| Autoimmune hemolytic anemia(AIHA)/Evans syndrome is a form of immune-related cytopenia characterized by autoantibodies directed against red blood cells and/or platelet. B lymphocytes play important roles in the pathogenesis of AIHA/Evans. CD5-B lymphocytes synthesis and secretion of autoantibodies, and CD5~+B lymphocytes are found in AIHA/Evans syndrome patients [1-3]. Previous studies have shown that the number of CD5~+B lymphocytes is correlated to the severity of AIHA/Evans syndrome [4]. Many studies have showed that CD5~+B lymphocytes have immune regulation function mainly through the secretion of IL-10[5-7]. Chronic lymphocytic leukemia(CLL) is a malignant tumor of CD5~+B lymphocytes, AIHA is the most commonly complication of CLL, the incidence is5%~25% [8-11]. The mechanism of CLL complicated by AIHA remains unknown.CD5~+B lymphocytes activation status, and whether its surface activation molecules are similar to CD5-B lymphocytes and clonal CD5~+B lymphocytes, and whether CD5~+B lymphocytes can secretion of IL-10 and TGF-β1 remain unclear in AIHA/Evans syndrome patients. In this study, we investigate the expression of activation molecules CD80, CD86, CD69 and CD40 on CD5~+B lymphocytes in peripheral blood(PB) of AIHA /Evans patients, and analyze its correlation with clinical parameters. At the same time, CD5~+B lymphocytes, which secrete IL-10 and TGF-β1, are detected. So this study is divided into two aspects of CD5~+B lymphocyte activation status and secretion function.Part 1 Expression of activated molecules on CD5~+B lymphocytes and its clinical significance in autoimmune hemolytic anemiaObjective:To investigate the expression of activation molecules CD80, CD86, CD69 and CD40 on CD5~+B lymphocytes in PB of AIHA /Evans patients, and analyze its correlation with clinical parameters. To understand the activation state of CD5~+B lymphocytes in PB of AIHA/Evans syndrome patients.Methods:15 untreated, 15 remission AIHA/Evans syndrome patients, 18 normal controls(NC) and nine CLL patients were enrolled in this study. CD80, CD86, CD69 and CD40 expression on CD5~+ and CD5-B lymphocytes, and percentage of CD19~+ and CD5~+ B lymphocyte were detected using flow cytometry in PB of AIHA/Evans syndrome patients.Results:1. The percentage and the quantity of B lymphocytes in newly diagnosed patients were(19.35±16.20%,(29.73±19.92)×107/L), significantly higher than those of remission patients(6.58±4.43%,(10.96±7.98)×107/L) and NC(6.69±2.12%,(9.92±4.03)×107/L)(P<0.05). The percentage and the quantity of B lymphocytes were negatively correlated with the level of HB, and were positively correlated with percentage of reticulocyte(Ret) and LDH. The percentage of CD5~+B lymphocytes in B lymphocytes of newly diagnosed patients was 22.01±15.04%, significantly higher than those of NC(12.47±6.47%, P=0.018).2. CD80 on CD5~+B lymphocytes of newly diagnosed patients was higher than that of remission patients, NC and CLL patients(P<0.05). CD80 on CD5~+B lymphocytes was lower than that on CD5-B lymphocytes in remission patients and NC(P<0.05).CD80 on CD5~+B lymphocytes was negatively correlated with HB, C3, C4(P<0.05)and positively correlated with Ret(P<0.05).3. CD86 on CD5~+B lymphocytes of untreated patients was higher than that of remission patients(P<0.05), NC(P<0.01). CD86 on CD5~+B lymphocytes of CLL was higher than that of NC, remission(P<0.05). CD86 on CD5~+B lymphocytes of untreated patients was higher than CD86 on CD5-B lymphocytes. CD86 on CD5~+B lymphocytes was negatively correlated with HB, C3, C4(P<0.05) and positively correlated with Ret(P<0.05).4. CD69 on CD5~+ and CD5-B lymphocytes of CLL was higher than those of AIHA/Evans patients and NC(P<0.05).5. CD40 on CD5~+B lymphocytes in four groups showed no significant difference.CD40 on CD5~+ and CD5-B lymphocytes showed no significant difference in newly diagnosed patients and NC, but was lower than that of CD5-B lymphocytes in remission patients(P=0.027).Conclusions: The proportion and quantity of B lymphocytes were increased, and CD5~+B lymphocytes are in activation state in PB with AIHA/Evans patients, the active molecules on CD5~+B lymphocytes differ from those on CD5- and clonal CD5~+B lymphocytes, and CD80, CD86 play an important role in the activation of CD5~+B lymphocytes.Part 2 Study on secretion function of CD5~+B lymphocytes in autoimmune hemolytic anemiaObjective: To understand the secretion function of CD5~+B lymphocytes in PB of AIHA/Evans syndrome patients.Methods: 25 untreated, 28 remission AIHA/Evans syndrome patients and 25 NC were enrolled in this study. CD5~+ B lymphocytes, which produce and secret IL-10 and TGF-β1, was detected by flow cytometry. B lymphocytes from PB were sorted by magnetic activated cell sorting(MACS). IL-10 and TGF-β1 m RNA were analyzed by real-time PCR(RT-PCR). The level of IL-10 and TGF-β1 in plasma was detected using Enzyme-linked Immunosorbent assay(ELISA).Results:1. The CD5~+CD19~+IL-10~+/CD5~+CD19~+ lymphocyte ratio in newly diagnosed patients was 82.18±14.78%, significantly higher than remission patients(56.68±24.39%) and NC(51.90±22.95%)(P<0.05). There was no significant difference of IL-10-producing CD5-B lymphocytes among the three groups. The ratio of CD5~+CD19~+IL-10~+/CD5~+CD19~+was negatively correlated with HB, Complement 3(P<0.05) and positively correlated with LDH, TBIL, DBIL(P<0.05).2. IL-10 m RNA of B lymphocyte in newly diagnosed patients(62.34±52.84) was higher than remission patients(4.33±3.83) and NC(1.78±1.66)(P<0.05).3. The level of IL-10 in serum in newly diagnosed patients(4.01±1.54pg/ml) was lower than remission patients(5.08±1.72pg/ml) and NC(5.70±1.60pg/ml)(P<0.05).4. The quantity of CD5~+ and CD5-B lymphocytes, which secretes TGF-β1, was no significant difference between the three groups. There was no significant difference of TGF-β1 m RNA or the level of TGF-β1 in serum within the three groups.Conclusions: B regulatory cells, which produce IL-10, increased and related with hemolysis and immune function in AIHA/Evans patients, but its secretory function were destroyed. B regulatory cells, which produce TGF-β1, were not related with AIHA/Evans patients. |
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