Role of the reticular cells during maturation process of the erythroblast. I. Denucleation of erythroblast by reticular cell; electron microscopic study.
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Animals↗
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Role of the reticular cells during maturation process of the erythroblast. II. Further observation on the denucleation process of erythroblast.
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Animals↗
[Abnormal erythroblastic island due to the development of intercellular junctions (erythroblastic synarthesis). A new mechanism of anemia. Problems posed by the diagnosis].
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Aged↗
[In vitro study of erythroblast precursors in Vasquez disease (polycythemia vera). Arguments in favor of a double population of erythroblastic stem cells in the bone marrow].
In vitro studies using the plasma clot culture system have been performed in order to compare the red cell pregenitors able to rise to erythrocytic colonies in 7 days (CFUE) in the bone marrow of polycythemia vera (PV), secondary polycythemias and normal subjects. In PV but never in normal individuals or secondary polycythemias, the bone marrow cells producing erythroid colonies without addition of erythropoietin were found. The erythropoietin dode response curves in PV is biphasic with a plateau up to a concentration of erythropoietin of 0.02-0.05 i.U./ml followed by a near normal response to erythropoietin at higher doses. Thus our results demonstrate that two populations of erythroid stem cells coexist in PV, one being abnormally sensitive to (or independant of) erythropoietin, the other normally responding to erythropoietin. After remission induced by P32 treatment, the abnormal population can disappear but the prognostic significance of this disappearance is uncertain. On the whole these results are in agreement with those of others laboratories using the plasma clot culture system. The reasons of the disagreement with the data published using the methylcellulose technic of culture are discussed.
Animals↗
Effect of surface digestion and metabolic inhibitors on appearance of ferritin in guinea pig erythroblasts in vitro: evidence for the production of apoferritin on the erythroblast cell membrane.
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Animals↗
[Study of anaemia using 3H-thymidine-labelled erythroblasts. 1. In vitro 3H-thymidine-positive values for erythroblasts in normal rabbits].
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Anemia↗
Aberrant iron accumulation and oxidized status of erythroid-specific delta-aminolevulinate synthase (ALAS2)-deficient definitive erythroblasts.
Alas2 encodes the erythroid-specific delta-aminolevulinate synthase (ALAS2 or ALAS-E), the first enzyme in heme biosynthesis in erythroid cells. Mice with the Alas2-null phenotype showed massive cytoplasmic, but not mitochondrial, iron accumulation in their primitive erythroblasts. Because these animals died by day 11.5 in utero, studies of iron metabolism in definitive erythroblasts were not possible using the in vivo model. In this study, embryonic stem (ES) cells lacking the Alas2 gene were induced to undergo differentiation to the definitive erythroblast stage in culture, and the phenotype of Alas2-null definitive erythroblasts was examined. Alas2-null definitive erythroblasts cell pellets were entirely colorless due to a marked deficiency of heme, although their cell morphology was similar to that of the wild-type erythroblasts. The level of expression of erythroid-specific genes in Alas2-null definitive erythroblasts was also similar to that of the wild-type erythroblasts. These findings indicate that Alas2-null definitive erythroblasts developed to a stage similar to that of the wild-type erythroblasts, which were also shown to be very similar to the bone marrow erythroblasts in vivo. In contrast, Alas2-null definitive erythroblasts contained 15 times more nonheme iron than did the wild-type erythroblasts, and electron microscopy found this iron to be distributed in the cytoplasm but not in mitochondria. Consistent with the aberrant increase in iron, Alas2-null definitive erythroblasts were more peroxidized than wild-type erythroblasts. These findings suggest that ALAS2 deficiency itself does not interfere with the development of definitive erythroid cells, but it results in a profound iron accumulation and a peroxidized state in erythroblasts.
5-Aminolevulinate Synthetase↗
The association of erythroblasts with macrophages promotes erythroid proliferation and maturation: a 30-kD heparin-binding protein is involved in this contact.
Although the association of erythroblasts with macrophages has been well documented in the human bone marrow, the function and identification of the intimate contacts occurring between the membranes of these two cell types in the physiology of erythropoiesis is not known. Using in vitro cultures of human peripheral blood derived erythroid progenitors, we have shown the presence of erythroblastic islands consisting of a central macrophage surrounded by a ring of erythroblasts that undergo terminal maturation leading to enucleation. However, when cultures were carried in the absence of intact macrophages, erythroid cells matured to the late erythroblast stage but failed to enucleate. Furthermore, the number of erythroid cells was markedly reduced in macrophage-depleted cultures, suggesting that the erythroblast-macrophage contact promotes proliferation and terminal maturation of erythroid cells leading to their enucleation. To examine the molecule(s) involved in the interaction between erythroblasts and macrophages, we have used a cell attachment assay involving incubation of solubilized surface-labeled erythroblasts with macrophage membrane proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Erythroblast surface proteins specifically attached to a 30-kD protein from macrophage membranes, whereas no adhesion was seen to the protein standards. An apparently similar protein of 30 kD was also detected on erythroblasts and was shown to mediate erythroblast-erythroblast contact in addition to the erythroblast-macrophage contact. The extraction of plasma membranes with Triton X-100 showed that the 30-kD protein is linked to the membrane skeleton via an integral membrane protein both in erythroblasts and macrophages. Furthermore, our results show that the cell:cell interactions mediated by the 30-kD protein are calcium-independent and could be specifically inhibited by heparin. We conclude that the association of erythroblasts with macrophages promotes erythroid proliferation and maturation leading to erythroblast enucleation and that a 30-kD heparin-binding protein present on the surface of macrophages and erythroblasts is involved in this contact. This protein is capable of binding homotypic and heterotypic cells.
Carrier Proteins↗
Characterization of first trimester fetal erythroblasts for non-invasive prenatal diagnosis.
Isolating fetal erythroblasts from first trimester maternal blood offers a promising non-invasive alternative for prenatal diagnosis. The aim of this study was to characterize the biological properties of first trimester primitive erythroblasts to facilitate their enrichment from first trimester maternal blood. Primitive erythroblasts were the predominant cell type until 12 weeks gestation, after which time their numbers declined steeply; 100% were epsilon-globin-positive versus <0.06% definitive erythroblasts. Buoyant densities of first trimester fetal erythroblasts ranged from 1.077 to 1.130 g/ml, and optimal recoveries were obtained with Percoll 1118. Although primitive erythroblasts carried a negative surface charge and were resistant to NH(4)Cl lysis, these properties had only a limited role in fetal cell enrichment. Immunophenotyping showed that primitive, like definitive, erythroblasts were GPA+, CD47+, CD45- and CD35-, whereas CD71 expression was weak/undetectable on primitive erythroblasts but strongly positive on 100% of definitive erythroblasts; primitive erythroblasts were also CD36- whereas definitive erythroblasts were CD36+. We therefore used CD45/GPA selection of Percoll 1118-separated cells to demonstrate successful enrichment of male epsilon-globin-positive fetal erythroblasts from model mixtures, and as proof of principle from some first trimester maternal blood samples. Fetal cell enrichment protocols based on first trimester epsilon-globin-positive primitive erythroblasts may allow reliable enrichment of fetal cells from maternal blood for early non-invasive prenatal diagnosis of genetic disorders.
Ammonium Chloride↗
Erythroblast iron metabolism and serum soluble transferrin receptor values in the anemia of rheumatoid arthritis.
OBJECTIVES: We have investigated in vitro erythroblast iron metabolism in the anemia of rheumatoid arthritis (RA). We also have examined the results in relation to bone marrow iron status in an attempt to explain the reported difference between serum soluble transferrin receptor (sTfR) values in anemia of chronic disease (ACD) and iron deficiency anemia (IDA) in patients with RA. METHODS: Bone marrow was examined in 29 anemic patients with RA, 9 healthy volunteers, and 6 patients with simple IDA. High purity erythroblast fractions were prepared from these bone marrow samples. Erythroblast surface TfR expression and iron uptake was assessed in vitro using (125)I-transferrin (Tf) and (59)Fe-Tf, respectively. The efficiency of erythroblast surface TfR function for Tf-iron uptake was determined by relating total iron uptake at 4 hours to surface TfR number. Serum sTfR values were measured for the RA anemia group, which was subdivided as RA-ACD (marrow iron present) or RA-IDA (marrow iron absent) on the basis of visible reticuloendothelial (RE) marrow iron stores. RESULTS: High purity (87 +/- 5%) erythroblast fractions were obtained from 35 of the 44 marrow samples. Erythroblasts obtained from patients with simple IDA showed a significant increase in surface TfR expression (P = 0.0003) and Tf-iron uptake (P = 0.001). RA anemia also led to a significant increase in erythroblast Tf-iron uptake (P = 0.016). This increase was not associated with an increase in surface TfR expression (P = 0.5), but was seen to occur as a result of a significant increase in the efficiency of surface TfR for Tf-iron uptake (P = 0.027). Within the RA anemia group, the increase in erythroblast Tf- iron uptake at 4 hours was more evident for RA-IDA (3.96 +/- 1.73 versus 1.66 +/- 0.66; P = 0.03) than for RA-ACD (2.69 +/- 1.18 versus 1.66 +/- 0.66; P = 0.057). This additional erythroblast response to absent RE iron stores led to a highly significant difference in serum sTfR values between RA-IDA and RA-ACD (40.2 +/- 14.0 versus 23.9 +/- 5.3 nmoles/liter; P = 0.001) CONCLUSIONS: An increase in erythroblast surface TfR efficiency for Tf-iron uptake compensates for the low plasma iron levels associated with anemia in RA and helps to maintain RA erythroblast iron uptake. With adequate RE iron stores, this increased efficiency limits intracellular iron deprivation and consequently reduces the need to increase surface TfR expression. As a result, serum sTfR levels in RA-ACD remain within the normal range. RA erythroblasts, however, are still able to respond to any additional worsening of the iron supply caused by absent RE iron stores. This additional response causes the highly significant increase in serum sTfR values seen between RA-IDA and RA-ACD.
Adult↗
Migration of erythroblastic islands toward the sinusoid as erythroid maturation proceeds in rat bone marrow.
It has been hitherto considered that mature erythroblasts migrate toward the sinusoid along the cytoplasmic processes of macrophages of erythroblastic islands in bone marrow. Our previous report, however, has demonstrated the morphological features of a mature erythroblastic island passing through the sinusoidal endothelium. In this study, the possibility of migration of erythroblastic islands toward the sinusoid was examined in rat bone marrow by light microscopical histoplanimetry. As a result, the more mature erythroblasts were not regularly arranged in the peripheral direction of the erythroblastic islands. Immature erythroblasts were populated more in the erythroblastic islands away from the sinusoid than in those islands neighboring the sinusoid, whereas mature erythroblasts were more in erythroblastic islands neighboring the sinusoid. These findings suggest that the formation of erythroblastic islands occurs in a region away from the sinusoid, and that erythroblastic islands migrate towards the sinusoids as erythroid maturation proceeds.
Animals↗
Temporal synthesis of band 3 oligomers during terminal maturation of mouse erythroblasts. Dimers and tetramers exist in the membrane as preformed stable species.
Band 3, the anion transport protein of the erythrocyte membrane, exists in the membrane as a mixture of dimers (B3D) and tetramers (B3T). The dimers are not linked to the skeleton and constitute the free mobile band 3 fraction. The tetramers are linked to the skeleton by their interaction with ankyrin. In this report we have examined the temporal synthesis and assembly of band 3 oligomers into the plasma membrane during red cell maturation. The oligomeric state of newly synthesized band 3 in early and late erythroblasts was analyzed by size-exclusion high-pressure liquid chromatography of band 3 extracts derived by mild extraction of plasma membranes with the nonionic detergent C12E8 (octaethylene glycol n-dodecyl monoether). This analysis revealed that at the early erythroblast stage, the newly synthesized band 3 is present predominantly as tetramers, whereas at the late stages of erythroid maturation, it is present exclusively as dimers. To examine whether the dimers and tetramers exist in the membrane as preformed stable species or whether they are interconvertible, the fate of band 3 species synthesized during erythroblast maturation was examined by pulse-chase analysis. We showed that the newly synthesized band 3 dimers and tetramers are stable and that there is no interconversion between these species in erythroblast membranes. Pulse-chase analysis followed by cellular fractionation showed that, in early erythroblasts, the newly synthesized band 3 tetramers are initially present in the microsomal fraction and later incorporated stably into the plasma membrane fraction. In contrast, in late erythroblasts the newly synthesized band 3 dimers move rapidly to the plasma membrane fraction but then recycle between the plasma membrane and microsomal fractions. Fluorescence photobleaching recovery studies showed that significant fractions of B3T and B3D are laterally mobile in early and late erythroblast plasma membranes, respectively, suggesting that many B3T-ankyrin complexes are unattached to the membrane skeleton in early erythroblasts and that the membrane skeleton has yet to become tightly organized in late erythroblasts. We postulate that in early erythroblasts, band 3 tetramers are transported through microsomes and stably incorporated into the plasma membrane. However, when ankyrin synthesis is downregulated in late erythroblasts, it appears that B3D are rapidly transported to the plasma membrane but then recycled between the plasma membrane and microsomal compartments. These observations may suggest novel roles for membrane skeletal proteins in stabilizing integral membrane protein oligomers at the plasma membrane and in regulating the endocytosis of such proteins.
Animals↗
Emperipolesis of erythroblasts within Kupffer cells during hepatic hemopoiesis in human fetus.
The state in which cells can inhabit other cells without damage is known as emperipolesis. Emperipolesis has been found in various physiological and pathological conditions. We performed a study of emperipolesis of erythroblasts within Kupffer cells in the human fetal liver. We found that Kupffer cells, identified by CD68 immunolabeling, contained 4-8 erythroblasts in a hypertrophic cytoplasm on light microscopy. Emperipoletic erythroblasts were present in various maturation stages from proerythroblast to reticulocyte. By electron microscopy, we found that erythroblasts occupied membrane-bound vacuoles that were separated from each other by thin partitions of Kupffer cell cytoplasm. Neither emperipoletic erythroblasts nor their Kupffer cell hosts showed evidence of damage. Emperipoletic cells in mitosis were found, which suggests the capacity for the proliferation of erythroblasts within Kupffer cells. Some Kupffer cells were seen to contain both emperipoletic cells and phagosomes, without evidence of interaction. Erythroblasts and other hemopoietic cells were also found to be closely associated with the sinusoidal surface of Kupffer cells. However, intercellular junctions, if present, were inconspicuous. On occasion, Kupffer cells engorged with erythroblasts nearly occluded the sinusoidal lumen. Our results demonstrate that emperipolesis of erythroblasts within Kupffer cells occurs in human fetal hepatic hemopoiesis. We suggest that emperipolesis may be one of the mechanisms that support the maturation of erythroblasts in the fetal liver.
Antigens, CD↗
Splenic erythroblasts in anemia-inducing Friend disease: a source of cells for studies of erythropoietin-mediated differentiation.
Splenic erythroblasts obtained from mice during the acute disease caused by either the polycythemia-inducing (FVP) or anemia-inducing (FVA) strain of Friend virus were examined for their degree of terminal differentiation. Morphology, benzidine staining, and heme synthesis kinetics showed that many erythroblasts from FVP-infected mice were undergoing terminal differentiation, while few erythroblasts from FVA-infected mice showed evidence of terminal differentiation. When cultured in methylcellulose medium, splenic erythroblasts from FVP-infected mice completed differentiation without the addition of erythropoietin (EP) to the medium. However, splenic erythroblasts from FVA-infected mice underwent terminal differentiation in vitro only when EP was added to the medium. From spleens of FVA-infected mice, a population of large, immature-appearing erythroblasts was obtained by separation with velocity sedimentation at unit gravity. Serial studies of the separated erythroblasts which were cultured with EP showed that despite some heterogeneity in their proliferative capacity, they were relatively homogeneous in their degree of differentiation in that they had not begun to synthesize heme or globin. Morphological changes and syntheses of heme and globins were monitored during terminal differentiation induced in vitro by EP. The accumulation of immature erythroblasts in vivo, their responsiveness in vitro to EP, and availability of large numbers of cells (10(8) or more) make the splenic erythroblasts of FVA-infected mice an ideal population of cells with which to study EP-mediated terminal differentiation. This erythroblast population should permit the biochemical and molecular studies in erythroid differentiation which heretofore had to be done with chemically induced erythroid differentiation in continuous cell lines.
Anemia↗
Erythroblastic islands: specialized microenvironmental niches for erythropoiesis.
PURPOSE OF REVIEW: This review focuses on current understanding of molecular mechanisms operating within erythroblastic islands including cell-cell adhesion, regulatory feedback, and central macrophage function. RECENT FINDINGS: Erythroblasts express a variety of adhesion molecules and recently two interactions have been identified that appear to be critical for island integrity. Erythroblast macrophage protein, expressed on erythroblasts and macrophages, mediates cell-cell attachments via homophilic binding. Erythroblast intercellular adhesion molecule-4 links erythroblasts to macrophages through interaction with macrophage alphav integrin. In intercellular adhesion molecule-4 knockout mice, erythroblastic islands are markedly reduced, whereas the erythroblast macrophage protein null phenotype is severely anemic and embryonic lethal. Retinoblastoma tumor suppressor (Rb) protein stimulates macrophage differentiation by counteracting inhibition of Id2 on PU.1, a transcription factor that is a crucial regulator of macrophage differentiation. Rb-deficient macrophages do not bind Rb null erythroblasts and the Rb null phenotype is anemic and embryonic lethal. Lastly, extruded nuclei rapidly expose phosphatidylserine on their surface, providing a recognition signal similar to apoptotic cells. SUMMARY: Although understanding of molecular mechanisms operating within islands is at an early stage, tantalizing evidence suggests that erythroblastic islands are specialized niches where intercellular interactions in concert with cytokines play critical roles in regulating erythropoiesis.
Animals↗
A novel bone marrow frozen section assay for studying hematopoietic interactions in situ: the role of stromal bone marrow macrophages in erythroblast binding.
Bone marrow macrophages are found in intimate contact with erythroblasts. Exact mechanisms and functions of this interaction are unclear. New insights into erythroblast binding were obtained using a newly developed bone marrow frozen section assay. This modified Woodruff and Stamper assay has some important advantages compared to other adhesion assays. Erythroblasts specifically adhered to bone marrow macrophages forming clusters, as appear in vivo. Selective depletion of bone marrow macrophages by intravenous injection of dichloromethyl-enediphosphonate resulted in a release of immature erythroid cells to the peripheral blood. Furthermore no erythroblasts adhered to bone marrow sections without macrophages. Evaluating the binding of erythroblasts to bone marrow macrophages we found that this binding is temperature and cation dependent. The receptor for erythroblasts present on macrophages recognizes a sialyated protein as ligand on erythroblasts, since neuraminidase treatment of erythroblasts resulted in a decrease in binding. A possible candidate for the erythroblast receptor on macrophages is the ED2 antigen. ED2 is a differentiation antigen present on resident macrophages that has some biochemical features characteristic of an adhesion molecule. Erythroblast binding to bone marrow was inhibited using a monoclonal antibody directed against ED2.
Animals↗
Prognostic significance of erythroblasts in burns.
Changes in hematopoiesis that occur in humans after a burn injury may have important effects on morbidity and mortality. In patients with a variety of severe diseases, the presence of erythroblasts in peripheral blood is known to be indicative of a poor prognosis. However, the prognostic significance of erythroblasts in peripheral blood of burn patients has not yet been estimated. This study included 464 consecutive burn patients, of whom 81 did not survive their injuries (17.5 percent). Together with erythroblasts in blood, data on age, sex, total burn surface area, third-degree burn, inhalation trauma, white blood cell count, C-reactive protein, and hemoglobin were studied. The mortality rate of patients with erythroblasts in peripheral blood (n = 53) amounted to 56.6 percent (n = 30; total burn surface area, 39 percent), which is significantly higher (p < 0.001) than the mortality rate of patients without erythroblasts (12.4 percent, n = 51; total burn surface area, 18.69 percent). None of the 10 patients with more than 1000 erythroblasts x 10/liter survived. The detection of erythroblasts in the peripheral blood of burn patients is highly predictive of death, with the odds ratio after adjustment for the other known prognostic factors being 8.3 (95 percent confidence interval, 4.5 to 15.3). Erythroblasts were detected for the first time on average 10 +/- 4 days (median, 6 days) after admission and 13 +/- 6 days (median, 7 days) before death. Detection of erythroblasts in burn patients is of high prognostic power with regard to in-hospital mortality, providing physicians with a strong prognostic method with which to identify seriously threatened patients. It seems attractive to think about an incorporation of erythroblasts into further refinements of burn scores.
Adult↗
[Scanning and transmission microscopic observations on circulating primitive erythroblasts of yolk sac origin in the mouse embryo].
The earliest hemopoietic tissues which appear during the ontogeny of mammals are the blood islands of the yolk sac, and the blood cells produced therein begin to circulate between the embryo and visceral yolk sac at the establishment of the circulatory system. Primitive erythroblasts derived from the yolk sac have a short life span of only several days, and they form a majority of the embryonic blood cells prior to the start of liver hemopoiesis. To clarify cell fragmentation of primitive erythroblasts at the ultrastructural level, using 18 embryos of ICR-mice at 10 and 11 days of gestation, we observed circulating erythroblasts by scanning and transmission electron microscopy. The circulating erythroblasts generally had an irregularly ovoid contour, and they showed a great deal of micropinocytosis on their cell surface. The nuclei of the erythroblasts were round and possessed one or two nucleoli which were in contact with the nuclear membrane. Their nuclear chromatin was dispersed, and the cytoplasm was rich in polyribosomes and mitochondria. The majority of circulating erythroblasts were at the stage of either basophilic or polychromatophilic erythroblasts. Cytoplasmic projections could occasionally be seen extending from the erythroblast surface, and some of the projections appeared to be liberated into the vascular lumen as cell fragments. On the basis of their size and shape, the cytoplasmic projections could be classified into three types; finger-like projections, vesicular projections and microvesicular projections. The finger-like projections were approximately 1 micron in diameter and 3 microns in length. The vesicular projections, connected with the cell by a narrow stalk, were teardrop in shape, and approximately 0.8 microns in diameter and 1.5 microns in length. The microvesicular projections were approximately 0.2 microns in diameter and 0.2-0.5 microns in length. The finger-like projections had micropinocytotic invaginations on their surface, but no invaginations could be seen on the vesicular and microvesicular projections. Not only the finger-like but also the vesicular projections contained cytoplasmic matrix with a few polyribosomes. The microvesicular projections, on the other hand, occasionally contained myelinic-like figures. These projections were seen on the surface of erythroblasts at various maturation stages. The cytoplasmic fragments released from the erythroblasts were engulfed and eliminated from the embryonic peripheral blood by intravascular macrophages. The fragmentation of cytoplasmic projections was considered to be related to the development of microfilaments involved in the cytoskeleton of the erythroid elements.
Animals↗