Effects Of Ctl-Mediated Cytotoxicity On The Onset Of ITP And Effects Of Plasma From ITP Patients On Megakaryocy Topoiesis And Platelet Production

Idiopathic thrombocytopenic purpura(ITP) is one of the most common forms of autoimmune disease characterized by a low platelet count and normal or increased number of megakaryocytes in bone marrow.It is usually a persisting disease,which relapses frequently and requires a long-term treatment.In some cases,it progresses rapidly and even threatens the patients' survival.The severe side effects of routine treatment lead to a poor prognosis.The pathogenic mechanism of ITP is not completely clear yet.The earliest studies have suggested that in the majority of patients,thrombocytopenia is mediated by antiplatelet autoantibodies,in which anti-GPâ…¡b/â…¢a and GPâ… b/â…¨antibodies are generally considered to be the most common ones.Binding of autoantibodies to these target antigens eventually results in platelet destruction by the reticuloendothelial system.Since platelet autoantibodies can be detected in only 50-70%of ITP patients and remission can occur despite the presence of platelet autoantibodies,there must be other mechanisms.Olsson et al reported several cytotoxic genes,such as Apo-1/Fas, granzyme B and perforin,together with genes involve in the Th1 cell response,such as interferon-γand interleukin-2 receptor-β,showed increased expression in the ITP patients.Most recently,in vitro studies suggested that cytotoxic T-lymphocyte(CTL, CD8⁺) may be involved in the pathogenesis of chronic ITP through cell-mediated destruction of autologous platelets.On the other hand,platelet kinetic studies and morphologic alterations of ITP marrow megakaryocytes suggest that megakaryocytopoiesis and thrombopoiesis may be disrupted.Since the target antigens are present on both platelets and their precursors,megakaryocytes,it is possible that megakaryocytopoiesis and thrombopoiesis are also impaired during ITP,which could further aggravate the thrombocytopenia caused initially by increased peripheral destruction of platelets. Recent in vitro studies,showing reduced megakaryocyte production and maturation in the presence of autoantibodies against platelet glycoproteins in ITP plasma,provide evidence for autoantibody-induced suppression of megakaryocytopoiesis.In normal physiology platelet production and mature megakaryocyte apoptosis are closely related events.In disease,however,the decreased megakaryocyte apoptosis might disrupt platelet formation.Growing evidence suggests that ITP megakaryocytes demonstrate predominantly characteristics of apoptosis-like programmed cell death which contribute to thrombocytopenia.Whether the difference exits in CTL-mediated cytotoxicity toward autologous platelets between ITP patients with and without anti-platelet antibodies remains unknown.In the present work,we evaluated autoantibodies against GPâ…¡b/â…¢a and GPâ… b/â…¨in ITP patients and observed the cytotoxic effect of CTL toward autologous platelets.In addition,we prospectively measured the effect of high dose dexamethasone(HD-DXM) on CTL-mediated cytotoxicity in ITP patients.Besides,we investigated the effect of plasma from patients with ITP on in vitro megakaryocyte production.â… Effects of CTL-mediated cytotoxicity on the onset of ITP and mechanism of action of dexamethasone.Objective:To investigate the difference in cytotoxicity toward autologous platelets between ITP patients with and without autoantibodies against GPâ…¡b/â…¢a and GPâ… b/â…¨,we measured platelet autoantibodies,and performed cytotoxic T cell assay in 48 patients. In addition,we prospectively measured CD8⁺ cytotoxic T-lymphocyte mediated cytotoxicity in patients during treatment with high dose dexamethasone.Methods:* 48 chronic ITP patients were enrolled in this study.Blood sampling was performed before and after treatment at the end of the second week with high dose dexamethasone.* Platelet autoantibodies against GPâ…¡b/â…¢a and GPâ… b/â…¨were detected by modified monoclonal antibody specific immobilization of platelet antigens(MAIPA) assay.* Platelets were prepared from peripheral blood.* CD8⁺T lymphocytes were positively selected using CD₈⁺ magnetic microbeads, according to the manufacture's recommendations(Miltenyi Biotec).* The CD8⁺T lymphocytes used as effector cells and autologous platelets used as target cells were incubated for 4 h.To stimulate cytolytic effector T-cells, anti-CD₃-antibody was added.* Cells were labeled with PEcy5-CD41a mAb,PEcyS-IgG1 used as isotype control, incubated with FITC-Annexin V mAb,and then analyzed with a Becton Dickinson FACScan flow cytometer.Logarithmic amplifiers were used for fluorescence signals, and 10 000 events were collected.CD41a⁺ cells were gated and recognized as platelets,and apoptotic platelets were Annexin V⁺ cells within that population.Results were expressed in percent and analyzed using the CellQuest software.The specific lysis was calculated according to the following equation:induced lysis - spontaneous lysis,and expressed in%.Results:* In the plasma of 48 patients with ITP,antibodies against GPâ…¡b/â…¢a and/or GPâ… b/â…¨were detected in 22 samples(groupâ… ).Negative reactions to both glycoproteins were displayed in the remainders(groupâ…¡).* Before HD-DXM treatment,positive platelet lysis was seen in 11 of groupâ… and 21 of groupâ…¡(groupâ… vs.groupâ…¡,P＜0.05),while 4 of groupâ… and 7 of groupâ…¡after treatment(groupâ… vs.groupâ…¡,no significant(NS);pretreatment vs.posttreatment in groupâ… ,P＜0.05,and in groupâ…¡,P＜0.01).On the other hand,before treatment,both groupâ… and groupâ…¡had increased platelet lysis compared with controls(groupâ… vs. controls,P＜0.05;groupâ…¡vs.controls,P＜0.01;groupâ… vs.groupâ…¡,P＜0.01),whereas platelet lysis was substantially decreased in both groups after treatment(pretreatment vs.posttreatment in groupâ… ,P＜0.01,and in groupâ…¡,P＜0.01).* Additionally,the platelet lysis was found to be negatively correlated with the platelet count in groupâ…¡(r=-0.439,P＜0.05),but not in groupâ… (r=-0.322,NS).Conclusions:We conclude that cytotoxicity toward autologous platelets is predominant in autoantibody-negative ITP patients and could be thwarted by HD-DXM,and protection against CTL-mediated destruction might be considered as a new therapeutic approach for ITP.â…¡Effects of plasma from patients with ITP on in vitro megakaryocytopoiesis and platelet production.Objective:To investigate the effects of plasma from patients with ITP on in vitro megakaryocyte production and platelet production.Methods:* Plasma samples were obtained from 49 patients with chronic ITP and 22 healthy blood donors.* IgG antibody was purified from plasmas by affinity chromatography.* Patient plasma was mixed with the washed control platelets and incubated.* CD34⁺ cells were purified from healthy umbilical cord blood mononuclear cells (MNCs) obtained after Ficoll-Hypaque gradient centrifugation by using a magnetic cell separation method.* CD34⁺ cells were cultured in medium containing thrombopoietin,stem cell factor, interleukin-3,and 10%plasma or purified IgG or antibody-adsorbed plasma from patients or controls.* Megakaryocytes were recognized as CD41a⁺ events by fluorescence-activated cell sorter(FACS).* Platelet count was analyzed in cultured cells.*Megakaryocyte ploidy was measured.CD41a⁺ cells were gated and ploidy distribution was assessed by the intensity of the PI fluorescence.* Apoptosis in megakaryocytes was measured using the Annexin V-FITC Apoptosis Detection Kit according to the manufacturer's instructions.* Bcl-xl expression in megakaryocytes was performed by first incubating the cells with PEcy5-conjugated CD41a mAb.After staining,cells were fixed in 1% paraformaldehyde,permeabilized with 0.1%saponin,and incubated with FITC-conjugated Bcl-xl.CD41a⁺ cells were gated and Bcl-xl expression was shown as mean fluorescence intensity(MFI) within that population.*To analyze Cyclin B1/Cyclin D3 expression in megakaryocytes,PEcy5-CD41a mAb labeled cells were incubated with FITC-Cyclin B1/FITC-IgG1,or FITC-Cyclin D3/ FITC-IgG1κ.The labeled cells were resuspended after washing and analyzed within two hours using flow cytometer.Results:* Role of plasma or purified IgG or antibody-adsorbed plasma in megakaryocyte productionWith twenty-six ITP plasma samples(group A),the number of megakaryocyte in the day- 15 culture was more than mean+1SD[(5.10+0.90)×10⁵]of control cultures. With fourteen ITP plasma samples(group B),the yield of megakaryocyte was less than mean-1SD[(5.10-0.90)×10⁵]of control cultures.With other nine ITP plasma samples(group C),the yield of megakaryocyte was between mean-1 SD and mean+1SD of control cultures.Megakaryocyte production was significantly suppressed in cultures containing IgG from both group A[(3.15±0.93)×10⁵]and B[(3.02±1.01)×10⁵]plasmas in comparison with cultures containing IgG from group C[(4.57±0.78)×10⁵]and control plasmas [(4.90±0.48)×10⁵]. After adsorption of autoantibody from patient plasmas,a significant increase in megakaryocyte number was seen in cultures with adsorbed group A plasmas [(7.85±1.30)×10⁵]than those cultured with control plasmas[(5.36±0.58)×10⁵].* More ITP plasmas boosted megakaryocyte mass with impaired maturation, decreased platelet production,and inhibited apoptosis.The percentage of polyploidy(N≥4) and the platelet count in cultures with both group A[16.35%±4.90%,(7.51±2.41)×10³],and B[16.11%±5.66%,(7.21±2.45)×10³]ITP plasmas were significantly lower than those in control[24.57%±2.83%, (11.21±1.82)×10³]and group C[24.66%±2.49%,(10.12±1.91)×10³]cultures (P＜0.05).On the other hand,reduced megakaryocyte apoptosis was observed in group A [21.88%±3.53%]cultures compared with that in group B[27.36%±4.31%],group C [28.21%±4.02%]and control[29.43%±3.80%]cultures(P＜0.05).Meanwhile, megakaryocytes in different group cultures all showed a degraded expression of Bcl-xL during the culture process with a significantly higher level in group A cultures (P＜0.05).* Patient IgG suppressed megakaryocyte yield,maturation and ability to produce platelet with no impact on apoptosis.A significant reduction in ploidy distribution and platelet release was seen in cultures containing IgG from both group A[14.12%±6.09%,(5.95±2.27)×10³]and B [15.68%±5.98%,(6.15±2.37)×10³]plasmas in comparison with cultures containing IgG from group C[23.14%±2.27%,(9.85±1.61)×10³]and control plasmas [23.98%±2.23%,(10.97±1.92)×10³].Besides,no significant difference in megakaryocyte apoptosis was seen between cultures containing patient IgG(from group A,B and C plasmas) and those containing control IgG.* More autoantibody-adsorbed plasmas increased megakaryocyte production not accompanied with more polyploidy cells and platelet produced,but inhibited apoptosis.After adsorption of autoantibody from patient plasmas,polyploidy percentage and platelet release rose to control level and even more[22.30%±2.86%,(9.77±2.28)×10³] than those cultured with control plasmas[23.42%±2.17%,(10.28±2.76)×10³].However,a significant reduced megakaryocyte apoptosis was observed in group A [22.44%±3.56%]cultures compared with that in group B[30.24%±3.71%],group C [30.08%±3.83%]and control[30.73%±3.99%]cultures(P＜0.05).* Abnormal expression of cyclin B1/D3 in megakaryocytes cultured with patient IgGCultured with control IgG,megakaryocytes expressed cyclin B1 with a reduction from 44.3%to 21.6%at day 9 and then began to express cyclin D3(3.5%).Whereas cultured with patient IgG(from group A and B),megakaryocytes expressed cyclin B1 at a persistently low level(20.5%) and almost did not express cyclin D3 during the whole culture time including day 12.Conclusions:Our findings show that IgG in some ITP plasmas suppresses in vitro megakaryocytopoisis and abnormal expression of cyclin B1/D3 might be the mechanism by which patient IgG inhibits megakaryocyte maturation.On the other hand,decreased apoptosis of megakaryocyte also contributes to in vitro dysmegakaryocytopoiesis and reduced platelet production,suggesting that inhibited megakaryocyte apoptosis may be involved in the pathogenesis of ITP.