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Transforming Growth Factor p (TGF-P), A Potent Inhibitor of
Erythropoiesis: Neutralizing TGF-/3 Antibodies Show Erythropoietin as a
Potent Stimulator of Murine Burst-Forming Unit Erythroid Colony
Formation in the Absence of a Burst-Promoting Activity
By lngunn Dybedal and Sten E.W. Jacobsen
Transforming growth factor p (TGF-p) is a bifunctional regulator of the growthof myeloid progenitors and is here dernonstrated t o directly inhibit the growth of primitive erythroid
progenitors by 95% t o 100% regardless of the cytokines
stimulating growth. Autocrine TGF-p production of primitive
hematopoietic progenitors has previously been reported. In
the present study, a neutralizing TGF-/3 antibody (anti-TGFp ) added t o serum-containing cultures, resulted in a 3-, 4-,
and 25-fold increase in burst-forming unit erythroid (BFUE) colony formation in response t o interleukin4 (IL-4) plus
erythropoietin (Epo), SCF plus Epo, and IL-l1 plus Epo, respectively. The growth of BFU-E progenitors has been suggested to require a burst-promoting activity in addition t o
Epo. Accordingly, we observed no BFU-E colony formation
in serum-containingcultures in reponset o Epo alone.In contrast, 50 BFU-Ecolonies were formed when anti-TGF-fi was
stimincluded in the
culture. In serum-free cultures, Epo also
ulated BFU-E colony formation in theabsence ofother cytokines, whereas anti-TGF-B had no effect on the number of
colonies formed.Quantitation of TGF-p1 in serum by an enzyme-linked immunosorbent assay method showed predominantly the presence of precursor (latent) TGF-p1, but
also showed active TGF-p1 at a concentration sufficient t o
potently inhibiterythroid colony formation. Thus, neutraliration of active TGF-p1in serum shows that Epo alone is sufficient t o stimulate the growthof murine BFU-E progenitors.
0 1995 by The American Societyof Hematology.
E
necrosis factor a (TNF-a), another inhibitor of erythroid
colony formation, has been suggested to act through an indirect r ~ u t e . * * ~ O . ~ ~
Autocrine TGF-P production by a subpopulation of human
hematopoietic stem cells has been shown by the use of antisense TGF-P oligonucleotides and neutralizing TGF-P antibodies.32Similarly, Ploemacher et al,33 byuse of a neutralizing TGF-P antibody, suggested that SCF-stimulated murine
hematopoietic progenitor cells could produce TGF-P as well.
Autocrine TGF-P production by hematopoietic stem cells
could potentially be one of the mechanisms involved in
maintaining the earliest stem cells in a quiescent state,32
because TGF-P is a potent inhibitor of primitive stem cells,
although it has little or no effect on more committed progenitors.21-2629
RYTHROPOIESIS, as part of the hematopoietic hierarchy, requires a highly complex series of cellular events
to keep the large number of mature erythroid cells constant.’.2
Both stimulatory and inhibitory cytokines have been shown
to be involved in the regulation of viability, proliferation, and
differentiation of erythroid progenitor cells.3 Erythropoietin
(Epo) is known to be the principal factor in regulating red
blood cell p r o d ~ c t i o n .In~ .addition
~
to being a viability factor
for erythroid progenitors, it is required for terminal differentiation of erythrocyte^.^.^ More primitive erythroid progenitors (BFU-E) are dependent on combined stimulation of Epo
and other hematopoietic growth factor^.^'^"-^ Stem cell factor
(SCF), interleukin-3 (IL-3), IL-6,IL-11, and IL-12 are glycoproteins shown to stimulate proliferation of primitive hematopoietic progenitors, granulocyte-monocyte (GM) precursors as well as primitive erythroid progenitors.lWl5IL-4, a
bifunctional regulator of the growth of GM progenitors,16is
a potent stimulator of BFU-E colony formation,” whereas
IL-9 enhances erythroid colony formation without affecting
GM
Transforming growth factor P (TGF-P) is a family of 25kD homodimeric proteins produced by many cell types with
bone and platelets being the main r e s e r v ~ i r . TGF-P
~ ~ , ~ ~has
been shown to be a bifunctional regulator of the growth
of hematopoietic progenitor cells in
The effects
(stimulatory or inhibitory) depend on the differentiation
stage of the target cell as well as the stimulatory cytokines
present in the
Specifically, TGF-P has been
shown to be a potent inhibitor of primitive myeloid progenitors,21.22.24,26,2Y and to inhibit erythroid progenitors such as
human and murine BFU-E and colony-forming unit-granulocyte, erythrocyte, megakaryocyte, macrophage and human
CFU-E colonies when stimulated with Epo plus SCF or Epo
plus IL-3.21-24.26.2y
However, murine CFU-E colony formation
has been shown to be unaffected by TGF-P t~-eatment.’~.’~
Although the ability of TGF-P to inhibit primitive murine
bone marrow (BM) progenitor cells has been shown to be
directly mediated,26it has not yet been established whether
the potent inhibition of erythroid progenitors by TGF-P is a
direct effect. This is of particular interest because tumor
Blood, Vol 86, No 3 (August l ) , 1995: pp 949-957
In the present study we used a population of Lin- murine
BM cells to investigate the effect of TGF-P1 on BFU-E
colony formation when cultured with stimulatory cytokines
not previously explored for their interaction with TGF-P1.
The studies also examined whether the effect of TGF-P1 on
the growth of BFU-E colony formation was directly mediated, and whether the inhibitory effects of TGF-P1 on Epo
plus SCF or Epo plus L-3-stimulated erythroid colony formation could be counteracted by combining multiple stimulatory cytokines. Finally, we explored whether autocrine
From the lnstitute of Cancer Research, University of Trondheim,
Trondheim, Norway; the Department of Immunology, lnstitute for
Cancer Research, The Norwegian Radium Hospital, Oslo, Norway;
and Hipple Cancer Research Center, 4100 S Kettering Blvd, Dayton,
OH.
Submitted October 7, 1994; accepted March 22, 1995.
Supported by the Norwegian Cancer Society.
Address reprint requests to Ingunn Dybedal, MD, Hipple Cancer
Research Center, 4100 S Kettering Blvd, Dayton, OH 45439-2052.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertiisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1995 by The American Society of Hematology.
oooS-4971/95/8603-0017$3.00/0
949
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950
DYBEDAL AND JACOBSEN
TGF-Pproductionbyerythroidprogenitorcellscouldbe
observed in vitro.
MATERIALS AND METHODS
Growth factors and antibodies. Purified recombinant rat (IT)
SCF and recombinant murine @Mu) granulocyte macrophage colony-stimulating factor (GM-CSF) were generously provided by Dr
Ian K. McNiece (Amgen Corp, Thousand Oaks, CA). Recombinant
human (rHu)Epo was kindly supplied by Hanne Krog Usland (Organon Teknika, Oslo, Norway) and rMuIL-4 was a generous gift
from Boehringer Mannheim (Mannheim, Germany). Purified rMuIL3 was purchased from Promega C o p (Madison, WI). rHu TGF-p1
was a gift from Tony Purchio, Oncogene Corp (Seattle, WA), rHuIL6, rMuIL-9, rHuIL-11, and rMuIL-12 were kindly supplied by Genetics Institute (Cambridge, MA), rHuG-CSF was generously provided by Unni Hjelmaas, (Roche, Oslo, Norway). A mouse IgG,
antibody that neutralizes murine and human TGF-PI and TGF-82
as well as chicken TGF-p3 was kindly provided byDr James R.
Dasch (Celtrix Laboratories, Palo Alto, CA).14The control antibody
used, was an unrelated murine IgG, antibody (6H8) directed against
a widely distributed human 180-kD membrane protein, kindly provided by B. Naume and T. Espevik (Institute of Cancer Research,
University of Trondheim, Trondheim, Norway).
Unless otherwise indicated, all growth factors and antibodies were
used at predetermined optimal concentrations: rrSCF, 100 ng/rnL;
rHuEpo, 5 U/mL; rMuIL-4, 10-20 ng/mL; rMu GM-CSF, 20 ng/
mL; rMuIL-3, 20 ng/mL; rHuTGF-01, 20 ng/mL; rHuIL-6, 50 ng/
mL; rMuIL-9, 50 UlmL; rHuIL-11, 50 ng/mL; rMuIL-12, 100 ng/
mL; rHuG-CSF, 50 ng/mL; Mu TGF-PI, p2, and P3 neutralizing
antibody, 3 pg/mL; 6H8, 3 pg/mL.
Separation of murine BM progenitor cells. To enrich the BM
in erythroid progenitor cells, Lin- BM cells were isolated from
normal BALB/c mice, according to a protocol described by Spangrude et al.’5 Briefly, light-density BM cells were obtained using
lymphocyte separation medium (Lymphoprep Animal; Nycomed,
Oslo, Norway). Cells werewashed twice and resuspended in Iscove’s-modified Dulbecco’s medium (IMDM; GIBCO-BRL, Paisley, UK) supplemented with 20% heat inactivated fetal calf serum
(FCS; Bio Whittaker, Walkersville, MD) 0.5 mg/mL glutamine and
0.04 mg/mL gentamycin (complete IMDM). The cells were incubated at 4°C for 30 minutes in a cocktail of predetermined optimal
concentrations of lineage-specific antibodies as previously described3? RB6-8C5 (Gr-l antigen; PharMingen, San Diego, CA),
RA3-6B2 (B220 antigen; PharMingen), L3T4 (CD4; PharMingen)
and MAC-l (Serotec, Oxfordshire, UK). For some experiments, Ly2 (CD8a; PharMingen) and Ter-l19 kindly provided by Dr Tatsuo
Kina (Chest Disease Research Institute, Kyoto University, Kyoto,
Japan) were added to the cocktail, as indicated. The cells were
washed twice and resuspended in complete IMDM. Sheep antirat
IgG (Fc)-conjugated immunomagnetic beads (Dynal, Oslo, Norway)
were added at a cellhead ratio of 1: 10 to 1:20 and incubated at 4°C
for 30 minutes. Labeled cells (Lin’) were removed by a magnetic
particle concentrator (Dynal), and the Lin-cells recovered from the
supernatant.
Semisolid colony assay. Lin- BM cells were plated inIMDM
and20% FCS unless otherwise indicated. In some experiments
IMDM and FCS were replaced by serum-free medium (x-vivo 15;
Bio Whittaker) supplemented with 1% bovine serum albumin (BSA;
HCC-9300; StemCell Technologies Inc, Vancouver, Canada), and
1.2% (final concentration) methylcellulose (Methocel, Fluka
Chemie, Buchs, Switzerland). Purified cytokines at predetermined
optimal concentrations were added to the cultures containing 0.5 X
IO“ to 2 X I 0“ cells/mL in Petri dishes. Cultures were incubated at
37°C in 5% CO2 in air for 7 to 9 days and scored for BFU-E and
CFU-GM colonies according to established riter ria.^^.^'
In addition, the erythroid nature of the colonies formed was verified by theuse of the Ter-I 19 antibody, which has beenshown
to be specific for cells of the erythroid lineage.3HTen micrograms
phycoerythrin-conjugated Ter-l19 (Ter-119-PE; Rat Ig G 2b; PharMingen), or 10 yg Gr-I-PE against a myeloid differentiation antigen
(Rat Ig G 2b; PharMingen), found to benegative on erythroid precursors and therefore, used as a control antibody,” were distributed in
200 pL IMDM, spread over the surface of cell cultures having been
incubated for 7 to 9 days in Petri dishes, incubated at 4°C for I
hour, and washed three times with cold phosphate-buffered saline.
Positive colonies were scored in a fluorescence microscope.
Single-cell proliferation assay. Lin.. cells were seeded in Terasaki plates (Nunc, Kamstrup, Denmark) at a concentration of one
cell per well in a volume of 20 pL complete IMDM with predetermined optimal concentrations ofpurified
rHuEpo, ITSCF,and
rHuTGF-81. Wells were scored for proliferation ( > l 0 cells) after
7 to 9 days of incubation at 37°C and 5% CO2 in air.
Estimation of average burst number and size per BFU-E colony
in serum-containing and serum-free cultures. To evaluate if there
were differences in the sizes of the BFU-E colonies formed in serumfree and serum-containing cultures, the number of bursts per BFUE colony as well as their size were measured. Measuring was done
by using a 21-mm ocular measuring scale (model El; Graticules
Ltd, Tonbridge Kent, UK) and an objective micrometer (model S8;
Graticules Ltd).
TGF-PI immunoassay. To measure the amount of TGF-p1 precursorflatent as well as active TGF-PI in FCS, we used a quantitative
sandwich enzyme-linked immunoassay (ELISA) technique (R&D
systems, Minneapolis, MN). Active TGF-PI was measured in 20%
and 100% FCS. To activate presursor/latent TGF-PI, 2.5 N acetic
acid/lO mol/L urea was incubated with the serum as well as IMDM
for 10 minutes. The samples were then neutralized with 2.7 N NaOHl
1 m o m HEPES, and final pH was measured to be 7.2 to 7.6.
The ELISA detects TGF-01 down to 5 pg/mL.
Statistical analysis. Unless otherwise indicated, all results were
expressed as the mean value t SEM of data obtained from three or
more separate experiments. The statistical significance of differences
between mean values of groups was determined using Student’s ttest for paired samples.
RESULTS
The effect of TGF-01
on erythroid progenitors was stimulated by differentcytokines.Inagreementwithprevious
studies, TGF-P1 almost completely inhibited BFU-E colony
formation in response to
SCF plus Epo or IL-3 plus Epo
(Table l).2’.24,2y
Notpreviouslyshown,wealsoobserved
complete inhibition of BFU-E colony formation induced by
IL-4 plus Epo (Table 1). In agreement with others, we found
IL-6 plus Epo, IL-9 plus Epo, and IL-I 1 plus Epo to be
poor stimulators of murine BFU-E colony formation when
cultured in FCSi’,14si8
(Table 1). However, synergistic effects
on BFU-E colony formation were observed when IL-9, IL6, or IL-l1 were combined with Epo plus IL-4 ( P 5 .01)
(Table l), andTGF-01
(20 ng/mL)almostcompletely
blocked the formation of BFU-E colonies in response to all
thesecytokinecombinations(Table
1). We haverecently
shown synergy betweenIL- 12 and SCF plus Epo in stimulating BFU-E-formed colonies,+” and TGF-01 potently inhibited this growth factor combination, as well (Table 1).
The inhibitory effect of TGF-01 on erythroid colonies is
directlymediated.
BecausebothTNF-aand
IFN-y can
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951
TGF-B IN SERUMSUPPRESSESBFU-EGROWTH
Table 1. Effects of TGF-p1 on Erythroid and GM
Colony
Formation of Murine Lin- BMCells
Colonies per 20,000Cells
BFU-E
Cytokines
EPO
EPO +
€PO +
Epo +
EPO
€PO +
EPO +
Epo +
EPO+
Epo
€PO +
€PO +
+
+
-
TGF-0
0
1L-9
IL-6
IL-12
IL-l1
1L-3
1L-4
SCF
IL-9 + IL-4
IL-6 1L-4
IL-l1 + IL-4
IL-12 + SCF
+
0
0
0
2 (1)
26 (5)
28 (1)
30 (1)
42 (3)
48 (3)
67 (4)
69 (2)
CFU-GM
+ TGF-0
- TGF-0
+ TGF-0
0
0
0
0
0
0
0
0
0
0
0
1 (0)
0
1 (0)
0
1 (0)
1 (1)
1 (0)
0
0
54 (2)
0
19 (3)
0
0
l(1)
31 (1)
0
0
12 (2)
0
1 (1)
0
0
0
0
Lin murine BM cells were separated as described in Materials and
Methods. Cells (20,000)were plated in 35-mm dishes with IMDM, 20%
FCS,1.2% methylcellulose, and cytokines at predetermined optimal
concentrations (Materials and Methods) in the absence or presence
of rHu TGF-01 (20 nglmL). Colonies were scored after 7 to 9 days
incubation at 37OC and 5% CO2 in air. The results represent the mean
values (SEM) of at least four independent experiments with triplicate
determinations.
inhibit erythroid colony formation and potentially be produced by accessory cells,”.3”..”.“’ weperformed a limiting
dilution study. Lin murine BM cells were seeded in methylcellulose with SCF plus Epo in 20% FCS in the absence or
presence of TGF-P1 at concentrations of 2,500, 5,000,
10,000. and 20,000 cells/mL, resulting in the formation of
4, 10, 20, and41 BFU-E colonies, respectively, in the absence of TGF-P1 (mean values of two separate experiments).
In the presence of TGF-PI, 0, 0, 2, and 2 BFU-E colonies
were observed at 2,500, 5,OOO. 10,000, and 20,000 celllmL,
respectively (data not shown). Lin- murine BM cells, stimulated with SCF, Epo, or SCF plus Epo were also seeded in
20% FCS at a concentration of one cell per well and 300
wells per group, in the presence or absence of TGF-01 . No
colonies were observed when cells were stimulated with Epo
or SCF alone. In the absence of TGF-PI, 7 2 2 colonies
(mean of five independent experiments) were observed in
response to the combination of SCF plus Epo, whereas no
colonies were formed in the presence of TGF-PI (data not
shown), suggesting that the inhibition was directly mediated
on the erythroid progenitor cells.
Multiple erythroid stimulators counteract TGF-PI-induced inhibition of RFU-E colony formation. It has recently been shown that a cocktail of multiple stimulatory
growth factors can counteract the inhibitory effects of TGF01 on primitive hematopoietic progenitors.“’ Here we
wanted to investigate whether multiple erythroid stimulatory
cytokines in a similar fashion could counteract the potent
TGF-PI-induced inhibition of BFU-E colony formation of
Lin- murine BM cells. TGF-01 inhibited only 54% of the
BFU-E colony formation when Lin- murine BM cells were
cultured with the combination of IL-3, IL-4, SCF, and Epo
(Fig l ) . Addition of IL-9, IL-l I , and IL-12 did not further
enhance the number of BFU-E colonies, either in the presence or absence of TGF-01 (Fig l ) . Interestingly, whereas
the cloning frequency of progenitors stimulated by SCF plus
IL-12 plus Epo and the cocktails of 4 and 7 stimulatory
cytokines wasnearly the same, the number of TGF-01unresponsive progenitors were enhanced 30-fold in response
to each of the cocktails compared with the SCF plus IL-12
plus Epo-induced progenitor cells (Fig l ) .
The EDso and maximuminhibitory concentration of TGFP on the multifactor, and SCF plus Epo and Epo plus IL3-stimulated BFU-E colony formation was similar, and consequently, increasing the concentration of TGF-P1 to 200
ng/mL did not further enhance the TGF-PI-induced inhibition (data not shown).
A neutralizing antibody against TGF-0 potently enhances
RFU-E colony formation in serum containing cultures. We
next examined whether a neutralizing TGF-0 antibody (antiTGF-P) could increase BFU-E colony formation of murine
progenitors in response to Epo alone, Epo plus IL-l I , Epo
plus IL-4, or Epo plus SCF (Table 2). Like other^,^.""." we
foundEpo alone to stimulate few or noBFU-E-formed
colonies in serum containing cultures (Tables 1 and 2). However, anti-TGF-fl enhanced Epo-stimulated colony formation in a concentration-dependent fashion withmaximum
effect observed at 3 pg/mL (Fig 2). resulting in an increase
in BFU-E colonies from 0 to 44 (Fig 2). In cultures with
1
0
0
0
- TGFQ 1
+ TGFQ 1
r
r
T
Fig 1. Effects of TGF-p1 on BFU-E colony formation stimulated by
different growth-factor combinations. Lin- BM cells, separated as
described in Materials andMethods, were plated at
20,000 cellsldish
in IMDM, 20% FCS, 1.2% methylcellulose and predetermined optimal
concentrations of cytokines as indicated. Cultures were grown in the
absence or presence of 20 ng/mL TGF-p1 and BFU-E colonies scored
after 7 t o 9 days’ incubation at37°C and 5% CO2in air. Results represent the mean values (SEMI of at least three independent experiments with triplicate determinations.
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DYBEDALANDJACOBSEN
952
Table 2. Effects of anti-TGF-P on BFU-E Colony Formation of
Murine Lin- BMCells Cultured in Serum-Containing
and Serum-Free Medium
BFU-E Colonies
20% FCS
Cytokines
~~
Serum-Free Medium
- anti-TGF-/3 + anti-TGF-0
- anti-TGF-/3
+ anti-TGF-/3
0
0
~~
Medium
0
0
EPO
10,000 cells
20,000 cells
EPO + IL-l1
10,000 cells
20,000 cells
EPO+ IL-4
10,000 cells
20,000 cells
Epo + SCF
5,000 cells
10,000 cells
20,000 cells
Lin- murine BM
cells, separated as described in Materials and Methods, were plated at 5,000, 10,000, and 20,000 cells in 35-mm dishes
in IMDM and 20% FCS or in serum-free medium (Materials and Methods), 1.2% methylcellulose, cytokines, and antibodies at predeterminedoptimal concentrations (Materials and Methods). Colonies
were scored after 7 to 9 days’ incubation at 37°C and 5%CO2 in
air. The results represent the mean values (SEM) of at least three
independent experiments with triplicatedeterminations. An unrelated
control lgGl antibody showed no effect on colony formationat 3 p g l
mL (data not shown).
SEM < 0.5.
20% FCS, IL-I 1 has little or no effect on BFU-E colony
formation when combined with Epo.’‘ However, in the present study, the number of BFU-E colonies formed in response
to Epo plus IL-I I, was enhanced from 3 to 74 when adding
anti-TGF-0 (Table 2). In addition, morepotent erythroid
stimulatory combinations such as IL-4 plus Epo and SCF
plus Epo gave a threefold and fourfold increase in BFU-E
colony formation, respectively, when anti-TGF-0 was
added (Table 2).
We were interested in examining whether the TGF-P activity present in our cultures was a component present in
serum or potently produced in an autocrine manner. Thus,
the growth of BFU-E colonies was examined in serum-free
medium as compared with FCS-supplemented IMDM. No
BFU-E colonies were observed in either serum-free or serum-containing medium or in either the absence or presence
of anti-TGF-P alone (Table 2). Whereas serum-containing
IMDM supported no erythroid colony formation in response
to Epo alone, 49 colonies were induced by Epo in the serumfree culture (Table 2). Similarly, the number of BFU-E colonies formed in serum-free medium in response to Epo plus
IL-4, Epo plus SCF, and Epo plus IL- I 1 were 3-, 4-, and 25fold higher than in FCS-supplemented cultures, respectively
(Table 2). Interestingly, nearly the same number of BFU-E
colonies were formed in the presence of anti-TGF-0 in
serum-supplemented cultures as in serum-free cultures without antibody (Table 2). Addition of anti-TGF-0 to the se-
rum-free cultures didnot further enhance thenumber of
BFU-E colonies in the presence of Epo, IL-4 plus Epo, or
SCF plus Epo (Table 2). suggesting that the TGF-P activity
in our system required the presence of serum.
BFU-E colonies were scored based on typicalmorphologic appearan~e.~~.~’
However, to confirm the erythroid nature of the colonies more specifically, we developed a
method to identify erythroid colonies based on Ter-I 19 expression, which is restricted to the erythroid lineage..” Linmurine BM cells cultured in serum-free medium with 1.2%
methylcellulose for 7 to 9 days in the presence of Epo, Epo
plus IL-4, or SCF plus G-CSF were stained with Ter-119PE or Gr-l-PE as described in Materials and Methods. Epoinduced colonies were completely negative for Gr- I staining,
whereas 95% to 100% colonies were Ter-l19 positive (Table
3). Epo plus IL-4-stimulated colonies were 90% to lot)%
Ter-l19 positive and also completely Gr-l negative. All GCSF plus SCF-stimulated colonies were Gr-l positive, and
Ter-l19 negative (Table 3). Erythroid colonies formed in
serum-free and serum-containing cultures in response to Epo
were also sampled from cultures, fixed, Giemsa stained, and
identified by typical cell morphology (data not shown). Finally, erythroid colonies formed in serum-free cultures were
also shown to be benzidine positive (data not shown) according to established criteria.“ Control colonies stimulated
with SCF plus G-CSF were benzidine negative.
6o
1
T
0
5 ~ 1 6
5x10”
~
1
3
5
10
anti-TGF-p1 (pglml)
Fig 2. Dose-response of anti-TFG-P on Epo-supported BFU-E colony formation of Lin- murine BM cells. Lin- BM cells, separated as
described in the Materialsand Methods, were platedat 20,000 cells/
dish in IMDM, 20% FCS,1.2% methylcellulose, 5 UlmL rHuEpo and
exposed t o increasing concentrations of M u anti-TGF-P as indicated.
BFU-E colonies were scored after 7 t o 9 days incubation at 37°C and
596CO2 in air. In the presence of 10 pg/mL of a control antibody
(murine lgG1; 6H8; Materials and Methods) combined with Epo, no
BFU-E colonies were observed (data not shown). The results represent themean values (SEM) of three independent experiments with
triplicate determinations.
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953
TGF-8 IN SERUM SUPPRESSESBFU-EGROWTH
m
Table 3. Expression of Ter 119 by BFU-E Colonies
Exp 2
Exp 1
FCS
Serum-free medium
% Positive Colonies
Exp 3
Ter-119-PE Gr-l-PE Ter-119-PE
Gr-1-PE
Ter-119-PE
J-
5ot
Gr-1-PE
v)
EPO
EPO + IL-4
G-CSF +
SCF
ND
90%
ND
0
100%
0
100%
100%
0
0
93%
ND
0
ND
0
100%
0
100%
Lin- murine BM
cells, separated asdescribed in Materials and Methods, were plated in serum-free medium with 1% BSA (Materials and
Methods), 1.2% methylcellulose, and cytokines at predetermined optimal concentrations (Materials and Methods). After 7 to 9 days incubation at 37°C and 5% CO2 in air, 10pg/mL Ter-119-PE (in 200 mL IMDM)
were spread over the surface of the cell culture of one dish in each
group. Gr-1-PE( I O pg/mL in 200 mL IMDM) was added to corresponding dishes. The plates were incubated for 1 hour on ice and washed
three times with coldPBS; PE-positive colonies were scored by using
a fluorescence microscope. A minimum of 40 colonies were scored
per dish.
Abbreviations: Exp, experiment; ND, not determined.
g 40"
0
0
C
8
30"
20"
10"
0
-
+
-
+
EPO
The studies in serum-free cultures suggested that a burstpromoting activity (BPA) is not required to stimulate BFU-E
colony formation. However, the serum-free medium contains
1% BSA (foreign protein) that could potently stimulate accessory cells to produce one or several of the cytokines
capable of synergizing with Epo. To exclude this possibility,
we next performed studies on Lin- cells plated at a density
of one cell per well in serum-free medium. To enhance the
cloning frequence, we also used the Ter-l19 antibody in the
lineage depletion. This antibody has been shown to detect
mature erythroid cells, but not erythroid progenitor cells.38
In serum-containing cultures, these Lin- cells formed no
colonies in response to Epo alone. However, 47 Epo-induced
clusters and colonies were formed in response to Epo alone
in serum-free cultures (Fig 3).
Next we investigated whether there was any difference in
the size of BFU-E colonies formed in serum-containing and
serum-free cultures. The number of bursts as well as the
size of individual bursts within each BFU-E colony were
measured. The size of individual bursts within each BFU-E
were comparable in serum-free and serum-containing cultures, and the number of bursts per BFU-E colony were
comparable for the Epo-stimulated colonies in the absence
and presence of serum (Table 4).In IL-4 plus Epo-stimulated
cultures, the number of bursts were slightly higher in serumcontaining cultures containing anti-TGF-P than in serumfree medium.
Quantitation of active and precursor (latent) TGF-PI in
FCS. Previous studies have shown the presence of TGF-01
in
To quantitatively determine the levels of active as
well as precursorfiatent TGF-01 in FCS, we used a TGF-P1
ELISA kit (Materials and Methods). The amount of presursor
TGF-01 was determined after acid activation and found to
be 8.2 t 0.6 ng/mL (mean of three experiments f SEM) in
100% FCS and active TGF-P1 to be 15 % 3 pg/mL (mean
of three experiments f SEM) in 100% FCS (Fig 4) Thus,
FCS contains high amounts of precursorfiatent TGF-01 as
Fig 3. A single-cell assay shows Epo aloneto be a potent stimulator of colony formation in serum-free medium. Lin- murine
BM cells,
separated as described in Materials and Methods,but with addkion
of Ter-l19 to increasethe cloningfrequence, were plated in microtiter
plates ata concentration of one cell perwell 20 pL in completeIMDM
(20% FCS) or x-vivo-l5 with 1% BSA in the presence or absence of
Epo 5 UlmL. Wells ware scored forcell growth (>l0 cells) &er 7 to
at 37°C and 5% COIin air.The results are presented
9 days incubation
as the maan number of colonies 1SEM) per 900 wells and represent
three separate experiments. *SEM <0.5.
well as low, but significant concentrations of active TGFPI.
Dose-response of TGF-PI on BFU-E colony formatiom
in serum-free cultures. Because active TGF-P1 was shown
in 100% FCS at a concentration of 15 pg/mL, resembling 3
pg/mL in complete IMDM (20% FCS), we next investigated
Table 4. A Comparison of the Burst Size and Numbers in BFU-E
Colonies Formed in Serum-Free and Serum-Containing Cultures
Mean No. of Bursts per
BFU-E Colony Formed
Serum-Free
Medium
20% FCS
Mean
Burst
Size
Serum-Free
Medium
(mm)
20% FCS
~
EPO
EPO + IL-4
13 (2)
20 (2)
17 (2)
31 (4)
0.040.03
(0.01)
0.09 (0.005)
(0.004)
0.07 (0.005)
Lin- murine BMcells 120,000), separated as described in Materials
and Methods, were cultured in 1.2% methylcellulose with Epo or Epo
plus IL-4 at predetermined optimal concentrations in serum-free medium or in IMDM supplemented with 20% FCS and 3 pg/mL antiTGF-p. After 8 days' incubation at 37°C and 5% COzin air, the number
of bursts and diameter of each burst within every BFU-E colony
formed were measured (Materials and Methods) and the mean burst
diameter per plate was estimated. The results represent the mean
burst diameter (SEM) and the mean number of bursts (SEM) per BFUE colony of two separate experiments with duplicate determinations.
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DYBEDAL AND JACOBSEN
954
ability of anti-TGF-P to enhance the clonal growth of erythroid progenitors, the addition of anti-TGF-P reduced GMCSF-induced CFU-CM colony formation by 33% (P < .OS)
(Fig 6). No significant difference (P = .2) was observed
in GM-CSF-stimulated colony formation in the absence or
presence of anti-TGF-P in serum-free medium (Fig 6). Although nearly the same number of colonies were formed in
serum-free medium as in 20%FCS,when GM-CSF was
combined with TGF-/? (P > .05), very few CFU-GM colonies were formed in serum-free medium in the presence of
GM-CSF alone (Fig 6).
Thus, the FCS-dependent TGF-0 activity can bidirectionally affect the growth of murine BM progenitor cells.
1
"""_
0.1
t
DISCUSSION
0
10
100
1000
TGFpl (pg/ml)
Fig 4. Quantitation of active and latentlprecursor
TGF-p1 in FCS.
A quantitative sandwich enzyme immunoassay techniquelELlSA kit
was used t o determine thepresence of active andprecursor TGF-p1
in FCS. To activate precursor TGF-p1, FCS was incubatedwith 2.5 N
acetic acidll0 mollLurea for l 0 minutes, and then neutralized with
2.7 N NaOHl1 mollL HEPES free acid. Because levels of active TGFp1 in FCS were expected t o be low, loo?& FCS was tested. On the
other hand, precursor TGF-p1 in serum wasexpected t o be high, and
was according t o recommendation diluted 1:30. A TGF-p1 standard
curve was generated by the use of a TGF-p1 standard at multiple
dilutions, the optical density by plotting
against the concentrations
of the standardTGF-p1. By comparing the optical density
of the test
samples t o this standard curve, the concentration of TGF-p1 could
be determined. Results are from one characteristic experiment (of
three). **, active TGF-p1; ***, precursor TGF-p1.
Previous reports have shown that TGF-P is a bidirectional
regulator of murine and human cytokine-induced hematopoiesis in vitro.'"2y The effect of TGF-P (stimulation or inhibition) appears to be dependent on the growth factors present,
and the nature of the target
Withregard to erythroid
progenitors, TGF-P inhibits SCF plus Epo and IL-3 plus
Epo-induced BFU-E and CFU-mix colonies.''~24~2h~'y
In the present study, we show for the first time that the
growth of erythroid progenitors stimulated by other factors
such as IL-4, IL-6, IL-9, IL-11, and IL-12 can be potently
inhibited by TGF-P1 as well.
A direct inhibitory effect of TGF-01 on very primitive
hematopoietic cells is previously shown.'"By theuseof
single-cell assays, we show here that the potent inhibitory
effects of TGF-P1 on BFU-E colony formation appear to be
directly mediated on the targeted erythroid progenitors as
-
J
Q)
0
8
._
zC
whether this concentration was sufficient to inhibit Epo-stimulated BFU-E colony formation. When 20,000 Lin- murine
BM cells were stimulated with Epo and increasing concentrations of TGF-P1 in serum-free medium, the maximum
inhibitory effect of TGF-P1 was obtained at 0.03 to 0.3 ng/
mL, whereas 3 pg/mL inhibited 75% of the BFU-E colony
formation (Fig 5). Thus, low levels of active TGF-P1 present
in serum can potently inhibit Epo-stimulated erythroid colony formation.
Anti-TGF-fl reduces GM-CSF-induced CFU-GM colony
jhrmation in serum-containing cultures. Although the effects of TGF-P on hematopoiesis are predominantly inhibitory, it has been shown that TGF-0 in combination with GMCSF can stimulate CFU-GM colony formation of murine BM
progenitors.'* Thus, Lin- cells were seeded in 20% FCS
and serum-free medium supplemented with GM-CSF in the
absence or presence of anti-TGF-P. In contrast with the
50
v)
40
30
0
-
8 20
z
10
0
0
3x10' 3x103
3x102
3x10"
3
30
TGFpl (ng/ml]
Fig 5. Dose-response of TGF-pl-induced inhibition of Epo-stimulated BFU-Ecolony formationin serum-free medium. Lin- murine BM
cells (20.000).separated as described in Materials andMethods, were
cultured in serum-free medium, 1.2% methylcellulose, 5 UlmL Epo,
and increasing concentrations of TGF-p1 for 8 days at 37°C and 5%
CO2 in air. The results represent the mean values of three independent experimentswith triplicatedeterminations; error bars show the
SEM. *SEM <0.5.
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TGF-0 IN SERUM SUPPRESSES BFU-E GROWTH
20% FCS
0
Serum-free medium
Fig 6. Effects of anti-TGF-p on GM-CSF-stimulated CFU-GM colony formation. Lin- BM cells, separatedas describedin Materials and
Methods, were plated at 5,000 cells/dish in IMDM with20% FCS or in
serum-free medium (Materials and Methods), 1.2% methylcellulose,
GM-CSF 20 ng/mL and exposed to either anti-TGF-pBpglmL or TGFp1 20 ng/mL. CFU-GM colonies were scored after 7 to 9 days' incubation at37°C and 5 % CO2in air. The results representthe mean values
(SEMI of three independent experiments with triplicate determinations.
well. This is of particular interest because accessory cells
might produce cytokines with both stimulatory and inhibitory effects on BFU-E colony f o r m a t i ~ n . ~ ~ . " ' . ~ ' . ~ ~
It has previously been shown that multiple stimulatory
cytokines can counteract TGF-P-induced growth inhibition
of very primitive murine hematopoietic progenitor celkd2
We show here that more than 40% of the BFU-E colonies
formed in response to a combination of four erythroid stimulatory factors (IL-3, IL-4, SCF, and Epo) were not inhibited
by TGF-01. In contrast, BFU-E colony formation in response to two- and three-factor combinations was almost
completely blocked. Because the cloning efficiencies of the
cocktail-induced progenitors were increased compared with
most of the 2 or 3 cytokine combinations, it could be argued
that TGF-P1 simply inhibits the additional recruited progenitor cells less efficiently. However, this seems unlikely
because the cloning frequency of SCF plus IL-12 plus Epostimulated BFU-E colonies was as high as for the multifactor-induced BFU-E colony formation in the absence of
TGF-PI, whereas the cloning frequency in the presence of
TGF-PI was 30-fold higher in response to the cocktail.
Autocrine production of TGF-P1 has been implicated in
human hematopoietic progenitor cells, ie, colony formation
of human CD34' progenitors in serum-containing cultures
was enhanced in the presence of antisense TGF-P1 oligonucleotides.3' Similarly, Ploemacher et
showed an increase
955
in CFU-C colonies when SCF-stimulated murine primitive
BM progenitors were seeded with anti-TGF-P both in cultures with FCS and in serum-free medium.
Wefoundthat
the addition of a neutralizing antibody
against TGF-0 enhanced the growth of murine erythroid
progenitor cells in serum-containing, but not serum-free cultures. Of particular interest was the fact that whereas Epo
alone stimulated no BFU-E colony formation in FCS-supplemented cultures, we found Epo to be a potent stimulator of
murine BFU-E colony formation in serum-free cultures when
acting alone. This was somewhat surprising, because there
seems to be a current opinion that the growth of BFU-E
progenitor cells requires the presence of a BPA in addition
to Epm?.5.7-Y.43Although some previous studies with more or
less unfractionated cell populations have shown formation
of BFU-E colonies in response to Epo alone, it has been
argued that the presence of accessory cells might have provided s,,&, activity.'.7'Y.'~.43.34.574" Because we reproduced the
effect of Epo alone in a single-cell assay as well, indirect
production of other cytokines seems unlikely.
The erythroid nature of the colonies formed in serum-free
medium in response to Epo alone, was confirmed by staining
positive with Ter-119, an antibody against an erythroid lineage-restricted antigen,3xas well as by erythroid cell morphology and benzidine ~taining.~'
The present studies in serum-free cultures suggest that a
BPA might not be required to stimulate the growth of BFUE progenitor cells, and offers an alternative explanation to
previous finding^.'^^^^^^^' Endogenous TGF-P might block the
growth of primitive BFU-Es in response to Epo alone,
whereas when they become more differentiated, they lose
the sensitivity to TGF-P. Similarly, the fact that only 20%
of BFU-Es have been shown to express Epo receptors4could
be explained by the potent ability of TGF-P to down-regulate
the expression of cytokine receptors.'"
As others," we found the stimulatory effect of IL-l I plus
Epo on BFU-E colony formation in serum-containing cultures to be very weak. However, anti-TGF-P increased the
BFU-E colony formation 25-fold. Even the BFU-E colony
formation in serum-containing cultures in response to more
potent erythropoietic combinations such as IL-4 plus Epo
and SCF plus Epo was enhanced threefold and fourfold when
anti-TGF-P was added. In contrast, the GM-CSF-induced
CFU-GM colony formation was reduced in the presence of
anti-TGF-P in serum-containing cultures. In addition, a very
low number of CFU-GM colonies was observed in serumfree medium, although in the presence of TGF-P1 the number of CFU-GM colonies was nearly the same as in FCS.
This observation is in agreement with the documented ability
of TGF-01 to synergize with GM-CSF to stimulate CFUGM colony formation of murine BM progenitors,'x and, in
fact, suggests thatthe ability of GM-CSF and TGF-B to
synergize is underestimated in FCS-containing cultures.
The potent effects of anti-TGF-0 in serum-containing
medium compared withno effects in serum-free cultures
could have several explanations. The presence of active
TGF-P in serum, was suggested by studies of Childs et aI4'
already in 1982. In agreement with this, we found active
TGF-01 in 20% FCS to be present at a concentration suffi-
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956
DYBEDAL AND JACOBSEN
cient to inhibit 75% of Epo-stimulated BFU-E colony formation. However, although this amount of active TGF-P1 can
potently inhibit erythroid colony formation, it is not sufficient to explain the complete absence of BFU-E colony formation in response to Epo in serum-containing cultures.
Therefore, alternative mechanisms might be involved. First,
FCS might contain low levels of other species of active TGFP, like TGF-03, which has been shown to be a particular
potent inhibitor of hematopoietic progenitor cell growth.”
Next, FCS might contain an activity capable of stimulating
production of additional active TGF-P by the target cells
population. Alternatively, the Lin- BM cells might produce
precursorAatent TGF-P, which requires an serum activity to
be activated. Finally, because other cell types have been
shown to be capable of activating latent TGF-P1,” itis
possible that Lin- cells might activate some of the abundant
latent TGF-P1 present in FCS. If active TGF-P1 is produced
by cells in serum-containing cultures, it mustinclude production by the progenitors themselves because anti-TGF-P enhances erythroid colony formation of Lin- cells plated individually. (I. Dybedal, unpublished observations, May 1994).
Regardless, the present data suggest that the addition of FCS
to cultures results in the presence of active TGF-P1 capable
of masking stimulatory and inhibitory effects of other cytokines. Furthermore, a serum-free culture system results in
erythroid progenitor cell growth similar to what is observed
in FCS-supplemented cultures in the presence of anti-TGF,B,and allows the growth of BFU-E colonies formed in response to Epo alone.
TGF-P in serum might of course be of importance in
regulating hematopoiesis in normal steady state as well as
pathologic conditions. TGF-P has been shown to be present
in plasma from normal
and increased serum
levels of TGF-P has been found in patients with thrombotic
thrombocytopenic
The present
well
as
as other
studies suggest that the use of anti-TGF-P might prove
useful in ex vivo expansion of BM progenitor cells.
In conclusion, TGF-P can directly and potently inhibit
murine BFU-E colony formation, and low levels of active
TGF-P1 present in serum can mask the effects of stimulatory
and inhibitory cytokines. Accordingly, serum-free cultures
show Epo alone as a potent stimulator of murine BFU-E
colony formation in vitro.
ACKNOWLEDGMENT
We thank Berit Sterdal and Randi Vik for skillful technical assistance, Jon Lamvik and Anders Waage for critical review of this
manuscript, and Emilio Barbera and Michael A. Palladino for helpful
suggestions and discussions.
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1995 86: 949-957
Transforming growth factor beta (TGF-beta), a potent inhibitor of
erythropoiesis: neutralizing TGF-beta antibodies show erythropoietin
as a potent stimulator of murine burst-forming unit erythroid colony
formation in the absence of a burst-promoting activity
I Dybedal and SE Jacobsen
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