Combined stimulation with the T helper cell type 2 cytokines interleukin (IL)-4 and IL-10 induces mouse mast cell apoptosis.

Mast cells are found in connective and mucosal tissues throughout the body. Their activation via immunoglobulin E (IgE)-antigen interactions is promoted by T helper cell type 2 (Th2) cytokines and leads to the sequelae of allergic disease. We now report a mechanism by which Th2 cytokines can regulate mast cell survival. Specifically, we find that interleukin (IL)-4 and IL-10 induce apoptosis in IL-3-dependent bone marrow-derived mast cells and peritoneal mast cells. This process required 6 d of costimulation with IL-3, IL-4, and IL-10, and expression of signal transducer and activator of transcription 6 (Stat6). Apoptosis was coupled with decreased expression of bcl-x(L) and bcl-2. While this process occurred independent of the Fas pathway, culture in IL-3+IL-4+IL-10 greatly sensitized mast cells to Fas-mediated death. Additionally, we found that IgE cross-linkage or stimulation with stem cell factor enhanced the apoptotic abilities of IL-4 and IL-10. Finally, IL-3-independent mastocytomas and mast cell lines were resistant to apoptosis induced by IL-3+IL-4+IL-10. These data offer evidence of Th2 cytokine-mediated homeostasis whereby these cytokines both elicit and limit allergic responses. Dysregulation of this pathway may play a role in allergic disease and mast cell tumor survival.

JNK and p38 are activated by erythropoietin (EPO) but are not induced in apoptosis following EPO withdrawal in EPO-dependent HCD57 cells.

Jun N-terminal kinase (JNK) and p38, members of the mitogen-activated protein kinase family of serine/threonine kinases, are activated as a result of cellular stress but may also play a role in growth factor-induced proliferation and/or survival or differentiation of many cells. A recent report has implicated JNK and p38 in the induction of apoptosis in the erythropoietin (EPO)-dependent erythroid cell line HCD57 following EPO withdrawal, whereas our previously reported data did not support a role for JNK in growth factor withdrawal-induced apoptosis in HCD57 cells. Therefore, further testing was done to see if JNK was activated in EPO withdrawal-induced apoptosis; the study was extended to p38 and characterized the effect of EPO on JNK and p38 activities. Treatment of HCD57 cells with EPO resulted in a gradual and sustained activation of both JNK and p38 activity; these activities decreased on EPO withdrawal. Transient activation of p42/p44 extracellular signal-related kinases (ERK) was also detected. Inhibition of ERK activity inhibited proliferation in EPO-treated cells but neither induced apoptosis nor activated JNK. Inhibition of p38 activity inhibited proliferation but did not protect HCD57 cells from apoptosis induced by EPO withdrawal. Treatment of HCD57 cells with tumor necrosis factor-alpha induced JNK activation but did not induce apoptosis. These results implicate JNK, p38, and ERK in EPO-induced proliferation and/or survival of erythroid cells but do not support a role for JNK or p38 in apoptosis induced by EPO withdrawal from erythroid cells.

Unraveling distinct intracellular signals that promote survival and proliferation: study of erythropoietin, stem cell factor, and constitutive signaling in leukemic cells.

This review summarizes selected recent studies of the intracellular signals that allow erythroid cells to survive and proliferate under the control of erythropoietin (EPO) and alteration in signals that contribute to EPO-independent survival and proliferation. The hypothesis explored is that the proliferation and survival signals are distinct and can be separately studied with the proper cell lines and growth factor stimulation. The anti- and pro-apoptotic proteins Bcl-XL and BAD are highly implicated in EPO-dependent survival of erythroid cells. Stat5 activity appears to be upstream of Bcl-XL expression such that pathologic, constitutive activation of Stat5 may be a common event in leukemic cells that become resistant to apoptosis by constitutive expression of Bcl-XL. Other signals apparently also control the expression of Bcl-XL, such as the expression of JunB which seem to be required to suppress Bcl-XL expression when EPO is withdrawn. Apoptosis may also be triggered by inactivation of Bcl-XL by BAD. Dephosphorylation of BAD as a result of withdrawal of survival factors converts prosurvival BAD to proapoptotic BAD. Phosphorylation of BAD at the serine 112 residue seems critical to promoting survival. Constitutive activation of a kinase that phosphorylates BAD serine 112 may, therefore, contribute to resistance to apoptosis in leukemic cells. We describe the resistance of erythroleukemic cells to apoptosis induced by EPO withdrawal apparently caused by constitutive BAD phosphorylation. The resistance to apoptosis in these cells is reversed by treatment with the PI3-kinase inhibitor, LY294002, suggesting that resistance to apoptosis in these cells likely results from constitutive P13-kinase that is an upstream activator of an S-112 BAD kinase. The MAP kinase cascade is apparently active in EPO-dependent and stem cell factor (SCF)-dependent proliferation but not survival. In addition, autocrine tumor necrosis factor-a! (TNF-alpha) may also be a proliferation factor not affecting survival. P13-kinase seems to be required for full EPO-dependent proliferation but is not required for EPO-dependent survival (but it can promote survival when activated).

Engagement of Gab1 and Gab2 in erythropoietin signaling.

Several signaling cascades are activated during engagement of the erythropoietin receptor to mediate the biological effects of erythropoietin. The members of the insulin receptor substrate (IRS) family of proteins play a central role in signaling for various growth factor receptors and cytokines by acting as docking proteins for the SH2 domains of signaling elements, linking cytokine receptors to diverse downstream pathways. In the present study we provide evidence that the recently cloned IRS-related proteins, Gab1 and Gab2, of the Gab family of proteins, are rapidly phosphorylated on tyrosine during erythropoietin treatment of erythropoietin-responsive cells and provide docking sites for the engagement of the SHP2 phosphatase and the p85 subunit of the phosphatidylinositol 3'-kinase. Furthermore, our data show that Gab1 is the primary IRS-related protein activated by erythropoietin in primary erythroid progenitor cells. In studies to identify the erythropoietin receptor domains required for activation of Gab proteins, we found that tyrosines 425 and 367 in the cytoplasmic domain of the erythropoietin receptor are required for the phosphorylation of Gab2. Taken together, our data demonstrate that Gab proteins are engaged in erythropoietin signaling to mediate downstream activation of the SHP2 and phosphatidylinositol 3'-kinase pathways and possibly participate in the generation of the erythropoietin-induced mitogenic responses.

Protein kinase B (c-Akt), phosphatidylinositol 3-kinase, and STAT5 are activated by erythropoietin (EPO) in HCD57 erythroid cells but are constitutively active in an EPO-independent, apoptosis-resistant subclone (HCD57-SREI cells).

We found that erythropoietin (EPO) and stem cell factor (SCF) activated protein kinase B (PKB/Akt) in EPO-dependent HCD57 erythroid cells. To better understand signals controlling proliferation and viability, erythroid cells that resist apoptosis in the absence of EPO were subcloned and characterized (HCD57-SREI cells). Constitutive activations of PKB/Akt, STAT5a, and STAT5b were noted in these EPO-independent cells. PI3-kinase activity was an upstream activator of PKB/Akt because the PI3-kinase inhibitor LY294002 blocked both constitutive PKB/Akt and factor-dependent PKB/Akt activity. The LY294002 study showed that proliferation and viability of both HCD57-SREI and HCD57 cells correlated with the activity of PKB/Akt; however, PKB/Akt activity alone did not protect these cells from apoptosis. Treatment of HCD57 cells with SCF also activated PKB/Akt, but did not protect from apoptosis. This result suggested that PKB/PI3-kinase activity is necessary but not sufficient to promote viability and/or proliferation. Constitutive STAT5 activity, activated through an unknown pathway not including JAK2 or EPOR, may act in concert with the constitutive PI3-kinase/PKB/Akt pathway to protect the EPO-independent HCD57-SREI cells from apoptosis and promote limited proliferation.

STAT6, NF-kappaB and C/EBP in CD23 expression and IgE production.

STAT6, NF-kappaB (p50) and C/EBPbeta transcription factors (TF) were examined with respect to CD23 regulation. Electrophoretic mobility shift assay (EMSA), competition and supershift analysis demonstrated that STAT6 binds the CD23a promoter but with a lower affinity than the consensus site. STAT6-/- mice were analyzed for CD23 levels and showed reduced expression after CD40 ligand trimer (CD40LT) stimulation. However, normal CD23 expression and even some IgE production was induced in STAT6-/- mice with CD40LT/IL-4. EMSA analysis indicated that the CD23a STAT site was bound by a protein in nuclear extracts from CD40+/-IL-4-stimulated STAT6-/-B cells. Western blot analysis of these nuclear extracts demonstrated the presence of STAT3 and STAT5, suggesting that these STATs can induce CD23 in this situation. Further supporting evidence was obtained by showing that IL-2 and IL-4 both synergize with CD40 in an identical manner for CD23 induction on STAT6-/- B cells. EMSA analysis of the two putative NF-kappaB sites confirmed binding to both, although one site bound with a higher affinity than the second. Analysis of p50-/-mice indicated that this subunit was not necessary for CD23 induction or CD40/IL-4-induced IgE production. Finally, no role for C/EBP was observed in CD23 induction by EMSA or by CD23 induction analysis in C/EBPbeta-/- mice, whereas the absence of C/EBP, did have an effect on IgE production and lipopolysaccharide-induced B cell proliferation. Based on these data, a model is presented which suggests that CD23 superinduction results from STAT and NF-kappaB interaction.

AP1 regulation of proliferation and initiation of apoptosis in erythropoietin-dependent erythroid cells.

The transcription factor AP1 has been implicated in the induction of apoptosis in cells in response to stress factors and growth factor withdrawal. We report here that AP1 is necessary for the induction of apoptosis following hormone withdrawal in the erythropoietin (EPO)-dependent erythroid cell line HCD57. AP1 DNA binding activity increased upon withdrawal of HCD57 cells from EPO. A dominant negative AP1 mutant rendered these cells resistant to apoptosis induced by EPO withdrawal and blocked the downregulation of Bcl-XL. JunB is a major binding protein in the AP1 complex observed upon EPO withdrawal; JunB but not c-Jun was present in the AP1 complex 3 h after EPO withdrawal in HCD57 cells, with a concurrent increase in junB message and protein. Furthermore, analysis of AP1 DNA binding activity in an apoptosis-resistant subclone of HCD57 revealed a lack of induction in AP1 DNA binding activity and no change in junB mRNA levels upon EPO withdrawal. In addition, we determined that c-Jun and AP1 activities correlated with EPO-induced proliferation and/or protection from apoptosis. AP1 DNA binding activity increased over the first 3 h following EPO stimulation of HCD57 cells, and suppression of AP1 activity partially inhibited EPO-induced proliferation. c-Jun but not JunB was present in the AP1 complex 3 h after EPO addition. These results implicate AP1 in the regulation of proliferation and survival of erythroid cells and suggest that different AP1 factors may play distinct roles in both triggering apoptosis (JunB) and protecting erythroid cells from apoptosis (c-Jun).

Polycythemia vera. V. Enhanced proliferation and phosphorylation due to vanadate are diminished in polycythemia vera erythroid progenitor cells: a possible defect of phosphatase activity in polycythemia vera.

Erythropoietin (EP) and stem cell factor (SCF) are essential growth factors for erythroid progenitor cell proliferation and differentiation in serum-free culture. It has been previously shown that burst-forming units-erythroid and colony-forming units-erythroid from patients with polycythemia vera (PV) have enhanced sensitivity to EP and SCF compared with normal erythroid progenitors, but little is known about the mechanism for this difference. In the present investigation, the effect of EP and SCF on protein tyrosine phosphorylation in day-8 normal and PV erythroid colony-forming cells, which give rise to colonies of 2-49 hemoglobinized cells, was studied. EP rapidly induced tyrosine phosphorylation of the EP receptor, whereas the most prominent phosphorylated protein induced by SCF was identified as the SCF receptor. No additional phosphorylated proteins were evident when PV cells were compared with normal cells. Culture of normal erythroid progenitors with orthovanadate, an inhibitor of protein tyrosine phosphatases, resulted in an increased number of erythroid colonies and enhanced protein tyrosine phosphorylation. However, in contrast, little enhancement was evident with PV cells. These results indicate that, although vanadate may be acting in normal erythroid progenitors as a phosphatase inhibitor that potentiates the kinase activity induced by SCF and EP, this function is diminished in PV cells. Because erythropoiesis is regulated by a balance between protein tyrosine kinase activity and protein tyrosine phosphatase activity, PV patients may have an abnormal phosphatase activity allowing increased cell proliferation.

Distinct signaling from stem cell factor and erythropoietin in HCD57 cells.

A recent report (Wu, H., Klingmuller, U., Besmer, P., and Lodish, H. F. (1995) Nature 377, 242-246) documents the interaction of the erythropoietin (EPO) receptor (EPOR) with the stem cell factor (SCF) receptor (c-KIT) and suggests that SCF acts through the EPOR. To elucidate the ability of SCF to affect the erythropoietin signaling pathway, we studied the effect of SCF on EPOR phosphorylation, SHC/ERK-1 activity, and cell proliferation and apoptosis in EPO-dependent HCD57 cells. Treatment of these cells with SCF resulted in phosphorylation of the EPOR. However, SCF-dependent phosphorylation of the EPOR did not initiate an EPO-like intracellular signal. SCF induced proliferation, SHC phosphorylation, and activation of ERK-1 but did not activate the JAK/STAT pathway. SCF stimulated SHC phosphorylation and ERK-1 activation independent of the EPOR in cells where the EPOR was down-regulated; the presence of the EPOR appeared to facilitate SCF activation of SHC and ERK-1. Furthermore, treatment of HCD57 cells with SCF increased cell number over a 3-day treatment, but apoptosis was observed in these cells. These data may illustrate two distinct pathways for erythroid cell proliferation and prevention of apoptosis in response to EPO, thereby providing a system to discriminate these intracellular signals.

Association of JAK2 and STAT5 with erythropoietin receptors. Role of receptor phosphorylation in erythropoietin signal transduction.

Cytokine receptors act at least partially by associating with Janus tyrosine protein kinases at the conserved box one motif of the receptor. These receptor-associated kinases then activate STAT transcription factors through phosphorylation. We found that the 78-kDa erythropoietin receptor (EPOR), a highly modified form of the 66-kDa receptor which is abundant in HCD57 cells, was phosphorylated on serine residues without EPO stimulation. Coprecipitation experiments showed the 78-kDa EPOR but not the more abundant 66-kDa EPOR was associated with JAK2, a Janus protein kinase, in both the presence and absence of EPO. Solubilized 78-kDa EPOR bound to purified, genetically engineered JAK2 better than the 62-76-kDa receptor proteins, and additional phosphorylation of tyrosine residues further increased the binding of the 78-kDa EPOR to JAK2-agarose beads. STAT5 DNA binding was activated by 10-100-fold lower concentrations of EPO in HCD57 cells than in primary erythroid cells, and STAT5 associated with the EPOR in an EPO-dependent manner. These data suggest that phosphorylation of either serine or tyrosine residues of the EPOR can enhance the association of the receptor with JAK2, possibly increasing the sensitivity to EPO.

Erythropoietin cell biology.

Erythropoietin (EPO) mediates its biologic actions in responsive cells through the 78 kDa cell surface form of the EPO receptor. The biosynthesis, activations, and destruction of EPO receptors are described. The binding of EPO to its receptor leads to the activation of a tyrosine protein kinase(s) that results in the phosphorylation of the receptor and other proteins.

Survival or death of individual proerythroblasts results from differing erythropoietin sensitivities: a mechanism for controlled rates of erythrocyte production.

Murine erythroid progenitors infected with the anemia-inducing strain of Friend virus (FVA cells) undergo apoptosis when deprived of erythropoietin (EPO). When cultured with EPO, they survive and complete terminal differentiation. Although cell volume is decreased and nuclear chromatin is condensed during both apoptosis and terminal differentiation, morphologic and biochemical distinctions between these two processes were observed. In apoptosis, homogeneous nuclear condensation with nuclear envelope loss occurred in cells that had not reached the stage of hemoglobin synthesis. In terminal erythroid differentiation, nuclear condensation with heterochromatin, euchromatin, and nuclear envelope preservation occurred simultaneously with hemoglobin synthesis. Cells with apoptotic morphology appeared asynchronously in EPO-deprived cultures, indicating that only a portion of the cells were undergoing apoptosis at any given time. The percentages of apoptotic cells and cleaved DNA increased with time in EPO-deprived cultures. Inhibition of DNA cleavage was directly proportional to EPO concentration over a wide physiologic range, demonstrating a heterogeneity in susceptibility to apoptosis based on variability in the EPO sensitivity of individual cells. A subpopulation of FVA cells with increased EPO sensitivity (decreased EPO requirement) was isolated from EPO-deprived cultures. This increased EPO sensitivity did not result from differences in EPO receptor number, affinity, or structure, suggesting that the differences are in the signal transduction pathway. These results indicate that control of red blood cell production involves both prevention of apoptosis by EPO and heterogeneity in the EPO requirement of individual progenitor cells.

The functional form of the erythropoietin receptor is a 78-kDa protein: correlation with cell surface expression, endocytosis, and phosphorylation.

An abundant 70- to 78-kDa form of the erythropoietin receptor (EPOR) was observed in HC-D57 murine erythroleukemia cells deprived of erythropoietin (EPO). In contrast to the 64- and 66-kDa EPOR proteins, these high molecular mass forms of EPOR (hmm-EPOR) correlated well with the number of binding sites and endocytosis of EPO. The hypothesis that hmm-EPOR are more highly glycosylated forms of the EPOR, appear on the cell surface, and represent at least one component of the biologically active EPOR was tested. Consistent findings were as follows. (i) Only hmm-EPOR increased following withdrawal of EPO from HC-D57 cells, correlating with a 10-fold increase in binding of 125I-labeled EPO. In addition, the EPO-dependent downregulation of 125I-EPO binding and disappearance of hmm-EPOR occurred in parallel while the amount of 66-kDa EPOR did not change. (ii) The 78-kDa EPOR was detected in COS cells expressing EPOR cDNA. (iii) Probing of the intact surface of these cells with anti-NH2-terminal antibody recovered only the 78-kDa EPOR. (iv) Enzymatic deglycosylation and dephosphorylation showed that hmm-EPOR apparently resulted from additional N-linked glycosylation of a 62-kDa EPOR. (v) The hmm-EPOR turnover in HC-D57 cells was accelerated 12-fold in the presence of EPO (half-life changed from 3 hr to 15 min). (vi) Anti-phosphotyrosine antiserum detected an EPO-dependent phosphorylation of the 78-kDa EPOR. The kinetics of tyrosine phosphorylation of a 97-kDa protein correlated with the occupancy and internalization of hmm-EPOR. In summary, we suggest that the 78-kDa EPOR is directly involved in the initial biological actions of EPO.

Metabolic adaptation during erythropoietin-mediated terminal differentiation of mouse erythroid cells.

Metabolic development was examined in erythroid precursor cells, which were isolated from the spleens of mice infected with the anemia-inducing strain of Friend virus (FVA cells). FVA cells undergo differentiation in vitro from the proerythroblast stage through the reticulocyte stage over a 48-hour period in the presence of erythropoietin. Concomitant with marked decreases in cellular size and energy demand, metabolic capacities of both glycolysis and oxygen consumption diminish after 48 hours in culture by 7- and 18-fold, respectively. Because the oxidative capacity decreases more than glycolytic ability does, the metabolic machinery increasingly shifts toward anaerobic metabolism. During the 48-hour period of differentiation, the 2,3-diphosphoglyceric acid (DPG) content per cell and 2,3-DPG mutase activity per cell increased eightfold and threefold, respectively. Freshly harvested FVA cells have adenosine triphosphate (ATP) levels of 7.23 +/- 2.52 mumol/10(10) cells or 3.76 +/- 1.31 mumol/mL cell water which are 12- or 2.3-fold higher, respectively, than the ATP levels of mature red blood cells. In the course of FVA cell differentiation, ATP content per cell decreases by fourfold, but ATP concentration in cell water remains unchanged because of a corresponding decrease in cellular size and water content during differentiation. These studies show that in the face of dramatic decreases in cell size and cellular energy demand, terminally differentiating erythroid cells maintain a constant ATP level by undergoing an involution of their glycolytic machinery as well as by losing their aerobic metabolic capacity.

Friend spleen focus-forming virus induces factor independence in an erythropoietin-dependent erythroleukemia cell line.

Erythroid cells from mice infected with the polycythemia-inducing strain of Friend spleen focus-forming virus (SFFVP), unlike normal erythroid cells, can proliferate and differentiate in apparent absence of the erythroid hormone erythropoietin (Epo). The unique envelope glycoprotein encoded by SFFV has been shown to be responsible for this biological effect. The recent isolation of an Epo-dependent erythroleukemia cell line, HCD-57, derived from a mouse infected at birth with Friend murine leukemia virus, afforded us the opportunity to study the direct effect of SFFVP on a homogeneous population of factor-dependent cells. The introduction of SFFVP in complex with various helper viruses into these Epo-dependent cells efficiently and reproducibly gave rise to lines which expressed high levels of SFFV and were factor independent. SFFV appears to be unique in its ability to abrogate the factor dependence of Epo-dependent HCD-57 cells, since infection of these cells with retroviruses carrying a variety of different oncogenes had no effect. The induction of Epo independence by SFFV does not appear to involve a classical autocrine mechanism, since there is no evidence that the factor-independent cells synthesize or secrete Epo or depend on it for their growth. However, the SFFV-infected, factor-independent cells had significantly fewer receptors available for binding Epo than their factor-dependent counterparts had, raising the possibility that the induction of factor independence by the virus may be due to the interaction of an SFFV-encoded protein with the Epo receptor.

Erythropoietin receptors in polycythemia vera.

The role of erythropoietin (EP) in polycythemia vera (PV) is controversial, with some experiments suggesting that erythroid progenitors in PV are exquisitely sensitive to EP and EP dependent, and others suggesting that PV progenitors are EP independent. We have examined the characteristics of the EP receptor (EP-R) on erythroid colony-forming cells (ECFC) from patients with PV. In contrast to normal ECFC, which have two classes of EP-R, with 20% showing high affinity (Kd = 0.13 nM; range, 0.04-0.20 nM) and the remainder lower affinity (Kd = 0.37 nM; range, 0.28-0.57 nM), PV ECFC show a single class of 851 low affinity EP-R with Kd = 0.72 nM (range, 0.36-0.85 nM). ECFC from patients with secondary (EP driven) polycythemia or anemia show two classes of EP-R (Kd = 0.18 and 1.10 nM, respectively). Attempts to remove tightly bound EP from putative high affinity EP-R in PV did not reveal any higher affinity receptors. Determination of molecular size by crosslinking showed two proteins of 90 and 100 kD similar to those seen with normal EP-R. These studies indicate the PV ECFC have EP-R that are structurally similar to normal EP-R but lack the higher binding affinity for EP.

Receptors for erythropoietin in mouse and human erythroid cells and placenta.

High and lower affinity receptors for erythropoietin (EP) were initially identified on a very pure population of EP-responsive erythroblasts obtained from the spleens of mice infected with anemia strain of Friend virus (FVA). The structure of the receptor for EP in these cells was determined to be proteins of 100 and 85 Kd by cross-linking 125I-EP. In this investigation, studies on the receptors for EP were extended to other mouse erythroid cells and human erythroid cells as well as to the placentas of mice and rats. Only lower affinity receptors for EP were detected on erythroblasts purified from the spleens of mice infected with the polycythemia strain of Friend virus and a murine erythroleukemia cell line, both of which are not responsive to EP in culture. Internalization of 125I-EP was observed in both groups of cells. The structure of the receptor determined by cross-linking 125I-EP was two equally labeled proteins of 100 Kd and 85 Kd molecular mass in all these mouse erythroid cells. The structure of the receptor was found to be very similar in human erythroid colony forming cells cultured from normal blood. These cells respond to EP with erythroid maturation and were previously shown to have high and lower affinity receptors. Placentas from mice and rats were found to have only lower affinity receptors for EP, and when placental membranes were cross-linked to 125I-EP, the same 100 Kd and 85 Kd bands were found as seen in mouse and human erythroid cells. The structure of the receptor was similar in cells that have high affinity receptors (FVA-infected and human erythroid colony-forming cells) and nonresponsive erythroid cells and placenta that have lower affinity receptors, but only the cells with the high affinity receptors respond to the addition of EP with erythroid maturation.

Human colony-forming units-erythroid do not require accessory cells, but do require direct interaction with insulin-like growth factor I and/or insulin for erythroid development.

The presence of heterogeneous erythroid progenitor cells, contaminant cells, or serum may alter erythroid colony development in vitro. To obtain highly purified colony-forming units-erythroid (CFU-E), we cultured partially purified human blood burst-forming units-erythroid (BFU-E) in methylcellulose with recombinant human erythropoietin (rHuEPO) for 7 d and generated cells that consisted of 30-60% CFU-E, but no BFU-E. A serum-free medium was used that allowed development of the same number of erythroid colonies as serum containing medium, but with a greater percentage of larger colonies. This medium consisted of delipidated crystalline bovine serum albumin, iron saturated transferrin, lipid suspension, fibrinogen, thrombin, Iscove's modified Dulbecco's medium/F-12[HAM], and insulin plus rHuEPO. When CFU-E were cultured in a limiting dilution assay and the percentage of nonresponder wells was plotted against cell concentration, both serum-free cultures and serum-containing cultures yielded overlapping straight lines through the origin indicating that CFU-E development did not depend on accessory cells and that insulin acted directly on the CFU-E. Human recombinant interleukin 3 (IL-3) and/or granulocyte-macrophage colony-stimulating factor had no effect on CFU-E growth, while they markedly enhanced BFU-E growth. Physiological concentrations of recombinant human insulin-like growth factor I (IGF-I) enhanced CFU-E growth in the absence of insulin and, together with rHuEPO in serum-free medium, provided a plating efficiency equal to that of serum-containing medium. Limiting dilution analysis in serum-free medium with IGF-I showed a straight line through the origin indicating that IGF-I also acted directly on the CFU-E and not through an effect on accessory cells. These data demonstrate that CFU-E do not require accessory cells, but do require IGF-I and/or insulin which act directly on the CFU-E.