A case of acquired pure red cell anemia studied by cloning of erythroid progenitor cells in vitro

A 70-year-old woman developed typical clinical symptoms of pure red cell anemia (PRCA) following a history of rheumatoid arthritis (RA). The patient's bone marrow erythropoietic progenitors cells were cloned in a micro agar culture system several times over a period of 11 months, revealing a diminished frequency of bone marrow erythroblasts paralleled by a markedly reduced number of CFU-e and BFU-e in vitro. No inhibitory activity in the patient's IgG fraction could be detected either by preincubation with IgG and/or rabbit complement, or in the continuous presence of IgG. Depletion of T lymphocytes from the patient's bone marrow cells led to an improved in vitro erythroid proliferation. Cytostatic therapy with cyclophosphamide clinically induced a marked increase in the bone marrow erythroblast and reticulocyte number, correlated in vitro by normalization of CFU-e levels and increase in the number of BFU-e. Nevertheless, BFU-e values never attained normal levels, which could be attributed to a reduced stem cell pool resulting from previous therapy with cyclophosphamide and/or antirheumatic drugs. Two independent factors, a reduced pool of committed stem cells as well as an autoimmune cell-mediated suppression, may both contribute to the pathomechanism of the disease in this patient.     

Contamination of erythropoietin by endotoxin: in vivo and in vitro effects on murine erythropoiesis.

Endotoxin was detected in all erythropoietin preparations tested and was removed from four lots, without loss of erythropoietic activity, by adsorption with limulus amebocyte lysate. Comparison of adsorbed (endotoxin-depleted) and nonadsorbed (endotoxin-containing) erythropoietin preparations demonstrated significant inhibition of CFU-e and BFU-e in vitro by nonadsorbed erythropoietin at concentrations higher than 0.25 U/ml and 2.0 U/ml, respectively. CFU-e and BFU-e were inhibited significantly by readdition in vitro of 10(-5)-10(-3) mug of endotoxin per unit of limulus-adsorbed erythropoietin. Administration of saline or 6 U of nonadsorbed or adsorbed erythropoietin twice a day for 4 days of CF1 mice resulted in reticulocyte counts of 2.1%, 9.9%, and 15.9%, respectively. Nonadsorbed erythropoietin resulted in a 29% decrease in erythropoiesis, a 42% decrease in CFU-e, and a 16% increase in granulopoiesis in the marrow, whereas adsorbed erythropoietin caused a 28% increase in erythropoiesis, no significant change in CFU-e and a 19% decrease in granulopoiesis in the marrow. Both preparations resulted in marked increases in splenic erythropoiesis and granulopoiesis. The effects of adsorbed erythropoietin are similar to those produced following stimulation of hematopoiesis by endogenous erythropoietin. Hemopoietic changes induced by nonadsorbed erythropoietin in vivo and in vitro are affected substantially by contamination of the erythropoietin preparations with endotoxin.     

Erythropoiesis in vitro. III. The role of potassium ions in erythroid colony formation.

The role of potassium as an essential promotor of erythroid progenitor growth (BFU-e & CFU-e) from normal murine hematopoietic tissues was studied. Dialyzed fetal calf serum, over a wide range of concentrations, was shown to reduce the numbers of BFU-e and CFU-e that could be cultured from normal murine bone marrow. A dose-dependent addition of 1 M KC1 restored erythroid progenitor colony growth to the levels generally seen when normal, non-dialyzed fetal calf serum was used. Furthermore, when [K] was increased in some human urinary and sheep plasma erythropoietin preparations, the number of erythroid progenitor cells cultured also increased. This influence is crucial to the differentiation of committed stem cells into the erythroid pathway and must therefore be considered in the development of serum-free growth media.     

Temporal response of murine pluripotent stem cells and myeloid and erythroid progenitor cells to low-dose glucan treatment

B6D2F1 female mice were intravenously administered 0.4 mg of glucan. 1, 5, 11, and 17 days later, the total nucleated cellularity (TNC) and the numbers of pluripotent hemopoietic stem cells (CFU-s), granulocyte-macrophage progenitor cells (GM-CFC), and erythroid colony-forming (CFU-e) and burst-forming (BFU-e) cells were assayed in the bone marrow and spleen. Bone marrow TNC was not altered, but splenic TNC increased approximately twofold on day 5 and remained increased on days 11 and 17 after glucan treatment. The concentrations of bone marrow and splenic CFU-s and GM-CFC both significantly increased (p less than 0.01) by 5 days after glucan administration; however, they returned to control levels by day 17. Splenic CFU-e concentration increased on days 5, 11, and 17, whereas splenic BFU-e concentration increased only on day 11 after treatment. By contrast, bone marrow CFU-e and BFU-e concentrations were either unaffected or slightly decreased by glucan treatment. When peripheral blood was assayed for CFU-s and GM-CFC, no detectable increase in the concentrations of these progenitors was noted at any time after glucan treatment. The relevance of these effects of low-dose (0.4 mg) glucan treatment is discussed with respect to previously reported effects of higher-dose (e.g., 4.0 mg) glucan treatment.     

Changes of the phenotype of erythroid progenitor cells (BFU-E, CFU-E) and their progenies during early steps of differentiation

Early differentiation processes of human erythroid progenitor cells (BFU-e, CFU-e) have been studied during in vitro proliferation using a panel of monoclonal antibodies with known reactivity on different levels of the erythroid cell line. Two antibodies recognizing structures on BFU-e (VIP-2b, BMA 021), two antibodies reactive with CFU-e and nucleated red cells (5F1, CLB-Ery-3) and one antibody directed against glycophorin A (VIE-G4) were used for this study. Normal human bone marrow cells were induced to proliferation in an erythroid progenitor cell assay and, after different periods of incubation, agar cultures were treated with these antibodies and complement. Thereafter, the remaining erythroid cells were incubated again to continue their proliferation with the same stimulators as before. The changes of the phenotype of BFU-e and CFU-e progenies during in vitro proliferation were determined by the reduction of colony formation in comparison with untreated control cultures. Our results indicate that the loss of HLA-DR antigens and the p45 structure is accompanied by the acquisition of structures recognized by the antibodies 5F1 and CLB-Ery-3. After 5-7 d of incubation BFU-e derived progenies exhibit the same antigenic structure as has been found for CFU-e. Glycophorin A expression could only be demonstrated at a late differentiation stage of the erythroid cell lineage.     

Further studies on the in vitro enhancement of erythroid stem cell (CFU-e and BFU-e) colony formation by ouabain

The cardiac glycoside ouabain has been shown to stimulate erythroid stem cell (CFU-e/BFU-e) colony formation while inhibiting both granulocyte/macrophage precursor cell (CFU-gm) and murine spleen stem cell (CFU-s) colony formation. We have examined the ability of ouabain to increase both CFU-e/BFU-e colony formation by performing time-delay and temperature-dependent studies. After pre-exposure of marrow cells to ouabain (10(-15) M) at temperatures of 27, 37, or 4 degrees C, erythropoietin (Ep) was added after time-delays ranging from 10 s to 60 min. The ouabain-induced increase in CFU-e/BFU-e colony formation was observed up to an Ep-delay of 20 s, after which time the enhancing effect of ouabain was diminished. This increase was also observed when marrow cells were first exposed to Ep prior to a similar delayed exposure to ouabain. The enhancement effect seen with ouabain was also temperature dependent, since at 37 degrees C an earlier time increase in CFU-e/BFU-e colony formation was observed when compared to identical studies performed at either room temperature or 4 degrees C. These studies suggest that ouabain-induced elevations in erythroid stem cell colony formation involve mechanisms that are both time and temperature dependent.     

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.     

Erythroid progenitor cell kinetics in chronic haemodialysis patients responding to treatment with recombinant human erythropoietin

The response of bone marrow and peripheral blood erythroid progenitors to human recombinant erythropoietin (rHuEPO) was studied in nine haemodialysed renal failure patients receiving this hormone for the correction of their anaemia. The haematocrit rose in all patients in response to thrice weekly injections of escalating rHuEPO doses (12-192 IU/kg). Both the numbers of CUF-e and BFU-e and their proliferative state in the bone marrow as well as BFU-e numbers in the peripheral blood were estimated before treatment and again after correction of the anaemia, at 16 h following an intravenous dose of rHuEPO. Following treatment bone marrow BFU-e numbers fell to a mean of 24.5% (P less than 0.01) of the pre-treatment values although there was no significant change in CFU-e or circulating BFU-e numbers. The mitotic rate (percentage S-phase cells) estimated by tritiated thymidine suicide rose from 45.2% to 68.4% (P less than 0.05) in the case of CFU-e and from 16.4% to 45.1% (P less than 0.05) for BFU-e following treatment with rHuEPO thus indicating in-vivo sensitivity of both the primitive as well as the mature erythroid progenitors to the hormone. The fall in BFU-e numbers in the bone marrow after several months of treatment may be due to a loss of cells from this progenitor pool by maturation that is uncompensated by replacement from the pluripotential stem cell compartment.     

In vivo erythropoietin requirements of regenerating erythroid progenitors (BFU-e, CFU-e) in bone marrow of mice.

Erythroid progenitors (B-8, B-4, CFU-e) in the femoral marrow of polycythemic mice were measured by in vitro culture assays after a single administration of BCNU or Myleran. BCNU reduced pluripotent stem cells to 40% and erythroid progenitors to less than 5% of normal. B-8, the earliest erythroid progenitors, regenerated without erythropoietin (Epo) completely within 5 days. At 14 days after BCNU, intermediate progenitors (B-4) attained 60% of their normal numbers and CFU-e attained approximately 30%. Daily injections of Epo promptly restored normal B-4 numbers and near-normal CFU-e numbers in BCNU-treated mice. After Myleran, CFU-s remained below 2% of normal for 14 days, and no regeneration of the B-8 occurred with or without daily Epo injections. The findings suggest that regneration of B-8 was dependent on cell inflow from the pluripotent stem cell compartment but was independent of the presence of Epo. Intermediate progenitors (B-4) required Epo and the presence of B-8 for complete and permanent regeneration. CFU-e were the most Epo-dependent of the three progenitors. B-4, recruited by Epo, required after their formation a second exposure to the hormone in order to progress into the CFU-e stage.     

Human erythropoiesis in vitro and the source of burst-promoting activity in a serum-free system

Bovine serum albumin (BSA) can markedly increase the number of size of erythropoietic bursts produced by mononuclear cells from human bone marrow and peripheral blood, and reduce the threshold amount of erythropoietin (Epo) required for initial burst formation. The purpose of this study was to determine a possible burst-promoting activity (BPA) of BSA. The experiments were performed in a miniaturized agar system, in which the addition of sheep Epo to cultures with or without BSA was delayed for five days. The results obtained have shown that, with or without BSA, Epo deprivation of up to five days (an epoprival state) did not markedly decrease the number of bursts produced by unfractionated peripheral mononuclear cells compared to the number produced in the presence of Epo from the beginning of culture. Similar results were found whether the fetal calf serum (FCS) concentration was 15% or 2%. The preservation of potential BFU-e formation during the epoprival state has therefore been attributed to the ability of T-lymphocytes and/or monocytes to supply BPA. In order to reduce the endogenous amount of BPA, a nonadherent, E-rosette-negative cell fraction was cultured in the presence of Epo, with or without BSA, in serum-free medium containing transferrin (TF). Under these conditions, an equal number of bursts was obtained in FCS and in serum-free medium containing Epo, BSA and TF, whereas no BFU-e growth was found in the presence of Epo and TF, but without BSA. If Epo was withheld for up to five days, the capacity to form erythroid colonies was still retained by the monocyte- and T-lymphocyte-depleted cell fraction in the continuous presence of BSA. However, BPA could not be detected in the BSA. This observation was further supported by experiments in serum-free medium using human recombinant Epo, in which no BFU-e colony formation could be detected in the presence of BSA. From our investigations carried out at limited cell density and in serum-free medium, it could be concluded that the crude Epo preparation was the source of BPA.     

Separation of erythroid progenitor cells in mouse bone marrow by isokinetic-gradient sedimentation.

Isokinetic-gradient sedimentation employing a shallow linear gradient of Ficoll in tissue culture medium was used to isolate erythroid progenitor cells (CFU-e) from mouse bone marrow. Following gradient sedimentation, 34% of the total nucleated cells and 48% of the CFU-e applied to the gradient were recovered, and three distinct modal populations of CFU-e could be distinguished. The slowest-migrating population did not require exposure to exogenous erythropoietin in order to form erythroid colonies in vitro. The other two modal populations of CFU-e required exposure to exogenous erythropoietin for differentiation. One of these, constituting 64% of the hormone-dependent CFU-e recovered, migrated with the bulk of the marrow cells, whereas the other migrated ahead of the bulk of the marrow cells. This latter population, which contained 34% of the CFU-e, was recovered with 11% of the marrow cells, representing a twofold to threefold enrichment. BFU-e migrated more slowly than the erythropoietin-dependent CFU-e. Resedimentation studies suggested that the two erythropoietin-dependent CFU-e populations were distinct modal populations. When cells from the fastest-migrating population of erythropoietin-dependent CFU-e were cocultured with unseparated marrow cells, a further twofold to threefold enhancement of erythroid colony formation was obtained. Comparison of isokinetic-gradient sedimentation with discontinuous and continuous albumin density-gradient sedimentation revealed that isokinetic-gradient sedimentation was a more efficient method than the former and a more rapid method than the latter for isolating CFU-e from mouse bone marrow.     

In vivo stimulation of murine granulopoiesis by human urinary extract from patients with aplastic anemia.

Significant in vivo stimulation of granulopoiesis was induced in mice by the administration of an extract from the urine of patients with aplastic anemia (AA). Sialic acid has been identified as an important molecular component for the in vivo biological activity of this granulopoietic factor, "granulopoietin," which is distinct and different from endotoxin. Urine from patients with AA was successively fractionated by Sephadex G-50 and DEAE-cellulose chromatography. The resultant extract, which we refer to as AA urinary extract, contained approximately equal to 44 international units of erythropoietin per A unit of protein and induced 15,000 colonies of granulocyte/macrophage precursor cells (granulocyte/macrophage colony-forming units, CFU-gm) per A unit of protein with mouse bone marrow. Eight daily intraperitoneal injections of this extract in mice induced a 6.2-fold increase in peripheral blood granulocytes and a 14.6-fold increase of splenic CFU-gm, with concomitant increases in the proliferation rates of CFU-gm in both bone marrow and spleen. Pretreatment of the AA urinary extract with sialidase significantly diminished these granulopoietic effects in vivo (P less than 0.001). In contrast, both extracts (i.e., native AA and sialidase-treated AA urinary extracts) revealed high granulocyte/macrophage colony-stimulating factor activity in vitro when clonal assays were performed with mouse bone marrow. Increased in vivo and in vitro granulopoietic activities were found in the concanavalin A "break-through" fraction, indicating that these activities were due to protein(s) that did not bind to the lectin. These results reveal that this urinary extract from patients with AA is capable of inducing significant granulopoiesis in mice and that sialic acid is an important component in the maintenance of this granulopoietic effect in vivo but not in vitro.     

In vitro myelotoxicity of 2',3'-dideoxynucleosides on human hematopoietic progenitor cells

Three nucleoside analogues, 2',3'-dideoxyadenosine (ddA), 2',3'-dideoxyinosine (ddI), and 2',3'-dideoxycytosine (ddC), were evaluated for their potential myelotoxic effects to normal human hematopoietic progenitor cells. The myeloid (granulocyte-monocyte colony-forming units, CFU-gm) and erythroid (erythroid burst-forming units, BFU-e: and erythroid colony-forming units, CFU-e) committed progenitor cells were exposed to the agents for a 1-h period prior to culture in a microcapillary assay or continuously exposed during the entire culture period. Both ddA and ddI (100 microM) were mildly toxic (less than 50% colony inhibition) to human CFU-gm, BFU-e, and CFU-e following either 1-h or continuous exposures. Marrow progenitor sensitivities to ddA and ddI were indistinguishable. Colony inhibition ranged from 47% to 67% for 1-h ddC exposure (100 microM), values that were comparable to ddA and ddI. Continuous exposure to ddC was highly myelotoxic to human hematopoietic progenitors, with concentrations of 10 and 100 microM suppressing colony formation by 79%-92% and 93%-97%, respectively. These results demonstrate that 1-h and continuous exposures to ddA and ddI were similarly myelotoxic to human hematopoietic cells, whereas a 1-h exposure to ddC was equivalent to ddA and ddI, yet continuous ddC exposure was extremely toxic to marrow cell progenitors.     

Stem Cell Migration Induced by Erythropoietin or Haemolytic Anaemia: the Effects of Actinomycin and Endotoxin Contamination of Erythropoietin Preparations

The injection of erythropoietin or the induction of anaemia with phenylhydrazine leads to changes in murine pluripotent and granulocyte-macrophage stem cells indicating migration from marrow to spleen. In order to evaluate the interrelationship between erythroid differentiation and stem cell migration we have selectively suppressed erythroid differentiation with actinomycin D. Anaemia or EP injection resulted in stem cell changes consistent with migration; actinomycin blocked these changes in anaemic but not EP injected mice while blocking erythropoiesis in both groups. The erythropoietin contained from 0.01 to 1000 microgram/ml of endotoxin as defined by the limulus test; it decreased marrow erythropoiesis and stimulated marrow granulopoiesis. Adsorption of the erythropoietin preparation with limulus lysate removed endotoxin without decreasing erythropoietin activity. Adsorbed erythropoietin stimulated erythropoiesis and not granulopoiesis, and stem cell changes induced by its administration were largely blocked by actinomycin, suggesting that endotoxin in the non-adsorbed erythropoietin caused the actinomycin resistant stem cell changes. The observation that actinomycin blocks both erythroid differentiation and stem cell migration suggests that these two physiologic events are closely linked. The effects of injected erythropoietin on murine haemopoietic stem cells may, to a significant extent, be secondary to the presence of endotoxin in the erythropoietin preparations.     

Endotoxin-induced suppression of erythropoiesis: the role of erythropoietin and a heme synthesis stimulating factor

The regulation of erythropoiesis is primarily controlled by erythropoietin (Ep). Recently, however, other factors that both stimulate and inhibit erythropoiesis have been reported. Using an in vitro liquid culture of bone marrow cells, a factor in normal mouse serum was demonstrated that markedly stimulated heme synthesis by marrow erythroid cells. In this study, the role of this heme synthesis stimulating factor (HSF) and Ep in the erythropoietic suppression caused by endotoxin administration to mice was examined. Although HSF levels did not alter appreciably after endotoxin injection, marrow erythroid cells from these animals became unresponsive to the factor. This could be reversed if Ep was added to the culture in vitro or if the hormone was injected into the mice 18 hr prior to harvesting the marrow. This marrow erythroid cell response is identical to that seen in animals in whom Ep levels are markedly reduced, such as that found in exhypoxic polycythemia, and suggest a decrease in the hormone following endotoxin administration. Additional studies demonstrated that when Ep was injected into mice 6 hr after endotoxin administration, an increase in femoral erythroid colony-forming units (CFU-E), proerythroblast number, and 59 Fe incorporation into femoral marrow cells could be demonstrated. These findings, together with the marrow erythroid cell response to the hormone, suggest that the mechanism for suppression of erythropoiesis after endotoxin injection is a reduction in the level of circulating Ep.     

Lnk inhibits erythropoiesis and Epo-dependent JAK2 activation and downstream signaling pathways

Erythropoietin (Epo), along with its receptor EpoR, is the principal regulator of red cell development. Upon Epo addition, the EpoR signaling through the Janus kinase 2 (JAK2) activates multiple pathways including Stat5, phosphoinositide-3 kinase (PI-3K)/Akt, and p42/44 mitogen-activated protein kinase (MAPK). The adaptor protein Lnk is implicated in cytokine receptor signaling. Here, we showed that Lnk-deficient mice have elevated numbers of erythroid progenitors, and that splenic erythroid colony-forming unit (CFU-e) progenitors are hypersensitive to Epo. Lnk(-/-) mice also exhibit superior recovery after erythropoietic stress. In addition, Lnk deficiency resulted in enhanced Epo-induced signaling pathways in splenic erythroid progenitors. Conversely, Lnk overexpression inhibits Epo-induced cell growth in 32D/EpoR cells. In primary culture of fetal liver cells, Lnk overexpression inhibits Epo-dependent erythroblast differentiation and induces apoptosis. Lnk blocks 3 major signaling pathways, Stat5, Akt, and MAPK, induced by Epo in primary erythroblasts. In addition, the Lnk Src homology 2 (SH2) domain is essential for its inhibitory function, whereas the conserved tyrosine near the C-terminus and the pleckstrin homology (PH) domain of Lnk are not critical. Furthermore, wild-type Lnk, but not the Lnk SH2 mutant, becomes tyrosine-phosphorylated following Epo administration and inhibits EpoR phosphorylation and JAK2 activation. Hence, Lnk, through its SH2 domain, negatively modulates EpoR signaling by attenuating JAK2 activation, and regulates Epo-mediated erythropoiesis.     

Influence of the tumour promoter TPA upon erythroid burst formation in vitro

A dose dependent effect of the tumour promoter TPA on burst formation by rabbit erythroid progenitors (BFU-e) was demonstrated in cultures deficient in the early erythroid regulator burst-promoting activity (BPA). In these culture conditions the burst number was highest (193% of controls) at 10(-9)M TPA and concentrations higher than 3 x 10(-9)M TPA were inhibitory. The degree of burst enhancement by TPA and bone marrow conditioned medium as a source of BPA was similar. The addition of optimal concentrations of both TPA and BPA simultaneously to cultures resulted in no further increase in burst number. Short-term incubation of bone marrow cells with TPA failed to enhance the percentage of S-phase BFU-e under conditions in which BPA significantly increases the number of BFU-e in the cell cycle. These results indicate that the same population of BFU-e responds to TPA and BPA, but TPA does not mimic the mitogenic effect of BPA upon BFU-e.     

In vitro erythropoiesis. II. Cytochemical enumeration of erythroid stem cells (CFU-e and BFU-e) from normal mouse and human hematopoietic tissues.

A procedure which provides more accuracy in the identification and scoring of in vitro derived erythroid colonies (i.e. BFU-e and CFU-e) from normal mouse and human marrow and spleen cells has been developed. Up to now identification of erythroid colonies has been based on staining with acidified benzidine which consistently displays "background staining" of granulocyte/macrophage colonies. The staining reaction with these reagents is such that identification of erythroid colonies is less than precise. The modified benzidine procedure described herein eliminates the staining reactivity of granulocyte/macrophage colonies and specifically reacts with only those cells that contain hemoglobin. Furthermore, individual colonies and/or cells may readily be counterstained and examined by high resolution light microscopy.     

Prolactin exerts hematopoietic growth-promoting effects in vivo and partially counteracts myelosuppression by azidothymidine.

Prolactin (PRL) is a neuroendocrine hormone that influences immune and hematopoietic development. The mechanism of action of this hormone in vivo remains unclear; therefore, we assessed the effects of PRL on hematopoiesis in vivo and in vitro. Normal resting mice were treated with 0, 1, 10, or 100 microg of recombinant human prolactin (rhPRL) for 4 consecutive days and euthanized on the fifth day for analysis of myeloid and erythroid progenitors in the bone marrow and spleen. Both frequencies and absolute numbers of splenic colony-forming unit granulocyte-macrophage (CFU-GM) and burst-forming unit-erythroid (BFU-e) were significantly increased in mice receiving rhPRL compared to the controls that had received saline only. Bone marrow cellularities were not significantly affected by any dose of rhPRL, but the absolute numbers and frequencies of bone marrow CFU-GM and BFU-e were augmented by rhPRL. These results suggest that rhPRL can promote hematopoiesis in vivo. Because rhPRL augments myeloid development in vivo, we examined the potential of the hormone to reverse the anemia and myelosuppression induced by azidothymidine (AZT). Mice were given rhPRL injections concurrent with 2.5 mg/mL AZT in drinking water. rhPRL partially restored hematocrits in the animals after 2 weeks of treatment and increased CFU-GM and BFU-e in both spleens and bone marrow. The experiments with AZT and rhPRL support the conclusion that the hormone increases myeloid and erythroid progenitor numbers in vivo, and they suggest that the hormone is clinically useful in reversing myelosuppression induced by AZT or other myeloablative therapies.     

Binding of iodinated erythropoietin to rat bone marrow cells under normal and anemic conditions

Specific binding sites for erythropoietin (Epo) were shown in normal and anemic rat bone marrow cells using [125I]labeled human recombinant Epo. When rats were treated once or several times with phenylhydrazine or malotilate, or by phlebotomy, the serum Epo level determined by RIA began to increase rapidly. Thereafter, both the number of erythroid colony-forming unit (CFU-E)-derived colonies and the Epo binding capacity of bone marrow cells increased almost simultaneously in response to induced anemic states, suggesting that the amount of Epo binding in bone marrow cells may reflect in vivo erythropoiesis. Scatchard analysis of the binding data from normal rats revealed the presence of a single class of binding sites (Kd = 0.18 +/- 0.04 nM, 38 +/- 5 sites/cell). In anemic states, the apparent average receptor number per cell increased (52-62 sites/cell) without changing in binding affinity toward Epo. Furthermore, [125I]Epo was cross-linked to the cell surface molecule of approximately 165 kd in nonreducing conditions and 75 kd in reducing conditions. Autoradiographic analysis indicated that Epo receptors were distributed on immature erythroid cells. Proerythroblasts were the most heavily labeled, whereas orthochromatic erythroblasts and cells of myeloid and lymphoid lineages were not labeled. Calculations based on Scatchard and autoradiographic analysis showed that proerythroblasts have 390 receptor sites per cell, twice as many as basophilic or polychromatophilic erythroblasts have. These results are consistent with the stage-specific action of Epo in physiological differentiation of erythroid cells.