~+B lymphocytes of hemolytic patients(1.19±1.46)was higher than that in remission patients(1.11±1.50)and healthy controls(0.22±0.17),but lower than that in the CLL group(4.08±1.75),but the difference was no statistically significant(P>0.05).Bcl-2 mRNA expression in CLL group was significantly higher than that in healthy group(P<0.05),and the difference was statistically significant.4.CD19~+CD5~+bcl-xl~+/CD19~+CD5~+cells in hemolytic group[(12.79±29.14)%]were higher than that in remission group[(12.79±29.14)%]and lower than that in healthy group[(15.94±10.56)%],and the difference was not statistically significant(P>0.05).CD19~+CD5~+bcl-xl~+/CD19~+cells in hemolytic group was higher than that in remission group[(0.01±0.03)%]and lower than that in healthy group[(1.36±4.10)%],there was no statistically significant difference among the three groups(P>0.05).5.CD19~+CD5~-bcl-xl~+/CD19~+CD5~-cells in hemolytic group[(1.05±3.87)%]and remission group[(0.02±0.06)%]and healthy group[(0.96±2.96)%]showed no significantly difference(P>0.05).CD19~+CD5~-bcl-xl~+/CD19~+cells in hemolytic group[(1.05±3.87)%]was higher than that in the remission group[(0.01±0.03)%]and the healthy control group[(0.49±1.84)%],there was no significant difference among the three groups(P>0.05).6.The expression level of bcl-xl mRNA in CD19~+B lymphocytes of hemolytic group(2.71±6.15)and remission group(2.27±2.97)were higher than that in healthy group(0.89±0.29),and lower than that in the CLL group(3.45±2.97).There was no significant difference among the four groups(P>0.05).7.CD19~+CD5~+apoptotic cells in hemolytic group[(20.16±26.26)%]was higher than that in remission group[(8.41±10.13)%]and healthy group[(5.24±5.71)%].There was a statistical difference between the hemolytic group and the healthy control group(P=0.05).CD19~+CD5~-apoptotic cells in hemolytic group[(1.77±4.19)%]and remission group[(1.57±4.43)%]and healthy group[(1.48±1.38)%]showed no significantly difference(P>0.05).The proportion of CD19~+CD5~+apoptotic cells in AIHA/Evans syndrome patients was higher than that in CD19~+CD5~-apoptotic cells,and the difference was statistically significant(P=0.004),while the apoptosis of CD5~+and CD5~-B lymphocytes in the remission group and the healthy control group showed no statistical difference(P>0.05).The number of CD19~+B apoptosis cells in hemolytic group was positively correlated with DBIL(P=0.0021).The apoptosis of CD19~+CD5~-B cells was positively correlated with DBIL(P=0.0025),but not with other clinical indicators.8.The activity of caspase 3 in CD19~+B cells of AIHA/Evans syndrome patients in the hemolytic group(23.32±21.76)was lower than that in healthy group(54.01±13.71).There was a statistical difference between hemolytic group and healthy group(P=0.026).Conclusion:The expression levels of bcl-2 and bcl-xl in CD19~+B lymphocytes in patients with AIHA/Evans syndrome were higher than those in healthy controls.Part 2 Proliferation of CD19~+B lymphocytes and its clinical significance in patients with AIHA/Evans syndromeObjective:The proliferation of CD19~+B lymphocytes in patients with AIHA/Evans syndrome was detected,and its correlation with clinical indicators was analyzed to understand the proliferation function of CD19~+B lymphocytes in patients with AIHA/Evans syndrome.Methods:9 AIHA/Evans syndrome patients and 6 healthy controls were enrolled in this study.CD19~+B lymphocytes were selected by MACS,and the proliferation of CD19~+B lymphocytes were detected by CCK-8 methods,and their correlation with clinical indicators was analyzed.Results:The OD value of CD19~+B lymphocyte proliferation in patients with AIHA/Evans syndrome(0.17±0.03)was higher than that in healthy controls(0.10±0.01,P=0.001).Conclusion:The proliferation of CD19~+B lymphocytes in patients of AIHA/Evans syndrome was higher than that in the healthy controls.Part 3 Metabolism of CD19~+B lymphocytes and its clinical significance in patients with AIHA/Evans syndromeObjective:The expression level of PKM2 in B1 lymphocyte and B2 lymphocyte in peripheral blood of AIHA/Evans syndrome patients was detected to understand the metabolism level of CD19~+B in peripheral blood of AIHA/Evans syndrome patients.Methods:11 hemolytic patients,8 remission patients and 10 healthy controls were enrolled in this study.The expression of PKM2 in B1 cells and B2 cells in peripheral blood of AIHA/Evans syndrome patients were detected by FCM.Results:1.The ratio of CD19~+CD5~+PKM2~+/CD19~+CD5~+in hemolytic group[(83.17±30.87)%]was higher than that in remission group[(76.41±37.31)%]and healthy group[(77.34±30.08)%],but there was no statistical difference(P>0.05).The ratio of CD19~+CD5~+PKM2~+/CD19~+in hemolytic group[(5.26±11.64)%],which was higher than that in remission group[(1.83±2.75)%]and healthy group[(2.09±1.69)%],there also was no statistical difference(P>0.05).The ratio of CD19~+CD5~+PKM2~+/CD19~+in hemolytic group was positively correlated with RBC,IgE,and IgA.The ratio of CD19~+CD5~-PKM2~+/CD19~+CD5~-among hemolytic group[(75.42±29.48)%],remission group[(66.20±41.45)%]and healthy group[(56.58±30.07)%]showed no differ statistically(P>0.05).The ratio of CD19~+CD5~-PKM2~+/CD19~+among hemolytic group[(69.13±35.45)%],remission group[(64.99±40.83)%]and healthy group[(52.98±31.25)%]was no significantly difference(P>0.05).2.PKM2 in CD5~+B lymphocytes were statistically lower than that in CD5~-B lymphocytes in three groups.Conclusion:The level of PKM2 in CD19~+B lymphocyte of hemolytic patients was higher than that in remission group and healthy group,suggesting that PKM2 may promote the proliferation of CD19~+B lymphocyte in AIHA/Evans syndrome patients through multiple ways.