K-252a-induced polyploidization and differentiation of a human megakaryocytic cell line, Meg-J: transient elevation and subsequent suppression of cyclin B1 and cdc2 expression in the process of polyploidization

Megakaryocytes are unique haemopoietic cells which undergo DNA replication, giving rise to polyploid cells. However, little is known about the mechanism of megakaryocytic polyploidization. To address this issue, we used the human megakaryocytic cell line Meg-J. In the presence of K-252a (an indolocarbasole derivative), Meg-J cells stopped proliferation and exhibited additional megakaryocytic features, including morphological changes, polyploidization, and increases in the levels of surface expression of platelet glycoprotein (GP) IIb/IIIa and GPIb. Thrombopoietin (TPO) promoted the K-252a-induced polyploidization and megakaryocytic differentiation. In the process of K-252a-induced polyploidization, levels of expression of both cdc2 and cyclin B₁ were elevated transiently and subsequently decreased. This suggested that the polyploidization process in Meg-J cells was at least in part associated with a transient elevation and subsequent decrease in the expression of cdc2/cyclin B₁ complex, a critical kinase involved in G2/M cell cycle transition.     

Megakaryocyte emperipolesis: a new frontier in cell-in-cell interaction

Abstract

Histology of bone marrow routinely identifies megakaryocytes that enclose neutrophils and other hematopoietic cells, a phenomenon termed emperipolesis. Preserved across mammalian species and enhanced with systemic inflammation and platelet demand, the nature and significance of emperipolesis remain largely unexplored. Recent advances demonstrate that emperipolesis is in fact a distinct form of cell-in-cell interaction. Following integrin-mediated attachment, megakaryocytes and neutrophils both actively drive entry via cytoskeletal rearrangement. Neutrophils enter a vacuole termed the emperisome which then releases them directly into the megakaryocyte cytoplasm. From this surprising location, neutrophils fuse with the demarcation membrane system to pass membrane to circulating platelets, enhancing the efficiency of thrombocytogenesis. Neutrophils then egress intact, carrying megakaryocyte membrane and potentially other cell components along with them. In this review, we summarize what is known about this intriguing cell-in-cell interaction and discuss potential roles for emperipolesis in megakaryocyte, platelet and neutrophil biology.     

Megakaryocyte progenitors in the bone marrow and peripheral blood of patients with myeloproliferative diseases

We studied the behavior in culture of megakaryocyte progenitor cells (CFU-Mk) from peripheral blood (PB) and bone marrow (BM) cells in eight patients with myeloproliferative diseases (MPD). In seven patients we observed megakaryocyte (Mk) colony formation from PB cells, which were generated in the absence of any added stimulator and which did not increase after the addition of a source of Mk-colony stimulating activity (CSA-Mk). The number of BM CFU-Mk was significantly higher in patients than in controls, and in seven out of eight patients the responsiveness to added CSA-Mk was retained. Plasma obtained from six patients did not stimulate normal donors' BM target cells to form Mk colonies. These data demonstrate an expansion of the CFU-Mk pool in MPD patients without increased plasma levels of CSA-Mk, and suggest that PB and BM CFU-Mk of MPD patients might have different kinetic properties.     

Megakaryocyte thromboxane production induced by platelet stimuli during in vitro culture

Megakaryocytes isolated from guinea pigs produced thromboxane (assayed by radioimmunoassay as thromboxane B2) in response to the platelet aggregatory stimuli arachidonic acid, thrombin, and the calcium ionophore A23187. The relative responses to these stimuli were similar in megakaryocytes and platelets from the same animals. When the megakaryocytes were maintained in short-term in vitro culture, all three stimuli still elicited thromboxane production. Following overnight in vitro culture, thromboxane production in response to thrombin decreased, overall, to under 60% of control values, increased approximately threefold in response to A23187, but did not show any alteration in response to arachidonic acid. Requirements for calcium were virtually unchanged. These results demonstrate that megakaryocytes contain all of the pathways needed for arachidonic acid mobilization from phospholipids in response to thrombin or A23187 and conversion of that arachidonate to thromboxane. These pathways are retained by the cells in short-term in vitro culture.     

Interleukin 10-induced thrombocytopenia in normal healthy adult volunteers: evidence for decreased platelet production

Recombinant human interleukin 10 (rhuIL-10) inhibits the production of proinflammatory cytokines and has shown promise in the treatment of inflammatory bowel disease. Clinical trials have been accompanied by a reversible decline in platelet counts. We conducted a randomized, double-blinded, placebo-controlled, parallel group trial in 12 healthy volunteers to investigate the aetiology of rhuIL-10-induced thrombocytopenia. Eight volunteers received 8 microg/kg/d of rhuIL-10 subcutaneously, while four subjects received a placebo alone for 10 d. A reversible decline in the platelet counts from a mean of 275 x 10(9)/l to 164 x 10(9)/l was observed in the IL-10-treated cohort (P = 0.012). A fall in the haemoglobin mean levels was also observed in the IL-10-treated cohort from 13.7 to 11.7 g/dl (P = 0.011). No significant change was observed in the bone marrow cellularity or myeloid/erythroid ratio or in the number of megakaryocytes per high-powered field (HPF). A fall was observed in the number of megakaryocyte colony-forming units (CFU-MKs) after the administration of IL-10 compared with those receiving the placebo (P = 0.068). No difference in the change in granulocyte-macrophage CFUs (CFU-GMs), mixed lineage CFUs (CFU-GEMMs) or erythroid burst-forming units (BFU-Es) was observed when comparing the IL-10- vs. placebo-treated groups (P > 0.465). Serum cytokine levels of thrombopoietin (TPO). IL-6 and granulocyte-macrophage colony stimulating factor (GM-CSF) were not decreased following IL-10 administration. In fact, both TPO and GM-CSF appeared to be slightly increased in the serum. All subjects underwent In111-labelled platelet survival studies with liver/spleen scans to assess splenic sequestration prior to and then on day 7 of treatment. A significant reduction in splenic sequestration of platelets (P =0.012) was observed in the IL-10-treated group, but not in the placebo-treated subjects.     

Intrinsically impaired platelet production in some patients with persistent or chronic immune thrombocytopenia

Persistent or chronic immune thrombocytopenias (P/C‐ITP) are acquired blood disorders lasting more than 3 months or 1 year, respectively. The pathogenesis of these disorders is thought to be immunological. We hypothesized that some patients with P/C‐ITP might have an intrinsic megakaryopoiesis defect. We identified a group of P/C‐ITP patients with acquired isolated mild thrombocytopenia (30–100 × 10⁹/l), undetectable anti‐platelet antibodies, negative autoimmune investigations and no need for treatment. We examined in vitro megakaryocyte differentiation and compared these patients' results with those of acute‐ITP patients and healthy controls. No difference in proliferation, ploidy or expression of surface markers was found. In contrast, P/C‐ITP patients had significantly fewer proplatelet‐forming megakaryocytes. This novel observation demonstrated that some patients diagnosed with P/C‐ITP have an intrinsic megakaryopoiesis defect independent of the bone‐marrow environment. Further investigations are needed to dissect mechanisms underlying this impaired proplatelet formation in these patients.     

Megakaryocyte ploidy in thrombocytosis: improved microdensitometric measurements with a new image analysis system

Megakaryocyte (MK) nuclear deoxyribonucleic acid (DNA) content was measured on a new image analysis system. Feulgen-stained bone marrow aspirate smears were analysed from 9 patients with thrombocytosis. Average optical density (OD) of stained nuclei was evaluated by pixel discrimination over 128 grey levels. Projected nuclear area was delineated manually. The product of these two parameters gave an index of nuclear DNA content. Neutrophils were used as 2N reference cells. Reproducibility of OD, nuclear area and their product was excellent for individual cells (CV less than 2.0% for MKs, less than 4.0% for neutrophils). The average CV for DNA content of 18 groups of 10 neutrophils was 5.5% (range 3.0-9.7%). A significant linear regression existed between MK nuclear area and DNA content (p much less than 0.001) for 627 MKs in essential thrombocythaemia (ET) and 346 MKs in reactive thrombocytosis (RT). Compared with RT, more 8N and 64N MKs were seen in ET (p less than 0.05). 128N MKs were unique to ET. Image analysis of MK ploidy may assist the clinical discrimination between primary and secondary thrombocytosis.     

Dopamine induces platelet production from megakaryocytes via oxidative stress-mediated signaling pathways

Dopamine (DA), a catecholamine neurotransmitter, is known to for its diverse roles on hematopoiesis, yet its function in thrombopoiesis remains poorly understood. This study shows that DA stimulation can directly induce platelet production from megakaryocytes (MKs) in the final stages of thrombopoiesis via a reactive oxygen species (ROS)-dependent pathway. The mechanism was suggested by the results that DA treatment could significantly elevate the ROS levels in MKs, and time-dependently activate oxidative stress-mediated signaling, including p38 mitogen-activated protein kinase, c-Jun NH2-terminal kinase, and caspase-3 signaling pathways, while the antioxidants N-acetylcysteine and L-glutathione could effectively inhibit the activation of these signaling pathways, as well as the ROS increase and platelet production triggered by DA. Therefore, our data revealed that the direct role and mechanism of DA in thrombopoiesis, which provides new insights into the function recognition of DA in hematopoiesis.     

Myocardial changes in pressure overload-induced left ventricular hypertrophy. A study on tissue composition, polyploidization and multinucleation.

Morphological changes in human myocardium associated with pressure overload-induced left ventricular hypertrophy were studied in 22 normal and 21 hypertrophic hearts obtained at autopsy. Samples were obtained from the left lateral ventricular wall, half way between the apex and the base. Myocyte dimensions, polyploidization, multinucleation and relative volume fractions were studied. Regression analysis in relation to indexed heart weight yielded statistically significant correlation coefficients for myocyte volume: r = 0.69 (P less than 0.001), for degree of polyploidization: r = 0.77 (P less than 0.001), for number of nuclei per myocyte: r = 0.47 (P less than 0.01) and for volume fraction of myocytes: r = 0.32 (P less than 0.05). Approximate numbers of myocytes and connective tissue cells per left ventricle were calculated. Correlation coefficients related to indexed heart weight were r = 0.34 (P less than 0.05) for the number of myocytes and r = 0.76 (P less than 0.001) for the number of connective tissue cells. Based on regression analysis in relation to indexed heart weight, we calculated that a doubling of indexed heart weight was associated with an increase in mean myocyte volume by 65%, degree of polyploidization by 24%, multinucleation by 7%, number of myocytes by 20% and number of connective tissue cells by 141%. The volume percentage of myocytes decreased by 6% in favour of the connective tissue fraction. These changes in myocardial composition indicate that the term 'hypertrophy' inadequately describes the actual myocardial changes in response to pressure overload.     

Megakaryocyte c-Mpl expression in chronic myeloproliferative disorders and the myelodysplastic syndrome: immunoperoxidase staining patterns and clinical correlates

The objectives of this study were to expand on recent observations that have suggested decreased thrombopoietin receptor (c-Mpl) expression in megakaryocytes of patients with polycythemia vera (PV) and agnogenic myeloid metaplasia (AMM). We applied an immunoperoxidase method with anti-c-Mpl antibody to 55 bone marrow sections from previously untreated patients with chronic myeloproliferative disorder (CMPD) or myelodysplastic syndrome (MDS). These included 8 patients with PV, 15 with AMM, 9 with essential thrombocythemia, 5 with chronic myelocytic leukemia, 9 with the 5q-syndrome and 9 with MDS with fibrosis. The findings were compared with those in four patients with reactive erythrocytosis (RE), six with immune thrombocytopenic purpura (ITP) and five normal controls. Staining intensity (SI) was moderate to strong both in normal controls and in patients with RE or ITP. In contrast, SI was weak in variable proportions of the megakaryocytes in every one of the aforementioned clonal myeloid disorders. The staining pattern (SP) was relatively uniform in MDS and heterogeneous in CMPD. Neither SI nor SP was significantly correlated with certain clinical or laboratory parameters. We concluded that altered megakaryocyte c-Mpl expression may be a nonspecific phenomenon in various subtypes of both CMPD and MDS. However, the characteristic staining patterns may complement the morphological distinction between clonal and reactive myeloproliferation.     

Stromal cell-derived factor-1 rs2297630 polymorphism associated with platelet production and treatment response in Chinese patients with chronic immune thrombocytopenia

Stromal cell-derived factor-1 (SDF-1), signaling through CXCR4, is implicated in megakaryopoiesis and platelet production. SDF-1 rs2297630 is a functional polymorphism in linkage disequilibrium with other functional variants in SDF-1. This study aimed to investigate the role of SDF-1 rs2297630 in chronic ITP. The genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism and confirmed by direct sequencing. Immature platelet fraction (IPF) was performed using Sysmex XE-2100. Anti-platelet autoantibodies were assayed by enzyme-linked immunosorbent assay. The main characteristics at diagnosis and the outcome of chronic ITP in 201 Chinese patients were retrospectively reviewed. There was no significant difference in either genotype or allelic distribution between ITP patients and the controls (p = 0.114; p = 0.787). However, both heterozygote (GA) and homozygote minor allele (AA) patients had significantly increased megakaryocyte quantity compared to homozygote genotype (GG) patients at diagnosis (p = 0.011). The mean IPF values of GA and AA genotype patients were higher than those observed in the GG genotype patients when platelet counts ≤50 × 10⁹/L at diagnosis (p = 0.007). Patients with GA and AA genotype showed a higher response rate to standard treatments than patients with GG genotype (p < 0.001). In particular, GA and AA genotype patients had a significantly increased chance of responding to steroids, intravenous immunoglobulin (IVIG), and thrombopoietin analogs (p = 0.007; p = 0.029; p = 0.034, respectively). No significant difference was found between anti-platelet antibodies and genotypes (p = 0.296). In summary, the SDF-1 rs2297630 was associated with platelet production and treatment response in Chinese patients with chronic ITP.     

Parasinusoidal location of megakaryocytes in marrow: A determinant of platelet release

Megakaryocytopoiesis occurs in the hematopoietic (extravascular) compartment of marrow. Thus, platelets must traverse the wall of the vascular sinuses of marrow to enter the circulation. We have examined mouse and rat marrow, fixed by rapid immersion so as to maintain anatomical relationships as close to the natural state as possible. Quantitative transmission electron microscopy (TEM) of random transections of femurs established that megakaryocytes reside less than 1 μ from a marrow sinus wall with a probability unlikely to be the result of chance (P < 0.001). An intimate relationship exists between the megakaryocyte periphery and the abluminal surface of the endothelial lining cell. At the time of platelet release megakaryocyte cytoplasm invaginates and penetrates the endothelial lining cell. The penetrating cytoplasm is detached and enters the marrow circulation. From their dimensions in comparison to circulating platelets, the released cytoplasm represents a packet of platelets that undergoes further fragmentation in the circulation. The parasinusoidal location of megakaryocytes and the process of sinus-wall penetration and platelet delivery was observed by TEM and scanning electron microscopy. These studies provided quantitative support for a specific anatomical arrangement of megakaryocytes in marrow. Moreover, the process of platelet release appears to be a physiological form of metastasis with invasion of vascular walls and vascular spread of cells, that are in this case amitotic.     

Thrombopoietin: its role from early hematopoiesis to platelet production

BACKGROUND AND OBJECTIVE: Thrombopoietin (TPO), also referred to as MpI ligand, is the most potent cytokine that physiologically regulates platelet production. With the availability of sufficient amounts of recombinant forms of the protein, the biological in vitro and in vivo activities of this cytokine have been extensively studied. The objective of this review is to summarize the published data focusing on TPO production and regulation and to discuss the pleiotropic biological action of this hormone. The review also highlights the results so far obtained in preclinical and clinical trials. EVIDENCE AND INFORMATION SOURCES: The material examined in this review includes data published by the author and articles or abstracts published in Journals covered by Medline. The author has contributed to the isolation of TPO, has been working in the field for several years and has contributed original papers on the TPO/MpI system in normal and pathologic situations. STATE OF THE ART: TPO is a hormone constitutively produced by the liver and kidneys. Plasma levels of TPO are regulated through receptor-mediated uptake, internalization and catabolism. First thought to be a lineage dominant factor promoting megakaryocytopoiesis, several lines of evidence indicate that TPO has pleiotropic effects on hematopoiesis. In vitro studies show that TPO alone, or in combination with early acting cytokines, stimulates the proliferation and enhances the expansion of primitive CD34+ CD38- hematopoietic progenitor cells. In vivo studies with c-mpl- and TPO-null mice reveal that the molecule sustains the survival and proliferation of early committed progenitor cells of various type. Preclinical and clinical trials indicate that recombinant TPO molecules increase platelet counts and megakaryocyte numbers in normal or mildly thrombocytopenic states. However, no significant effects of TPO administration on platelet recovery have so far been reported in patients subjected to intensive chemotherapy regimens. Recombinant molecules appear to be safe to administer and very little toxicity is reported. TPO augments the number of erythroid and myeloid committed progenitor cells in marrow, and mobilized stem cells in peripheral blood. PERSPECTIVES: The potential clinical use of TPO is still unclear. With the increased knowledge of the multiple effects of TPO on hematopoiesis, it is expected that future carefully monitored clinical trials will provide more information regarding the eventual benefits of this cytokine in the treatment of thrombocytopenia. At present, one successful application of TPO appears to be its addition in cytokine cocktails used to expand hematopoietic stem cells ex vivo.     

CD226 is expressed on the megakaryocytic lineage from hematopoietic stem cells/progenitor cells and involved in its polyploidization

OBJECTIVES: In this study, we examined the expression of CD226 on megakaryocytic, granulocytic and erythroid lineage from hematopoietic stem cells/progenitor cells in adult and fetus and its potential role in megakaryocytic maturation. METHODS: CD34(+) cells from adult and fetus were induced to differentiate toward the megakaryocytic lineage by thrombopoietin (TPO) and the granulocytic lineage by granulocyte colony-stimulating factor (G-CSF), respectively. Mononuclear cells from fetal liver and CD34(+) cells from adult were induced to differentiate toward erythroid-lineage by erythropoiesis (EPO). We investigated the expression of CD226 and lymphocyte function associated antigen-1 (LFA-1) (CD11a) during hemopoiesis. We also studied the effect of CD226 monoclonal antibody (MoAb) and LFA-1 MoAb on megakaryocyte with antibody cross-liking technique. RESULTS: CD34(+) cells from adult and fetus and TPO-induced CD41(+) cells all expressed CD226 molecule. CD226 was not expressed on erythroid progenitor cells and erythroblasts and most cells of granulocytic lineage although G-CSF induced a significant increase of the expression of CD226 on CD34(+) cells in early period of time. CD226 MoAb acts on megakaryocytes by inducing intracellular calcium mobilization. The expression of LFA-1 decreased significantly at late stage of differentiation and maturation of fetal megakaryocytes whereas the expression of LFA-1 on adult megakaryocytes retained at a high level. CD226 MoAb in combination with LFA-1 MoAb shifted the ploidy of generated megakaryocytes from adult-derived CD34(+) cells to higher classes significantly although CD226 and LFA-1 MoAb slightly increased the ploidy of the generated megakaryocytes individually. CD226 MoAb or LFA-1 MoAb or CD226 MoAb plus LFA-1 MoAbs did not increase the ploidy of the generated megakaryocytes from fetus-derived CD34(+) cells. CONCLUSION: CD226 molecules play an important role in maturation of the megakaryocytes in combination with LFA-1.     

The size and number of bone marrow megakaryocytes in malignant lymphoma and their relationship to abnormalities in platelet count

Platelet counts were measured routinely in 718 volunteer blood donors and 124 patients with malignant lymphoma. When considered by gender, the mean platelet counts of the patients with malignant lymphoma were significantly increased when compared to controls (P less than 0.01). In addition, 9% of males and 4% of females with lymphoma exhibited a thrombocytosis, in which their platelet counts exceeded the upper limit of the normal platelet count for control subjects (defined as 2 SDs in excess of the mean value). Megakaryocyte whole size, cytoplasmic and nuclear sizes, and megakaryocyte numbers were also quantified in bone marrow obtained from the iliac crest of 5 hematologically normal subjects and 26 patients with malignant lymphoma. Mean megakaryocyte sizes were significantly greater in subjects with lymphoma, when compared to control values (P less than 0.05). No significant differences in megakaryocyte numbers in bone marrow were observed between the two groups. For subjects with malignant lymphoma, platelet count was found to be significantly positively correlated with megakaryocyte size. The results of this study suggest that the elevated platelet count associated with malignant lymphoma is mediated by an increase in the size of the megakaryocytes in the bone marrow.