alpha-Amino-N-butyric acid stimulates fetal hemoglobin in the adult

The effect of alpha-amino-N-butyric acid (alpha ABA) on fetal hemoglobin production in the adult was examined in vivo after being administered to normal and anemic baboons and in erythroid progenitor cell cultures. Infusion of alpha ABA for five days resulted in four- to fivefold increases in the level of F reticulocytes of normal or chronically anemic baboons. The induction of HbF by alpha ABA was strikingly enhanced by the administration of 5-azacytidine. The addition of alpha ABA in culture produced a concentration-related increase of HbF in baboon CFUe and e-cluster colonies. In addition to the induction of HbF, alpha ABA stimulated the growth of all classes of erythroid progenitors in vivo or in culture. The activation of gamma-globin gene expression by alpha ABA is attributed to an interaction between regulatory sites of globin chromatin modified by alpha ABA and the immature intracellular environment of the expanding erythropoiesis. The combination of chromatin modification, DNA methylation, and the immature intracellular environment of rapid erythroid regeneration may explain the synergistic induction of HbF by alpha ABA and 5-azacytidine.     

Effects of interleukin-3 and erythropoietin on in vivo erythropoiesis and F-cell formation in primates

To test the in vivo cooperativity between interleukin-3 (IL-3) and erythropoietin (Epo) in stimulating erythropoiesis and hemoglobin F (HbF) production in primates, we administered recombinant human IL-3 and recombinant human Epo to baboons and macaques. The effect of these treatments was assessed by serial bone marrow cultures and by measuring HbF production in the progeny of bone marrow progenitors and in peripheral-blood reticulocytes. Administration of IL-3 alone to hematologically normal or anemic baboons produced an early increase in erythroid colony-forming units (CFUe) and erythroid clusters (e-clusters) with an increase in reticulocyte counts and a late increment in the relative frequency of erythroid burst-forming units (BFUe). In parallel to the increase in peripheral-blood reticulocytes, IL-3 increased the frequency of F reticulocytes in the normal and anemic animals. When administration of IL-3 was followed by administration of Epo, expansion in all classes of erythroid progenitors and increase in reticulocytes occurred, beyond the levels observed when the animals were treated with Epo alone. The combination of IL-3 and Epo, however, did not increase consistently the rate of F reticulocytes beyond the level induced by Epo alone. These results suggest that IL-3 enhances the effect of Epo on erythropoiesis, but the combination of the two growth factors does not lead to a preferential and significant enhancement of HbF production.     

Hemoglobin switching in sheep: a comparison of the erythropoietin- induced switch to HbC and the fetal to adult hemoglobin switch

Stimulation of sheep erythropoietic progenitor cells by erythropoietin (epo) has been studied with regard to its effect on the pattern of hemoglobin production. An analysis of hemoglobin (Hb) synthesis in BFU- E- and CFU-E-derived colonies from fetuses either homozygous for HbA (AA) (homozygous also for the beta c gene responsible for HbC production) or HbB (BB) (lacking the beta c gene) indicated the following. Colonies derived from precursor cells from 51- and 89-day fetuses exhibited small but detectable increments of HbB synthesis with prolonged incubation in vitro. This response was not dependent on the epo concentration. Erythropoietic precursor cells from a 124-day BB fetus were already committed to HbB synthesis, since HbF production was replaced by HbB on successive days in vitro as erythroid colonies matured; this switch was not affected by varying the epo concentration. In contrast, progenitor cells from a 124-day AA fetus responded to higher doses of epo by forming colonies in which more HbC was made at the expense of both HbF and HbA. Erythropoietic stress did not result in induction of HbF in vivo or in erythroid colonies derived from CFU-E in young adult BB sheep, whereas our prior studies had shown induction of HbC synthesis under analogous conditions in colonies derived from young adult AA sheep. We conclude that the epo-induced HbF (or HbA) to HbC switch and the fetal to adult hemoglobin switch are regulated by different mechanisms.     

Hemopoietic stress and fetal hemoglobin synthesis: comparative studies in vivo and in vitro.

Baboons exposed to acute hemolytic stres increase their production of fetal hemoglobin (HbF). Although the maximal in vivo HbF levels attained in 5 treated animals varied from 6.4% to 34.8%, their cultured bone marrow erythroid cells reverted to the fetal pattern of hemoglobin synthesis. These data suggest that HbF synthesis is modulated by the interaction of inhibiting and promoting factors, which is different among animals in vivo but equal in the cultures of their bone marrow erythroid cells.     

Erythroid progenitors in paroxysmal nocturnal haemoglobinuria

In vitro colony formation of bone-marrow erythroid progenitor cells in patients with paroxysmal nocturnal haemoglobinuria (PNH) was examined. The numbers of early and late erythroid progenitors (BFU-E and CFU-E) showed wide variations; two cases out of eight cases of PNH showed decreased erythroid colony formation, but other cases showed normal or rather increased colony formation of BFU-E and CFU-E. The number of erythroid progenitors in patients with PNH may be related to the marrow cellularity.     

Effects of recombinant erythropoietin on the concentration and cycling status of human marrow hematopoietic progenitor cells in vivo

The concentration of human marrow progenitors CFU-E, BFU-E, CFU-GM, and CFU-Mk and the percentage of these progenitor cells in DNA synthesis were studied in nine patients with transfusion-dependent anemia of end- stage renal failure before and 2 weeks after treatment with human recombinant erythropoietin (Epo) at a dose of 150 to 300 U/kg intravenously three times per week. The concentration of CFU-E in the posttreatment marrow increased by a mean of 4.15-fold, BFU-E by 3.37- fold, CFU-GM by 1.86-fold, and CFU-Mk by 1.96-fold as compared with their respective concentrations in the pretreatment marrows. This increase in the concentrations of marrow progenitors was accompanied by almost a doubling of the percentage of these cells in DNA synthesis as assessed by the 3H-thymidine suicide technique. These observations demonstrate that at the progenitor cell level the human marrow responds to therapeutic doses of Epo as an organ rather than by a selective expansion of the erythroid cell line.     

Characterization of the potentiation effect of activin on human erythroid colony formation in vitro

Activin, also named FSH-releasing protein, was previously shown to induce hemoglobin accumulation in K562 cells and potentiate the proliferation and differentiation of CFU-E in human bone marrow cultures. Present studies indicate that the potentiation effect of activin is lineage specific. In addition to CFU-E, activin caused an increase in the colony formation of BFU-E from either bone marrow or peripheral blood. It had little effect on the colony formation of CFU- GM and the mixed colonies from CFU-GEMM. In serum-depleted culture, the effect of activin was shown to be dose-dependent with doses effective at picomolar concentrations. The potentiation effect of activin was exerted indirectly through mediation of both monocytes and T lymphocytes. Activin was also found to increase specifically the proportion of DNA-synthesizing erythroid progenitors from both bone marrow and peripheral blood. It had little effect on DNA synthesis in CFU-GM and in mitogen-stimulated lymphocytes. Addition of the monocytes or T lymphocytes to their respective depleted subpopulations of mononuclear cells reconstituted the enhancing effect of activin on the colony formation and DNA synthesis of erythroid progenitors. These results strongly suggest a specific role of activin in potentiating the proliferation and differentiation of erythroid progenitors in vitro.     

Enhanced colony formation of human hemopoietic stem cells in reduced oxygen tension

In general, cell cultures, including hemopoietic stem cells, are produced in an atmosphere of various CO2 concentrations in air, although most cells in vivo proliferate and differentiate at lower oxygen tensions. We therefore investigated the effect of reduced oxygen tension on the in vitro colony growth of committed and multipotential hemopoietic progenitor cells from human bone marrow. All hemopoietic progenitor cells (CFU-mix, BFU-E, CFU-E, and CFU-GM) investigated showed enhanced colony growth at lower oxygen tension. CFU-E showed the highest enhancement, followed in order by BFU-E, CFU-mix and CFU-GM. At reduced oxygen tension, the sensitivity of early and late erythroid progenitor cells to erythropoietin was significantly increased, and this can be one of the mechanisms for the enhanced colony growth of erythroid progenitors. In the colony growth of CFU-GM, plating efficiency was also enhanced by the predominant increment of neutrophilic colonies. The lowering of oxygen tension would presumably reduce oxygen toxicity and result in the increased colony growth of human bone marrow stem cells, although the precise mechanisms of oxygen toxicity at the level of hemopoietic stem cells have yet to be elucidated. However, this clonal culture system, using a low oxygen tension, can be a useful means for elucidating the regulatory mechanisms involved in the proliferation and differentiation of hemopoietic progenitor cells in physiological and pathological conditions.     

Monoclonal antibodies detecting antigenic determinants with restricted expression on erythroid cells: from the erythroid committed progenitor level to the mature erythroblast

Two new cell surface antigens specific for the erythroid lineage were defined with cytotoxic IgM monoclonal antibodies (McAb) (EP-1; EP-2) that were produced using BFU-E-derived colonies as immunogens. These two antigens are expressed on in vivo and in vitro derived adult and fetal erythroblasts, but not on erythrocytes. They are not detectable on resting lymphocytes, concanavalin-A (Con-A) activated lymphoblasts, granulocytes, and monocytes or granulocytic cells or macrophages present in peripheral blood or harvested from CFU-GM cultures. Cell line and tissue distributions distinguish McAb EP-1 and EP-2 from all previously described monoclonal antibodies. McAb EP-1 (for erythropoietic antigen-1) inhibits the formation of BFU-E and CFU-E, but not CFU-GM, colonies in complement-dependent cytotoxicity assays. By cell sorting analysis, about 90% of erythroid progenitors (CFU-E, BFU-E) were recovered in the antigen-positive fraction. Seven percent of the cells in this fraction were progenitors (versus 0.1% in the negative fraction). The expression of EP-1 antigen is greatly enhanced in K562 cells, using inducers of hemoglobin synthesis. McAb EP-2 fails to inhibit BFU-E and CFU-E colony formation in complement-dependent cytotoxicity assays. EP-2 antigen is predominantly expressed on in vitro derived immature erythroblasts, and it is weakly expressed on mature erythroblasts. The findings with McAb EP-1 provide evidence that erythroid progenitors (BFU-E and CFU-E) express determinants that fail to be expressed on other progenitor cells and hence appear to be unique to the erythroid lineage. McAb EP-1 and EP-2 are potentially useful for studies of erythroid differentiation and progenitor cell isolation.     

Pure red cell aplasia: lymphocyte inhibition of erythropoiesis

The pathogenesis of pure red cell aplasia (PRCA) was studied in a patient who had no evidence of malignancy. In marrow culture, no erythroid colonies (from late erythroid progenitors [CFU-E]) but normal numbers of well-haemoglobinized erythroid bursts (from early erythroid progenitors [BFU-E]) were found, indicating that BFU-E existed in the patient but that their subsequent in vivo differentiation was inhibited. Autologous coculture studies suggested that inhibition was mediated by the patient's ER+ lymphocytes. After remission was induced with cyclophosphamide, autologous ER + cells no longer suppressed in vitro erythropoiesis. However, cryopreserved ER + cells, obtained with anaemia, suppressed BFU-E growth from remission marrow. An expanded population of large granular lymphocytes (LGL) with ER +, F_cγ+. T3 +, T8 +, HNK-1 +, Ia —, M1 — phenotype and no functional natural killer (NK) cell activity was noted during PRCA that reverted to normal with remission. For this patient, both in vivo and in vitro evidence demonstrates a cellular inhibition of erythropoiesis at the level of differentiation between BFU-E and CFU-E.     

Factor-independent erythropoietic progenitor cells in leukemia induced by the myeloproliferative leukemia virus

The myeloproliferative leukemia virus (MPLV), a novel murine retroviral complex that does not transform fibroblasts, has been shown to cause an acute leukemia in adult mice accompanied by a progressive polycythemia. The present study demonstrates that, on in vivo inoculation, MPLV induces a rapid suppression of growth factor requirement for in vitro colony formation by both the late and the primitive erythroid progenitor cells. CFU-e-derived erythrocytic colonies developed and differentiated in semi-solid medium without the addition of erythropoietin (Epo). In addition, the formation of CFU-e colonies was not altered by the presence of specific neutralizing Epo antibodies. In the spleen, the CFU-e pool size increased rapidly up to 30-fold. By day 6 postinfection, 100% of these progenitor cells were Epo-independent. The in vivo effects of MPLV-infection on early erythroid progenitor cell compartments were examined in cultures grown for seven days. The concentration of erythroid progenitor cells was twofold elevated in spleen from MPLV-infected mice. As early as day 4 postinfection, 50% of these progenitors produced fully hemoglobinized colonies in serum-free cultures without the addition of interleukin-3 (IL-3) and Epo. Most spontaneous colonies were large and contained up to 10(5) cells per colony. They were composed of either erythroblasts only (16%) or erythroblasts and megakaryocytes (70%); few of them were multipotential (14%). In the marrow, the total number of BFU-e was reduced and only few factor-independent bursts were observed, suggesting a rapid migration of infected progenitors from marrow to spleen. Furthermore, the data show that abnormal erythropoiesis was due to the replication defective MPLV information and was not influenced by the Fv-2 locus.     

The Cellular Basis for the Defect in Haemopoiesis in Flexed-Tailed Mice

Three assays for erythropoietic progenitor cells have been applied to mice of genotype f/f and to nearly congenic +/+ controls. When f/f mice were tested for their ability to generate transient endogenous erythroid spleen colonies 4-6 days after 800 rads and 10 units of erythropoietin, the numbers of such colonies detected were greatly reduced, although normal numbers of spleen colonies appeared at later times (9-12 days) postirradiation. In contrast, cells capable of erythropoietic colony formation in culture (CFU-E) were present within the normal range in both f/f spleen and marrow and their sensitivity to erythropoietin in culture was the same as that found previously for CFU-E in the marrow and spleen of +/+ mice. Transfusion-induced plethora reduced the number of CFU-E in marrow to a similar extent in both f/f and +/+ mice; likewise, subsequent administration of 10 units of erythropoietin induced a rapid return in the number of marrow CFU-E in both genotypes. In the spleen, CFU-E numbers were approximately three-fold lower in f/f mice in each group. These results support the view that the 5 day assay for transient endogenous spleen colonies detects cells (TE-CFU) that are different from both CFU-E and pluripotent stem cells (CFU-S), although possibly overlapping to some extent with the immediate progenitors of CFU-E. The results also indicate that the generation or maturation of TE-CFU represents a primary site of expression of the f/f defect.     

Prospective isolation and global gene expression analysis of the erythrocyte colony-forming unit (CFU-E)

The erythrocyte colony-forming unit (CFU-E) is a rare bone marrow (BM) progenitor that generates erythrocyte colonies in 48 hours. The existence of CFU-Es is based on these colonies, but CFU-Es have not been purified prospectively by phenotype. We have separated the "nonstem," "nonlymphoid" compartment (lineage marker [lin]-c-Kit+Sca-1-IL-7Ralpha-) into interleukin 3 receptor alpha negative (IL-3Ralpha-) and IL-3Ralpha+ subsets. Within IL-3Ralpha- but not IL-3Ralpha+ cells we have identified TER119-CD41-CD71+ erythrocyte-committed progenitors (EPs). EPs generate CFU-E colonies at about 70% efficiency and generate reticulocytes in vivo. Depletion of EPs from BM strongly reduces CFU-E frequencies. EPs lack potential for erythrocyte burst-forming unit, megakaryocyte, granulocyte (G), and monocyte (M) colonies, and for spleen colony-forming units. Chronically suppressed erythropoiesis in interferon consensus sequence-binding protein (ICSBP)-deficient BM is associated with reduced frequencies of both the EP population and CFU-E colonies. During phenylhydrazine-induced acute anemia, numbers of both the EP population and CFU-E colonies increase. Collectively, EPs (lin-c-Kit+Sca-1-IL-7Ralpha-IL-3Ralpha-CD41-CD71+) account for most, if not all, CFU-E activity in BM. As a first molecular characterization, we have compared global gene expression in EPs and nonerythroid GM progenitors. These analyses define an erythroid progenitor-specific gene expression pattern. The prospective isolation of EPs is an important step to analyze physiologic and pathologic erythropoiesis.     

Haemoglobin synthesis in CFU-E-derived erythroid colonies during sheep ontogeny

The patterns of haemoglobin synthesis in erythroid colonies grown from sheep marrow CFU-E of different ontogenic stages have been reported previously (Darbre et al , 1979) but only at a single fixed time point in culture. Culture of adult and late fetal progenitors enhanced γ chain synthesis whereas culture of earlier progenitors during the'fetal switch'seemed to increase β-chain synthesis. In order to try to explain these findings, patterns of growth and of haemoglobin synthesis in the colonies were examined over a time-course of 6 d for all developmental stages. Colonies assessed later in vitro made proportionately less γ chain at every age. It was concluded that because fetal colonies mature more quickly in vitro than later and adult counterparts, and also because γ and β chain synthesis appeared to be asynchronous during sheep colony maturation, the results of assay at a single time point are not strictly comparable. The relevance of these findings to the study of the switch from fetal to adult haemoglobin synthesis is discussed.     

On the induction of fetal hemoglobin by butyrates: in vivo and in vitro studies with sodium butyrate and comparison of combination treatments with 5-AzaC and AraC

To obtain information on the cellular mechanism of induction of fetal hemoglobin (HbF) by sodium butyrate (NaB), we treated adult baboons with NaB and assessed its effects on HbF expression. Infusion of NaB increased F reticulocytes and F-positive CFUe and e-cluster colonies without induction of reticulocytosis or increase in progenitor cell numbers. Addition of NaB in bone marrow cultures increased the frequency of F-positive CFUe and e-clusters without increasing progenitor cell numbers. NaB induced HbF in human adult BFUe cultures and increased the gamma/gamma + beta globin chain and mRNA ratios in short-term incubations of culture-derived erythroblasts. There was a synergistic induction of HbF by NaB and 5-azacytidine (5-azaC), but not when the animal was treated with NaB and cytarabine (AraC). Our results suggest that the activation of gamma-globin expression by NaB reflects an action of this compound on globin genes or globin chromatin.     

Phenotyping of peripheral blood hemopoietic progenitor cells--in vitro cultures using CD34-/CD33-immunomagnetic purging.

In contrast to many detailed studies on the antigenic profile of hemopoietic progenitor cells from human bone marrow, sparse information, so far, has been gathered with regard to the antigen expression of hemopoietic progenitors present in peripheral blood. Previous studies by multiparameter flow-cytometry have revealed substantial differences of the coexpression of the CD33-, CD19-, and CD74- antigens, respectively, on CD34-positive cells from blood versus those from bone marrow, respectively. Immunomagnetic purging with monoclonal antibodies detecting the CD34-, and the CD33- antigen, respectively, has been used to further characterize the expression of these antigens on day 8 and d-14 granulocyte/macrophage and erythroid colonies as grown from circulating progenitor cells. Purging with CD34 monoclonal antibody abrogated all colony formation, whereas purging with CD33 antibody led to differential inhibition of the various progenitors. Purging bone marrow cells with CD34 antibody, an inhibition of only about 25% was observed with regard to erythroid colonies, whereas an inhibition of about 85% was observed for CFU-GM. These findings reinforce the view that circulating progenitor cells represent relatively immature stages of differentiation, when compared to bone marrow progenitors. Particularly, d-8 erythroid colonies from blood do not represent the equivalent of the genuine CFU-E as described from bone marrow, but they seem to be early stages of BFU-E development.     

Megaloblastic Change is a Feature of Colonies Derived from an Early Erythroid Progenitor (BFU-E) Stimulated by Monocytes in Culture

S ummary . The morphology of stained preparations of cells from human bone marrow and peripheral blood erythroid colonies cultured in methylcellulose, were examined by light microscopy. Although the morphology of 7 d erythroid colonies (CFU-E) was largely normoblastic, bone marrow and peripheral blood erythroid bursts (BFU-E) showed a variable degree of megaloblastic and dyserythropoietic change. This was not due to nutritional deficiencies of the culture system and the deoxyuridine suppression test demonstrated active thymidine synthesis.
Megaloblastic morphology was correlated with the growth induced by the addition of monocytes to erythroid progenitors. It was concluded that megalo-blastosis was a feature of the erythroblasts derived from an early BFU-E which required monocytes for their development.     

Fetal hemoglobin synthesis in vivo: direct evidence for control at the level of erythroid progenitors

To test directly whether the control of fetal hemoglobin (HbF) in the adult takes place at the level of erythroid progenitors or at the level of erythroblasts, we treated animals with high doses of erythropoietin and examined the effects of this manipulation on the globin gene programs of erythroid progenitors. We found that administration of erythropoietin produced a rapid expansion of all classes of erythroid progenitors. Almost all the expansion of colony-forming units-erythroid and 46-56% of erythroid clusters was due to the increase of HbF-programmed erythroid progenitors. The expansion of HbF-programmed erythroid progenitors was followed, 2-3 days later, by a wave of reticulocytes containing HbF in the peripheral blood. These results provide direct in vivo evidence that fetal-globin expression in the adult is controlled at the level of erythroid progenitors.