Influence of Recombinant Hematopoietins and of Fetal Bovine Serum on the Globin Synthetic Pattern of Human BFUe

By Anna Rita Migliaccio,* Giovanni Migliaccio, * Martha Brice, Pantelis Constantoulakis, George Stamatoyannopoulos, and Thalia Papayannopoulou

We have studied the effects of recombinant hematopoietic growth factors, granulocyte-macrophage colony-stimulat- ing factor (GM-CSF) and/or interleukin-3 (IL-3) on the globin program of adult human erythroid progenitors (BFUe) stimulated to terminal differentiation by erythropoietin under fetal bovine serum (FBSI-supplemented or FBS- deprived culture conditions. Fetal globin production by BFUe-derived erythroblasts was assessed at the protein and mRNA level and its cellular distribution was evaluated by immunofluorescence. Although hemoglobinization and maturation of BFUe-derived erythroblasts was by and large comparable in FBS-replete versus FBS-deprived cultures, the latter had significantly less (up to 20-fold) y-globin and y-globin mRNA levels. Reduced y-globin in serum-deprived cultures was also reflected by a smaller proportion of erythroblasts with detectable y-globin by immunofluores-

EVERAL PREVIOUS studies have shown that fetal S hemoglobin (Hb F) is activated during the in vitro terminal erythroid differentiation of normal erythroid progen- itors (BFUe, CFUe), so that levels produced in vitro are much higher compared with in vivo ones from the same individual.'-' This in vitro enhancement of H b F synthesis has been attributed to an intrinsic potential of adult hematopoie- tic progenitors, manifested under a permissive in vitro environment. Several parameters have been found to influ- ence the final levels of H b F synthesized in vitro: the maturation stage of erythroid cells a t the time of testing (partially v completely hem~globinized~.~); the time in cul- ture required for full development of a BFUe (early v late maturing BFUe)7; and factors present within the culture medium components or generated by accessory cells in c ~ l t u r e . ~ . ' ~ The above variables have been shown to either increase or decrease the proportion of H b F synthesized. The best example of a negative influence is represented by factor(s) present in fetal sheep serum or plasma that sup- presses the in vitro activation of H b F synthesized by adult normal or hereditary persistence of fetal hemoglobin (HPFH) erythroid progenitor cell^.^,'^ In contrast, factors present in most batches of fetal bovine serum (FBS) and removed by charcoal treatment seem to exert a positive influence on H b F in In addition to factors present in serum, putative cytokines produced by accessory cells in vitro seem to

From the Divisions of Hematology and Medical Genetics. Depart- ment of Medicine. University of Washington, Seattle, WA.

Submitted November 9. 1989; accepted May 29, 1990. *Present address: New York Blood Center, New York, NY 10021. Address reprint requests to Thalia Papayannopoulou, MD.

DrSci, Division of Hematology, RM-IO. University of Washington. Seattle, WA 981 95.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact.

0 1990 by The American Society of Hematology. 0006-4971/90/7606-0018$3.00/0

cence. Erythroid bursts induced by either GM-CSF or IL-3 produced similar levels of y-globin both in FBS-supple- mented and in FBS-deprived cultures. These results, ob- tained even in cultures of highly enriched BFUe, suggest that GM-CSF and IL-3, although they significantly increase the number and size of erythroid bursts, do not by them- selves exert a direct influence on the level of fetal globin synthesis. By contrast, factor(s1 present in FBS appear to exert a dominant influence on fetal globin synthesis in vitro. Although FBS-deprived conditions appear to largely abrogate the in vitro activation of fetal hemoglobin (Hb F) in normal samples, they do support increased Hb F production in samples from patients with hereditary persistence of fetal hemoglobin or from cord blood. 0 1990 by The American Society of Hematology.

enhance Hb F ~ynthesis.""~ Since these products of acces- sory cells or cell lines are, in addition, capable of potentiating the growth of erythropoietic progenitors in vitro and since many of them have been molecularly cloned and are avail- able in pure form, one has the opportunity to test whether these defined factors directly modulate fetal globin synthesis in vitro. Furthermore, since it is known from previous studies that the effects of recombinant growth factors on erythroid development may differ in serum-containing versus serum- deprived c ~ l t u r e s , l ~ ~ ' ~ their influence may vary depending on culture conditions. We have designed experiments to study the direct influence of the recombinant cytokines granulocyte- macrophage colony-stimulating factor (GM-CSF) and inter- leukin-3 (IL-3) on Hb F in vitro, both in serum-containing and serum-deprived cultures. We have found that the final high levels of H b F synthesized in vitro are primarily dependent on the presence of FBS, since in serum-deprived cultures activation of H b F was suppressed. In these experi- ments, recombinant cytokines, IL-3 and GM-CSF, alone and in combination, failed to show a direct effect on in vitro H b F synthesis.

MATERIALS AND METHODS

Cell Preparation Marrow and peripheral blood specimens were obtained from

normal volunteers with informed consent through a protocol ap- proved by the institution's Human Subjects Review Committee. The marrow was aspirated from the posterior iliac crests into heparinized syringes and diluted 1:l with IMDM (Gibco, Grand Island, NY). The light density cell fraction (d = 1.077) of marrow and peripheral blood was separated by Ficoll (Organon Teknika Co, Durham, NC) gradient centrifugation (400g for 20 minutes). Adherent cells were removed by a double 1-hour adherence incubation to plastic flasks containing lo6 cells/mL of IMDM supplemented with 10% (vol/vol) FBS (Hyclone, Logan, UT) at 3 7 T in 5% CO, in air. T cells were removed by a modification" of the soybean agglutinin method of Reisner et aI.l9 In one experiment from normal peripheral blood, highly enriched BFUe fractions were prepared. For this purpose peripheral blood mononuclear cells (PBMC) were first labeled with monoclonal antibodies (MoAbs) CamPath 1 ,20 58.8;' 5F1 ,22 react-

1150 Blood, Vol 76, No 6 (September 15). 1990: pp 1150-1 157

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CYTOKINES AND HBF IN SERUM-FREE CULTURES 1151

ing with lymphocytes, granulocytes, and/or monocytes. Following labeling, the cells were “panned” on anti-mouse IgG-coated plates. Nonadherent cells were incubated overnight in 20% FBS-containing medium and subsequently labeled with MoAb My-10 (CD34) and “panned” on plates coated with anti-mouse IgG. Adherent cells were flushed from plates and used for cultures.

Hematopoietic Growth Factors The purified recombinant human hematopoietic growth factors

used in these experiments included erythropoietin (Epo) (1.5 U/ mL = 6 x lo-’’ m ~ l / L ) ’ ~ and GM-CSFZ4 (100 U/mL = 4.5 x IO-’’ mol/L). Conditioned medium from COS-1 cells transfected with a plasmid designed for expression of human IL-3 cDNAZ5 (from Genetics Institute, Cambridge, MA) was used as the source of IL-3. The concentration of IL-3 was lo4 U/mL of conditioned medium and the specific activity was IO4 U/mg of protein.I7 IL-3 was used at a concentration of 2 x lo-’’ mol/L. The concentrations of E p , GM-CSF, and IL-3 used have been previously shown to induce maximal growth of erythroid bursts in both FBS-supplemented and FBS-deprived culture conditions.” Phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM; 5%, vol/vol), prepared serum free,” was used as a source of GM-CSF and IL-3 in some FBS-supplemented cultures.

Cell Culture Light density mononuclear bone marrow or peripheral blood cells,

prepared as above, were inoculated at 3 x IO4 to 3 x 10’ cells per plate. Highly enriched progenitors prepared with additional panning steps as described above were inoculated at 1,000 cells per plate. Cells were cultured in duplicate 35-mm plates either in FBS- deprived or in FBS-supplemented semisolid media. Each I-mL dish contained the following components in IMDM: methylcellulose (0.8% wt/vol, final concentration); j3-mercaptoethanol (7.5 x mol/L); antibiotics (100 U of penicillin, 250 ng of amphotericin B, and 100 pg of streptomycin); and either heat-inactivated FBS (40%, vol/vol) or a mixture of nutrients including deionized bovine serum albumin (BSA) (2 x mol/L), BSA-absorbed cholesterol (4 pg/mL), soybean lecithin (12 pg/mL), iron-saturated human trans- ferrin (9 x mol/L), insulin (1.7 x mol/L), nucleosides (10 pg/mL each), inorganic salts, sodium pyruvate mol/L), and L-glutamine (2 x mol/L) as All chemicals for this mixture were obtained from Sigma (St Louis, MO).

Plates were incubated at 37°C in a fully humidified incubator with 5% CO, in air and scored with a dissecting microscope at 12 to 14 days for the presence of erythroid bursts. Erythroid bursts (contain- ing >200 cells) were identified on the basis of their orange-red color and plucked from the culture dishes for hemoglobin analysis.

Hemoglobin Analysis Globin analysis was performed both at the protein and mRNA

level. For the protein analysis two approaches were used: immunoflu- orescence using specific anti-y and anti-8 chain antibodies and isoelectric focusing of metabolically labeled (3H-leucine) globin chains. mRNA was assessed by a slot/blot assay following hybridiza- tion to specific y or @ probes.

Immunofluorescence. Cytocentrifuged preparations from pooled erythroid bursts lifted from methylcellulose cultures were prepared and fixed with methanol. Direct or indirect immunofluorescence analysis of the smears was performed using anti-y or anti+ chain antibodies as previously described.’

Erythroid bursts individu- ally collected from the cultures were pooled and incubated overnight with tritiated leucine (Amersham, Arlington Heights, IL; specific activity = 126 Ci/mmol/L) in leucine-free medium. The cells were

Isoelectric focusing of globin chains.

washed, lysed, and the cell lysate subjected to isoelectric focusing.2’ After focusing, the gel was fixed, treated with En’Hance (Dupont, Boston, MA), dried, and subjected to fluorography and densitometry as previously described.?

Pooled bursts lifted from methylcellulose cul- tures were washed twice in phosphate-buffered saline (PBS) and resuspended in cold TE buffer (10 mmol/L Tris, 0.1 mmol/L EDTA) to which 20 U of RNase inhibitor was added (RNasin, Promega, Madison, WI). The cells were then lysed with 5% NP-40 and following centrifugation the lysates were transferred to tubes containing SSC (0.15 mol/L NaCI, 0.015 mol/L trisodium citrate) and formaldehyde (6 x SSC, and 8% formaldehyde final), dena- tured at 65OC for 15 minutes and used for slot blotting immediately,28,29 or stored at -70°C for later analysis. The blots were hybridized with 50 ng of in vitro transcribed radioactive (32P-UTP) RNA probes. For detection of &globin mRNA the 0.7-kb Pst IIEcoRI fragment containing the 3’ end of the human genomic @-globin gene was subcloned in the antisense orientation in a T7-plasmid. For y-globin mRNA, the 0.6-kb EcoRIIHindIII fragment of the 3’ end of Gy-globin gene was subcloned in a Sp6 plasmid vector. Hybridization conditions were as previously described” and the relative amounts of y- and &globin mRNAs were calculated either by directly measuring the radioactivity (cpm) of every slot or by densitometric scanning of developed gels.

RNA analysis.

RESULTS

Bone Marrow Cultures

All bone marrow cell cultures were done with light density mononuclear cells depleted of adherent and/or T cells (Tables 1 and 2). In these cultures Epo alone induced more than 50% of the maximal colony growth, both under FBS- supplemented or FBS-deprived conditions (Table 1). The addition of GM-CSF and IL-3 increased not only the number, but the size of erythroid bursts in these cultures (Table 1 ) . Cultures in which only GM-CSF was added (in addition to Epo) consistently had colonies of smaller size than the ones obtained with IL-3 in both serum-supple- mented and serum-deprived cultures (data not shown). Hb F synthesis (y/y + (I ratio) was evaluated in all these cultures by isoelectric focusing. Consistently higher levels of y/y + (I were observed in FBS( +) versus FBS( -) conditions (Table 1 ) . The addition of GM-CSF and/or IL-3 had no significant effect on these ratios.

Peripheral Blood Cultures

When PBMC were used for culture and Epo was the only exogenous growth factor, colony growth did not vary signifi- cantly between FBS( +) and FBS( -) conditions (Table 2). However, both the number and size of colonies (bursts) increased when IL-3 or GM-CSF was added, especially in cultures depleted of adherent cells (eg, Table 2, PB-VI to PB-VII), under both FBS( +) and FBS( -) conditions. As in bone marrow experiments, globin biosynthesis showed a marked difference (from twofold to sevenfold) between FBS( +) and FBS( - ), regardless of the presence or absence of IL-3 or GM-CSF (Fig 1). Evaluation of y/y + (I ratios by RNA hybridization gave similar results (Fig 2). The lower levels of H b F synthesized in FBS(-) cultures were also reflected, in the few cases tested, by the smaller proportion of cells with detectable H b F by immunofluorescence (Fig 3). In

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MlGLlACClO ET AL 1152

Table 1. Bone Marrow Erythroid Cultures Under Serum-Containing and Serum-Deprived Conditions: Globin Synthesis in the Presence of Hematopoietic Growth Factors (Epo, GM-CSF, IL-3)

Growth BFUe*/3 x lo4 y-Globin Cells/ Sample Culture Factor Cells YIY + 8' ( + ) % Cells Burst

- BM-l Ad- FBS (+) EPO 47 0.1 1 20.0 Epo + IL-3 68 0.15 35.0 -

FBS ( - 1 EPO 34 0.07 5.0 - EPO + IL-3 51 0.06 11.0 -

10 0.13 - - EPO + GM-CSF 22 0.10 - - EPO + PHA-LCM 25 0.17 - -

5 0.07 - -

BM-ll Ad- FBS(+) EPO

FBS ( - ) EPO - - EPO + GM-CSF 25 0.7

BM-Ill Ad- FBS (+) EPO 36 0.10 - 5,300 EPO + IL-3 + GM-CSF 55 0.04 - 17,300

FBS ( - ) EPO 35 0.03 - 5,300

BM-IV Ad -T- FBS (+I EPO 26 ND - 5,000 EPO + IL-3 + GM-CSF 94 0.42 - 14,000

EPO + IL-3 + GM-CSF 53 0.13 - 6,000 300

EPO + PHA-LCM 146 0.17 - 990 670

EPO + IL-3 + GM-CSF 186 0.02 - 2,300

EPO + IL-3 + GM-CSF 49 0.04 - 9,100

FBS ( - 1 EPO 19 ND - 2,000

66 0.12 - BM-V Ad-T- FBS ( + I EPO

- FBS(- ) EPO 58 0.05

Abbreviations: BM, bone marrow; Ad-, minus adherent cells; Ad-T-, minus adherent cells and minus T cells: ND, Not done. *Determined by isoelectric focusing of globin chains.

Table 2. Peripheral Blood Erythroid Cultures Under Serum-Containing and Serum-Deprived Conditions: Globin Synthesis in the Presence of Recombinant Growth Factors (Epo, GM-CSF. IL-3)

y-Globin YIY +8 Growth BFUel3 x lo5 Sample Culture Factor Cells Protein. mRNA ( + ) % Cells

83 0.20 0.220 - 60 NM 0.008 - 80 0.12 0.190 - 59 0.01 0.006 -

82 0.15 0.260 - 61 0.02 0.01 1 -

PB-l FBS (+) EPO FBS ( - ) EPO

PB-ll FBS (+) EPO FBS ( -1 EPO

PB-Ill FBS ( + I EPO FBS(-) EPO

- - PB-IV FBS (+) EPO 91 0.13 FBS ( - ) EPO 104 0.07

PB-V FBS ( + I EPO 40 0.31 EPO + IL-3 80 0.50

FBS ( - ) EPO 40 0.04 EPO + IL-3 70 0.07

PB-VI Ad - FBS (+) EPO 11 0.07

EPO + GM-CSF 52 0.07 EPO + IL-3 + GM-CSF 80 0.07

- - - - - -

- - - - - -

4 2 5

4

1.5

EPO + IL-3 77 0.05 - - -

FBS (-1 EPO 0 EPO + IL-3 36 0.06 -

- - EPO + GM-CSF 16 0.06 EPO + IL-3 + GM-CSF 79 0.06

EPO + IL-3 82 - 27 34 EPO + GM-CSF 36

EPO + IL-3 + GM-CSF 98 - 36

12 EPO + IL-3 69 EPO + GM-CSF 17 - 14

22 EPO + IL-3 + GM-CSF 87

- - - - PB-VI1 Ad- FBS (+) EPO 9 -

- - -

- - - FBS ( - ) EPO 2 - - -

- -

Abbreviation: PB. peripheral blood; N M , not measurable. *Determined by isoelectric focusing of globin chains.

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CYrOKlNES AND HBF IN SERUM-FREE CULTURES 1163

F i g l . b # k o n l C ~ d g k b i r , c h s l n s t r o m ~ hklod aqthroid knn moa (orpuh”: P6-I). tQkMn woo reduced from 0.31 IA) and 0.60 (B) to 0.04 (D) and 0.07 (C), rmpodwly (moo Tablo 2). when tho cdlr -0 culturod in FBS( - 1 onvironnwm.

only one experiment (Table 2. PB-V) addition of IL-3 increased y-globin further. especially in the presence of FBS.

To test the direct effect of IL-3 and/or GM-CSF we prepared populations of PBMC highly enriched in BFUe (Table 3). Proportion of bursts was from 4.7% to 14.7% of cells plated. depending on the culture conditions u d . The number of bursts doubled when cytokines were added in addition to Epo; the fraction of large sire bursts also increased. Fetal globin synthesized by these bursts was from 4.2% to 9.8% in FBS( +) cultures and was reduced to 0.396 to 0.9% in FBSl-) cultures.

Fb2. Lynt00fr0mpOd.d

roprondng from 60.000 to

tor immobilimtion) with radio- active 7 and B prok. ond pro- coasodforautorodiography b o Matoriolr and Mothodd. Thrw oxp.rim.ntr from PB cukuros (Tablo 2, PB-I to PB-110 aro rhown. (A) Tho f and fl rignolo o b t a i d from ouum-contain- ing c u h o s . (E) Tho f and fl rigruls obtoinod from an Monti- ul numbor ol d l s littod from urum-doprkod cukuror.

k*m In IncrUBlng dihnkm.

2,000 COHS, -0 hykMiZd Id-

To test whether FBS( -) conditions allow the expression of Hb F in individuals with increased Hb F in vivo (ie, HPFH subjects) we cultured peripheral blood from two HPFH heterozygotes (Table 4) and one cord blood. Although significant differences in Hb F protein and mRNA were again found between FBS( + ) versus FBS( - ) conditions, the diflcrences were much less pronounced. In HPFH samples the levels under FBS(-) were much higher than in the experiments with normal samples. presumably because the HPFH chromosome and normal chromosome (of an HPFH heterozygote) are affected differently by the FBS( -) condi- tions. As in virtually all other experiments. addition of cytokines did not significantly affect Hb F levels. Of interest. levels synthesized with adult serum or with charcoal-treated FBS were lower than those with untreated FBS (same serum lot, Table 4). as previously documented.” Hb F present in bursts from the cord blood sample was very high under FBS( -) conditions. as evidenced by proportion of y( +) erythroblasts with immunofluorescence (FBSI + 1: 178 BFUe/ IO‘. 91% y ( + ) normoblasts; FBSl -1: 202 BFUe/lO‘. 83% y [ +] normoblasts). These data indicate that FBS( -) condi- tions do not inhibit expression of high levels of Hb F in cases where high Hb F is expressed in vivo.

DISCUSSION

Although Epo is necessary for manifestation of the tetmi- nal erythroid phenotype. numerous previous reports have shown that for optimal growth of human early erythroid progenitors in vitro, other factors in addition to Epo are required.”~’*~”‘” Two factors with significant effect on eryth- roid burst development or with potent burst promoting activity (BPA) arc the cytokines IL-3 and GM-CSF. Both have been shown to achieve their effect by acting directly on erythroid progenitors.”.’5 They act on partially overlapping subsets of erythroid progenitors. with IL-3 recruiting a wide spectrum of both early and late stages of erythroid progeni- tors while GM-CSF and Epo affect mainly a subset of BFUe. Thus, by using combinationsofcytokinesonecan investigate- globin synthetic patterns of distinct pools of BFUes in vitro. However. whether or not distinct cytokines influence Hb F

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Fig 3. Roportkn of HbF-poJtirn colla by ontl-7 gkbln-FITC in FBS(+) cu)hr.r (A. C, ond E) c0mpor.d with FBSl-I cukurr (E. 0. ud FI. In A ond B bum growth wom nimuf.t.d by Epo ond IL-3, in C ond D by Epo and GM-CSF, and in E ond F by Epo 0)om ( o x p . r h n n : B M I ond PB-VI1 Ad - 1. In all FBS( - ) cukuros tho popartion of HbF( + ) d i m wom ot loon twofold bwu.

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CMOKINES AND HBF IN SERUM-FREE CULTURES 1155

Table 3. Culture of Enriched BFUe From a Normal Peripheral Blood: Effect of IL-3 or GM-CSF

~

Y1-r + B Culture Growth Factor BFUe/105 RNA Protein

FBS(+) Epo 6,820 0.071 10.03 EPO + 11-3 12,160 0.062 10.03 Epo + GM-CSF (50 U/mL) 14,740 0.098 10.03 Epo + GMCSF (100 U/mL) 11,420 0.061 t0.03 Epo + GM-CSF (200 U/mL) 10,940 0.044 0.04 EPO + GM-CSF 13,840 0.042 0.03

(200 U/mL) + IL-3 FBS(-) EPO 4,700 0.003 N M

EPO + IL-3 7,500 0.005 N M Epo + GMCSF (50 UImL) 7,240 0.007 N M Epo + GM-CSF (100 U/mL) 7,080 0.006 N M Epo + GM-CSF (200 U/mL) 5,560 0.007 N M EPO + GM-CSF 8,240 0.009 N M

(200 U/mL) + IL-3

Abbreviation: NM: not measurable.

synthesized by BFUe progeny in vitro can be reliably addressed only under conditions where endogenous produc-

dently regulated, in agreement with previous interpreta- tion~.’!’~

One can only speculate about the mechanisms responsible for the low levels of H b F in the serum-deprived cultures. Since, under serum-containing conditions, the levels of H b F are severalfold higher than those observed in vivo for each particular individual, one may suggest that a H b F inducer is present in these cultures, but absent from the FCS-deprived cultures. As a result, H b F cannot be activated under these conditions and levels close to in vivo levels are observed. Since the levels without serum are higher in cultures of cord blood and HPFH subjects, it is unlikely that an inhibitor is present or that solely a negative regulator is elaborated under these culture conditions. Furthermore, since the effect of FBS was also observed in highly enriched progenitor popula- tions, it is unlikely that it is mediated by accessory cells, as was previously ~uggested.~’ The possibility that under serum- deprived conditions erythroid maturation is enhanced, thus lowering the synthetic ratios between y- and j3-globin indi- rectly, could also be considered. This possibility appears unlikely since (a) from morphologic evaluation and cytochem- ical staining there were no significant or consistent differ-

tion of cytokines is minimized and when putative factors or cofactors present in FBS are largely eliminated. Therefore, to study the effect of cytokines or serum in vitro on H b F synthesis we have used either cell populations depleted of adherent cells or of both adherent and T cells, or highly enriched target cells (BFUe) in both serum-containing and serum-deprived environments.

The most striking finding in our experiments is that irrespective of the cytokines present in each culture, the levels of y-globin synthesized were from 2- to over 20-fold lower in serum-deprived cultures compared with similarly stimulated serum-supplemented cultures. It would appear that in vitro activation of H b F is by and large abrogated in serum-deprived conditions. Since different populations of bursts in the presence of the same stimulus combination (IL-3 or GM-CSF) may have different proliferative capaci- ties (ie, burst size) yet exhibit similar low levels of H b F, the two parameters (ie, burst size and H b F) must be indepen-

ences in maturation between serum-containing and serum- deprived conditions (Fig 3); (b) immunofluorescent studies, which are dependent on globin accumulation and not on synthetic ratios, showed low numbers of y-globin-positive cells in FBS(-) conditions in both mature and immature colonies (data not shown); and (c) the proportion of total globin mRNA per cell (as cpm/cell) or j3 only mRNA/cell was comparable in both culture conditions (Table 4). There- fore, the contribution of maturation differences to these data cannot be significant. Suppression of H b F activation in vitro has been previously reported in cultures containing, instead of FBS, fetal sheep serum or plasma* and in cultures in which FBS has been previously treated with activated c h a r c ~ a l . ~ ~ ’ ~ Whether the mechanism(s) lowering H b F levels in all these culture settings are similar (ie, absence of activators) is unclear a t present.

The experiments reported here were mainly designed to evaluate a direct effect of exogenously added cytokines. In

Table 4. Peripheral Blood Erythroid Cultures From Two HPFH Patients Under Serum Control V Serum-Deprived Conditions

Sample Culture Growth Factor

YIY + B cpm1Cell BFUe/105 Cells Protein mRNA YlB

HPFH-II Experiment 1 FSB( + )

C-FBS ( + 1 i AS +

C-AS + C-FBS ( + ) FBS( -1

Experiment 2

Greek HPFH ( - 117) Experiment 3 C-FBS ( + 1

FBS ( -1

EPO EPO EPO EPO

EPO

Epo+lL-3+GMCSF

EPO + IL-3 + GMCSF EPO + IL-3

EPO + 11-3 + GMCSF EPO EPO + IL-3 + GMCSF EPO + IL-3

27 38 22 20 24 25 32 32

23 27 25 36

0.37 0.14 0.20 0.22 0.20 0.15 0.22 0.2 1

0.25 0.09 0.10 0.08

0.135 0.078 0.164 0.125

0.166 0.055 0.06 1 0.049

c

61217,234 1.556/7.937 1,160/8,125

374/6.452 44116,788 43517,215

Abbreuiations: C-FBS, charcoal-treated FBS; AS, normal adult AB serum; C-AS, charcoal-treated AS. *Cell counts (to derive cpm/cell) were not done in these samples.

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1156 MlGLlACClO ET AL

contrast to recent suggesting a stimulatory effect of IL-3 or GM-CSF on H b F, we failed to see such direct effects. It is likely that the composition of our FBS(-) components are different from those used in other laborato- ries. Our data, however, are in agreement with those of

effects through residual accessory cells activated by culture components present in some but not in others39 (and present report) cannot be excluded. Further experi- ments with unicellular cultures and addition of cytokines and/or accessory cells may resolve Some of these issues.

ACKNOWLEDGMENT We wish to thank Dr H. Waldmann for his generous gift of

CamPath 1 . We also thank S. Brenner for exuert secretarial

Fujimori et a139 who also failed to detect any IL-3 effects. The reasons for the disparate results of Gabbianelli et compared with Fujimori et a139 and ours are not readily apparent. The possibility that the cytokines exert indirect assistance.

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PapayannopoulouAR Migliaccio, G Migliaccio, M Brice, P Constantoulakis, G Stamatoyannopoulos and T the globin synthetic pattern of human BFUeInfluence of recombinant hematopoietins and of fetal bovine serum on 

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