cells Erythropoietin receptor characteristics on ...

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1991 77: 2583-2590

Erythropoietin receptor characteristics on primary human erythroid cells VC Broudy, N Lin, M Brice, B Nakamoto and T Papayannopoulou

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Erythropoietin Receptor Characteristics on Primary Human Erythroid Cells By Virginia C. Broudy, Nancy Lin, Martha Brice, Betty Nakamoto, and Thalia Papayannopoulou Erythropoietin (EP) exerts its effects on erythropoiesis by binding to a cell surface receptor. We examined EP receptor expression during normal human erythroid differentiation and maturation from the burst-forming unit-erythroid (BFU-E) to the reticulocyte level. In contrast t o previous studies, we assessed EP receptor number and affinity in erythroid precursors immunologically purified from fresh bone marrow aspirates or fetal liver samples and in reticulocytes purified from peripheral blood. EP receptors were quantitated by equilibrium binding experiments with ‘=IEP. We found that purified primary erythroblasts from both adult and fetal sources exhibited a single high-affinity (kd 100 pmol/L) binding site for EP under our experimental conditions, and 135 or 250 receptors per cell, respectively. Reticulocyteswere devoid of EP receptors. We compared these data t o in vitro-derived BFU-E progeny at both early and late stages of maturation. Cultured BFU-E progeny also displayed a single class of

receptors of slightly lower affinity (210 to 220 pmol/L). Preparations enriched in colony-forming units-erythroid (CFU-E) and proerythroblasts (day 9 BFU-E progeny) displayed approximately 1,100 receptors per cell, whereas populations containing mature erythroblasts (day 14 BFU-E progeny) exhibited approximately 300 receptors per cell. Furthermore, information from binding experiments was complemented by autoradiography in both enriched BFU-E preparations. cultured BFU-E progeny (days 9 and 14). and marrow mononuclearcells. These studies are consistent with a peak in EP receptor expression at the CFU-E/proerythroblast stage and a decrease with further maturation to undetectable levels at the reticulocyte stage. These data exarnining EP receptor characteristics on freshly isolated erythroid precursor cells complement previous data on EP receptor biology using cutture-derived erythroblasts. 0 1991by The American Society of Hematology.

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samples were obtained in the Clinical Research Center at the University of Washington (Seattle). Human fetal liver cells (gestational age 55 to 80 days) were obtained from therapeutic abortions. Consent was obtained for the use of these tissues, and these studies were approved by the Institutional Review Board at the University of Washington. Purified populations of normal human erythroid precursor cells were isolated from adult marrow bufFy coat cells or from fetal liver cells by direct immune adherence with monoclonal antibody (MoAb) EP-1.” The antigen recognized by EP-1 is preferentially expressed on late erythroid progenitor cells and erythroid precursor cells.” The entire immune adherence procedure was complete within 2 hours after obtaining the marrow aspirate or fetal liver sample. Isolation of reticulocytes. Reticulocytes were isolated from the peripheral blood of a normal adult and a patient with sickle cell disease by Percoll density centrifugation (Sigma, St Louis, MO).I9 Brilliant cresyl blue staining demonstrated that the cell preparations varied between 20% (normal donor) and 90% (sickle cell patient) reticulocytes. Isolation of BFU-E progeny. Peripheral blood (50 to 100 mL) was obtained from healthy adult volunteers, and mononuclear cells were isolated by Ficoll-Hypaque density centrifugation (Organon Teknika Corp, Durham, NC). The mononuclear cells were cultured at a density of 5 x 105/mLin Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 30% heat-inactivated fetal calf serum (FCS; Hyclone, Logan, UT), 1% deionized bovine serum albumin (BSA, Reheis, Phoenix, AZ),1.5 U/mL recombinant human EP, and 2 U/mL purified recombinant human interleu-

RYTHROPOIETIN (EP) plays a central role for the survival, proliferation, and maturation of erythroid cells.’ The biologic effects of E P are mediated through binding to a specific cell surface receptor. Understanding the cellular distribution and binding characteristics of E P receptors during normal erythroid differentiation may provide insights into the mechanisms of EP action, and the pathophysiology of familial or acquired erythrocytosis. E P receptor expression in human cells has been examined using human erythroid cell lines2 or culture-derived erythroid precursor^.'^^ Although these populations have advantages in terms of numbers of cells available and purity, they are either generated under high E P stimulus (culture-derived erythroblasts) or they are nonresponsive to EP (many erythroleukemia lines) and therefore may not reflect the receptor characteristics present in bone marrow erythroblast^.^^'" The binding parameters of the E P receptor on normal human or murine cells remains controversial, with reports of one4*”l5 or classes of binding sites. We purified fresh erythroid precursor cells from bone marrow aspirates or fetal liver by immunologic means and quantitated E P receptors on these cells. We also performed parallel experiments with culture-derived erythroblasts. Our results show that primary erythroblasts purified from adult marrow exhibit a single class of high-affinity EP receptors (kd 100 pmol/L) with approximately 135 receptors per cell. Cultured cells (burst-forming unit-erythroid [BFU-E] progeny) display a single class of binding sites of slightly lower affinity (kd 220 pmol/L), possibly due to exposure to high concentrations of E P in vitro. Preparations enriched in colony-forming unit-erythroid (CFU-E)/ proerythroblasts exhibit approximately 1,100 receptors per cell, whereas preparations containing mainly late erythroblasts had approximately 300 receptors per cell, demonstrating a decrease in receptor expression with terminal erythroid maturation. MATERIALS AND METHODS

Isolation of erythmidprecursor cellsfrom bone marrow or fetal liver. Normal human bone marrow cells were aspirated from the posterior iliac crest of consenting healthy adult volunteers. The marrow Blood, Vol77, No 12 (June 15). 1991: pp 2583-2590

__ From the University of Washington, Seattle. Submitted July 17,1990; accepted February 7, 1991. Supported by National Institutes of Health Grant DIG1232 and an American Cancer Society Junior Faculty Research Award. Also to supported by a National Institutes of Health Grant Mol “037 the Clinical Research Center. Address reprint requests to Vi.gnia C. Broudy, MD, Division of Hematology, RM-IO, Department of Medicine, University of Washington, Seattle, WA 981 95. The publication costs of this article were defrayed in part by page chaTe payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 1991 by The American Society of Hematology. 0006-4971/9117712-0010$3.OOlO ~~

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kin-3 (IL-3; Genetics Institute, Cambridge MA), and made semisolid with 1.4% methylcellulose (Dow, Midland, MI). The EP used to culture cells was partially purified (6,000 Uimg) from the supernatants of BHK cells transfected with the human EP gene?O The plates were incubated in a humidified atmosphere containing 5% CO,. Erythroid colonies were identified by characteristic morphology under the inverted microscope. Under these conditions the plates contained less than 10% myeloid colonies. To obtain erythroid populations comprised mainly of proerythroblasts, individual colonies were plucked early (day 8 or -9 of culture). Mature, well-hemoglobinized bursts cultured in parallel from the same normal donor were plucked later (day 13 or 14 of culture). In some experiments, the mature bursts were plucked on day 12 and placed in suspension culture for 1 to 2 days to permit further maturation to occur. The suspension culture medium consisted of IMDM supplemented with 10% FCS, 1% BSA, 1.5 U/mL EP, and 1 U/mL IL-3. This approach yielded up to 15 x 106 cells from the day 9 colonies, and 30 X lo6 cells from the mature bursts. For each experiment, cytopreparations of cells were stained with benzidine" and were evaluated in conjunction with binding data. To determine whether CFU-E were present, cells plucked on day 8 were cultured in plasma clots. The cells (104imL) were cultured in IMDM supplemented with 30% FCS, 1% BSA, 10% beef embryo extract (GIBCO, Grand Island, NY), 1 U/mL EP, 1 UimL IL-3, and 10% bovine citrated plasma. After a 5-day incubation at 37%, the plasma clots were harvested, stained with benzidine, and erythroid colonies of t 8 cells were counted. IdentiFcution of equilibrium binding conditions. We examined the time course of '=I EP binding to normal marrow cells at 15°C. Because the rate of binding may be slower with lower concentrations of ligand,z2these experiments were performed with 60 pmol/L '=I EP. Plateau binding was achieved by a 6-hour incubation at 15°C; continuing the incubation up to 24 hours did not significantly increase the amount of cell-surface-associated EP. Internalization of EP at 15°C was evaluated by using glycine-HCI to strip the cell s ~ r f a c e . ' The ~ . ~ ~results of these experiments showed that 85% of the cell-associated EP was surface bound after a 6-hour incubation at 15°C. Quuntitution of EP receptors. EP receptors were measured by incubating cells with '"I EP (300 to 900 Ci/mmol; Amersham, Arlington Heights, IL) with or without a 100-fold molar excess unlabeled purified human recombinant EP (129,000 Uimg; AMGEN, Thousand Oaks, CA) in binding buffer consisting of RPMI supplemented with 1% BSA and 50 mmoliL HEPES (pH 7.4) as previously described? The concentration of I2'I EP varied from 16 pmoliL to 2.6 nmol/L. The cells were added to a premixed solution of binding buffer containing 12'1 EP with or without unlabeled EP at 4°C. The final concentration of cells was 0.2 to 1.5 X lo6 per 100 pL. The cell suspension was incubated for 6 hours at 15°C in a Dubnoff shaking incubator. At the conclusion of the incubation, the cell suspension was gently layered onto 150 ILLof phthalate oil at 4°C (di-n-butylphthalate:dinonylphthalate, 3:2) and centrifuged for 1minute in a Beckman Microfuge 11 operated in the cold room. The cell pellets were clipped off and I2'I was measured in both pellets and supernatants using a Packard 5330 gamma counter with an efficiency of 66%. The clonogenic potential of the cells at the conclusion of the equilibrium binding period was evaluated by culturing human peripheral blood mononuclear cells as described previously before and after the 6-hour incubation at 15°C. Erythroid colony growth was undiminished at the conclusion of the eq~iiibriumbinding period (70 colonies and 83 colonies per 3 X 10' cells plated before and after the 15°C incubation, respectively), and the viability of the cells was determined to be 100% by trypan blue exclusion. Erythroid precursor cells would be expected to have their cell

surface receptors partially occupied by E P bound in vivo or acquired during culture, interfering with quantitation of receptors. To circumvent this problem, the cells were incubated in EP-free medium for 90 minutes at 37°C to permit internalization and degradation of surface-bound EP, as previously described.'" Datu analysis. Equilibrium binding data were analyzed using the ligand program.24Additionally, the data were analyzed by the method of Lipkin et alZ as previously de~cribed.~ The two methods gave similar results, and binding parameters estimated by the ligand program are presented in this report. htoradiogruphy. The cellular distribution of EP receptors in normal human marrow and the expression of receptors during differentiation of peripheral blood BFU-E were examined by autoradiography. Marrow mononuclear cells were obtained by Ficoll-Hypaque density centrifugation. BFU-E were enriched from normal human peripheral blood nononuclear cells by negative selection with a cocktail of MoAbs, including immunoadherence and complement-dependent lysis followed by positive selection with MoAb My10 as described.26The enriched BFU-E were further cultured in IL-3 in suspension for 3 days before autoradiographic analysis of '''I EP binding. Under these conditions, we have previously determined that CFU-E first appear on day 4 in vitro:' consistent with prior results.28The percentage of BFU-E in the day 3 cells was determined by plasma clot assay. To examine EP receptor display during late erythroid maturation, peripheral blood mononuclear cells were cultured in EP plus IL-3 and individual erythroid colonies were plucked on day 9 or day 14. The cells were incubated with high specific activity ','I EP (3,000 Ciimmol; Amersham) for 1 hour at 37°C in the presence of 0.2% azide. The final concentration of ''I EP was 1 nmoliL. At the conclusion of the incubation, the cell suspension was gently layered on Percoll (density 1.028 g/mL) and centrifuged for 1 minute at 4°C in a Beckman Microfuge 11to separate cell-associated '=I EP from free EP. Cytopreparations were made in a Shandon Southem cytospin. The slides were processed as described" and exposed for 3 to 4 weeks. Specific binding was determined by performing grain counts on marrow mononuclear cells incubated with '=I EP with or without a 100-foldmolar excess of unlabeled EP; the results of both measurements are presented. Specific binding for the enriched BFU-E and for the day 9 and day 14 BFU-E progeny was determined by counting grains overlying the cells and in an area immediately adjacent to each cell; the grain count in the adjacent area was then subtracted. RESULTS

EP receptors on purified primary erythroid cells. Normal human erythroblasts were isolated from adult marrow or fetal liver by direct immune adherence with EP-1. Up to 40 x lo6 erythroid precursor cells would be isolated with EP-1 within 2 hours after the fetal liver or adult marrow specimen was obtained. These populations of cells were greater than 95% erythroid and consisted mainly of late erythroblasts (Fig 1). Although EP-1 recognizes antigenic determinants present on BFU-E as well as CFU-E and erythroblasts, less than 1% of the cells were CFU-E, and no BFU-E were identified by colony assay, suggesting that the EP-1 antigen must be expressed at low density on BFU-E. Equilibrium binding analysis of E P receptors on purified adult marrow or fetal liver erythroblasts showed a single class of high-affinity sites (Figs 1 and 2). In four experiments, fetal erythroblasts were found to have 250 35 receptors per cell with a kd of 100 f 35 pmol/L (mean f SEM). Primary adult marrow erythroblasts exhibited 135 2

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Flg 1. EP Mndlng to prim.~ nurrow erythroMasta. (A) Adult marrow huffy coat cells. (E) Erythroblasts Isolated from marrow atffv coat cells by direct Immune adherence with EP-1. (A and E) Benzidine stain with W r i g M C k m u counterstain, original magntfkation x40. (C) Primary marrow erythroblasts, 1.2 x lo', were incubated with 'IEP (2.3 pmol/L to 2.6 nmollL) with or without a 1oO.fold excess of unlabeled EP for 6 hours at 15°C. The average of duplicate points is shown.

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2586

BROUDY ET AL

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Erythropoietin Bound (pM) Flg 2 EP binding to fatal l i m erythroblats h o W dimt h m u m .dh.mnm wtlh EP-1. Primary fetal h e r erythroblasts, 0.8 x lV, were incubated with 'YEP (16 pmollL to 2.6 nmollL) for 6 houn at 15°C. The averageof hiplieate points is presented.

20 receptors pcr ccll, kd IO8 2 40 pmol/L (mcan 2 SEM of thrcc cxpcrimcnts). Rcticulocytcs did not have detcctable binding of '"I EP (two cxpcrimcnts). EP receptotx on in vitro RFU-E ptqeny. Pcriphcral blood mononuclcar cclls wcrc cultured in scmisolid mcdium supplcmcntcd with EP and IL-3, and erythroid colonics wcrc pluckcd on day 9 or day 14. Day 9 cclls wcrc largc palc carly crythroblasts (Fig 3). lmmunofluorcsccnt staining showcd that 100% of the day 9 cclls wcrc positivc with EP-I.'" Plasma clot assay showcd that 9.2% of day 8 cclls wcrc CFU-E. By day 14, thc population includcd smaller well-hcmoglobinizcd latc erythroblasts (Fig 3). Both populations of cclls cxhibitcd a singlc class of EP rcccptors (Fig 3). In this cxpcrimcnt. the carly crythroblasts displaycd 1.740 receptors pcr ccll with a kd of 180 pmolk, thc latc erythroblasts had 580 rcccptors pcr cell with a kd of 215 pmol/L. In two additional cxpcrimcnts. day 9 cclls cxhibitcd an avcragc of 800 rcccptors pcr ccll with a kd of 270 pmol/L. Thc day 14 cclls displaycd 235 2 50 rcccptors pcr ccll, kd Z10 2 5 pmol/L (mcan 2 SEM of thrcc additional cxpcrimcnts). A summary of EP rcccptor numbcrs and binding affinity in normal human cclls is presented in Tables 1 and 2.

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Flg 3. EP bindingto BN.Epl0g.n~. (A) Mphoml Mood B N - E were c u l t u d for 9 day. (left panel) 01 14 day. (right panel); Wright-Giemu stain, original magnification x90. (6) Day 9 cells ( 0 )or day 14 cells (0).0.3 x 1V. wem incubated with "1 EP with or without unlabeled EP for 6 houn at 15%. The overage

of duplicate determination8 ir shown.

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ERYTHROPOIETIN RECEPTORS ON HUMAN CELLS 20

Table 1. EP Receptors on Purified Primary Erythroblasts

18

Cells

Experiments

Receptors1 Cell

kd (pmollL)

Adult marrow erythroblasts Fetal liver erythroblasts

3 4

135 f 20 250 2 35

108 f 40 100 -+ 35

No. of

Cellular distribution of EP receptors. Autoradiographic analysis of '=I EP binding to bone marrow mononuclear cells showed that only erythroblasts and megakaryocytes display EP receptors. No specific binding of '''I E P to myeloid or lymphoid cells was detected. Grain counts showed a progressive decrease in the binding of E P in conjunction with erythroid maturation (Fig 4). Proerythroblasts displayed 15- to 20-fold more EP binding than orthochromatic erythroblasts. Peripheral blood preparations enriched for BFU-E also bound 1251EP (Fig 5). In this experiment, 45% of the cells (88 of 194 cells analyzed) were noted to have specific binding of Iz5IEP. When these cells were replated in plasma clots, there were 31 BFU-E per 100 cells cultured (average of duplicate clots). No CFU-E were observed. The slightly higher fraction of cells binding E P than percentage of BFU-E detected by colony assay could be due to a plating efficiency less than 100%. Culture-derived BFU-E progeny displayed specific binding of '''I EP (Fig 6). The day 9 cells exhibited an average of 3.5 grains per cell (grain count performed on 86 cells), whereas the day 14 cells displayed an average of 0.8 grains per cell (grain count performed on 156 cells). DISCUSSION

Prior studies of E P receptors on normal human cells have used in vitro generated erythroid cells. Because these cells were generated under nonphysiologic high levels of EP, they may not reflect the true number or affinity of E P receptors on native erythroid precursors. Therefore, we examined EP receptor expression in erythroid cells freshly purified from both adult and fetal sources. A single class of high-affinity binding sites was identified on all of the normal human erythroid precursor cells studied under the conditions we used (equilibrium binding at 15°C). This finding held true whether the data were analyzed by the ligand methodz4or by the method of Lipkin et al." Although a two-site model could be fitted to the data using the ligand program in approximately 30% of the experiments, the one site model was preferred whether the experiments were analyzed individually or pooled (F-test). We interpret these data to show that the high-affinity site predominates on normal human erythroid precursors. The binding affinity of the EP receptor was similar in adult and fetal erythroid cells (108 f 40 pmol/L and 100 f 35 pmol/L, respectively), despite the observation Table 2. EP Receptors on Cultured BFU-E Progeny ~

Cells

Day 9 erythroblasts Day 14 erythroblasts

~~

No. of Experiments

Receptorsl Cell

kd (pmollL)

3

1,100 f 350 300 100

210 f 30 220 2 10

4

16 -1 J

14

4

2

Fig 4. Autoradiographic analysis of lz51 EP binding to human marrow cells. (0).Binding in the presence of 1 nmol/L "51 EP; (MI, binding in the presence of 1 nmol/L '=I EP plus 100 nmol/L unlabeled EP. Grain counts were performed on 28 proerythroblasts, 35 basophilic erythroblasts, 63 polychromatophilic erythroblasts, and 104 orthochromatic erythroblasts.

that E P may play a unique role in fetal erythropoiesis. Fetal erythroid progenitor cells (BFU-E) can differentiate into CFU-E in the presence of EP alone in single-cell cultures under serum-free conditions?' In contrast, BFU-E from adults require both EP and IL-3 to undergo the same differentiation ~ t e p s . However, ~ , ~ ~ erythroblasts obtained by culturing BFU-E displayed a lower affinity E P receptor (kd 210 to 220 pmol/L). EP levels in normal adults are 20 to 40 mU/mL, whereas levels of E P used for cell culture are usually 1 to 2 U/mL, 50-fold higher. Exposure to a high E P level or a change in membrane composition during culture may result in the expression of a lower affinity E P receptor. Local alterations in membrane proteins may affect E P receptor-binding affinity?2 Boussios et a133 showed that receptor affinity may change with the developmental stage during yolk sac erythropoiesis. However, human embryonic cells have not been available for study, and this phenomenon has not yet been found during the late stages of fetal or adult erythroid maturation. E P receptor display is not a static property of the cell, but changes with cellular differentiation and maturation. The present report provides several pieces of evidence to support this concept. Fetal liver erythroblasts exhibited a higher number of receptors per cell than did adult marrow erythroblasts, reflecting the greater number of early erythroblasts among the fetal cells. Our data show that the final stages of normal erythroid maturation are accompanied by a decrease in E P receptor expression: equilibrium binding experiments showed that day 9 erythroblasts expressed threefold more receptors per cell than did day 14 erythroblasts, and reticulocytes were devoid of E P receptors. Autoradiographic analysis of '*'I E P binding to cultured BFU-E progeny showed that day 9 erythroblasts had fourfold more grains per cell than day 14 erythroblasts, confirming and strengthening the Scatchard analysis results. Autoradiography of '"I E P binding to marrow mononuclear cells verified the decrease in E P binding with terminal erythroid maturation, in accord with prior rep o r t ~ We . ~ demonstrate herein that a preparation of cells containing BFU-E without CFU-E27displays specific bind-

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2588

BROUDY ET AL

Fig 5. Autdiography of u( EP bindlng to entichod peripheral blood BFU-E. OrigInaI magnHk.tion x90.

A

B

Fig 6. Autdiography of '1EP binding to BFU-E Day 9 eqthroblnt.. (B) Day 14 aqthroblasts. Original magnification x90 in (A) and (B).

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ERYTHROPOIETIN RECEPTORS ON HUMAN CELLS

ing of EP, and a previous report showed that binding of E P continuously increases as BFU-E mature to CFU-E in vitro.” Taken together, these studies show that EP receptor expression peaks at the CFU-E/proerythroblast stage and decreases with terminal erythroid maturation. Autoradiography shows that megakaryocytes are rich in EP receptors, and quantitative studies indicate that megakaryocytes display 6,500 receptors per cell.” Although megakaryocytes clearly express E P receptors, the biologic significance of these receptors remains unclear.35The use of EP in humans has not been associated with clinically significant thrombocyt~sis.~~ Prior reports describe high-affinity and low-affinity sites

on erythroid precursor cells isolated from mice infected with the anemia strain of the Friend virus, enriched populations of murine CFU-E, and normal human erythroid However, other investigators have found one class of sites on normal murine or human erythroid cells.4J 1-1538 The conditions used to achieve equilibrium binding (prolonged incubation at 4°C v higher temperatures) may partially explain the discrepancy in results. We find predominantly the high-affinity E P receptor which, from a physiologic point of view, is probably the most relevant one. The function of the lower-affinity receptor in normal human erythropoiesis remains unclear at present.

REFERENCES

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