Defective delivery of iron to the developing red cell of the Belgrade laboratory rat

Erythroid cell iron and transferrin uptake and release was studied in the anemia of the Belgrade laboratory rat (gene symbol, b), an autosomal recessive trait characterized by hypochromia and hyperferrinemia. When reticulocyte-rich red cells were incubated in vitro with doubly (59Fe, 125I) labeled transferrin, b/b cells demonstrated a significantly higher uptake of transferrin (164% of control at 60 min), and a significantly lower uptake of iron (21% of control at 60 min) than control cells. These findings with b/b cells were simulated by sodium-fluoride-treated control cells, but not by trypsin-treated control cells. When reticulocytes exposed to doubly labeled transferrin were incubated in normal rat plasma, there was a substantial loss of 125I from both the b/b cells (mean 71%) and control cells (mean 49%), but only a loss of 59Fe from the b/b cells (mean 21%). These findings suggest a defect in the delivery of iron to the b/b reticulocyte, which is distal to the binding of transferrin to its cell surface receptor.     

Evidence for the synthesis of embryonic globin chains in adult erythroid progenitor cells

Embryonic globin chains were found to be synthesized in vitro by the BFU-E colonies derived from adult sickle cell anemia (SS) patients, their heterozygous relatives as well as a few normal controls. In the absence of sufficient material for conducting direct structural analyses of these peptides, they were confirmed by evaluating the co-migration of the epsilon and zeta-chains with the corresponding structurally characterized globin chains obtained from K562 cell lysates on a reversed phase high performance liquid chromatogram. The presence of zeta-chain was also confirmed using an immunologic procedure. Furthermore, significant 35S-methionine incorporation peak was observed corresponding to the zeta-chain synthesized by the BFU-E-derived colonies implying an active synthesis of this embryonic globin chain in BFU-E cells obtained from hemopoietically adult persons.     

Ferritin synthesis and Iron uptake in developing erythroid cells

The effect of maturation on ferritin synthesis and iron uptake by erythroid precursor cells was studied in vitro in rabbit bone marrow cells synchronized by actinomycin D. Early mononuclear precursors were compared with basophilic normoblasts and reticulocytes. Apoferritin synthesis was measured by following the incorporation of ³H-amino acids into purified ferritin, and this was correlated with total protein synthesis and with radioactive iron uptake by whole cells and radioactive iron incorporation into heme and ferritin. With increasing cellular maturation, a progressive reduction in both apoferritin and total protein synthesis has been observed, undifferentiated cells synthesizing 12 times more protein and 20 times more ferritin than reticulocytes. The rate of ferritin synthesis was inversely related to the rate of radioactive iron uptake by whole cells and radioactive iron incorporation into heme; lowest rates of radioactive uptake and heme synthesis were found in undifferentiated cells, and highest rates in reticulocytes. Radioactive iron incorporation into ferritin was higher in basophilic normoblasts than in undifferentiated cells but was very low in reticulocytes. This sequence of events indicates that in developing erythroid cells the phase of maximal ferritin synthesis precedes the phase of maximal iron uptake. The accumulation of newly formed apoferritin is followed by an increase in cellular iron uptake and an increased retention of iron in ferritin. Finally, with continuing cellular maturation, ferritin iron and iron derived directly from transferrin are utilized for hemoglobin synthesis.     

Abnormal megakaryocytopoiesis in the Belgrade laboratory rat

The Belgrade laboratory rat (b/b rat) has hereditary, hypochromic, microcytic anemia with a variety of red cell abnormalities. Although this anemic syndrome has been recently ascribed to the defective delivery of iron to the developing red cell, the basic hematopoietic defect is still unknown. In this article we present evidence that the b/b rat has an additional hematologic defect. We have found that the megakaryocyte number in the marrow of the b/b rat is decreased to one half that of the normal rat, but the maturation rate of recognizable megakaryocytes is accelerated and the size is increased. The platelet count is moderately reduced. These findings indicate that megakaryocytopoiesis in the anemic b/b rat is abnormal and suggest that the genetic defect may involve the progenitors of the megakaryocyte cell lineage. Alternatively, the megakaryocytic abnormalities may be secondary to the severe anemia.     

Abnormal megakaryocytopoiesis in the Belgrade laboratory rat

The Belgrade laboratory (b/b) rat has a hereditary hypochromic microcytic anemia because of defective transmembrane iron transport into erythroblasts. The present study was prompted by our previous work in which we showed that the b/b rat has hypomegakaryocytic thrombocytopenia associated with increased megakaryocyte size. To define the basic mechanism underlying this abnormality in the b/b rat we have studied both megakaryocytopoiesis and granulopoiesis in anemic b/b rats, chronically transfused b/b rats, iron-treated b/b rats, and controls. We have found decreased concentrations of megakaryocyte and granulocyte progenitors in the marrow of b/b rats. Full correction of the severe anemia by chronic transfusion resulted in normalization of megakaryocyte progenitors, small acetylcholinesterase positive cells, megakaryocyte size, and platelet counts, along with granulocyte progenitors. In contrast, the partial correction of anemia obtained by iron treatment resulted in improvement, but not normalization, of these parameters. These findings indicate that abnormal megakaryocytopoiesis in the b/b rat can be best interpreted as a consequence of hypoxia because of the severe anemia. Because we have recently shown that the number of erythroid progenitors in b/b rats is also low, we propose that abnormal megakaryocytopoiesis in this animal is a reflection of an acquired stem cell disorder induced by the prolonged hypoxia resulting from the severe anemia.     

Fluorescence probe measurement of the pH of the transferrin microenvironment during iron uptake by rat bone marrow erythroid cells

Fluorescence probe measurements of the transferrin micro-environment during iron uptake by rat erythroid cells revealed that part of the transferrin is taken up in an acidic environment. The pH of this intracellular transferrin environment is 5.7. When rat erythroid cell precursors are incubated with diferric transferrin then in the incubation medium monoferric transferrins TfNFe and TfFeC appear. In view of the known instability of TfNFe at acidic pH, TfNFe cannot arise after endocytosis of Tf2Fe in acid vesicles at pH below 6.0. The results support the existence of a mechanism other than endocytosis in the iron uptake process in rat erythroid cells.     

Glucose uptake by rat erythroid cells: the effects of erythropoietin and dexamethasone.

Erythropoietin (Ep) stimulated glucose uptake by erythroid progenitor cells in the liver of fetal rats, as measured by [3H]2-deoxy-D-glucose uptake. This dose-dependent stimulation was maximal at 0.2 U/ml Ep and decreased during cell differentiation. Dexamethasone (DEX) inhibited glucose uptake by erythroid progenitors; this dose-dependent effect was maximal at 10(-7) M DEX. Ep partly counteracted the inhibitory effect of DEX. This antagonism may contribute to the overall antagonism between Ep and glucocorticoids in the development of erythroid tissue in vivo and in vitro.     

Uptake of transferrin-bound and nontransferrin-bound iron by reticulocytes from the Belgrade laboratory rat: comparison with Wistar rat transferrin and reticulocytes.

The mechanism underlying the impaired uptake of iron from transferrin by reticulocytes from the Belgrade laboratory rat was investigated using 125I- and 59Fe-labeled transferrin isolated from homozygous Belgrade rats and from Wistar rats, nontransferrin-bound Fe(II) in an isotonic sucrose solution, and reticulocytes from Belgrade and Wistar rats. The Belgrade rat transferrin had the same molecular weight and net charge as Wistar rat transferrin, donated iron equally well to both types of reticulocytes, and competed equally for transferrin binding sites on the cells. Hence, the defect in iron uptake by Belgrade rat reticulocytes could not be attributed to an abnormality of the transferrin molecule. The rate of uptake of Fe(II) from sucrose into the cytosolic and stromal fractions of Belgrade rat reticulocytes was only about 35% as great as that by Wistar rat reticulocytes. With both types of cells, the uptake process was saturable, suggesting the presence of a carrier-mediated process. It was therefore concluded that the defect in iron uptake by Belgrade rat erythroid cells is probably the consequence of a deficiency in a membrane carrier for iron.     

Induction of globin mRNA transcription by erythropoietin in differentiating erythroid precursor cells

The effects of erythropoietin (epo) on the proliferation of late erythroid progenitor cells (CFU-E) and on the formation of hemoglobin and of globin mRNA in these cells are described. CFU-E were isolated from thiamphenicol-pretreated anemic mice by elutriation and Percoll density gradient methods. These CFU-E are restricted in their capacity to proliferate in vitro without added epo. The epo dependence in vitro was not absolute. With no epo in the culture medium the first cell division was unimpaired, whereas the third division was only 1%-2% of the control. In the absence of epo the synthesis of hemoglobin is very low in CFU-E, but is increased significantly after about 5 h of incubation with epo present. In epo deprived cells there was considerable hemoglobin formed at about 14 h, but not earlier. The presence as detected by the Northern blot technique of globin mRNA, isolated from CFU-E, was variable, probably depending on the presence of some more mature erythroid cells. By an extrapolation method we show evidence that pure CFU-E would have virtually no detectable globin mRNA. The production of globin mRNA is rapidly (2 h) induced in cells incubated with epo. We conclude that epo, besides having a mitogenic effect on CFU-E, induces the rapid expression of the globin genes.     

Calmodulin antagonists inhibit and phorbol esters enhance transferrin endocytosis and iron uptake by immature erythroid cells

Seven antagonists of the calcium-binding protein calmodulin were found to inhibit iron and transferrin uptake by reticulocytes. This inhibition could be completely accounted for by inhibition of the endocytosis and exocytosis of transferrin. When four of the antagonists were tested with the nucleated erythroid cells from the liver of the fetal rat, inhibition of iron uptake was also observed but at higher concentrations than required for the same degree of inhibition with reticulocytes. The tumor promoters phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDB) were shown to increase the rates of iron and transferrin uptake by reticulocytes and fetal liver erythroid cells by accelerating the rates of transferrin endocytosis and exocytosis. Since these substances are known to stimulate the calcium-activated enzyme protein kinase C while calmodulin antagonists are inhibitory, it is concluded that this enzyme plays an important role in the endocytosis and intracellular cycling of transferrin, and iron uptake by immature erythroid cells. However, the possibilities that calmodulin is also involved or that the inhibitory effects of the calmodulin antagonists are due to nonspecific actions on the cell membrane cannot be excluded.     

Defective organization of the erythroid cell membrane in a novel case of congenital anemia

In the present paper, we demonstrate the erythroid cell membrane unique properties in a previously characterized case of hemoglobin-H disease, associated with congenital dyserythropoietic anemia type-I features. In order to explain the patient's cell membrane distortions and the high affinity for the various intracellular inclusions, we studied its composition and structure in comparison to other anemic and non-anemic cases. Red cells from peripheral blood were fractionated into cellular, membrane and protein extracts. Membrane attached immunocomplexes were separated and collected by immunoprecipitation. The subcellular fractions were analyzed by SDS-PAGE electrophoresis and immunoblotted against a variety of erythroid-specific antibodies. The protein composition of the membrane was characterized by immunogold electron microscopy. In the membrane of the CDA-associated case, we identified sialic acid and protein deficiencies, formation of protein crosslinkings, excesses of bound globin and immunoglobulins and aberrant peptides. In contrast to the typical hemoglobin-H disease, the ghost-bound globin exhibited preferential attachment to the skeletal proteins than the band 3 and the skeleton-bound globin consisted not only of beta- but also of alpha-globin chains. Another hallmark, probably associated with the CDA defect, was the participation of glycophorins in the membrane-bound immunocomplexes and the pathological clustering of the latter in the membrane. This study strongly suggests that the result of the combinatorial effects on the diseased membrane created a unique profile, quite distinct from the one observed in several typical hemoglobinopathies. Our observations shed light into critical membrane alterations leading to hemolysis in the novel CDA-associated disease and probably into the CDA-I or CDA-I-like diseases.     

Defective erythroid progenitor differentiation system in congenital hypoplastic (Diamond-Blackfan) anemia

To explore the etiology of congenital hypoplastic or Diamond-Blackfan anemia (DBA) we investigated in vitro erythropoiesis in nine patients. Of the nine, seven were clinically responsive to prednisone. Four were infants evaluated at the time of diagnosis. Six were never or were only minimally transfused. Those for whom prednisone had been prescribed had discontinued the drug a minimum of five months prior to study. The bone marrows of these nine patients were compared with those of hematologically normal individuals and with those of four patients with transient erythroblastopenia of childhood (TEC) whose erythroid aplasia was as severe as that of the patients with DBA. Using the plasma clot semisolid culture technique to enumerate erythroid progenitors and to evaluate the growth characteristics of the colonies to which they give rise, we concluded that at the onset of DBA: (a) erythroid progenitor frequency does not correlate with the degree of anemia and erythroblastopenia; (b) erythroid progenitor differentiation may in some cases be abnormally insensitive to crude preparations of erythropoietin; and (c) progenitor erythropoietin insensitivity in vitro does not necessarily indicate prednisone insensitivity in vivo. Thus, DBA does not appear to be solely the result of deficient formation of erythroid progenitors but is, in addition, a disorder that is due to defective progenitor differentiation in vivo.     

Imbalanced globin chain synthesis determines erythroid cell pathology in thalassemic mice

Background

β-thalassemia occurs from the imbalanced globin chain synthesis due to the absence or inadequate β-globin chain production. The excessive unbound α-globin chains precipitate in erythroid precursors and mature red blood cells leading to ineffective erythropoiesis and hemolysis.

Design and Methods

In vitro globin chain synthesis in reticulocytes from different types of thalassemic mice was performed. The effect of imbalanced globin chain synthesis was assessed from changes of red blood cell properties including increased numbers of red blood cells vesicles and apoptotic red blood cells, increased reactive oxygen species and decreased red blood cell survival.

Results

The α/β-globin chain ratio in β^IVSII-654-thalassemic mice, 1.26±0.03, was significantly higher than that of wild type mice, 0.96±0.05. The thalassemic mice show abnormal hematologic data and defective red blood cell properties. These values were improved significantly in doubly heterozygous thalassemic mice harboring 4 copies of human β^E-globin transgene, with a more balanced globin chain synthesis, 0.92±0.05. Moreover, transgenic mice harboring 8 extra copies of the human β^E-globin transgene showed inversely imbalanced α/β-globin synthesis ratio, 0.83±0.01, that resulted in a mild β-thalassemia phenotype due to the excessive β-globin chains. The degree of ineffective erythropoiesis also correlated with the degree of imbalanced globin chain synthesis. Bone marrow and splenic erythroid precursor cells of β^IVSII-654-thalassemic mice showed increased phosphatidylserine exposure in basophilic and polychromatophilic stages, which was restored to the normal level in doubly heterozygous mice.

Conclusions

Imbalanced α/β-globin chain as a consequence of either reduction or enhancement of β-globin chain synthesis can cause abnormal red blood cell properties in mouse models.     

Gamma-interferon alters globin gene expression in neonatal and adult erythroid cells

Interferons have the ability to enhance or diminish the expression of specific genes and have been shown to affect the proliferation of certain cells. Here, the effect of gamma-interferon on fetal hemoglobin synthesis by purified cord blood, fetal liver, and adult bone marrow erythroid progenitors was studied with a radioligand assay to measure hemoglobin production by BFU-E-derived erythroblasts. Coculture with recombinant gamma-interferon resulted in a significant and dose-dependent decrease in fetal hemoglobin production by neonatal and adult, but not fetal, BFU-E-derived erythroblasts. Accumulation of fetal hemoglobin by cord blood BFU-E-derived erythroblasts decreased up to 38.1% of control cultures (erythropoietin only). Synthesis of both G gamma/A gamma globin was decreased, since the G gamma/A gamma ratio was unchanged. Picograms fetal hemoglobin per cell was decreased by gamma-interferon addition, but picograms total hemoglobin was unchanged, demonstrating that a reciprocal increase in beta-globin production occurred in cultures treated with gamma-interferon. No toxic effect of gamma-interferon on colony growth was noted. The addition of gamma-interferon to cultures resulted in a decrease in the percentage of HbF produced by adult BFU-E-derived cells to 45.6% of control. Fetal hemoglobin production by cord blood, fetal liver, and adult bone marrow erythroid progenitors, was not significantly affected by the addition of recombinant GM-CSF, recombinant interleukin 1 (IL-1), recombinant IL-2, or recombinant alpha-interferon. Although fetal progenitor cells appear unable to alter their fetal hemoglobin program in response to any of the growth factors added here, the interaction of neonatal and adult erythroid progenitors with gamma-interferon results in an altered expression of globin genes. This supports the concept that developmental globin gene switching can be regulated by environmental factors.