| Idiopathic thrombocytopenic purpura (ITP) is one of the most common forms of autoimmune disease affecting both adults and children, 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 pathogenetic mechanism of ITP is not completely clear yet. It has long been believed that thrombocytopenia is mediated by autoantibodies that are directed against various platelet membrane receptors, including platelet glycoproteins such as glycoprotein II b/IIIa (GPIIb/IIIa) or GP I b/IX complexes. Binding of autoantibodies to these target antigens eventually results in platelet destruction by the reticuloendothelial system.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. This hypothesis that megakaryocytopoiesis and thrombopoiesis may be disrupted in ITP has been supported by platelet kinetic studies and morphologic alterations of ITP marrow megakaryocytes. Recently, Chang et al reported that plasma from patients with childhood ITP suppressed in vitro megakaryocyte production. Similarly, McMillan et al studied the effect of plasma from adult patients with chronic ITP on in vitro megakaryocyte production. Their further study proved the suppression was mediated by plasma autoantibodies. Taken together, these data indicate that autoantibodies not only are involved in platelet destruction, but may also contribute to the inhibition of platelet production.However, these mechanisms cannot account for all observations made in this disorder. Some ITP patients' platelets are absent of detectable antigen-specific autoantibodies and remission in ITP can occur despite the presence of platelet autoantibodies. It is difficult to interpret all the impairment of platelet with antibody-mediated immunity. These phenomena indicate the presence of other mechanisms in ITP.It has been well known that T-lymphocyte abnormalities may have pathogenetic importance in ITP patients. Mature reactive T-cell clones have also been shown to be deleted peripherally through activation-induced cell death (AICD). AICD is induced in T cells via different death pathways, of which Fas/FasL is the best characterized. Yoshimura et al found a higher level of soluble Fas which protects cells from undergoing FasL induced apoptosis in patients with chronic ITP. Furthermore, Olsson et al reported that apoptotic resistance of T cells in patients with active ITP may lead to defective clearance of potentially pathogenic reactive T cells through AICD and consequently, may allow continuing autoimmune platelet destruction, i.e. platelet antibody production and cell-mediated cytotoxicity. 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 plateletsIn 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. Triggers for the different cell death pattern are largely unknown. To understand the contribution of CD8+ T cells in bone marrow to the pathogenesis of ITP, we investigated the effect and mechanism of the CD8+ T cells of patients with ITP on autologous megakaryocytopoiesis.I Effects of CD8+ T cells from patients with ITP on quantity of in vitro megakaryocytopoiesisObjective: To investigate the effects of CD8+ T lymphocytes in bone marrow from patients with chronic ITP on quantity of in vitro autologous megakaryocytopoiesis.Methods: * 10 mL bone marrow and 30 mL blood from 15 chronic ITP patients and 13 controls were collected respectively.* Mononuclear cells (MNCs) were prepared.* CD8+T/CD3+ T ratio in MNCs was detected.* CD8+ T lymphocytes were positively selected using CD8+ magnetic microbeads, according to the manufacture's recommendations.* Platelets were prepared from peripheral blood.* Proliferative responses by CD8+ T cells in bone marrow to autologous platelets were performed.* The prepared bone marrow cells were divided into four groups: MNCs were cultured directly (group MNC); CD8+T depleted MNCs were cultured (group CD8+T-dep); purified CD8+ T cells were 1:1 added to autologous CD8+T-dep MNCs in coculture (group coculture); and dexamethasone was added to coculture (group DEX) and adjusted final concentration to 1.0×10-6 mol/L.*The prepared cells were planted in semi-solid and liquid culture systems.*Megakaryocytes were recognized as CD41a+ events by fluorescence -activated cell sorter (FACS).* Megakaryocyte colony forming units (CFU-MK) were quantitated. Results: * The ratio of CD8+T/CD3+T in ITP bone marrow was 59.94%±5.15%, which was significantly higher than that in ITP peripheral blood (49.33%±3.70%; p<0.05) and in normal bone marrow (38.70%±3.43%; p<0.05).* CD8+ T cells derived from patients with chronic ITP showed vigorous proliferation after being incubated with autologous platelets for 7 days, but CD8+ T cells from controls did not show reactivity (p<0.05).* The counts of CFU-MK and megakaryocytes were very similar among group MNC, group CD8+T-dep and group coculture, which were not influenced by the depletion or addition of autologous CD8+T cells from normal bone marrow.* When numbers of CFU-MK of ITP were compared, there was no statistical difference between group MNC and group CD8+T-dep (55.87±18.40 vs. 59.40±12.76; p>0.05), between group CD8+T-dep and group coculture (59.40±12.76 vs. 54.47±21.48; p>0.05),nor between group MNC and group coculture(55.87±18.40 vs. 54.47±21.48; p>0.05). CFU-MK formation of chronic ITP patients was not influenced by the depletion or addition of autologous CD8+ T cells. When megakaryocyte counts were compared, the depletion of CD8+ T cells caused a significant reduction in megakaryocytes when compared with group MNC, the addition of CD8+ T cells increased megakaryocytes when compared with group CD8+T-dep, and DEX decreased megakaryocytes when compared with group coculture. Also, megakaryocyte count of group coculture was maximal in coculture containing the most numerous CD8+T cells.Conclusion: ITP CD8+ T cells increased megakaryocytes, and the increased megakaryocyte count was not mediated by accelerated proliferation of megakaryocyte progenitors.II Effects of CD8+ T cells from patients with ITP on quality of in vitro megakaryocytopoiesisObjective: To investigate the connection between the increased megakaryocytes mediated by ITP CD8+ T cells and the suppressed apoptotic megakaryocytes and platelet production.Methods: * 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 manufacturers 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.* Fas expression in megakaryocytes was performed by sequentially incubating with PEcy5-conjugated CD41a mAb and FITC-conjugated Fas mAb. CD41a+ cells were gated and Fas expression was reported as MFI within that population.* The levels of soluble Fas (sFas) and soluble TRAIL (sTRAIL) of cell-free supernatants of megakaryocytic cultures were quantified by the respectiveenzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's instructions.Results: * The megakaryocytic characteristics were very similar among group MNC, group CD8+T-dep and group coculture in controls. The depletion or addition of autologous CD8+ T cells from normal bone marrow did not affect apoptotic megakaryocytes and platelet production.* A significant reduction in platelets was noted in cocultures when compared with CD8+T-dep cultures and MNC cultures. The count of platelets in group CD8+T-dep was remarkably higher than that of group MNC. When the yield of megakaryocytes was maximal in cocultures, in contrast, the platelet production reached the minimum, indicating the ability of platelet production by megakaryocytes dropped, though the quantity of megakaryocytes increased.* The percentage of polyploidy (N≥4) megakaryocytes in group CD8+T-dep was significantly higher than that in group MNC (p<0.05). The addition of CD8+ T cells decreased the percentage of polyploidy megakaryocytes when compared with group CD8+T-dep (p<0.05).* The percentage of apoptotic megakaryocytes was significantly higher in group CD8+T-dep than that in group MNC (p<0.05). The addition of CD8+T cells decreased the percentage of apoptotic megakaryocytes when compared with group CD8+T-dep (p<0.05).* The expression of Bcl-xl was significantly lower in group CD8+T-dep than that in group MNC (p<0.05). The addition of CD8+T cells increased the expression of Bcl-xl when compared with group CD8+T-dep (p<0.05).* The expression of Fas was significantly higher in group CD8+T-dep than that in group MNC (p<0.05). The addition of CD8+ T cells decreased the expression of Fas when compared with group CD8+T-dep (p<0.05).* The sFas level of the group CD8+T-dep (415.01±178.47) was significantly lower than that of the group MNC (977.46±287.31) and the group coculture (1424.88±469.93, both P<0.05). sTRAIL level was too low to be detected by ELISA.* Compared with the ITP group coculture, the platelet numbers and the percentage of polyploid nuclei of the group DEX increased largely(16.36±6.86 and 21.37±4.23 respectively, both P<0.05), the percentage of apoptotic CD41a+ cells and the Fas expression level increased(19.04±3.41 and 26.57±4.63 respectively, both P<0.05), the Bcl-xl expression level reduced(30.70±5.88, P<0.05), and the sFas level reduced(719.13±220.53, P<0.05).Conclusion: CD8+ T cells in bone marrow of patients with chronic ITP might suppress megakaryocyte apoptosis leading to the increased megakaryocytes and the impaired platelet production. Megakaryocyte apoptosis would be a novel target for the management of ITP.
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