Activity of nucleolar organizers in central macrophage culture of bone marrow erythroblastic islands

The erythroblastic islands of the bone marrow are morphofunctional units of erythropoiesis. In this work the functional state of erythroblastic islands' cells of the bone marrow, for the first time, was defined by the estimation of the activity of the nucleolar organizers of central macrophages in the erythroblastic islands, cultivated during 24 and 48 hours with the presence of various doses of erythropoietin. The findings indicated that the increase in doses of erythropoietin was accompanied by the corresponding increase of the activity of nucleolar organizers in central macrophages of erythroblastic islands. The nucleolar organizers of central macrophages in cultures of erythroblastic islands responded to very small doses of erythropoietin by their activation.     

the effect of colonystimulating macrophage factor on activity of the nuclear organisers in central macrophages of the cultures of the bone marrow erythroblastic islands

Erythroblastic islands of the bone marrow are morpho-functional units of erythropoiesis. The functional state of erythroblastic islands' cells of the bone marrow was for the first time defined by estimation of activity of the nuclear organisers of the central macrophages in the erythroblastic islands cultivated for 24 hrs in presence of various doses of the colony-stimulating macrophage factor. The findings indicate that increased doses of the colonystimulating macrophage factor was accompanied by a respective enhancement of the activity of nucleolar organisers in central macrophages of erythroblastic islands.     

Antibodies against interspecies erythrokaryocyte antigen in patients with partial red cell aplasia of the bone marrow

Immunofluorescense and cytotoxicity test in vitro were used to demonstrate specific antibodies in sera of 11 out of 19 patients with partial red cell aplasia (PRCA). The antibodies reacted with erythroblast cells from embryos and adult men, with bone marrow cells from a female patient suffering from acute erythroleukemia, with erythrokaryocytes of mouse embryos and cells of Rauscher's viral erythroleukemia. The results of cross adsorption and blockade of the immunofluorescent reaction of the sera of PRCA patients with antibodies against mouse erythroblast antigen bearing an interspecies determinant suggest that in the pathogenesis of PRCA there takes part an autoimmune reaction against specific interspecies antigen to erythrokaryocytes. This antigen is apparently similar to antigen against mouse erythroblast cells.     

Changes in various links of metabolism and the antioxidant system in the bone marrow erythroid cells of the pig during the neonatal period]

The age dynamics of activities of enzymes which catalysis several stages of metabolism (hexokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase, 2,3-diphosphoglycerate mutase, glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase and cytochrome oxidase) and antioxidant system (superoxide dismutase, glutathione peroxidase and glutathione reductase) was studied in the bone marrow erythroid cells of pig during the 10-day period after birth as well as in the cells of 30 days old animals. It was established that in the neonatal period of development the reorganization of energy metabolism in pig bone marrow erythrokaryocytes took place. It consisted in the intensification of oxidative processes and in a great measure was directed on the activation of 2,3-diphosphoglycerate mutase formation in the nature red cells. During the early period after birth the activation of antioxidant system in erythroid cells of pig bone marrow was observed.     

Rubidium uptake in single cells

Summary Rubidium uptake was measured in single erythroid and myeloid cells of rabbit by means of X-ray microanalysis. It was found in the nucleated bone marrow cells that after incubation in rubidium the sums of potassium and rubidium concentrations were similar to the original potassium concentrations, indicating that there was one-to-one replacement of potassium by rubidium. Although the nuclear potassium and rubidium concentrations were higher than those in the cytoplasm, the nuclear and cytoplasmic ratios of K/Rb were similar. This implies that the potassium in both compartments exchanged freely with rubidium. In the erythroid line of cells there was a continuous reduction of potassium transport activity during the maturation process as indicated by the decrease in rubidium uptake rates. The uptake was measured in seven groups of cell types that could be distinguished on the basis of morphology and chemical composition. The order of the groups from high to low rubidium uptake were: esosinophilic myelocyte > early erythroblast and thinrimmed erythroblast > late erythroblast > early bone marrow red cell > late bone marrow red cell > peripheral blood red cell. Thus, there is a continuous decrease in rubidium transport as the erythroid cells mature.     

Effects of various concentrations of erythropoietin on cultured erythroblast islands

Proliferative effects of increasing concentrations of erythropoietin were studied in vitro. The maximal amount of erythroblastic islands consisting of residual macrophages and erythroid cells was observed with the doses 0.5 to 1.5 ME/ml of erythropoietin. Smaller doses stimulated the reconstruction of erythroblastic islands.     

人红细胞生成素的分离、纯化及鉴定

 用自制的苯基-琼脂糖CL-4B和羟基邻灰石等层析材料,从再生障碍性贫血病人尿中分离、纯化制得了红细胞生成素(EPO)。用多血小鼠红细胞~(56)Fe参入法测定该制品在体内的生物活力。用小鼠与人骨髓红系祖细胞培养法测其在体外的生物活力。实验结果说明,我们自制的EPO制品,不仅能用于动物,也能用于人骨贿红系祖细胞的培养。用Azocoll法测该制品中蛋白水解酶活力为阴性。     

Induction of erythropoietin responsiveness in vitro by a distinct population of bone marrow cells.

Bone marrow contains a small population of primitive erythroid progenitor cells which can be detected by their capacity to form large numbers of erythroid progeny in viscous cultures containing erythropoietin (EP). These cells have been termed erythroid 'burst-forming units' (BFUe). The present study demonstrates that expression of the erythroid differentiation potential of BFUe requires the presence of an activity additional to EP. This activity has been designated as BFA (burst feeder activity). It is shown that the number of BFUe detected and their apparent sensitivity to EP are directly related to the BFA concentration of the cultures. BFA was found to be associated with a population of bone marrow cells of high buoyant density and small volume, which are sensitive to irradiation. The radiation dose-effect curve provided strong evidence that bone marrow BFA is independent of cell proliferation; this was supported by showing that BFA is unaffected by in vivo treatment with hydroxyurea. The findings are compatible with a two-step regulation model for erythroid differentiation in which BFA-induced progeny of BFUe acquire sensitivity to EP.     

INDUCTION OF ERYTHROPOIETIN RESPONSIVENESS IN VITRO BY A DISTINCT POPULATION OF BONE MARROW CELLS

Bone marrow contains a small population of primitive erythroid progenitor cells which can be detected by their capacity to form large numbers of erythroid progeny in viscous cultures containing erythropoietin (EP). These cells have been termed erythroid ‘burst-forming units’(BFUe). The present study demonstrates that expression of the erythroid differentiation potential of BFUe requires the presence of an activity additional to EP. This activity has been designated as BFA (burst feeder activity). It is shown that the number of BFUe detected and their apparent sensitivity to EP are directly related to the BFA concentration of the cultures. BFA was found to be associated with a population of bone marrow cells of high buoyant density and small volume, which are sensitive to irradiation. The radiation dose-effect curve provided strong evidence that bone marrow BFA is independent of cell proliferation; this was supported by showing that BFA is unaffected by in vivo treatment with hydroxyurea. The findings are compatible with a two-step regulation model for erythroid differentiation in which BFA-induced progeny of BFUe acquire sensitivity to EP.     

Antigenic marker of human erythrokaryocytes similar to mouse erythroblastosis antigen]

Identical antigenic determinants are discovered on the surface of human erythrokaryocytes with antibodies against specific antigen of murine erythroblasts (Ag-Ed), previously revealed in study of Rauscher leukemia, in the immunofluorescent and cytotoxic tests. The antigen is present on the membranes of the majority of human embryonic liver and adult bone marrow nuclear erythroid cells, but is not found in fetal thymocytes, newborn kidney cells, adult human hepatic cells and in peripheral blood erythrocytes. Ag-Eb appears to possess an inter-species determinant, shared by mammalian nuclear erythroid cells, and may be used as their specific marker.     

Erythropoietin stimulates 45Ca2+ uptake in Friend virus-infected erythroid cells

It has been shown previously in this laboratory that in vitro infection of mouse bone marrow cells with the anemia strain of Friend leukemia virus leads to growth of large bursts of erythroid cells which are arrested in development prior to hemoglobin synthesis but can respond to erythropoietin (EP) to complete the late stage of erythroblast differentiation. In this study, the effect of EP on the metabolism of 45Ca2+ in these cells was examined. At 4 degrees C, an increased rate of 45Ca2+ uptake and efflux as well as an increase in the steady state level of 45Ca2+ in treated cells was observed. Exchange of 45Ca2+ from preloaded cells at 4 degrees C indicated that treatment with EP increased the size of a rapidly exchanging pool of 45Ca2+ from 5 to 12% of total 45Ca2+ in the cell. The effect of treatment with EP can be seen as increased exchange of extracellular 45Ca2+ with cellular Ca2+; however, an effect of EP on the net level of Ca2+ in these cells cannot be excluded. This investigation demonstrates one of the earliest effects of EP on erythroid cells and suggests that alterations in Ca2+ metabolism may contribute to the progression of erythroid cells to their final development.     

Production of erythropoietic cells in vitro for continuous culture of Plasmodium vivax

Plasmodium vivax cannot be maintained in a continuous culture. To overcome this major obstacle to P. vivax research, we have developed an in vitro method to produce susceptible red blood cell (RBC) precursors from freshly isolated human cord hematopoietic stem cells (HSCs), which were activated with erythropoietin to differentiate into erythroid cells. Differentiation and maturation of erythroid cells were monitored using cell surface markers (CD71, CD36, GPA and Fy6). Duffy⁺ reticulocytes appeared after 10 days of erythroid cell culture and exponentially increased to high numbers on days 14–16. Beginning on day 10 these erythroid cells, referred to as growing RBCs (gRBCs), were co-cultured with P. vivax-infected blood directly isolated from patients. Parasite-infected gRBCs were detected by Giemsa staining and a P. vivax-specific immunofluorescence assay in 11 out of 14 P. vivax isolates. These P. vivax cultures were continuously maintained for more than 2 weeks by supplying fresh gRBCs; one was maintained for 85 days before discontinuing the culture. Our results demonstrate that gRBCs derived in vitro from HSCs can provide susceptible Duffy⁺ reticulocytes for continuous culture of P. vivax. Of particular interest, we discovered that parasites were able to invade nucleated erythroid cells or erythroblasts that are normally in the bone marrow. The possibility that P. vivax causes erythroblast destruction and hence inflammation in the bone marrow needs to be addressed.     

Stimulation of adenylate cyclase activity by catecholamines and prostaglandins E during differentiation of rabbit bone marrow erythroid cells

After fractionation of rabbit bone marrow into erythroid cells at different developmental stages adenylate cyclase activity of membrane ghosts was assayed in the presence of sodium fluoride, catecholamines or prostaglandins E. Both basal and fluoride-stimulated adenylate cyclase decreased continuously during differentiation. Only catecholamines having beta 2-adrenergic activity stimulated adenylate cyclase and their effect was restricted to the most immature cells, the proerythroblasts and, to a lesser extent, the basophilic erythroblasts. Thus, uncoupling of beta-adrenergic receptors occurs early in erythroblast development and hormone responsiveness is lost before the final cell division. Prostaglandin E receptors and adenylate cyclase remain coupled throughout erythroid cell development.     

THE ROLE OF ERYTHROPOIETIN IN REGULATION OF POPULATION SIZE AND CELL CYCLING OF EARLY AND LATE ERYTHROID PRECURSORS IN MOUSE BONE MARROW

This study was designed to determine the stage in haemopoietic cell differentiation from multipotential stem cells at which erythropoietin becomes physiologically important. The responses of haemopoietic precursor cells were monitored in the bone marrow of mice under conditions of high (after bleeding) and low (after hypertransfusion) ambient erythropoietin levels. The number of relatively mature erythroid precursors (CFU-E), detected by erythroid colony formation after 2 days of culture, increased three-fold in marrow by the fourth day after bleeding, and decreased three-fold after hypertransfusion. Assessed by sensitivity to killing by a brief exposure to tritiated thymidine (³H-TdR) in vitro, the proliferative activity of CFU-E was high (75% kill) in untreated and bled animals, and was slightly lower (60% kill) after hypertransfusion. The responses of more primitive erythroid progenitors (BFU-E), detected by erythroid colony formation after 10 days in culture, presented a contrasting pattern. After hypertransfusion they increased slightly, while little change was noted until the fourth day after bleeding, when they decreased in the marrow. The same response pattern was observed for the progenitors (CFU-C) detected by granulocyte/macrophage colony formation in culture. The sensitivity of BFU-E to ³H-TdR was normally 30%, and neither increased after bleeding nor decreased after hypertransfusion. However, in regenerating marrow the ³H-TdR sensitivity of BFU-E increased to 63%, and this increase was not affected by hypertransfusion. These results are interpreted as indicating (1) that physiological levels of erythropoietin do not influence the decision by multipotential haemopoietic stem cells to differentiate along the erythroid pathway as opposed to the granulocyte/macrophage pathway; (2) that early erythroid-committed progenitors themselves do not respond to these levels of erythropoietin, but rather are subject to regulation by erythropoietin-independent mechanisms; and (3) that physiological regulation by erythropoietin commences in cells at a stage of maturation intermediate between BFU-E and CFU-E.     

Kupffer cells support extramedullary erythropoiesis induced by nitrogen-containing bisphosphonate in splenectomized mice

Our previous study indicated that injecting nitrogen-containing bisphosphonate (NBP) induced the site of erythropoiesis to shift from the bone marrow (BM) to the spleen. This was due to the depletion of BM-resident macrophages, which support erythropoiesis. In this study, we examined NBP treatment-induced extramedullary hematopoiesis in splenectomized mice, focusing on hepatic hematopoiesis. NBP-treated mice did not display anemia or significant change in erythropoietin production, while megakaryopoiesis and erythropoiesis were constantly observed in the liver. Erythroblastic islands were detected in the sinusoidal lumen. Kupffer cells expressed VCAM-1 following NBP treatment, which is an important factor for erythroblast differentiation. Cl₂MBP-liposome treatment depleted the erythroblastic islands, and decreased the number of hematopoietic cells in the liver, as determined by colony forming assays. Together, these results indicate that Kupffer cells support erythropoiesis, acting as stromal cells in the liver, and that they might act as a niche for hematopoietic precursor cells in an emergency.     

Src transduces erythropoietin-induced differentiation signals through phosphatidylinositol 3-kinase

In this study, we examined the molecular mechanism of erythropoietin-initiated signal transduction of erythroid differentiation through Src and phosphatidylinositol 3-kinase (PI3-kinase). Antisense oligonucleotides against src but not lyn inhibited the formation of erythropoietin-dependent colonies derived from human bone marrow cells and erythropoietin-induced differentiation of K562 human erythroleukaemia cells. Antisense p85alpha oligonucleotide or LY294002, a selective inhibitor of PI3-kinase, independently inhibited the formation of erythropoietin-dependent colonies. In K562 cells, Src associated with PI3-kinase in response to erythropoietin. Antisense src RNA expression in K562 cells inhibited the erythropoietin-induced activation of PI3-kinase and its association with erythropoietin receptor. PP1, a selective inhibitor of the Src family, reduced erythropoietin-induced tyrosine phosphorylation of erythropoietin receptor and its association with PI3-kinase in F-36P human erythroleukaemia cells. The coexpression experiments and in vitro kinase assay further demonstrated that Src directly tyrosine-phosphorylated erythropoietin receptor, and associated with PI3-kinase. In vitro binding experiments proved that glutathione S-transferase-p85alpha N- or C-terminal SH2 domains independently bound to erythropoietin receptor, which was tyrosine-phosphorylated by Src. Taken together, Src transduces the erythropoietin-induced erythroid differentiation signals by regulating PI3-kinase activity.     

The effect of erythropoietin on the growth and development of spleen colony-forming cells

The progressive growth and development of spleen colonies was studied in heavily irradiated host mice in which erythropoiesis was modified by various procedures. Erythropoietic activity in non-polycythemic hosts bearing spleen colonies was not increased by injections of exogenous erythropoietin. Detectable levels of erythropoietin were found in the heavily irradiated host mice suggesting that the failure of exogenous erythropoietin to modify erythropoiesis was because the host mice were already maximally stimulated by the high endogenous erythropoietin levels. Spleen colonies do not become erythroid in polycythemic mice. The injection of exogenous erythropoietin into heavily irradiated polycythemic hosts did not decrease the total number of spleen colonies produced by a given bone marrow transplant, as would be expected if erythropoietin acted directly on the colony-forming cells. Comparison of growth curves for colony-forming cells in the spleens of polycythemic hosts either receiving or not receiving erythropoietin indicated that the overall doubling time of colony-forming cells during the first ten days after transplantation was not changed by the daily injection of erythropoietin. These experiments are consistent with the concept that erythropoietin is necessary for the development of erythroid colonies. Erythropoietin acts upon some progeny of the colony-forming cell rather than the colony-forming cell itself.     

Differentiation of chick blood island erythroblasts into erythrocytes and stability in long-term culture

Clusters of 20-70 erythroblasts from blood islands of early chick blastoderm were cultured in serum-free chemically defined medium for a 3-month period. The erythroblast cluster produces erythroid cells and hemoglobins characteristic of the primitive and definitive erythroid cell lines. It seems there is a progenitor erythroid cell(s) in the erythroblast cluster which starts and/or continues maturing along various pathways of hemopoietic differentiation under simple culture conditions. The erythroid character of these cells is stable during the 3-month culture period.