Changes in enzyme activities involved in the degradation of 1,3-bisphosphoglycerate during erythropoiesis in rat bone marrow

Rat bone marrow cells have been fractionated by density gradient in Percoll. Differential counting of erythroid cells, haemoglobin concentration and bisphosphoglycerate mutase and phosphoglycerate kinase activities have been determined in cellular fractions. As shown by means of a statistical approach, an increase in bisphosphoglycerate mutase activity and a slight decrease in phosphoglycerate kinase activity is found in erythroid cells as their haemoglobin content increases. Our results suggest that there is a synthesis of 2,3-bisphosphoglycerate during the erythropoietic process which parallels the synthesis of haemoglobin.     

Pathogenic mitochondrial DNA-induced respiration defects in hematopoietic cells result in anemia by suppressing erythroid differentiation

Anemia is a symptom in patients with Pearson syndrome caused by the accumulation of mutated mitochondrial DNA (mtDNA). Such mutated mtDNAs have been detected in patients with anemia. This suggested that respiration defects due to mutated mtDNA are responsible for the anemia. However, there has been no convincing experimental evidence to confirm the pathophysiological relation between respiration defects in hematopoietic cells and expression of anemia. We address this issue by transplanting bone marrow cells carrying pathogenic mtDNA with a large-scale deletion (ΔmtDNA) into normal mice. The bone marrow-transplanted mice carried high proportion of ΔmtDNA only in hematopoietic cells, and resultant the mice suffered from macrocytic anemia. They show abnormalities of erythroid differentiation and weak erythropoietic response to a stressful condition. These observations suggest that hematopoietic cell-specific respiration defects caused by mtDNAs with pathogenic mutations are responsible for anemia by inducing abnormalities in erythropoiesis.     

Endothelial Cell-Selective Adhesion Molecule Expression in Hematopoietic Stem/Progenitor Cells Is Essential for Erythropoiesis Recovery after Bone Marrow Injury

Numerous red blood cells are generated every second from proliferative progenitor cells under a homeostatic state. Increased erythropoietic activity is required after myelo-suppression as a result of chemo-radio therapies. Our previous study revealed that the endothelial cell-selective adhesion molecule (ESAM), an authentic hematopoietic stem cell marker, plays essential roles in stress-induced hematopoiesis. To determine the physiological importance of ESAM in erythroid recovery, ESAM-knockout (KO) mice were treated with the anti-cancer drug, 5-fluorouracil (5-FU). ESAM-KO mice experienced severe and prolonged anemia after 5-FU treatment compared to wild-type (WT) mice. Eight days after the 5-FU injection, compared to WT mice, ESAM-KO mice showed reduced numbers of erythroid progenitors in bone marrow (BM) and spleen, and reticulocytes in peripheral blood. Megakaryocyte-erythrocyte progenitors (MEPs) from the BM of 5-FU-treated ESAM-KO mice showed reduced burst forming unit-erythrocyte (BFU-E) capacities than those from WT mice. BM transplantation revealed that hematopoietic stem/progenitor cells from ESAM-KO donors were more sensitive to 5-FU treatment than that from WT donors in the WT host mice. However, hematopoietic cells from WT donors transplanted into ESAM-KO host mice could normally reconstitute the erythroid lineage after a BM injury. These results suggested that ESAM expression in hematopoietic cells, but not environmental cells, is critical for hematopoietic recovery. We also found that 5-FU treatment induces the up-regulation of ESAM in primitive erythroid progenitors and macrophages that do not express ESAM under homeostatic conditions. The phenotypic change seen in macrophages might be functionally involved in the interaction between erythroid progenitors and their niche components during stress-induced acute erythropoiesis. Microarray analyses of primitive erythroid progenitors from 5-FU-treated WT and ESAM-KO mice revealed that various signaling pathways, including the GATA1 system, were impaired in ESAM-KO mice. Thus, our data demonstrate that ESAM expression in hematopoietic progenitors is essential for erythroid recovery after a BM injury.     

Indicators of erythrocyte formation and degradation in rats with either vitamin A or iron deficiency

Vitamin A deficiency produces anemia and altered iron status. In this study with rats we tested two hypotheses regarding vitamin A deficiency: (1) that it impairs erythropoiesis, leading to an increased red cell turnover, and (2) that it inhibits the glycosylation of transferrin. Erythropoietic activity was assessed indirectly by determining the myeloid:erythroid ratio in bone marrow smears, the number of erythroid colonies in the red pulp of spleen, the blood reticulocyte index, and zinc protoporphyrin and plasma transferrin receptor concentrations. Transferrin glycosylation was assessed by measuring the sialic acid content of transferrin. The effects of vitamin A deficiency were compared with those of iron deficiency. Iron deficiency produced anemia and low iron levels in organs. Vitamin A deficiency produced low levels of plasma and hepatic retinol, and it induced decreased plasma total iron-binding capacity and raised iron levels in tibia and spleen. Short- but not long-term iron deficiency reduced the number of erythroid colonies in spleen; vitamin A deficiency had no influence. Neither iron nor vitamin A deficiency influenced the myeloid:erythroid ratio in bone marrow smears and the blood reticulocyte production. Plasma transferrin receptor and erythrocyte zinc protoporphyrin concentrations were not affected by vitamin A deficiency but increased with iron deficiency. Vitamin A deficiency did not stimulate erythrocyte breakdown, as indicated by unaltered plasma lactate dehydrogenase activity and reduced plasma total bilirubin levels. Both vitamin A and iron deficiencies raised the proportion of multiple sialylated transferrins in plasma. Thus, we have not found evidence that vitamin A deficiency affects erythropoiesis and erythrocyte turnover. The iron accumulation in spleen and bone marrow may be related to reduced iron transport due to inhibition of transferrin synthesis rather than inhibition of transferrin sialylation.     

Transferrin binding capacity as a marker of differentiation and maturation of rat erythroid cells fractionated by counter current distribution in aqueous polymer two-phase systems

Rat bone marrow cell populations, containing different proportions of erythroid cells, have been fractionated by counter-current distribution in the non-charge-sensitive dextran/polyethyleneglycol two-phase systems on the basis of hydrophobic cell surface properties. Cell fractions with a low distribution coefficient, which contain non-erythroid cells and early erythoblasts, showed a low transferrin binding capacity and a low haemoglobin/cell ratio whereas cell fractions with a high distribution coefficient, which contain intermediate-late erythroblasts and mature red cells, showed an elevated transferrin binding capacity and the highest haemoglobin/cell ratio. These results support transferrin binding capacity as a good marker parameter for the erythroid bone marrow cell differentiation and maturation processes.     

Reticulocyte changes after experimental anemia and erythropoietin treatment of horses.

Availability of recombinant human erythropoietin (EPO) has facilitated use to enhance red blood cell production, and therefore aerobic performance, in human and equine athletes. Recombinant human EPO promotes growth and differentiation of equine erythroid precursor cells, but in some horses repeat administration induces immune interference with endogenous EPO resulting in fatal anemia. Although blood reticulocyte parameters acquire unique changes in humans treated with EPO, with manual enumeration methods, horses were not considered to release reticulocytes from the bone marrow into circulation, even under severe erythropoietic stress. The goals of this study were to determine whether reticulocytes could be detected and characterized in horses that are anemic or have been treated with EPO using a modern hematology analyzer. Anemia was induced in six horses by removal of 30 ml of blood/kg of body wt over 24 h. After 28 days, the horses were treated twice with 55 U/kg of EPO (Eprex), and after 65 days they were treated thrice with 73 U/kg of EPO. Blood samples were analyzed with the ADVIA120 instrument every 3-5 days and bone marrow samples 7 days after anemia and EPO treatments. Analysis of blood reticulocyte parameters by ANOVA in a randomized complete block design determined that anemia and EPO induced significant (P < or = 0.05) increases in red cell distribution width and reticulocyte mean cell volume. Parameters changed only after EPO treatment were cellular hemoglobin concentration mean, mean cell volume, reticulocyte concentration, proportion of macrocytic reticulocytes, and reticulocyte cellular hemoglobin. These findings indicate that horses under erythropoietic stress and after EPO treatment release reticulocytes with unique characteristics into circulation.     

E-cadherin is functionally involved in the maturation of the erythroid lineage

Differentiation and proliferation of hematopoietic progenitors take place in the bone marrow and is a tightly controlled process. Cell adhesion molecules of the integrin and immunoglobulin families have been shown to be involved in these processes, but almost nothing was known about the involvement of the cadherin family in the hematopoietic system. A PCR screening of RNA of human bone marrow mononuclear cells with specific primers for classical cadherins revealed that E-cadherin, which is mainly expressed by cells of epithelial origin, is also expressed by bone marrow cells. Western blot analysis and immunofluorescence staining of bone marrow sections confirmed this unexpected finding. A more detailed analysis using immunoaffinity columns and dual color flow cytometry showed that the expression of E- cadherin is restricted to defined maturation stages of the erythropoietic lineage. Erythroblasts and normoblasts express E- cadherin, mature erythrocytes do not. A functional role of E-cadherin in the differentiation process of the erythroid lineage was indicated by antibody-inhibition studies. The addition of anti-E-cadherin antibody to bone marrow mononuclear cultures containing exogeneous erythropoietin drastically diminished the formation of erythropoietic cells. These data suggest a non-anticipated expression and function of E-cadherin in one defined hematopoietic cell lineage.     

Bone marrow stromal cells selectively stimulate the rapid expansion of lineage-restricted myeloid progenitors

Bone marrow stromal cells serve hematopoietic microenvironments where different blood cells are controlled in their growth and differentiation. To characterize functions of stromal cells, 33 bone marrow stromal cells including preadipocytes, endothelial cells, and fibroblasts were established from transgenic mice harboring temperature-sensitive SV40 T-antigen gene and their selective stimulatory abilities to support large colony formation of lineage-specific hematopoietic progenitor cells (erythroid, monocyte/macrophage, granulocyte, and monocyte-granulocyte) were examined. Among established stromal cells, 27 clones showed erythropoietic stimulatory activity in the presence of erythropoietin. On myeloid progenitors, the stromal cells showed lineage-restricted stimulatory activity and a reciprocal relationship was observed between granulocyte formation and macrophage formation, but these activities were not dependent on the amount of produced colony-stimulating factors (CSFs). Our present study with many stromal cells established from bone marrow indicated that each stromal cell in the bone marrow may provide the preferable microenvironment for a rapid expansion of the lineage-restricted progenitor cells in combination with CSFs. © 1995 Wiley-Liss, Inc.     

Alterations in Bone and Erythropoiesis in Hemolytic Anemia: Comparative Study in Bled,Phenylhydrazine-Treated and Plasmodium-Infected Mice

Sustained erythropoiesis and concurrent bone marrow hyperplasia are proposed to be responsible for low bone mass density (BMD) in chronic hemolytic pathologies. As impaired erythropoiesis is also frequent in these conditions, we hypothesized that free heme may alter marrow and bone physiology in these disorders. Bone status and bone marrow erythropoiesis were studied in mice with hemolytic anemia (HA) induced by phenylhydrazine (PHZ) or Plasmodium infection and in bled mice. All treatments resulted in lower hemoglobin concentrations, enhanced erythropoiesis in the spleen and reticulocytosis. The anemia was severe in mice with acute hemolysis, which also had elevated levels of free heme and ROS. No major changes in cellularity and erythroid cell numbers occurred in the bone marrow of bled mice, which generated higher numbers of erythroid blast forming units (BFU-E) in response to erythropoietin. In contrast, low numbers of bone marrow erythroid precursors and BFU-E and low concentrations of bone remodelling markers were measured in mice with HA, which also had blunted osteoclastogenesis, in opposition to its enhancement in bled mice. The alterations in bone metabolism were accompanied by reduced trabecular bone volume, enhanced trabecular spacing and lower trabecular numbers in mice with HA. Taken together our data suggests that hemolysis exerts distinct effects to bleeding in the marrow and bone and may contribute to osteoporosis through a mechanism independent of the erythropoietic stress.     

Anethole prevents hydrogen peroxide-induced apoptosis and collagen metabolism alterations in human skin fibroblasts

Thyroid hormone stimulates erythropoietic differentiation. However, severe anemia is sometimes seen in patients with hyperthyroidism, and the mechanisms have not been fully elucidated. Bone marrow is comprised about 2–8 % oxygen, and the characteristics of hematopoietic stem cells have been shown to be influenced under hypoxia. Hypoxia-inducible factor-1 is a critical mediator of cellular responses to hypoxia and an important mediator in signal transduction of thyroid hormone [triiodothyronine (T3)]. The aim of this study was to investigate the effect of T3 on erythropoiesis under hypoxia mimicking physiological conditions in the bone marrow. We maintained human erythroleukemia K562 cells under hypoxic atmosphere (2 % O₂) and examined their cellular characteristics. Compared to that under normal atmospheric conditions, cells under hypoxia showed a reduction in the proliferation rate and increase in the hemoglobin content or benzidine-positive rate, indicating promotion of erythroid differentiation. T3 had no effect on hypoxia-induced erythroid differentiation, but significantly inhibited activin A/erythroid differentiation factor-induced erythroid differentiation. Moreover, GATA2 mRNA expression was suppressed in association with erythroid differentiation, while T3 significantly diminished that suppression. These results suggest that T3 has a direct suppressive effect on erythroid differentiation under hypoxic conditions.     

A mathematical model of erythropoiesis in mice and rats. Part 4: Differences between bone marrow and spleen

In a preceding analysis we hypothesized that the most important parameter controlled by erythropoietic regulation in vivo is the degree of amplification (number of cell divisions) in the CFU-E and erythroblast cell stages. It was concluded that erythropoietic amplification in vivo is controlled according to a sigmoidal dose-response relationship with respect to the control parameter which is the haematocrit (or haemoglobin concentration). Here, this hypothesis is extended to include the differences in murine bone marrow and splenic erythropoiesis that are described and quantified by different dose-response relationships. Comparing several sets of experimental data with mathematical model simulations, this approach leads to the following conclusions: (i) in the unperturbed normal steady state at least one extra erythropoietic cell division takes place in the spleen compared with the bone marrow; (ii) a strong erythropoietic stimulus, such as severe bleeding or hypoxia, can induce five to six additional cell divisions in the spleen but only two to three additional divisions in the bone marrow; this results in a considerable increase in the spleen's contribution to erythropoiesis from about 10% in normal animals to over 40% during strong stimulation; (iii) under erythropoietic suppression, such as red cell transfusion, a similar number of cell divisions is skipped in both organs and the splenic contribution to erythropoiesis remains unchanged. In conclusion, the concept that bone marrow and spleen microenvironments differ in the dose-response relationship for erythropoietic regulation provides an explanation for the changing contribution of splenic murine erythropoiesis following a variety of experimental treatments.     

Myelosuppressive effects in vivo of purified recombinant murine macrophage inflammatory protein-1 alpha.

Purified recombinant murine macrophage inflammatory protein-1 alpha (rmuMIP-1 alpha), a cytokine with myelopoietic activity in vitro, was assessed in vivo by injection into C3H/HeJ mice for effects on proliferation (percentage of cells in S phase DNA synthesis of the cell cycle) and absolute numbers of granulocyte-macrophage, erythroid, and multipotential progenitor cells in the femur and spleen, and on nucleated cellularity in the bone marrow, spleen, and blood. rmuMIP-1 alpha rapidly decreased cycling rates (at 2 to 10 micrograms/mouse i.v.) and absolute numbers (at 5 to 10 micrograms/mouse i.v.) of myeloid progenitor cells in the marrow and spleen. These effects were dose- and time-dependent and reversible. Suppressive effects were noted within 3 to 24 h for cell cycling and absolute numbers of progenitor cells in the marrow and spleen, and by 48 h for circulating neutrophils. A study comparing the effects of i.v. injection of rmuMIP-1 alpha versus rmuMIP-1 beta, a biochemically similar molecule but with no myelosuppressive effects in vitro, demonstrated myelosuppression in vivo by rmuMIP-1 alpha, but not by rmuMIP-1 beta. The results suggest that rmuMIP-1 alpha has myelosuppressive activity in vivo and offers the possibility that it may be a useful adjunct to treatments involving cytotoxic drugs because of its reversible suppressive effects on normal progenitor cell cycling.     

A mathematical model of erythropoiesis in mice and rats. Part 4: Differences between bone marrow and spleen

Abstract. In a preceding analysis we hypothesized that the most important parameter controlled by erythropoietic regulation in vivo is the degree of amplification (number of cell divisions) in the CFU-E and erythroblast cell stages. It was concluded that erythropoetic amplification in vivo is controlled according to a sigmoidal dose-response relationship with respect to the control parameter which is the haematocrit (or haemoglobin concentration). Here, this hypothesis is extended to include the differences in murine bone marrow and splenic erythropoiesis that are described and quantified by different dose-response relationships. Comparing several sets of experimental data with mathematical model simulations, this approach leads to the following conclusions: (i) in the unperturbed normal steady state at least one extra erythropoietic cell division takes place in the spleen compared with the bone marrow; (ii) a strong erythropoietic stimulus, such as severe bleeding or hypoxia, can induce five to six additional cell divisions in the spleen but only two to three additional divisions in the bone marrow; this results in a considerable increase in the spleen's contribution to erythropoiesis from about 10% in normal animals to over 40% during strong stimulation; (iii) under erythropoietic suppression, such as red cell transfusion, a similar number of cell divisions is skipped in both organs and the splenic contribution to erythropoiesis remains unchanged. In conclusion, the concept that bone marrow and spleen microenvironments differ in the dose-response relationship for erythropoietic regulation provides an explanation for the changing contribution of splenic murine erythropoiesis following a variety of experimental treatments.     

Nicotinic acetylcholine receptors alpha4beta2 and alpha7 regulate myelo- and erythropoiesis within the bone marrow

Nicotine consumed upon smoking affects numerous physiological processes through nicotinic acetylcholine receptors, which mediate cholinergic regulation by the neuronal and endogenous acetylcholine. Consequently, nicotinic receptors are expressed in many non-excitable tissues including the blood. In spite of the documented effect of nicotine on hematopoiesis, little is known about the expression and role of nicotinic receptors in the course of blood cell differentiation. The aim of the present study was to investigate whether and how nicotinic receptors are involved in the development of myeloid and erythroid cells within the bone marrow. The presence of nicotinic receptors containing alpha4(beta2) and alpha7 subunits in the bone marrow cells of C57Bl/6 mice was shown by the binding of [125I]-alpha-bungarotoxin or [3H]-Epibatidine and by flow cytometry with subunit-specific antibodies or fluorescein-labeled alpha-cobratoxin. Both TER119+ (erythroid) and CD16+CD43med (myeloid) progenitor cells bound more alpha4-specific antibodies than their mature forms, while the binding of alpha-cobratoxin and alpha7-specific antibodies was also high in mature cells. According to morphological analysis, either the absence of alpha7-containing nicotinic receptors in knockout mice or their desensitization in mice chronically treated with nicotine decreased the number of myeloid and erythroid progenitors and junior cells. In contrast, the absence of beta2-containing receptors favored myelocyte generation and erythroid cell maturation. It is concluded that the development of both myeloid and erythroid cell lineages is regulated by endogenous cholinergic ligands and can be affected by nicotine through alpha7- and alpha4beta2-containing nicotinic receptors, which play different roles in the course of the cell maturation.     

Variable in vitro erythropoiesis in patients with transient erythroblastopenia of childhood

Transient erythroblastopenia of childhood (TEC) is a pure red cell aplasia which primarily affects children in the infant and toddler age group. The clinical syndrome of TEC is well defined and is characterized by moderate to severe anemia with reticulocytopenia, selective aplasia of the erythroid bone marrow elements, and spontaneous recovery, usually within a month of presentation. We utilized the plasma clot tissue culture technique to explore the defect of erythropoiesis in seven patients with TEC. Culture of bone marrow at diagnosis in four patients revealed an increased erythroid proliferative capacity in one and a decreased capacity in three. The former patient plus three additional patients were found to have a transient serum inhibitor of erythroid colony formation in autologous and allogeneic systems. The three patients with diminished erythroid proliferative capacity had no demonstrable serum inhibitor, and in one patient studied the erythroid proliferative capacity became supernormal after recovery. We conclude that although TEC has a characteristic clinical picture, in vitro studies reveal a variable expression of the erythropoietic defect and support the hypothesis of a heterogeneous pathogenesis of this disorder.     

Effect of blood transfusion on erythropoiesis of homozygotic beta-thalassemia patients

The influence of blood transfusion on erythropoiesis (bone marrow erythroblasts, peripheral blood erythroblasts and reticulocytes) has been studied in 20 non splenectomized homozygous beta thalassaemia patients aged 3 to 16 years and in 10 splenectomized patients aged 8 to 24 years affected with the same disease. The number of reticulocytes was the same in the two groups but the number of erythroblasts in the splenectomized group was higher than in the other group. There was no correlation between the erythroblasts and the reticulocytes of the peripheral blood on one hand and the haemoglobin level proceeding from the same sample on the other hand. In the non splenectomized group of patients, an inverse relationship was found between the percentage of bone marrow erythroblasts and the mean annual haemoglobin level (r = -0.71; p less than 0.01). These results demonstrate the effect of blood transfusion on the erythroid cell line in homozygous beta thalassaemia and the delay between the transfusions and the medullary erythroblastic response.     

Heme-regulated eIF2alpha kinase (HRI) is required for translational regulation and survival of erythroid precursors in iron deficiency.

Although the physiological role of tissue-specific translational control of gene expression in mammals has long been suspected on the basis of biochemical studies, direct evidence has been lacking. Here, we report on the targeted disruption of the gene encoding the heme-regulated eIF2alpha kinase (HRI) in mice. We establish that HRI, which is expressed predominantly in erythroid cells, regulates the synthesis of both alpha- and beta-globins in red blood cell (RBC) precursors by inhibiting the general translation initiation factor eIF2. This inhibition occurs when the intracellular concentration of heme declines, thereby preventing the synthesis of globin peptides in excess of heme. In iron-deficient HRI(-/-) mice, globins devoid of heme aggregated within the RBC and its precursors, resulting in a hyperchromic, normocytic anemia with decreased RBC counts, compensatory erythroid hyperplasia and accelerated apoptosis in bone marrow and spleen. Thus, HRI is a physiological regulator of gene expression and cell survival in the erythroid lineage.     

Impairment of erythroid and megakaryocytic differentiation by a leukemia-associated and t(9;9)-derived fusion gene product, SET/TAF-Ibeta-CAN/Nup214

SET-CAN associated with the t(9;9) in acute undifferentiated leukemia encodes almost the entire sequence of SET and the C-terminal two-third portion of CAN, including the FG repeat region. To clarify a role(s) of SET-CAN in leukemogenesis, we developed transgenic mice expressing SET-CAN under the control of the Gata1 gene hematopoietic regulatory domain that is active in distinct sets of hematopoietic cells. SET-CAN transgenic mice showed anemia, thrombocytopenia, and splenomegaly. A significant number of transgenic mice started dying after 6 months post-birth, being in good agreement with the fact that red blood cells and platelets decreased. We found that a significant number of c-kit+ myeloid cells appeared in peripheral blood in transgenic mice. Characterization of the bone marrow cells of transgenic mice indicated impairment in hematopoietic differentiation of erythroid, megakaryocytic, and B cell lineages by SET-CAN. Transgenic mice, in particular, exhibited a high population of the c-kit+Sca-1+Lin- fraction in bone marrow cells compared with that of the control littermates. Our results demonstrate that SET-CAN blocks the hematopoietic differentiation program--one of the characteristics of acute myeloid leukemia.