Ex Vivo Effect Of RhIL-11 On Bone Marrow Megakaryocyte Progenitors Of Children With Idiopathic Thrombopenia Purpura And Induces The Expression Of Transcription Factors In Dami Cells

Idiopathic thrombocytopenic purpura(ITP) is the most common hemorrhagenicdisease in children. There are two types of ITP: acute ITP(AITP) and chronic ITP(CITP). Some data indicated that the pathologic mechanism was different betweenthem. While maturity disturbance of megakaryocyte(MK) and impaired productionof platelet have been regarded as the main pathogenesis of ITP, the mechanismremains still unclear. Megakaryocytopoiesis involves the proliferation anddifferentiation of megakaryocytic progenitor cells into immature MK, and thedifferentiation of immature MK to mature MK which produce platelets. The formeris regulated mainly by thrombopoietin(TPO) and to a lesser degree by othercytokines such as interleukin-1(IL-1), IL-3, the latter by TPO and probably IL-6 andIL-11. A number of transcription factors have been implicated in the control of MKdifferentiation. In recent few years, the transcription factor GATA-1 and nuclearfactor erythroid 2 (NF-E2) have been paid increasing attention for their involvementin megakaryocytic hematopoiesis: the former was thought to play an important rolein proliferation and differentiation of MK, while the latter was found to be essential for late MK maturation and platelet formation. Recombinant human interleukin 11(rhIL-11) is a thrombopoietic growth factor, rhIL-11 can promote the proliferationand maturation of MK progenitors in patients with bone marrow failure (BMF)caused by radiotherapy and chemotherapy. However, there was no reliable dataabout the effect of rhIL-11 on ITP. Whether IL-11 takes part in the transcriptionalregulation ofmegakaryocytopoiesis or not remains unknown.In order to preliminary analyse the mechanism of ITP and the action of rhIL-11,we firstly observed the morphological characteristics and hematopoietic function ofbone marrow MK in children with ITP. Moreover, bone marrow mononuclear cells(MNC) were cultured in serum-free medium with rhTPO, rhIL-3, rhIL-6 and rhIL-11,in order to elucidate further the effects of rhIL-11 in children with CITP.Furthermore, we investigated the effects of rhIL-11 on mRNA and proteinexpressions of megakaryocyte-associated transcription factors GATA-1 and NF-E2in a human megakaryocytic cell lines Dami.PartⅠStudy on megakaryocyte progenitors of bone marrow in childrenwith idiopathic thrombocytopenic purpura and its clinical significanceSubjects and Methods1. Bone marrow were respectively aspirated from 27 children with AITP and25 children with CITP. Prednisone were used with 2.0mg/(kgd) in the CITP groupfor less than 4 weeks. 17 cases with CITP undergoing prednisone were observed toshow good recovery. While others children with CITP had no response. 10comparably normal children were controls. 2. MK were detected in bone marrowsmears. 3. Ficoll density gradient method was used to purify MNC from bonemarrow. 4. MNC (2×10~5/ml) were cultured in a collagen-based system containedserum-free medium with rhTPO, rhlL-3, rhlL-6 about 14 days. 5. The numbers ofmegakaryocyte colony forming unit (CFU-MK), MK burst forming unit (BFU-MK)were measured at day 14 by CD41 linked to a secondary biotinylatedantibody-alkaline phosphatase-anti-alkaline phosphatase (APAAP) detection system. 6. Statistical assay: the data were dealt with SPSS 13.0 statistical software.Results1. MK in bone marrow smears: The number of MK in children with AITP werehigher than the control group (P＜0.05). The number of MK in children withCITP were higher than the control group (P＜0.05). There was no significantdifference between the CITP and the AITP groups (P＞0.05). MK in 17 childrenwith CITP responded to prednisone were higher than controls (P＜0.05). In 8children with CITP, who had no response to prednisone, MK were lower thananother CITP group (P＜0.05) and the control group (P＜0.05).2. Megakaryocyte progenitors in serum-free medium with collagen: CFU-MK andBFU-MK in children with AITP were higher than the control group (P＜0.05).There was no significant difference between the CITP and the control group(P＞0.05). CFU-MK and BFU-MK of 17 children with CITP were higher thanthe control group (P＜0.05), while CFU-MK and BFU-MK of 8 children withCITP were lower than the control group (P＜0.05).3. Correlativity assay: BFU-MK and MK of the AITP group were positivelycorrelated (γ_s=0.65, P＜0.01). CFU-MK and MK of the AITP group werepositively correlated (γ_s=0.71, P＜0.01). CFU-MK and BFU-MK of the AITPgroup were also positively correlated (γ_s=0.74, P＜0.01). The same results couldbe found in the CITP group, respectively (γ_s=0.90, P＜0.01;γ_s=0.98, P＜0.01;γ_s=0.92, P＜0.01).PartⅡEffect of rhlL-11 on bone marrow megakaryocyte progenitors ofchildren with chronic idiopathic thrombocytopenic purpura in vitroSubjects and Methods1. Bone marrow were respectively aspirated from 23 children with CITPcome from PartⅠ, including 15 cases responded to prednisone and 8 easesnon-responded to prednisone. 10 comparably normal children from PartⅠwere controls. 2. Ficoll density gradient method was used to purify MNC from bonemarrow. 3. MNC (2×10~5/ml) were cultured in a collagen-based system containedserum-free medium with rhTPO, rhIL-3, rhIL-6 about 14 days. rhIL-11 wereadditionally added or not into the medium. 4. The numbers of CFU-MK andBFU-MK were measured at day 14 by CD41 linked to APAAP detection system. 5.MNC of bone marrow were cultured in serum-free medium with rhTPO, rhIL-3,rhIL-6, rhIL-11 were additionally added or not into the medium. 6. The numbers ofMNC were measured at day 0, 7 and 14. 7. CD41~+ cells were measured at day 14 byflow cytometer. 8. Statistical assay: the data were dealt with SPSS13.0 statisticalsoftware.Results1. The proliferation effects of CFU-MK and BFU-MK by rhIL-11: In 15 childrenwith CITP responded to prednisone, CFU-MK and BFU-MK in medium withrhTPO, rhIL-3, rhIL-6, rhIL-11 were higher than in medium without rhIL-11(P＜0.01). The same results could be observed in the control group (P＜0.01). Butin 8 children with CITP non-responded to prednisone, there was no significantdifference between the group with rhlL-11 and the group without rhlL-11(P＞0.05). In three groups, there were not CFU-MK and BFU-MK could befound in medium with rhIL-11.2. The proliferation effects of MNC by rhIL-11: In three groups, MNC had nosignificant proliferation effects by rhIL-11 combined rhTPO, rhIL-3, rhIL-6 atday 0,7,14, compared with no-rhIL-11 cytokine cocktail (P＞0.05).3. The proliferation effects of CD41~+ cells by rhIL-11: In 15 children with CITPand the control group, CD41~+ cells could be efficiently increased with thecytokine combination including rhIL-11 at day 14 (P＜0.01). However, the samephenomenon could not be observed in 8 children with CITP non-responded toprednisone (P＞0.05). In three groups, there were not CD41~+ cells could be foundin medium with rhIL-11. PartⅢrhIL-11 induces the expression of transcription factor GATA-1and NF-E2 in Dami cellsSubjects and Methods:1. The growth curve wre made according to the numbers of Dami cells from theday 0～7.2. Cell Counting Kit-8 (CCK-8) was used to detected the effect of rhIL-11on the proliferation of Dami cells. 3. Immunohistochemitry was used to detected theexpression of IL-11 receptorα(IL-11Rα) in Dami cells. 4. Western Blot was usedto detected the protein expression of IL-11Rαin Dami cells. 5. Western Blot wasused to detected the protein expression of GATA-1 and NF-E2 in Dami cellsincubated with rhIL-11 (50ng/ml) at 0h, 1h, 2h, 4h. 6. RT-PCR was used to detectedthe mRNA expression of GATA-1 and NF-E2 in Dami cells incubated with rhIL-11(50ng/ml) at 0h, 1h, 2h, 4h. 7. Statistical assay: the data were dealt with SPSS13.0statistical software.Results:1. The effect of rhIL-11 on the proliferation of Dami cells: rhIL-11 (50ng/ml or100ng/ml) has no positive effect on the proliferation of Dami cells (P＞0.05).2. The expression of IL-11Rαin Dami cells: Western Blot andimmunohistochemitry demonstrated that Dami cells express IL-11 Rαand its.protein.3. The protein expression of GATA-1 and NF-E2 in Dami cells: Western Blotdemonstrated that rhIL-11 (50ng/ml) up-regulates the protein expression ofGATA-1 and NF-E2 in Dami cells. Compared to 0h, the protein expression ofGATA-1 were increased at 1h (P＜0.01), 2h (P＜0.01) and 4h (P＜0.01),respectively. And the protein expression of NF-E2 were also increased at 1h(P＜0.01), 2h (P＜0.01) and 4h (P＜0.01), respectively.4. The mRNA expression of GATA-1 and NF-E2 in Dami cells: RT-PCRdemonstrated that rhIL-11 (50ng/ml) up-regulates the mRNA expression of GATA-1 and NF-E2 in Dami cells. The mRNA expression of GATA-1 wasincreased at 1h compared to 0h (P＜0.01). Then the expression of GATA-1 weresignificantly reduced at 2h and 4h compared to 0h (P＜0.01). Compared to 0h,the mRNA expression of NF-E2 were increased at 1h (P＜0.01), 2h (P＜0.01) and4h (P＜0.01), respectively.Conclusions1. To study MK in bone marrow and megakaryocyte progenitors through culture invitro, the different phases may be diagnosed during the early stage of ITP.2. Some CITP children with lower megakaryocyte progenitors have no response toprednisone, their MK might be abnormal.3. In some children with CITP, MK progenitors could be efficiently increased byrhIL-11 combined rhTPO, rhIL-3, rhIL-6, compared with no-rhIL-11 cytokinecocktail, rhIL-11 may have activity in these patients. But there is no significanceof rhIL-11 on megakaryocyte progenitors in children with CITP non-respondedto prednisone.4. rhIL-11 increases the protein and mRNA expression of GATA-1 and NF-E2 inDami cells. It may take part in the transcriptional regulation ofmegakaryocytopoiesis. Dami cells express IL-11Rα. rhIL-11 might affect Damicells by combining with the receptor on the cell membrane to regulate the signaltransduction pathway in the cytoplasm.