The Effects Of B Cell Activating Factor In Immune Thrombocytopenia

英文摘要ⅠThe effects of BAFF and BAFF-R-Fc fusion protein in immune thrombocytopeniaImmune thrombocytopenia (ITP) is an autoimmune disorder in which the patients'immune system is activated by platelet autoantigens resulting in immune-mediated platelet destruction and/or suppression of platelet production. In addition, several abnormalities involving the cellular mechanisms of immune modulation such as Th1 bias, the decreased number or defective function of regulatory T cells and the platelet destruction by cytotoxic T cells (CTL) have been described.B-cell activating factor (BAFF) (also known as BlyS, TALL-1, THANK, zTNF4 and TNFSF13B) belonging to the family of tumor necrosis factor (TNF) ligands is critical for the maintain of normal B-cell development, homeostasis, autoreactivity and T cell costimulation. In addition, it also augments certain Th1-associated inflammatory responses. BAFF binds to three receptors:B-cell maturation antigen (BCMA, TNFRSF17), transmembrane activator and calcium-modulating cyclophilin ligand (CAML) interactor (TACI, TNFRSF13B) and BAFF receptor (BR3/BAFF-R, TNFRSF13C). BR3, identified as the crucial receptor for B-cell survival, is expressed on a wide range of B-cell subsets, including immature, transitional, mature, memory and germinal center B cells, as well as on plasma cells. Furthermore, BAFF binding to BR3 on T cells has been shown to costimulate T-cell proliferation both in vitro and in vivo.Several lines of evidence suggested that BAFF may play an important role in autoimmunity. Autoantigen-binding B cells may have an increased dependence on the BAFF survival signal. In addition, elevated BAFF plasma level was observed in many patients with autoimmune diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Sjogren's syndrome (SS), and multiple sclerosis (MS). Inhibition of BAFF signaling is a potentially therapeutic option for treatment of B cell-mediated autoimmune conditions. Data from animal tests and clinical trials had proved that blockade of BAFF by blocking reagents (TACI-Ig, BAFF-R-Ig, BR3-Fc) was effective therapeutic approach for some autoimmune diseases.Objective:To investigate the possible mechanism of BAFF and therapeutic effect of BR3-Fc in ITP, we measured the expression of plasma BAFF and BAFF mRNA, and the effects of rhBAFF and BR3-Fc on B cells, T cells, platelets, secretion of IFN-γand IL-4 were measured by flow cytometry and ELISA.Methods:◆Forty-five patients diagnosed with ITP were selected for detection of plasma BAFF and BAFF mRNA. Of these patients, twenty-five patients were active ITP patients who had not been treated with GCs for at least one month prior to sampling whereas twenty patients were in remission. Twenty-four healthy controls matched for sex and age with the study population were voluntary blood donors.◆The expression of plasma BAFF and BAFF mRNA in ITP patients and controls were measured by ELISA and RT-PCR, the levels of plasma anti-platelet autoantibodies (GPIIb/IIIa and GPIb/IX) were measured by MAIPA.◆Peripheral blood mononuclear cells (PBMCs) and platelets from additional eighteen ITP patients with active disease and fifteen controls were selected for detection of apoptosis on CD19+, CD4+, CD8+ cells, platelets, secretion of cytokines (IFN-y and IL-4) and autoantibodies by flow cytometry and ELISA.Results:◆Elevated levels of plasma BAFF and BAFF mRNA in active ITP patientsThe level of plasma BAFF in ITP patients with active disease was significantly higher (mean±SD,593.1±219.0 pg/ml) than that in patients in remission (432.5±121.4pg/ml, P＜0.05) and controls (454.4±132.5pg/ml, P＜0.05). The relative amount of BAFF mRNA in patients with active disease was increased 3.1 and 2.5-fold compared to patients in remission (P＜0.01) and healthy controls (P<0.01), respectively. No significant difference of plasma BAFF or BAFF mRNA between patients in remission and healthy controls was found (P>0.05).In addition, no association was found between the levels of BAFF and anti-platelet autoantibodies (P>0.05).◆RhBAFF promotes the survival of CD19+ cells, BR3-Fc corrects its effectRhBAFF significantly decreased the annexin V% of CD19+ cells in ITP patients (groupⅡ:7.0±4.3% vs groupⅠ:13.2±7.4%, P＜0.05) but not in controls. BR3-Fc corrected the effect of rhBAFF on apoptosis of CD19+ cells (groupIII:11.2±4.1% vs groupⅠ:13.2±7.4%, P>0.05).◆RhBAFF promotes the survival of CD8+ cells, BR3-Fc corrects its effectRhBAFF significantly decreased the annexin V% of CD8+ cells in both ITP patients and controls. BR3-Fc corrected the effect of rhBAFF on apoptosis of CD8+ cells only in ITP patients. The annexin V% on CD8+ cells in ITP patients in group I, group II and group III were 6.5±3.2%,4.4±2.2%and 6.3±2.9%, respectively, The annexin V% on CD8+ cells in controls in group I, group II and groupIII were 10.5±2.7%,8.3±3.2%and 8.9±4.0%, respectively.◆No significant effect of rhBAFF and BR3-Fc on apoptosis of CD4+ cellsThere was no significant effect of rhBAFF on annexin V% of CD4+ cells in both ITP patients and controls (P>0.05). The annexin V% of CD4+ cells in ITP patients in group I, group II and group III were 8.6±4.5%,5.3±1.8% and 8.2±3.8%, respectively.◆RhBAFF promotes the apoptosis of platelets, BR3-Fc corrects its effectRhBAFF significantly increased apoptosis of platelets in ITP patients (group II: 8.4±4.9% vs group I:3.2±1.0%, P＜0.05) but not in controls. BR3-Fc corrected the effect of rhBAFF on apoptosis of platelets(group III:5.0±3.0% vs group II:8.4± 4.9%, P＜0.05). In order to further confirm the results, we also measured the apoptosis of platelets by mitochondrial membrane potential assay kit with JC-1 which was a more precise method for detection of platelet apoptosis. Similar results were found.◆Effects of rhBAFF and/or BR3-Fc on secretion of cytokines by PBMCsThe levels of IFN-γand IL-4 in supernatant were measured by ELISA. RhBAFF promoted the secretion of IFN-γin the presence of PHA (10μg/ml) (P<0.05) in ITP patients but not controls, and combination of BR3-Fc and rhBAFF reduced the level of IFN-γcompared with group rhBAFF (20ng/ml) (P<0.05). The mean±SD of group I was 74.0±12.5pg/ml, and it increased to 95.1±25.7pg/ml in group II, and reduced to 82.4±17.4pg/ml in groupⅢ, similar to that in group I. There was no detectable level of IFN-y when incubating cells without PHA. The level of IL-4 was below the detectable limit of the assay used.Conclusions:◆The expression of BAFF is elevated in active ITP patients, and the expression of BAFF correlates to disease activity.◆BAFF may play a pathogenic role in ITP by promoting the survival of CD19+ and CD8+ cells, increasing the apoptosis of platelets and the secretion of IFN-γ.◆Blockade of BAFF by BR3-Fc might be a promising therapeutic approach for ITP. 英文摘要ⅡHigh dose dexamethasone inhibit BAFF expression in patients with immune thrombocytopeniaThe treatment regimens for ITP include glucorticosteroids (GCs), intravenous immunoglobulin (IVIg), intravenous anti-D immunoglobulin, splenectomy, danazol and other immunosuppressive drugs in which GCs have been widely recognized as the most appropriate first line treatment. Recently, HD-DXM has been used as the first-line therapy for adult patients with ITP, which has a higher response rate than conventional prednisone doses Some studies have showed that the treatment with GCs induces a marked decrease in BAFF levels in patients with SLE and bullous pemphigoid. Recently, a study about the inhibition of DXM on BAFF in fibroblast-like synoviocytes from patients with RA has been reported, which showed DXM inhibited the expression of BAFF in a dose-and time-dependent manner. Up to date, there is no data about the effect of HD-DXM on BAFF expression.Objective:To investigate the effects of HD-DXM on BAFF expression and its possible mechanismMethods:◆20 acitive ITP patients were enrolled in this study. Blood sampling was performed before and after treatment at the end of the second week with HD-DXM. Twenty-four healthy controls were voluntary blood donors.◆The expression of plasma BAFF and BAFF mRNA in ITP patients and controls were measured by ELISA and RT-PCR.◆Moreover, we evaluated the effects of DXM on BAFF expression, secretion of IFN-γand IL-4, and proliferation of lymphocytes by ELISA, real time quantitative PCR and cell proliferation assay [cell counting kit-8 (CCK-8)] respectively in in vitro experiment. Results:◆Clinical therapeutic effect of HD-DXMResponses were reached in patients:CR in 16 (80%), R in 3 (15%), and NR in 1 (5%). The mean platelet count was 165×109/L (range 23 to 332×109/L) two weeks after the initiation of treatment.◆Decreased expression of BAFF in active ITP patients with HD-DXM treatmentBoth plasma BAFF and its mRNA expression are elevated in active ITP patients before HD-DXM treatment. And plasma BAFF correlated with its mRNA levels in active ITP patients (r=0.45, P＜0.01).After administration of HD-DXM, plasma BAFF levels in ITP patients were significantly decreased (297±120 pg/ml) compared with pretherapy (597±197 pg/ml, P＜0.001), and lower than that of the normal controls (454±132 pg/ml, P＜0.001).Similar results were found on BAFF mRNA levels. After administration of HD-DXM, the relative amount of BAFF mRNA was 0.32 fold and 0.13 fold of that of healthy controls (P<0.001) and untreated patients respectively (P<0.001).◆Changes of BAFF correlated with clinical responsesAfter administration of HD-DXM, eighteen of twenty patients had reduced plasma BAFF level, with two patients who relapsed shortly after discontinuation of the therapy and later received splenectomy had similar or even higher expression.◆Expression of BAFF is inhibited by DXM in vitroBecause BAFF is elevated in untreated ITP patients, and after administration of HD-DXM, it reduced significantly, we investigated whether DXM, a most used glucocorticoids for ITP therapy, is able to inhibit the expression of BAFF. We cultured PBMCs from 10 untreated ITP patients with active disease and 11 controls were adjusted to 1×106/ml in RPMI-1640 culture medium, cultured at a density of 1×106 cells/well in a 24-well culture plate and incubated with different concentrations of DXM at 37℃with 5% CO2, after 72h, cells were harvested for RT-PCR.The results showed that DXM suppressed the expression of BAFF in a dose-dependent manner.◆Effects of DXM on the expression of IFN-γand IL-4PBMCs from active ITP patients and healthy controls were cultured with different concentrations of DXM (OnM,7.8nM,15.6nM,31.3nM,62.5nM,125nM, 250nM) at 37℃with 5% CO2, with 10μg/ml phytohemagglutinin (PHA). After 72h, supernatants were collected and levels of IFN-y and IL-4 were measured. When PHA was added, levels of IFN-y were available in all samples. A dose-dependent inhibition of IFN-y by DXM was obvious. The levels of plasma IL-4 were lower than the detection limits.◆Effects of DXM on the proliferation of PBMCsAfter cultured with different concentrations of DXM (OuM,0.125uM,0.25uM, 0.5uM, 1uM,2uM and 4uM) for 72h, proliferation in active ITP patients and controls was assayed by CCK-8. The results showed DXM inhibited the proliferation of PBMCs of both patients and controls, reaching about 50% of inhibition with 0.5uM DXM, and about 75% inhibition with 2uM DXM in active ITP patients. Similar results in healthy controls were observed.◆Correlation of BAFF with clinical and laboratory parameters in ITP patientsCorrelations between levels of BAFF and platelet counts were analyzed in ITP patients, and no significant associations were found (P>0.05). There was no significant correlation between changes of BAFF and platelets.Conclusions:◆HD-DXM inhibits the expression of plasma BAFF and its mRNA levels in ITP patients;◆DXM inhibits the expression of BAFF, IFN-y and proliferation of lymphocytes in vitro. 英文摘要ⅢElevated interleukin-21 correlated to Th17 and Th1 cells in patients with immune thrombocytopeniaIL-21, a recently discovered cytokine, was originally thought to be restricted to CD4+ T cells (Th1 and Th2 cells) and NKT cells, but it is now clear that IL-21 is also produced by Th17 cells. IL-21/IL-21R system is involved in the regulation of central functions of the immune system, including promoting T-cell-mediated humoral immune responses and antibody production, increasing the cytolytic potential of NK cells, promoting the differentiation of naive Th cells into Th17 cells. Data from mouse models have suggested that IL-21 promotes autoimmunity and blocking the action of IL-21 holds promises in a number of disease settings. IL-21 and/or IL-21 R are also implicated in the pathogenesis of some autoimmune diseases such as SLE, RA, SS in humans.Up to date, there is no data about IL-21 in patients with ITP. To further investigate the possible role of IL-21 in the pathogenesis of ITP, we measured the levels of IL-21 and correlated its levels to Th17 cells, Th1 cells and Tel cells. Objective:To investigate the possible role of IL-21, Th17, Th1 and Tc1 cells, as well as their relationship in the pathogenesis of ITP, and evaluated their clinical relevanceMethods:◆Patients and controls:Twenty-six adult chronic ITP patients were enrolled by diagnostic criteria for ITP. The control group consisted of twenty-one adult healthy volunteers matched for sex and age with the study population.◆We examined the levels of CD3+CD8-IL-21+, CD3+CD8+IL-21+,Th17,Th1 and Tc1 cells in peripheral blood which was activated in vitro by PMA/ionomycin in short-term cultures in ITP patients and controls by flow cytometry through intracellular cytokines analysis. Th17 cells and Th1 cells were identified as those that were CD3+CD8-IL-17A+and CD3+CD8-IFN-γ+, and Tc1 cells were those that were CD3+CD8+IFN-γ+.◆ELISA was used to detect the expression of IL-21 in plasma.◆Anti-platelet autoantibodies were detected using the modified MAIPA assay.Results:◆Elevated IL-21 in active ITP patientsIL-21 was expressed on both CD3+CD8-T cells and CD3+CD8+T cells. The percentage of CD3+CD8-IL-21+T cells was significantly elevated in active ITP patients (mean±SD,4.4±0.8%) compared to healthy controls (2.3±0.8%, P＜0.001). Similarly, elevated percentage of CD3+CD8+IL-21+T cells was found in active ITP patients (0.8±0.5%) compared to healthy controls (0.5±0.3%, P＜0.05)We investigated plasma IL-21 by ELISA. The level of IL-21 was significantly increased in active ITP patients (148.3±124.2pg/ml) compared to controls (78.0±32.0pg/ml, P＜0.05).◆Increased expression of Th17, Th1 and Tc1 cells in active ITP patients treatmentIL-17 was expressed on CD3+CD8-T cells and IFN-y was expressed on both CD3+CD8-T cells and CD3+CD8+T cells. ITP patients had significantly increased percentage of Th17 cells, Thl cells and Tc1 cells. The percentage of Th17 cells was significantly increased in ITP patients (2.1±0.8%) compared to controls (1.2±0.5%, P＜0.05). Similarly, we found significantly increased percentage of Thl cells and Tc1 cells in ITP patients (Th1 cells:18.6±2.3%, Tc1 cells:21.4±8.4%) compared to controls (Th1 cells:10.9±2.6%, Tc1 cells:11.4±3.8%).◆Correlation between CD3+CD8-IL-21+T cells, CD3+CD8+IL-21+T cells, Th17 cells, Th1 cells and Tc1 cells in active ITP patientsWe observed that the percentage of CD3+CD8-IL-21+T cells positively correlated to Th17 cells (P＜0.01) and Thl cells (P＜0.01). No significant correlation between CD3+CD8+IL-21+T cells and Tc1 cells was found (P>0.05).◆Correlation of cells with autoantibodies in ITP patientsThe levels of CD3+CD8-IL-21+T cells, CD3+CD8+IL-21+T cells, Th17 cells, Th1 cells and Tc1 cells were not significantly different for patients who had positive MAIPA test compared to those with negative MAIPA test (P>0.05).Conclusions:◆ITP patients have elevated IL-21 level;◆Our results indicated a possible role of IL-21 in ITP patients correlated to Th17 and Th1 cells and blockade of IL-21 may be a reasonable therapeutic strategy for ITP especially those with active disease.