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.     

Fetal calf serum contains activities that induce fetal hemoglobin in adult erythroid cell cultures.

Cultures of peripheral blood or bone marrow erythroid progenitors display stimulated production of fetal hemoglobin. We investigated whether this stimulation is due to factors contained in the sera of the culture medium. Comparisons of gamma/gamma + beta biosynthetic ratios in erythroid colonies grown in fetal calf serum (FCS) or in charcoal treated FCS (C-FCS) showed that FCS-grown cells had significantly higher gamma/gamma + beta ratios. This increase in globin chain biosynthesis was reflected by an increase in relative amounts of steady-state gamma-globin mRNA. In contrast to its effect on adult cells, FCS failed to influence gamma-chain synthesis in fetal burst forming units-erythroid (BFU-E) colonies. There was a high correlation of gamma-globin expression in paired cultures done with C-FCS or fetal sheep serum. Dose-response experiments showed that the induction of gamma-globin expression is dependent on the concentration of FCS. These results indicate that FCS contains an activity that induces gamma-globin expression in adult erythroid progenitor cell cultures.     

Erythropoiesis following bone marrow transplantation from donors heterozygous for β-thalassaemia

This study shows a marked and protracted activation of HbF synthesis in homozygous beta.-thalassaemia patients transplanted from HLA identical siblings heterozygous for beta-thalassaemia, as compared to patients transplanted from normal donors. HbF synthesis in recipients was much higher in relation to the corresponding bone marrow donor values either normal or heterozygous for beta thalassaemia. gamma-chain synthesis and G gamma/A gamma ratio were also studied in peripheral blood BFU-E from recipients and their donors. BFU-E from donors heterozygous for beta-thalassaemia showed higher gamma chain synthesis as compared to normal donors. Peripheral blood BFU-E gamma/beta + gamma ratios and G gamma percentage were higher in recipients than in their corresponding donors both normal or heterozygotes. The marked and protracted reactivation of HbF synthesis in recipients of heterozygous beta-thalassaemia bone marrow most likely results from an increased erythropoietic stress on erythroid progenitors. In order to obtain adequate Hb levels heterozygous beta-thalassaemia bone marrow should produce more red blood cells to compensate for the low MCH. The magnitude of activation of HbF synthesis was very variable. This variability may result from inherited differences in the capacity of reactivation of HbF synthesis of red cell progenitors from heterozygous beta-thalassaemia under stressed erythropoiesis.     

Sodium butyrate enhances fetal globin gene expression in erythroid progenitors of patients with Hb SS and beta thalassemia

Increasing the expression of the gamma globin genes is considered a useful therapeutic approach to the beta globin diseases. Because butyrate and alpha-amino-n-butyric acid (ABA) augment gamma globin expression in normal neonatal and adult erythroid progenitors, we investigated the effects of sodium butyrate and ABA on erythroid progenitors of patients with beta thalassemia and sickle cell anemia who might benefit from such an effect. Both substances increased fetal hemoglobin (Hb F) expression in Bfu-e from 7% to 30% above levels found in control cultures from the same subjects with sickle cell anemia. The fraction of cultured erythroblasts producing Hb F increased more than 20% with sodium butyrate treatment in 70% of cultures. In most cultures, this produced greater than 20% total Hb F and greater than 70% F cells, levels which have been considered beneficial in ameliorating clinical symptoms. Alpha: non-alpha (alpha-non-alpha) imbalance was decreased by 36% in erythroid progenitors of patients with beta thalassemia cultured in the presence of butyrate compared with control cultures from the same subjects. These data suggest that sodium butyrate may have therapeutic potential for increasing gamma globin expression in the beta globin diseases.     

Analysis of hemoglobin F production in Saudi Arabian families with sickle cell anemia

Erythrocytes and progenitor-derived erythroblasts of sickle cell anemia patients from the Eastern Province of Saudi Arabia contain increased fetal hemoglobin and G gamma globin. A distinctive DNA polymorphism haplotype in the beta globin gene cluster (++- +-), tightly coupled to a C----T substitution at position -158 5' to the cap site of the G gamma globin gene, is strongly associated with sickle cell disease in this region. To determine whether the increased fetal hemoglobin production and/or elevated G gamma globin content are tightly linked to this haplotype, we studied 55 members of five Saudi families in which sickle cell disease is present. The results did not suggest a tight linkage of the haplotype to increased fetal hemoglobin production. On the other hand, several sickle trait family members heterozygous for the haplotype had normal fetal hemoglobin production in culture but elevated G gamma to A gamma ratios in peripheral blood. This observation suggests that in this genetic background increased expression of the G gamma globin gene may occur without a measurable increase in total fetal hemoglobin production. The family studies also clearly demonstrate that increased fetal hemoglobin production by erythroid progenitors is dependent on zygosity for the sickle gene in this population. These findings strongly suggest that other factors, such as the products of genes stimulated by hemolytic stress or other genetic determinants associated with the Saudi beta S chromosome, may interact with the -158 C----T substitution and influence gamma globin gene expression in this population.     

Proportion of fetal hemoglobin synthesis decreases during erythroid cell maturation.

Peripheral blood mononuclear cells from pregnant and postpartum women were cultured in vitro with erythropoietin. Burst-forming unit (BFU-E)-derived erythroid colonies composed of immature erythroblasts with low hemoglobin contents were observed by day 8 of culture. By day 12 of culture, numerous BFU-E-derived erythroid colonies with high hemoglobin contents were present. The gamma/(gamma + beta) globin synthetic ratio was approximately 12% in the early cultures and 6% in the late cultures, indicating that the proportion of fetal hemoglobin synthesis decreases during erythroid cell maturation. These studies also reveal that the capacity fof fetal hemoglobin production by peripheral blood BFU-E in vitro is not altered during pregnancy.     

Adult''fetal-like''erythropoiesis characterizes recovery from bone marrow transplantation

The transient fetal-like erythropoiesis which appears during recovery from bone marrow transplantation has now been examined at the level of erythroid progenitor cells. A 7-year-old boy with beta +-thalassaemia major was studied during engraftment from his beta-thalassaemia trait sister. Hb F and i antigen rose as expected. Macrocytosis never developed, but red cell size distribution became very heterogeneous. Bone marrow CFU-E and BFU-E were detected by 30 d, prior to the appearance of reticulocytes. Marrow erythroid progenitor cell numbers were normal by 146 d, while those in the blood became normal by 360 d. After transplantation globin synthesis ratios in erythroid colonies were diagnostic of thalassaemia trait, indicating engraftment. Individual erythroid colonies derived from both blood and marrow at all times during reconstitution showed no correlation of G gamma and gamma. Thus the fetal-like stress erythropoiesis of marrow expansion following transplantation was derived from adult and not fetal progenitor cells.     

Influence of hydroxyurea on fetal hemoglobin production in vitro

Cytotoxic drugs increase circulating fetal hemoglobin levels. We examined the mechanism by measuring the fetal hemoglobin produced per BFU-E-derived erythroblast following hydroxyurea treatment in vivo and in vitro. Treatment of four sickle cell patients increased the percentage of circulating F reticulocytes. The frequencies of bone marrow or peripheral blood BFU-E or CFU-E-derived colonies and their fetal hemoglobin content were unaffected. In all cases, the number of erythroid cells/progenitor-derived colony increased. To explore further the effect of hydroxyurea on fetal hemoglobin production, we added 50 mumol/L hydroxyurea to cultures of peripheral blood BFU-E-derived erythroblasts on 1 of 9 days (day 5 through 13) to nine samples. These BFU-E were derived from the peripheral blood of normal donors, sickle trait donors, and sickle cell anemia patients and from the bone marrows of monkeys. This concentration of hydroxyurea was selected so that the frequency of BFU-E and their size was moderately decreased. Addition of hydroxyurea to these progenitor-derived erythroid cells had no effect on fetal hemoglobin content per cell. Neither did transient exposure of progenitors to hydroxyurea prior to culture in nontoxic concentrations (0 to 500 mumol/L) result in a significant increase in fetal hemoglobin content in progenitor-derived erythroblasts. These data suggest that hydroxyurea does not directly alter the HbF program expressed by progenitor-derived erythroid cells. Instead, it enhances hemoglobin F content secondarily, possibly by inducing alterations in erythropoiesis.     

Patterns of Globin Chain Synthesis in Erythroid Colonies Grown from Sheep Marrow of Different Developmental Stages

Erythroid colonies were grown from fetal sheep bone marrow at different stages of development and from adult marrow. Colony numbers increased with erythropoietin concentration, but fetal erythroid cells were more sensitive to the hormone than adult cells. Haemoglobin synthesis was characterized in the colonies and compared to that in control marrow incubations. No beta chain synthesis was detectable in marrow incubations or erythroid colonies from fetal marrow at 87-96 d gestation. Incubations of 115-120 d marrow shoed 5% beta chain synthesis while erythroid colonies synthesised up to 20% beta chains. Incubations and erythroid colonies from 125 d marrows showed about 27% beta chain synthesis, despite a similar increase in colony numbers with erythropoietin as the other marrows. Later in gestation, beta chain synthesis was lower in erythroid colonies than in marrow incubations and declined further in erythroid colonies as erythropoietin concentration increased. Adult marrow showed no detectable gamma chain synthesis but erythroid colonies produced up to 12% gamma chains, the identity of which was confirmed by peptide mapping. All changes in globin synthesis in vitro were correlated with colony numbers. In adult marrow, the maximal level of gamma chain production was inversely related to the cloning efficiency of the marrow samples studied. Potential uses of this model for studying globin gene expression are discussed.     

Adult and neonatal patterns of human globin gene expression are recapitulated in liquid cultures.

We have recently described a two-step liquid culture system that supports the proliferation and maturation of human erythroid progenitors. Several days after the addition of erythropoietin, the cultures undergo erythroid differentiation in a synchronized fashion. The purpose of the present study was to determine detailed kinetics of globin gene expression at the mRNA level in adult and newborn erythroid cells. Our results show that in cultures derived from normal adult peripheral blood, the mRNA levels of alpha- and beta-globin genes increased throughout most of the culture period, whereas gamma-globin mRNA remained at a low level. In contrast, high expression of all three globin genes, alpha, beta, and gamma, was observed in cultures derived from cord blood. The results demonstrate that the populations of erythroid progenitors in cord blood and in adult peripheral blood are fundamentally different, suggesting that this culture system recapitulates the normal pattern of globin gene expression, providing a valuable tool in the investigation of the regulation of the switch from fetal to adult hemoglobin.     

β Globin messenger RNA content of bone marrow erythroblasts in heterozygous β-thalassemia

RNA from bone marrow erythroblasts and peripheral blood reticulocytes of patients with heterozygous β-thalassemia was analyzed for relative content of α and β globin messenger RNA by molecular hybrization. Erythroblasts from nonthalassemic patients exhibited approximately the same α and β globin mRNA content (β/α mRNA ratio = 0.8–1.0) as circulating reticulocytes (β/α mRNA ratio = 0.74–1.2). The mRNA ratios corresponded well to levels of globin synthesis observed in bone marrow and peripheral blood. Erythroblasts from four patients with heterozygous β-thalassemia also exhibited approximately the same β/α mRNA ratios in bone marrow erythroblasts (0.34–0.59) as in reticulocytes (0.34–0.4): β globin mRNA was clearly deficient in bone marrow erythroblasts. Globin biosynthesis by erythroblasts of β-thalassemia heterozygotes was balanced despite the mRNA deficiency (β/α = 0.9–1.0), suggesting that post-translational phenoma (eg, proteolysis of free globin chains), rather than instability of β mRNA, accounts for the balanced globin chain synthesis frequently observed in bone marrow erythroblasts of patients with β-thalassemia trait.     

Globin synthesis in erythroid bursts that mature sequentially in culture. I. Studies in cultures of adult peripheral blood BFU-Es

To study whether the culture time at which the burst populations mature influences the expression of fetal hemoglobin in bursts, we measured hemoglobin synthesis in cohorts of fully hemoglobinized erythroid bursts maturing sequentially in cultures of adult peripheral blood BFU- Es. In 13 of 15 experiments, a decline in gamma/gamma + beta ratio was noted as the culture time advanced. On the average, erythroid bursts that mature during the third culture week showed lower levels of fetal Hb synthesis compared to bursts that are already mature in the second culture week. The decline of gamma/gamma + beta ratio with culture time was also noted in erythroid bursts composed of immature erythroblasts. The enhanced HbF formation in peripheral blood BFU-E cultures is thus most pronounced among the bursts that become hemoglobinized early, and there is a tendency for normalization of HbF synthesis in bursts that mature in late culture days. These results can be interpreted by several alternatives, including the possibility that the expression of high HbF levels in the early days of adult BFU-E cultures is a reflection of premature commitment to terminal differentiation of progenitors that possess an active HbF program. The present data indicate that the variation of HbF synthesis with culture time should be taken into consideration when the influence of various culture conditions of HbF synthesis is studied in BFU-E cultures.     

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.     

Increased synthesis of mouse minor hemoglobin in erythroid colonies: a cellular model for hemoglobin regulation

Globin synthesis was examined in mouse erythroid colonies. Bone marrow cells from DBA/2J adults were cultured in methylcellulose and labeled with 3H-leucine; globin synthesis ratios were determined following electrophoresis of lysates on polyacrylamide gels containing urea, acid, and Triton X-100. Colonies derived from the immature progenitor cells, BFU-E and CFU-E, produced close to 40% beta mi of total beta, while cluster-forming units, erythroblasts, and reticulocytes synthesized approximately 30% beta mi. Thus, beta mi synthesis decreased with increasing maturity of the erythroid compartment being examined, qualitatively resembling the decrease in fetal hemoglobin between BFU-E and erythrocytes in human adults. The mouse system described here thus provides a small animal model for studies of changes in hemoglobin expression during erythroid 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.     

Differentiation kinetics and globin gene expression by circulating human BFUe in suspension cultures

We studied the kinetics of erythroid differentiation and the globin synthetic patterns of circulating early erythroid progenitors (erythroid burst-forming units, BFUe) stimulated to differentiate in suspension cultures in the presence of interleukin 3 (IL-3) and erythropoietin. Erythroid progenitor cells present at the onset of culture and on successive days (2-12) thereafter were quantitatively assessed by clonal assays, whereas globin synthesis was measured sequentially in aliquots from the suspension culture. Although BFUe numbers increased to a peak value by day 4, the number of progenitors generating larger bursts was progressively decreasing with a concomitant increase in the number of smaller sized bursts. Erythroid colony-forming units (CFUe) and erythroid clusters were first detected by day 4 and peaked on day 6. Proerythroblasts were morphologically identifiable on day 4, and they progressively increased in number and maturity so that, at culture days 10 and 12, 51% and 59% of the culture cells were erythroblasts, respectively. In keeping with the morphologic changes during the liquid culture, globin mRNA was first detected on day 4. gamma/gamma + beta mRNA ratios were highest on days 4 and 6 and declined thereafter. Our results show that circulating BFUe (at least the majority of them) can differentiate and mature as a cohort in suspension cultures, providing terminal progeny with accelerated kinetics compared to semisolid, clonal cultures. In this system the same cohort of cells can be easily sampled throughout the culture for molecular studies on erythroid differentiation.     

Embryonic----Fetal Hb switch in humans: studies on erythroid bursts generated by embryonic progenitors from yolk sac and liver.

The synthesis of embryonic (zeta, epsilon), fetal (alpha, gamma), and adult (beta) globin was evaluated in human yolk sacs (YS) and livers at different ontogenic stages (i.e., from 6 through 10-12 wk of age) by means of analytical isoelectric focusing. Globin production was comparatively evaluated in vivo (i.e., in directly labeled erythroblasts from YS and liver) and in vitro [i.e., in erythroid bursts generated in culture by erythroid burst-forming units (BFU-E) from the same erythropoietic tissues]. Erythroid bursts generated in vitro by BFU-E from 6-wk livers and YS show essentially a "fetal" globin synthetic pattern: this is in sharp contrast to the "embryonic" pattern in corresponding liver and YS erythroblasts directly labeled in vivo. The invitro phenomenon suggests that (i) 6-wk BFU-E constitute a new generation of progenitors, which have already switched from an embryonic to a fetal program, and/or (ii) expression of their fetal program is induced by unknown in vitro factor(s), which may underlie the in vivo switch at later ontogenic stages. It is emphasized that 6- to 7-wk BFU-E are endowed with the potential for in vitro synthesis of not only epsilon- and gamma-chains but also some beta-globin. In general, we observed an inverse correlation between the levels of epsilon- and beta-chain synthesis. These results, together with previous studies on fetal, perinatal, and adult BFU-E, are compatible with models suggesting that in ontogeny the chromatin configuration is gradually modified at the level of the non-alpha gene cluster, thus leading to a 5'----3' activation of globin genes in a balanced fashion.