ALTERATIONS IN POLYRIBOSOMES DURING ERYTHROID CELL MATURATION

This communication presents a morphological study of the changes in ribosome content and organization which occur during the maturation of erythroid cells of the phenylhydrazine-treated rabbit. Electron micrographs of thin sectioned nucleated and non-nucleated erythroid cells have been subjected to a quantitative analysis of the distribution of ribosomes as polyribosomes of various sizes and as single ribosomes. The ribosomes of nucleated erythroid cells of marrow are virtually all arranged in the polyribosome configuration consisting of clusters of 2 to 6 individual ribosomes. These cells are the most active in the erythroid series in protein biosynthesis. During maturation to the non-nucleated reticulocyte stage, found in the circulating blood, there is a decrease in protein synthesizing capacity, a fall in total ribosome content, and, more significantly, a decrease in the number and size of polyribosomes. Maturation to the ribosome-free erythrocyte, either under in vitro or in vivo conditions, entails a further decrease in protein synthesis which correlates with a progressive disaggregation of the biosynthetically active polyribosomes into smaller clusters and inactive single ribosomes. Possible models which may account for the stability of the polyribosome and for the mechanism of polyribosome dissociation are discussed.     

Ultrastructural Aspects of Erythropoietic Differentiation in Long-term Bone Marrow Culture

Long-term liquid cultures of mouse bone marrow produce stem cells (CFU-S) and differentiated granulocytes for many months. Addition of AMS (anaemic mouse serum) to the cultures almost entirely eliminates the granulopoietic activity and stimulates erythropoiesis, with full erythroid maturation and the production of adult haemoglobin. Ultrastructural analsysis of in situ fixed material reveals the cell shape and surface morphology of the erythroid maturation series, and the generation of erythroblastic islands in vitro. Each erythroblastic island consists of one or more synchronously maturing cohorts of erythroid cells undergoing four or five divisions between proerythroblast and normoblast. Each island is centered on a macrophage, which interacts with the developing erythroid population in several ways. Expelled nuclei are phagocytosed by the macrophage, which also has large areas of closely apposed membrane with the erythroid cells, gap junctions, and possible reciprocal vesicular activity. Changes in the adherent layer (stromal cells) also occur with the transition from granulopoiesis to erythropoiesis. There is a reduction in the endothelial cell cover, and mobilisation of lipid from the granulopoietic associated apidocytes.     

Penetration of maturating red blood cells by Toxoplasma gondii

Penetration of Toxoplasma gondii tachyzoites was studied in vitro using murine erythroid cells at different stages of development. Toxoplasma gondii penetrated nucleated erythroblasts and macroreticulocytes from foetal mouse liver and the circulating erythrocytes of foetal, neonatal or severely anaemic adult mice. Immature reticulocytes were more susceptible to penetration than mature ones, indicating that some change in their membrane properties occurred during maturation. The present results confirmed our previous finding that the major erythrocyte membrane-specific proteins do not prevent erythrocyte penetration since these proteins are known to be present in the reticulocyte membrane.     

Transferrin receptors and iron uptake during erythroid cell development

Experiments were performed to determine the level of transferrin receptors and rate of transferrin-bound iron uptake by various immature erythroid cell populations. Developing erythroid cells from the rat and mouse foetal liver at various stages of gestation were studied. In addition Friend leukaemic cells grown in culture were examined. The transferrin receptor level of Friend cells was similar to that of erythroid cells from the mouse foetal liver. During erythroid cell development the transferrin receptor level increased from about 300,000 per cell at the early normoblast stage to reach a maximum of about 8000,000 per cell on intermediate normoblasts. Further maturation of intermediate normoblasts was accompanied by a decline in the number of transferrin receptors, reaching a level of 105,000 in the circulating reticulocyte. The rate of iron uptake from transferrin during erythroid cell development was found to correlate closely with the number of transferrin receptors. In each of the immature erythroid cell populations studied the rate of iron uptake was about 36 iron atoms per receptor per hour. These results indicate that the level of transferrin receptors may be the major factor which determines the rate of iron uptake during erythroid cell development.     

An electron-microscope autoradiographic study of transferrin endocytosis by immature erythroid cells

The receptor-mediated endocytosis of 125I-transferrin by immature erythroid cells was studied using the technique of quantitative electron microscope autoradiography. Morphometric analysis of the grain distribution in erythroid cells from the foetal rat liver revealed that the 125I-transferrin radioactivity was localized mainly to intracellular vesicles (61%) and the cell membrane (25%) after 20 min incubation at 37 degrees C. No activity was found associated with the nucleus or mitochondria and only a small amount with the cytosol (13%). In erythroid cells which possessed a prominent Golgi complex, most of the autoradiographic grains were associated with vesicles located in this region, giving rise to a polar distribution of the 125I-transferrin. Uptake of transferrin was found to be maximal at the basophilic normoblast stage of development and then declined progressively during maturation to the reticulocyte. The kinetics of endocytosis of 125I-transferrin by rabbit reticulocytes was also studied by electron microscope autoradiography. Up to 30% of the cell-bound transferrin was internalized almost immediately upon incubation at 37 degrees C. After 30 sec incubation, 42% of the cell-bound 125I-transferrin was estimated to be internal and this rose to almost 70% at steady state between the binding and release of transferrin after 12 min incubation.     

The effects of repeated bleedings on bone marrow and blood morphology in adult laboratory rats

There were slight and rapidly disappearing changes in the bone marrow and blood morphology of laboratory rats after repeated bleedings on consecutive days, except marked increases in the counts of circulating reticulocytes. However, the significantly elevated reticulocyte count was not accompanied by the similar increase in the count of marrow erythroid nucleated cells. Marked increments could be found in the counts of splenic erythroid nucleated cells and, therefore, it is possible for rat circulating reticulocytes to originate following repeated bleedings from the spleen. The validity of investigations of bone marrow and blood morphology for detecting haemorrhage is discussed with a view to preclinical safety drug evaluations performed on adult laboratory rats.     

Synthesis and assembly of membrane skeletal proteins in mammalian red cell precursors

The synthesis of membrane skeletal proteins in avian nucleated red cells has been the subject of extensive investigation, whereas little is known about skeletal protein synthesis in bone marrow erythroblasts and peripheral blood reticulocytes in mammals. To address this question, we have isolated nucleated red cell precursors and reticulocytes from spleens and from the peripheral blood, respectively, of rats with phenylhydrazine-induced hemolytic anemia and pulse-labeled them with [35S]methionine. Pulse-labeling of nucleated red cell precursors shows that the newly synthesized alpha- and beta-spectrins are present in the cytosol, with a severalfold excess of alpha-spectrin over beta-spectrin. However, in the membrane-skeletal fraction, newly synthesized alpha- and beta-spectrins are assembled in stoichiometric amounts, suggesting that the association of alpha-spectrin with the membrane skeleton may be rate-limited by the amount of beta-spectrin synthesized, as has been shown recently in avian erythroid cells (Blikstad, I., W. J. Nelson, R. T. Moon, and E. Lazarides, 1983. Cell, 32:1081-1091). Pulse-chase experiments in the rat nucleated red cell precursors show that the newly synthesized alpha- and beta-spectrin of the cytosol turn over coordinately and extremely rapidly. In contrast, in the membrane-skeletal fraction, the newly synthesized polypeptides of spectrin are stable. In contrast to nucleated erythroid cells, in reticulocytes the synthesis of alpha- and beta-spectrins is markedly diminished compared with the synthesis and assembly of proteins comigrating with bands 2.1 and 4.1 on SDS gels. Thus, in nucleated red cell precursors, the newly synthesized spectrin may be attached to the plasma membrane before proteins 2.1 and 4.1 are completely synthesized and incorporated in the membrane.     

Unique monoclonal antibodies specifically bind surface structures on human fetal erythroid blood cells

Background

Continuing efforts in development of non-invasive prenatal genetic tests have focused on the isolation of fetal nucleated red blood cells (NRBCs) from maternal blood for decades. Because no fetal cell-specific antibody has been described so far, the present study focused on the development of monoclonal antibodies (mAbs) to antigens that are expressed exclusively on fetal NRBCs.Methods: Mice were immunized with fetal erythroid cell membranes and hybridomas screened for Abs using a multi-parameter fluorescence-activated cell sorting (FACS). Selected mAbs were evaluated by comparative FACS analysis involving Abs known to bind erythroid cell surface markers (CD71, CD36, CD34), antigen-i, galactose, or glycophorin-A (GPA). Specificity was further confirmed by extensive immunohistological and immunocytological analyses of NRBCs from umbilical cord blood and fetal and adult cells from liver, bone marrow, peripheral blood, and lymphoid tissues.Results: Screening of 690 hybridomas yielded three clones of which Abs from 4B8 and 4B9 clones demonstrated the desired specificity for a novel antigenic structure expressed on fetal erythroblast cell membranes. The antigenic structure identified is different from known surface markers (CD36, CD71, GPA, antigen-i, and galactose), and is not present on circulating adult erythroid cells, except for occasional detectability in adult bone marrow cells.Conclusions:The new mAbs specifically bind the same or highly overlapping epitopes of a surface antigen that is almost exclusively expressed on fetal erythroid cells. The high specificity of the mAbs should facilitate development of simple methods for reliable isolation of fetal NRBCs and their use in non-invasive prenatal diagnosis of fetal genetic status.     

Distribution of the inducer of differentiation bis-acetyl-diaminopentane in murine erythroleukemia cells

Exposure of murine leukemia cells in culture to bis-acetyl-diaminopentane (BADP) caused erythroid maturation as measured by the accumulation of hemoglobin in treated cells. The appearance of differentiated cells in cultures exposed to BADP occurred 18 to 20 hours earlier than in those treated with dimethylsulfoxide (DMSO), a standard inducer of differentiation in this system. Studies with [³H]BADP indicated the occurrence of relatively rapid association of the inducer with cells, and subsequent linear accumulation. Fractionation of cellular components and measurement of radioactivity from BADP therein demonstrated that this agent preferentially associates with a fraction enriched for plasma membrane. In addition, [³H]BADP was capable of binding to the plasma membrane-enriched fraction isolated from murine erythroleukemia cells as measured by gel filtration. These findings support the concept that interaction of inducers of murine erythroleukemia differentiation such as BADP with components of the surface membrane may be important in the cascade of events that lead to the erythroid maturation of these leukemic cells.     

Hereditary erythroblastic anaemia in the laboratory mouse

Hereditary erythroblastic anaemia was found in a homozygous mutant (hea/hea) of an inbred strain CFO, which originated from noninbred CF#1 mice from Carworth Inc. This newly-described anaemia is inherited as an autosomal recessive and is lethal at 15-25 days of age. Erythrocytes of anaemic mice show striking polychromasia, anisocytosis, and poikilocytosis. One of the most marked features of this anaemia is the presence of large numbers of nucleated cells, mainly orthochromatophilic erythroblasts and myeloid cells, in the circulation. They also include immature erythroid and myeloid cells. Many naked nuclei appear on smears from circulating blood of anaemic infants. Erythrocytes, haematocrit percentage, and haemoglobin content of older anaemic infants were only about 50% of those of the normal. Formation of erythroid, myeloid cells, and megakaryocytes in the bone marrow seems to be progressively affected by mutant alleles in the anaemic infants.     

The effect of progesterone on hemoglobin synthesis in suspension cultures of fetal erythroid cells from calf liver

When fetal calf liver erythroid cells were incubated in the presence of small amounts of progesterone (10(-7)-10(-8) M), the hemoglobin synthesis in these cells was significantly increased. The increase in the amount of radioactivity in de novo synthesized hemoglobins could be demonstrated when techniques such as isoelectric focusing, chromatography on DEAE-cellulose and gel chromatography on Sephadex G-100 were used to isolate the hemoglobin fraction. Using the latter technique, it was shown that the synthesis of cytoplasmic non-hemoglobin proteins in erythroid-cell lysates was also stimulated by progesterone. The presence of hepatocytes in culture nullified the hormone action. It was necessary that progesterone was present during the first hours of culture. Delayed addition of the steroid to the cells had no effect on hemoglobin synthesis. Erythropoietin was necessary to obtain stimulation by progesterone. These results suggest that the target cell of the hormone is an erythropoietin-sensitive cell. High concentrations of progesterone (10(-4) M) strongly inhibited hemoglobin synthesis in fetal calf erythroid cells. Culture of cells under this condition, however, gives rise to a cell population that preferentially synthesizes adult hemoglobin. Our results suggest that in the erythropoietic calf liver, high concentrations of progesterone may preferentially stimulate adult hemoglobin synthesis, or that those cells which have a high capacity to synthesize adult hemoglobins are less sensitive to toxic concentrations of the hormone. The effects of stimulation of hemoglobin synthesis in fetal calf erythroid cells occur at hormone concentrations that suggest a possible physiological role of progesterone in fetal, and eventually also in maternal, erythropoiesis.     

Fetal erythropoiesis in steel mutant mice : I. A morphological study of erythroid cell development in fetal liver

A method of definitive identification of mutant (S1/S1^d) and wild-type (+/+) mouse embryos in segregating litters is described, based on the total number of circulating erythrocytes in a unit volume of embryonic blood and the relative proportion of nonnucleated vs. nucleated red blood cells. Evidence is presented that from days 13–17 of gestation, S1/S1^d embryos have many fewer fetal liver derived nonnucleated erythrocytes whereas the number of yolk sac-derived nucleated red blood cells is similar between S1/S1^d and +/+. Erythroid precursor cells at various stages of maturation in mutant fetal livers are studied by light and electron microscopy, and their fine structure is found to be identical to those present in normal embryos. The number of hemoglobin-containing mature erythroblasts in mutant fetal livers is far fewer than that of the normal, whereas the number of immature erythroid precursors present in a unit area of fetal liver is not significantly different between S1/S1^d and +/+. It is suggested that the mutant S1 gene product(s) interferes with or fails to support the differentiation of immature erythroid precursors into hemoglobin synthesizing cells.     

Five-dimensional flow cytometry as a new approach for blood and bone marrow differentials

We have used five independent variables on a flow cytometer to discriminate and to quantify the cellular components within both blood and bone marrow aspirates. The signals were stored in list mode by which a five-dimensional space was created. The cells--differentiated into: 1) erythrocytes, 2) reticulocytes, 3) nucleated erythroid cells, 4) platelets, 5) lymphocytes, 6) monocytes, 7) neutrophils, 8) eosinophils, and 9) immature leukocytes--had to meet unique criteria with regard to their characteristics in the created five-dimensional space in order to be classified in a specific cell category. Forward and orthogonal light-scattering signals were matched with three fluorescence variables to obtain discrimination without necessitating erythrocyte lysis. Thiazole orange (binding predominantly to RNA) and LDS-751 (principally detecting DNA) were used to differentiate erythrocytes, platelets, reticulocytes, and nucleated cells. A monoclonal antibody, CD45, conjugated with phycoerythrin, was used to aid in discriminating between lineages of nucleated cells.     

Cell-type-related segregation of surface galactosyl-containing components at an early developmental stage in hemopoietic bone marrow cells in the rabbit

The avidin-biotin complex was used for the selective ultrastructural labeling of terminal cell surface galactosyl residues. Rabbit bone marrow cells were treated with the enzyme galactose oxidase in the presence of biotin hydrazide. Subsequent treatment with ferritin-avidin conjugates enabled the electron microscopic visualization of terminal membrane-based galactose and/or N-acetylgalactosamine on these cells. All stages of erythroid development were characterized by high levels of exposed cell surface galactose, whereas all leukoid cells in the same preparations were virtually unlabeled by the above method. Modulations in the distribution of these surface determinants during differentiation and maturation of rabbit erythroid cells were found to concur in inverse fashion with respect to that of terminal sialic acids. Neuraminidase treatment, before the above labeling procedure, resulted in the exposure of additional galactosyl residues on the surface of all bone marrow cell types. The results indicate that a galactose-bearing glycoconjugate(s) may comprise an erythroid-specific membrane constituent of rabbit bone marrow cells. The high density of galactose on the surface of even the earliest erythroid precursors may eventually enable the identification and isolation of a stem cell, which already contains the erythroid-specific galactoconjugate(s). The results suggest that variations in the spectrum of cell surface carbohydrates may serve as recognition signals in the complex set of intercellular interactions which occur during the development and maturation of the erythrocyte. The occurrence of similar but species- specific variations in the complement of surface heterosaccharides during erythroid development of humans and other mammals supports this contention.     

Vimentin downregulation is an inherent feature of murine erythropoiesis and occurs independently of lineage.

In mammalian erythropoiesis, the mature cells of the primitive lineage remain nucleated while those of the definitive lineage are anuclear. One of the molecular and structural changes that precedes enucleation in cells of the definitive lineage is the cessation in the expression of the gene for the intermediate filament (IF) protein vimentin and the removal of all vimentin filaments from the cytoplasm. We show here that in immature primitive cells vimentin is synthesized and forms a cytoplasmic network of IFs. As differentiation proceeds in vivo, vimentin gene expression is downregulated in these cells; this is accompanied by the loss of vimentin filaments from the cytoplasm. This loss temporally coincides with the nucleus becoming freely mobile within the cytoplasm, suggesting that, while IF removal is not directly linked to the physical process of enucleation, it may be a prerequisite for the initiation of nuclear mobility in both lineages. These changes are also observed in early primitive cells cultured in vitro, suggesting that they constitute an intrinsic part of the murine erythroid differentiation program independent of lineage and hematopoietic microenvironment.     

The role of oxidant injury in the pathophysiology of human thalassemias

The anemia in beta-thalassemia major is caused by a combination of hemolysis and ineffective erythropoiesis, with the latter being more important. Studies of the underlying cause of the hemolysis have indicated that oxidant injury to circulating red blood cells (RBCs) was of critical importance, with evidence of oxidant damage to RBC membrane proteins 4.1 and band 3. Therefore, it seemed reasonable that oxidant damage to thalassemic erythroid precursors would cause their accelerated apoptosis and ineffective erythropoiesis. However, direct analysis showed that the apoptotic programs turned on in thalassemics were not those triggered by oxidative damage but were dependent on activation of FAS/FAS-Ligand interaction. Thus, destruction of thalassemic erythroid precursors may involve different mechanisms from those that cause RBC hemolysis.     

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.     

Further studies on the rabbit erythroid cell plasma membrane transferrin receptor

Studies reproted here indicate that a major cell surface glycoprotein of the rabbit bone marrow erythroid cell may be involved in binding transferrin. The glycoprotein has an apparent molecular weight of 18,000. It is suggested that bone marrow erythroid cells may provide an invaluable source of the red cell membrane transferrin receptor for future studies.