Effects Of Plasma From ITP Patients And CD8~+T Cells From Spleen Of ITP Mouse On In Vitro Megakaryocytopoiesis And Platelet Production And Related Mechanisms

Primary immune thrombocytopenia (ITP) is a common immune-mediated hematologic disorder characterized by a low platelet count, detectable autoantibodies binding to platelet glycoproteins and normal or increased number of megakaryocytes in bone marrow. Recently, although ITP patients have received new treatments such as rituximab and thrombopoietin(TPO) receptor agonists, about30%failed to respond or relapsed shortly after the treatment.The pathogenic mechanism of ITP is still unclear right now. It has been long believed that autoantibodies produced by autoreactive B cells against self-antigens, specifically immunoglobulin G (IgG) antibodies against glycoprotein(GP)Ⅱb/Ⅲa and/or GPIb/Ⅸ, are considered to play a crucial role. As a result of the accelerated destruction, platelet production is thought to compensatorily increase. However, platelet turnover studies reported that two-thirds of ITP patients showed decreased or normal platelet production, suggesting that impaired platelet production may also contribute to thrombocytopenia.Recent in vitro studies, showing reduced megakaryocyte production and maturation in the presence of autoantibodies against platelet glycoproteins in ITP plasma, have provided evidence for autoantibody-induced suppression of megakaryocytopoiesis. However, reduced platelets cannot be ascribed to decreased megakaryocyte production in ITP patients with normal or increased bone marrow megakaryocytes. It has been known that platelets are formed from mature megakaryocytes and arise from the development of long and thin cytoplasmic extensions called proplatelets. Several reports indicated a close relationship among apoptosis, megakaryocyte maturation, and platelet release. Therefore, impaired megakaryocyte apoptosis may lead to persistent platelet reduction in ITP patients.Besides, T-cell-mediated immune abnormalities have been considered equally important in the pathogenesis of ITP. It has been reported that in the peripheral blood of ITP patients, CD8+T cells increased, CD4+/CD8+ratio decreased. Olsson et al reported several cytotoxic genes together with genes involved in the Thl cell response showed increased expression in 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. Platelets are formed from mature megakaryocytes. Megakaryocytes have same surface markers as that on the platelets. Cytotoxic T-lymphocytes may also directly destroy megakaryocytes.To investigate the effects of plasma from ITP patients on magakaryocyte maturation and platelet production, we obtained CD34+cells from healthy umbilical cord blood and cultured them with plasma from either chronic ITP patients or healthy subjects. We also incubated CD8+T cells from the spleen of ITP mouse together with BALB/c mouse bone marrow megakaryocytes or with a macrophage cell line(PU5-1.8cells) to investigate the cytotoxic effects of CD8+T cells on megakaryocytes.I Effects of plasma from ITP patients on in vitro megakaryocytopoiesis and platelet production and related mechanisms Objective:To investigate the effects of plasma from ITP patients on in vitro megakaryocytopoiesis and platelet production, we cultured umbilical cord blood derived CD34+cells together with plasma from ITP patients or healthy controls. We measured megakaryocyte and platelet numbers, megakaryocyte apoptosis and several proteins(Fas,FasL,TRAIL,TRAIL-R2,Caspase-3,Caspase-8) that related to megakaryocyte apoptosis. We also measured TPO,IL-11,sFas,sTRAIL in the plasma and cultural supernatant.Methods:◆Plasma samples were obtained from49patients with chronic ITP and22healthy blood donors.◆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, and10%plasma from patients or controls.◆Megakaryocyte and platelet numbers, megakaryocyte apoptosis and the expression of Fas,FasL,TRAIL,TRAIL-R2,Caspase-3,Caspase-8in megakaryocytes were measured by flow cytometry.◆The levels of TPO,IL-11,sFas,sTRAIL in the plasma and cell cultural supernatant from both ITP and control group were measured by ELISA.Results:◆Most ITP plasma inhibited megakaryocyte apoptosis, boosted megakaryocyte mass and depressed platelet production in vitroITP patients can be divided into3groups according to the effect that their plasma had on megakaryocyte production after15days of coculture. Compared with healthy controls (mean±SD,5.10±0.90×105), the number of megakaryocytes in group A (26ITP patients) was significantly increased (7.13±0.86×105, P<0.0001), whereas that in group B (14ITP patients) was markedly decreased (2.83±1.02×105; P<0.0001). However, the plasma of9ITP patients in group C did not significantly affect the number of megakaryocytes (5.20±0.66×105; P=0.776).Reduced megakaryocyte apoptosis was observed in group A (21.88%±3.53%) compared with that of controls (29.43%±3.80%; P<0.0001) on day15. Megakaryocyte apoptosis did not differ significantly among group B (27.36%±4.31%), group C (28.21%±4.02%), and controls.The platelet release values in groups A, B, C, and control cultures were7.51±2.41×103,7.21±2.45×103,10.12±1.91×103, and11.21±1.82×103, respectively. The platelet counts in groups A and B were significantly lower than those in controls (P<0.0001).◆Abnormal expression of TRAIL, caspase-8, and caspase-3in megakaryocytes cultured with group A patient plasmaAt day6, TRAIL, caspase-3, and caspase-8expression in megakaryocytes did not differ significantly among the4groups, whereas at days10and14, the expression of TRAIL, caspase-3, and caspase-8in group A was remarkably lower than that of controls, but there was no significant difference among groups B, C, and the controls. On the other hand, the decreased expression of TRAIL, caspase-3, and caspase-8in group A was consistent with inhibited megakaryocyte apoptosis and decreased platelet production at days10and14of the culture process.The expression of Fas and FasL was low and showed no significant difference on the surface of megakaryocytes among the4group cultures at any detecting points. The expression of TRAIL-R2on megakaryocytes and the expression of TRAIL on produced platelets were relatively stable and showed no significant difference among the4groups at any detecting points.◆Abnormal expression of Bcl-xL in megakaryocytes cultured with group A patient plasmaOn day8and15, Bcl-xL expression in megakaryocytes in group A(82.29%±5.02%.72.57%±5.28%) were significantly higher when compared with that in the control group(51.02%±4.77%,47.34%±5.87%; P<0.0001). ◆The levels of IL-11and TPO showed no difference in ITP plasmaPlasma IL-11concentration in group A (126.74±44.23pg/ml) was remarkably higher than that in controls (31.19±9.20pg/ml; P<0.0001), but there was no significant difference among groups A, B and C. TPO levels in plasma did not differ significantly among the4groups.◆Decreased sTRAIL expression in plasma and cell culture supernatants of group APlasma sTRAIL concentration in group A (2343.24±1155.81pg/ml) was remarkably lower than that of controls (5653.37±1583.32pg/ml; P<0.0001). And at days6,10, and14, sTRAIL concentrations in cell culture supernatants of group A (1200.67±321.49,585.59±277.98, and373.65±272.51pg/ml) were remarkably lower than those in controls(2347.18±479.40,1257.13±329.24, and852.32±307.32pg/ml; P<0.0001). sTRAIL levels in plasma and cell culture supernatants showed no remarkable differences among groups B, C, and controls at any detecting points.The plasma levels of sFas in groups A, B, C, and controls showed no significant difference, whereas the sFas levels in cell culture supernatant of group A (127.38±18.28and137.72±25.67pg/ml) were significantly lower than those in control supernatants (163.75±23.21and178.68±17.40pg/ml) at days6and10, respectively (P<0.0001). However, no difference was found at day14. There were no remarkable differences among groups A, B, and C at any detecting points.Conclusions:◆Most ITP plasma inhibited megakaryocyte apoptosis, boosted megakaryocyte mass and depressed platelet production in vitro.◆The boosted megakaryocyte mass was not related to the levels of TPO and IL-11in ITP plasma.◆The abnormal expression of TRAIL and Bcl-xL in megakaryocytes may play a role in the pathogenesis of impaired megakaryocyte apoptosis in ITP. II The cytotoxic effects of CD8+T cells from spleen of ITP mouse on in vitro megakaryocytesObjective:To investigate the cytotoxic effects of CD8+T cells from the spleen of ITP mouse on in vitro megakaryocytes or PU5-1.8(a macrophage cell line), we cultured the effecter cells(CD8+T cells) together with the target cells(megakaryocytes or PU5-1.8) in different ratios for4hours in vitro. The death rates of the target cells were measured by flow cytometry.Methods:◆Bone marrow cells were obtained from the BALB/c mice and then cultured in the megakaryocyte medium with TPO for7-8days in vitro. The expression of CD61and H2-Kd on the surface of BALB/c mice megakaryocytes was measured by flow cytometry.◆PU5-1.8cells and EL-4cells were cultured in vitro. The expression of CD61and MHC I molecule(H2-Kd/H2-Kb) on the surface of PU5-1.8cells and EL-4cells were measured by flow cytometry.◆Create ITP mouse model:platelets from wild type BALB/c mice or wild type C57BL/6mice (100×106) were transfused into BALB/c CD61KO mice by tail vein once a week for2-4weeks. Immunization was monitored by measuring IgG antiplatelet antibody production in the sera of CD61KO mice by flow cytometry. After well immunized, the spleen cells (15×103) from CD61KO mice were then transferred to SCID(Severe Combined Immunodeficient) mice. Platelet counts and the bleeding symptoms of the SCID mice should be monitored every week.◆CD8+T cells and/or CD19+B cells were separated from the splenocytes of ITP mice by positive selection. These splenocytes were used as effecter cells in the cytotoxicity assay.◆Cytotoxicity assay:the in vitro cultured megakaryocytes from the bone marrow of BALB/c mice or PU5-1.8cells were used as target cells in the cytotoxicity assay. The effecter cells in our assay included splenocytes from BALB/c platelet immunized ITP mice(non-depleted splenocytes, CD8+T-depleted splenocytes and CD19+B-depleted splenocytes) and splenocytes from C57BL/6platelet immunized ITP mice. The splenocytes from the wild type BALB/c mice were used as controls. The effecter cells and the target cells were cultured in different E:T ratios (E:T=20:1,10:1,5:1,2.5:1) for4hours in vitro. The death rates of the target cells were measured by flow cytometry.Results:◆Mature polyploidy megakaryocyes were observed in the culture system. CD61and H2-Kd were highly expressed on the surface of these megakaryocytesBone marrow cells from BALB/c mice were cultured in the megakaryocyte medium with TPO for7days in vitro. Most of the bone marrow cells developed into mature polyploidy megakaryocyes(N>4). CD61and H2-Kd were highly expressed on the surface of these megakaryocytes. The level of CD61on megakaryocytes was85.70%and that of H2-Kd was99.69%.◆CD61and H2-Kd/H2-Kb were highly expressed on the surface of PU5-1.8cells and EL-4cellsCD61and H2-Kd were highly expressed on PU5-1.8cells independent of platelet engulfment or whether the engulfed platelets were thrombin-activated or not. CD61and H2-Kb were also expressed on EL-4cells. The level of CD61on EL-4cells was48.87%and that of H2-Kb was99.33%.◆Splenocytes from BALB/c platelet immunized ITP mice showed cytotoxic effects on in vitro megakaryocytes and PU5-1.8cells.More target cells were killed by splenocytes from BALB/c platelets immunized ITP mice in comparison with that by splenocytes from naive BALB/c mice. However, splenocytes from C57BL/6platelets immunized ITP mice showed less killing. We further depleted CD8+T cells or CD19+B cells from the splenocytes of BALB/c platelets immunized ITP mice and discovered less killing in CD8+T depleted group but not much change in CD19+B depleted group, which indicated that CD8+T cells played a key role in the cytotoxicity assay.Conclusions:◆Splenocytes from BALB/c platelet immunized ITP mice showed direct cytotoxic effects on in vitro megakaryocytes.◆CD8+T cells played a key role in the cytotoxicity assay.