ERYTHROPOIETIN-INDEPENDENT REGENERATION OF ERYTHROID PROGENITOR CELLS FOLLOWING MULTIPLE INJECTIONS OF HYDROXYUREA

It was shown previously that colony formation in vitro by early erythroid progenitor cells (BFUe) requires sequential stimulation with a specific glycoprotein termed BFA and erythropoietin (EP). The action exerted by BFA was characterized as induction of proliferation in BFUe resulting after several cell divisions in EP-responsive progeny. The present study is directed at detection of EP-independent regulation of erythroid progenitor cells in vivo. Haemopoietic regeneration was induced by multiple administrations of hydroxyurea (HU). The femoral regeneration patterns of haemopoietic stem cells (CFUs), granulocyte/macrophage progenitor cells (CFUgm) and erythroid progenitor cells (BFUe, day 3 BFUe and CFUe) were studied in hypertransfused mice in comparison to nontransfused controls. The results show that (1) the phase of exponential regeneration of none of the cell populations studied is affected by hypertransfusion; (2) each of these cell populations exhibit a distinct regeneration pattern, indicating that they behave as separate functional entities; and (3) the three erythroid cell populations are suppressed by hypertransfusion in the post-exponential phase of regeneration in contrast to CFUs and CFUgm. The results support a two-regulator model of erythropoiesis.     

The relative spatial distribution of erythroid progenitor cells (BFUe and CFUe) in the normal mouse femur

Abstract. Femoral mouse bone marrow cells were separated into axial and marginal fractions, in order to investigate the relative concentration of erythroid progenitor cells (BFUe and CFUe) with respect to their location across the diameter of the femur. Two areas of high incidence of early progenitor cells (BFUe) were identified: one lying near the bone surface with a peak at about 410 μ m radial distance from the axis of the bone; the other nearer the centre of the bone with a peak at about 270 μ m. The more immature BFUe were found in higher proportion in the marginal peak. In contrast, CFUe, apart from very low concentration values in the vicinity of the bone surface, demonstrated a fairly uniform distribution throughout the marrow. The present results indicate that the distribution of erythroid progenitor cells within the bone marrow is not random. The haemopoietic tissue seems to exhibit a well-defined structure that may be relevant in regulating proliferation and differentiation processes.     

PARTICLE-INDUCED ERYTHROPOIETIN-INDEPENDENT EFFECTS ON ERYTHROID PRECURSOR CELLS IN MURINE BONE MARROW

A possible regulatory action of phagocytic cells on erythropoiesis was investigated by infusion of inert polystyrene latex particles (LAT). LAT appeared to induce changes in the femoral content of erythroid progenitor cells. These changes were most pronounced in primitive erythroid progenitor cells (BFUe) and appeared to be gradually damped in more differentiated populations (CFUe and erythroblasts). LAT did not influence granulocyte/macrophage progenitor cells (CFUc). The effects of LAT could not be attributed to changes in the systemic erythropoietin (EP) concentration. Administration of dexamethason nullified the effect of low doses of LAT, suggesting that phagocytosis of the particles is essential to the observed effects. Erythroid burst formation was previously found to be dependent on a bone marrow associated activity, termed BFA (burst feeder activity). BFA acts as an in vitro inducer of EP-responsiveness in BFUe. In this study it was found that LAT-induced changes in femoral erythroid progenitor cell content were characteristically preceded by corresponding changes in BFA. It was concluded that BFA-associated cells probably play a role in vivo in the early differentiation of erythroid progenitor cells. The present data are interpreted as direct in vivo evidence supporting a two-step regulatory model operating in erythropoiesis and provide evidence that phagocytic cells are a component of the erythroid haemopoietic inductive micro-environment.     

Effect of an anti-murine transferrin receptor-ricin A conjugate on bone marrow stem and progenitor cells treated in vitro

A monoclonal antibody with specificity for murine transferrin receptor was conjugated with the toxic A subunit of ricin. The dose range, specificity, and kinetics of inhibition of protein synthesis of the conjugate were determined on the murine T-lymphoma cell line, BW5147. When toxin was present throughout the period of culture, in vitro myeloid (CFUc) and erythroid (CFUe and BFUe) bone marrow colonies were inhibited by doses of conjugate comparable to those that inhibit protein synthesis in murine cell lines (IC50 of 5 X 10(-11)M). Bone marrow exposed briefly (30 min to 6 h) to anti-transferrin receptor antibody-ricin A conjugate was assayed for myeloid (CFUc) and erythroid (CFUe and BFUe) progenitors in vitro and for in vivo spleen colony formation (CFUs). Only CFUe were depleted by this pulse exposure, consistent with the higher frequency of proliferating cells and transferrin receptor expression in the CFUe population relative to other progenitors.     

Erythroid precursors studied by mouse bone marrow cultivation in a plasma clot]

Two types of erythroid precursors were found by cultivation of the mouse bone marrow in the plasma clot with mouse serum and without adding exogenic erythropoietin to the culture medium. The first precursor had properties similar to the erythroid colony-forming unit (CFUe) previously described while the second resembles in its properties the erythroid burst-forming unit (BFUe). Optimal concentration of mouse serum in the culture medium was 10-15%. Clone nature of the colonies and bursts described is confirmed by linear dependence of their number on the cell concentration in the culture.     

DIFFERENTIATION OF MURINE MARROW MEGAKARYOCYTE PROGENITORS (CFUM): HUMORAL CONTROL IN VITRO

Differentiation of mouse marrow megakaryocyte progenitors (CFUm) was studied in vitro by a colony assay using a plasma clot system. Erythropoietin (EPO) from sheep plasma (6 units/mg protein) in doses from 1 to 5 units/ml induced a linear increase in CFUm to a maximum of 20 colonies/10⁵ cells plated. Human urinary EPO also induced a dose-responsive increase in CFUm, but the maximum was 9 colonies/10⁵ with 2·0 units/ml of EPO and there was a decrease in colonies above that concentration. Thrombocytopoiesis-stimulating factor (TSF) derived from human embryonic kidney culture supernatant fluids induced a dose-responsive increase in CFUm in concentrations from 0·01 to 0·32 mg protein/ml in the absence of added EPO. TSF did not support the growth in vitro of erythroid colonies from mouse marrow (CFUe and BFUe) indicating an absence of EPO activity. In these studies sheep EPO appeared more effective in supporting CFUe growth than human EPO. TSF also had a stimulatory function in megakaryocyte differentiation at a precursor level. Multiple humoral factors play a role in megakaryocytopoiesis in vitro.     

Development of early erythroid precursors (BFUe) from mouse bone marrow in a serum-free culture media. Effect of hemin

In culture medium containing albumin, iron saturated transferrin, phospholipids, cholesterol and erythropoietin, BFUe growth requires foetal Calf serum. Without serum the BFUe development is advantageously restored by the addition of hemin associated with spleen conditioned medium. In erythropoietin supplemented serum-free medium the growth of primitive erythroid precursor cells is closely dependent on growth factor supply found in spleen conditioned medium. Using this medium allows us to clearly distinguish between the respective effects of erythropoietin and of BPA on erythroid progenitor cells.     

INDUCTION OF ERYTHROPOIETIN RESPONSIVENESS IN VITRO BY A DISTINCT POPULATION OF BONE MARROW CELLS

Bone marrow contains a small population of primitive erythroid progenitor cells which can be detected by their capacity to form large numbers of erythroid progeny in viscous cultures containing erythropoietin (EP). These cells have been termed erythroid ‘burst-forming units’(BFUe). The present study demonstrates that expression of the erythroid differentiation potential of BFUe requires the presence of an activity additional to EP. This activity has been designated as BFA (burst feeder activity). It is shown that the number of BFUe detected and their apparent sensitivity to EP are directly related to the BFA concentration of the cultures. BFA was found to be associated with a population of bone marrow cells of high buoyant density and small volume, which are sensitive to irradiation. The radiation dose-effect curve provided strong evidence that bone marrow BFA is independent of cell proliferation; this was supported by showing that BFA is unaffected by in vivo treatment with hydroxyurea. The findings are compatible with a two-step regulation model for erythroid differentiation in which BFA-induced progeny of BFUe acquire sensitivity to EP.     

Induction of erythropoietin responsiveness in vitro by a distinct population of bone marrow cells.

Bone marrow contains a small population of primitive erythroid progenitor cells which can be detected by their capacity to form large numbers of erythroid progeny in viscous cultures containing erythropoietin (EP). These cells have been termed erythroid 'burst-forming units' (BFUe). The present study demonstrates that expression of the erythroid differentiation potential of BFUe requires the presence of an activity additional to EP. This activity has been designated as BFA (burst feeder activity). It is shown that the number of BFUe detected and their apparent sensitivity to EP are directly related to the BFA concentration of the cultures. BFA was found to be associated with a population of bone marrow cells of high buoyant density and small volume, which are sensitive to irradiation. The radiation dose-effect curve provided strong evidence that bone marrow BFA is independent of cell proliferation; this was supported by showing that BFA is unaffected by in vivo treatment with hydroxyurea. The findings are compatible with a two-step regulation model for erythroid differentiation in which BFA-induced progeny of BFUe acquire sensitivity to EP.     

Erythroid colony formation by fetal rat liver and spleen cells in vitro: inhibition by a low relative molecular mass component of fetal spleen.

Liver and spleen hematopoietic cell suspensions from 20-day-old-fetal rats were fractionated on Percoll gradients. A granulocyte-rich splenic fraction inhibited CFUe production by cultures of a CFUe-enriched liver fraction, and by cultures of unfractionated liver and spleen hematopoietic cells. Conditioned medium from the spleen cell fraction contained an inhibitor of relative molecular mass, Mr, 25-35 x 10(3). The sensitivity of spleen cells to the inhibitor varied with the age of the fetus from which they were derived (20-day-old less than 18-and 19-day-old). No such age-dependence was found for liver cells. The inhibitor affects cycling CFUe, blocks the lethal effect of AraC, does not appear to be lineage-specific and its influence can be reversed by washing.     

ERYTHROID AND GRANULOCYTE-MACROPHAGE PROGENITOR CELLS IN PRE-NATAL 'STEEL' (SlJ/SlJ) ANAEMIC MICE

The erythropietin sensitivities of dissociated cell cultures and explanted fragments of fetal livers of congenitally anaemic Sl^J/Sl^J mice, and their normal littermates, have been compared. The erythropoietin responsiveness of Sl^J/Sl^J foetal liver cells is deficient in both types of culture. The maximum liver complement of erythroid colony forming cells (CFUe) occurs on the 16th day of development when ‘normal’ livers contain approximately 6 × 10⁵ erythroid colony forming cells/liver. In Sl^J/Sl^J fetuses the maximum reached is only 1 × 10⁵. Granulocyte-macrophage colony forming cells (CFU_C) in Sl^J/Sl^J fetal livers are also reduced to approximately 60% of normal numbers. Erythroid colony forming cells are also reduced in the spleen and femoral bone marrow of Sl^J/Sl^J mice in the 2–3 days preceding birth. Granulocyte-macrophage colony forming cells are rare in the femoral marrow of pre-natal Sl^J/Sl^J mice, but their production in the Sl^J/Sl^J pre-natal spleen appears unaffected.     

Erythropoietin effects on iron metabolism in rat bone marrow cells

This paper describes a study of the incorporation of ^{5 9}Fe from ^{5 9}Fe-labelled rat transferrin into rat bone marrow cells in culture. ^{5 9}Fe was found in both stroma and cytoplasm of marrow cells, and the cytoplasmic ^{5 9}Fe separated by polyacrylamide gel electrophoresis, into ferritin, haemoglobin and a low molecular weight fraction.The incorporation of ^{5 9}Fe into all three cytoplasmic fractions, but not into the stroma, increased progressively with time. Erythropoietin stimulated the increase of ^{5 9}Fe in ferritin within 1 h, the earliest time examined, and more than 3 h later in the stroma and haemoglobin.A proportion of the ⁵⁹Fe incorporated into the stroma and low molecular weight iron fractions during a 1 h incubation with ⁵⁹Fe-labelled transferrin was mobilised into ferritin and haemoglobin during a subsequent 4-h “cold-chase”. Erythropoietin, when present during the “cold-chase”, did not influence these ⁵⁹Fe fluxes. The erythropoietin stimulation of ⁵⁹Fe incorporation into ferritin, one of the earliest erythropoietin effects to be recorded, was therefore considered to be due to an increase of ⁵⁹Fe uptake by the hormone-responsive cells rather than a direct effect on ferritin synthesis.20-h cultures containing erythropoietin when incubated with ⁵⁹Fe-labelled transferrin for 4 h, showed dose-related erythropoietin stimulation of ⁵⁹Fe incorporation into haemoglobin only.In the presence of 10 mM isonicotinic acid hydrazide, ⁵⁹Fe incorporation into haemoglobin was inhibited, as in reticulocytes (Ponka, P. and Neuwirt, J. (1969) Blood 33, 690–707), while that into the stroma, ferritin and low molecular weight iron fractions, was stimulated; there were no reproducible effects of erythropoietin.     

Haemopoietic modulation in tumour-bearing animals: enhanced progenitor-cell production in femoral marrow

Altered haematopoiesis in the femoral marrow was observed in mice bearing the Lewis lung carcinoma (LLca). During tumour growth, a marked reduction was observed in the myeloperoxidase-positive cells (granulocytes) of the marrow 7 days after inoculation of the LLca tumour reaching a nadir (17% of control) by day 28. Accompanying this suppression of mature white cells was a gradual expansion of the CFUc-GM compartment followed by an increase in the number of femoral CFUs. Humoral-stimulating activity (HSA) increased through day 14 in the serum of these animals; then returned to control levels by day 28. During this same interval, the more primitive erythroid progenitor (BFUe) compartment expanded to 168% of control, while the more differentiated (CFUe) compartment was reduced (45% of control at day 28). Reductions in both 59Fe-incorporation and erythroblasts/femur confirmed the suppression of erythroid differentiation in marrow during tumour growth. Similar results were observed following the daily injection (188 mg equivalent dose; q 24 hr X 10) of the supernatant prepared from LLca tissue. Marked differences were observed between the response of the spleen and the marrow to the supernatant. The data suggest that the growth of the LLca tumour results in a dissociation of the normal continuity of haematopoietic steady-state differentiation in the marrow of tumour-bearing animals.     

THE ROLE OF ERYTHROPOIETIN IN REGULATION OF POPULATION SIZE AND CELL CYCLING OF EARLY AND LATE ERYTHROID PRECURSORS IN MOUSE BONE MARROW

This study was designed to determine the stage in haemopoietic cell differentiation from multipotential stem cells at which erythropoietin becomes physiologically important. The responses of haemopoietic precursor cells were monitored in the bone marrow of mice under conditions of high (after bleeding) and low (after hypertransfusion) ambient erythropoietin levels. The number of relatively mature erythroid precursors (CFU-E), detected by erythroid colony formation after 2 days of culture, increased three-fold in marrow by the fourth day after bleeding, and decreased three-fold after hypertransfusion. Assessed by sensitivity to killing by a brief exposure to tritiated thymidine (³H-TdR) in vitro, the proliferative activity of CFU-E was high (75% kill) in untreated and bled animals, and was slightly lower (60% kill) after hypertransfusion. The responses of more primitive erythroid progenitors (BFU-E), detected by erythroid colony formation after 10 days in culture, presented a contrasting pattern. After hypertransfusion they increased slightly, while little change was noted until the fourth day after bleeding, when they decreased in the marrow. The same response pattern was observed for the progenitors (CFU-C) detected by granulocyte/macrophage colony formation in culture. The sensitivity of BFU-E to ³H-TdR was normally 30%, and neither increased after bleeding nor decreased after hypertransfusion. However, in regenerating marrow the ³H-TdR sensitivity of BFU-E increased to 63%, and this increase was not affected by hypertransfusion. These results are interpreted as indicating (1) that physiological levels of erythropoietin do not influence the decision by multipotential haemopoietic stem cells to differentiate along the erythroid pathway as opposed to the granulocyte/macrophage pathway; (2) that early erythroid-committed progenitors themselves do not respond to these levels of erythropoietin, but rather are subject to regulation by erythropoietin-independent mechanisms; and (3) that physiological regulation by erythropoietin commences in cells at a stage of maturation intermediate between BFU-E and CFU-E.     

Haematopoiesis In Mice Heterozygous For the W Trait: Defective Formation of Transient Endogenous Spleen Colonies

It has been determined that W/+ and W^v/+ heterozygous mice, as compared with normal +/+ homozygous littermates, form significantly lower numbers of transient 5-day endogenous spleen colonies in response to X-irradiation. This defect was evident for doses of irradiation between 2–6 Gy (200–600 rad) and was associated with a slightly increased radiosensitivity of the assayed precursor cells (TE-CFU) in W heterozygotic mice. Moreover, the defect was transplantable, i.e., intrinsic to the marrow cells and not to the microenvironment, and was not associated with a similar decrease in cells which form erythropoietic bursts in vitro (BFUe). This study provides a cellular basis for increased radiosensitivity of W/+ and W^v/+ mice and suggests that the ‘W’ mutation is semi-dominant, both with respect to the white spotting and TE-CFU formation.     

Dietary Protein-induced Changes of the Erythropoietin Level in Rat Serum

Erythropoietin, a glycoprotein, is the primary regulator of erythropoiesis. The most convenient and sensitive assay for active erythropoietin is to measure its stimulatory effect on in vitro ³H-thymidine incorporation into DNA of erythropoietin-responsive cells. An attempt with this method to estimate the erythropoietin level in rat serum, however, was unsuccesful because of the presence of inhibitory substance(s) and non-erythropoietic factor(s) stimulating ³H-thymidine incorporation. Pretreatment of the serum by heating, extraction of erythropoietin from denatured-protein aggregates, and subsequent concentration of erythropoietin in the extract with alcohol precipitation made it possible to measure the serum erythropoietin levels. Rabbit anti-erythropoietin antibody was used for a quantitative estimation of erythropoietin in the concentrated extracts. Erythropoietin levels in sera of rats fed on varied amounts of casein for 7 days were measured with these procedures to find if the impairment of erythropoiesis upon protein deprivation was due to changes in the erythropoietin level. We found that the level in protein-deprived rats was less than 1/8 that of 20% casein-fed rats, a level undetectable by the present assay, and that the serum erythropoietin increased as the protein content in the diet was increased up to 20%, then leveled off. The erythropoietin in serum decreased rapidly after protein deprivation; the level at 12hr after deprivation began was about 1/5 that in 20% casein-fed rats. Thus, the depression of erythropoiesis upon protein deprivation is primarily caused by the lowered level of erythropoietin.     

Neuroprotective cytokines repress PUMA induction in the 1-methyl-4-phenylpyridinium (MPP(+)) model of Parkinson's disease

The hematopoietic cytokines erythropoietin (Epo) and granulocyte-colony stimulating factor (G-CSF) provide neuroprotection in several in vitro and in vivo models of Parkinson’s disease (PD). The molecular mechanism by which Epo and G-CSF signals reduce the neuronal death in PD is not clear. Here, we show that in rat pheochromocytoma PC12 cells, Epo and G-CSF efficiently repressed the 1-methyl-4-phenylpyridinium (MPP⁺)-induced expression of the proapoptotic protein PUMA (p53 up-regulated modulator of apoptosis). Accordingly, Epo and G-CSF treatment reduced the PC12 cell fraction that underwent apoptosis by MPP⁺ treatment and thus improved cell viability. Downregulation of PUMA expression by Epo and G-CSF in MPP⁺-treated PC12 cells seems to be mediated by repression of p53, as the expression of p53 was increased by MPP⁺-treatment and reduced by Epo and G-CSF. Together, these results suggest that the neuroprotective activities of Epo and G-CSF in an experimental model of PD involve the repression of the apoptosis-inducing action of PUMA.     

Stimulation of porphyrin accumulation in chick embryo liver cell cultures by partially purified erythroprotein

Step III and Step IV erythropoieten derived from sheep plasma stimulated the accumulation of porphyrins in cultured chick embryo liver cells. Increased porphyrin accumulation occurred within hepatocytes. It was not accompanied by increased hemoglobin formation. Stimulation of porphyrin accumulation was inhibited by hematin but was unimpaired by heating erythropoietin to 60°C for 10 min or preincubating it with trypsin. A more highly purified preparation of erythropoietin from human urine had no effect on porphyrin accumulation. The data indicate that a component in partially purified sheep erythropoietin can increase levels of a heme precursor in non-erythroid tissue. The participation of such a component should be considered when interpreting biochemical effects observed with crude erythropoietin preparations, other than ⁵⁹Fe incorporation into red cells or heme.     

Effects of dexamethasone on the levels of plasma corticosteroid binding globulin in rats and monkeys.

Rat marrow cells were preincubated for 45 hours with 5.5 × 10^?4M sodium hexachloroiridate. This treatment abolished DNA synthesis whilst improving cell survival over that of controls. The synthesis of RNA, protein and glycoprotein continued and could be further increased by the addition of erythropoietin for up to 44 more hours. Heme synthesis also continued in the absence of DNA synthesis but could not be stimulated by erythropoietin.