0021-972X/92/7306-0447$03.

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Journal of Clinical Endocrinology and Metabolism Vol. 74, No. 2
Copyright 0 1992 by The Endocrine Society Printed in U.S.A.

Comparative Studies of the Erythroid-Potentiating

Effects of Biosynthetic Human Insulin-Like
Growth Factors-I and -II*
SHOSHANA MERCHAV, ILANA SILVIAN-DRACHSLER, ILANA TATARSKY,
MATS LAKE, AND ANNA SKOTTNER
Department of Hematology, Technion Faculty of Medicine (SM., IS-D., I.T.), Rappaport Family Institute
for Research in the Medical Sciences (S.M.), Haifa, Israel; and KabiGen (M.L.) and the Department
of Pharmacology, Kabi Peptide Hormones (A.S.), Stockholm, Sweden

ABSTRACT. The erythroid-potentiating effects of biosyn-
thetic human insulin-like growth factor-I (IGF-I) and IGF-II
were evaluated and compared in serum-free cultures of human
marrow erythroid progenitors. IGF-I and IGF-II enhanced the
in uitro growth of relatively mature (CFU-E) and primitive
(BFU-E) erythroid progenitors at similar dose-dependent mag-
nitudes. Significant elevations in erythroid colony counts were
detected at 0.2-0.6 ng/mL of both peptides, with a maximal
increase in CFU-E and BFU-E numbers detected at 2-6 ng/mL.
Similar enhancement of erythroid progenitor cell growth by both
peptides was also detected in cultures of marrow cells that had
been depleted of accessory cells or in cultures of highly enriched
hemopoietic progenitors. IGF-I and IGF-II enhanced erythroid
progenitor cell growth at both limiting and saturating concen-
trations of recombinant human erythropoietin or granulocyte-
macrophage colony-stimulating factor, but did not alter the
sensitivity of CFU-E and BFU-E to their respective hemopoietic
regulators. The erythroid-potentiating effects of IGF-I and IGF-
II were completely abrogated by monoclonal antibodies directed
against IGF-I membrane receptors. IGF-I and IGF-II thus ap-
pear to exert their effects on human marrow erythroid progeni-
tors via a direct mechanism involving the type I IGF receptor.
(J Clin Endocrinol Metab 73: 441-452, 1992)

T HE INSULIN -like growth factors (IGFs) are pep-

tides structurally similar to proinsulin (l-3). IGF-I
is the presumed mediator of GH in peripheral tissues (4),
erythrocytes (17, 18) and erythroleukemic
While the in vitro growth-promoting
on human primitive (BFU-E)
cells (19, 20).
effects of each IGF
and relatively mature
and IGF-II, whose function in the adult is presently (CFU-E) erythroid progenitors have been independently
undefined, has been proposed as a fetal growth regulator investigated (21-25), comparative analysis of the effects
(5). Both IGFs are potent mitogens for various cultured of both IGFs on these precursors has not yet been per-
mammalian cells (6); their effects are mediated via well formed. Furthermore, whereas the erythroid-promoting
characterized membrane receptors. The IGF-I (type I) effects of IGF-I are completely abrogated by antibodies
receptor resembles that of insulin, consisting of two directed against the type I receptor (24), the mechanism
subunits and possessing tyrosine kinase activity (7). The by which IGF-II enhances erythropoiesis in vitro is pres-
IGF-II receptor is a single polypeptide without kinase ently unknown.
activity (8), recently shown to be identical to the man- The present study was aimed at measuring and com-
nose-6-phosphate receptor (9). While apparent cross- paring the in vitro effects of biosynthetic human IGF-I
and IGF-II on human marrow erythroid progenitors. The
reactivity between IGF-I, IGF-II, and their receptors
role of the type I receptor as mediator of IGF-II was
exists for various cells (5, lo-12), recent evidence sug-
evaluated by using the anti-IGF-I receptor antibody aIR-
gests that both IGFs act predominantly via the IGF-I
3 (26).
receptor (13-16).
IGF-I and IGF-II receptors are present on human
Materials and Methods
Received October 19,199O. Preparation of marrow cells
Address all correspondence and requests for reprints to: Dr. Shos-
hana Merchav, Haemopoiesis Unit, Rappaport Family Institute for Human bone marrow aspirates from hematologicaly normal
Research in the Medical Sciences, Technion-Israel Institute of Tech-
nology, P.O.B. 9697, Haifa 31096, Israel. donors were diluted 3-fold in Hanks’ Balanced Salt Solution
* This work was supported in part by the Mitchell Copp Research and subjected to density gradient centrifugation. The mono-
Fund. nuclear cells (cl.077 g/cm3) were collected and washed.

447

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448 MERCHAV ET AL. .JCE & M. 1992
Voll4*No2

Removal of marrow accessory cells Chapel Hill, NC), was added at culture nitiation at a final
concentration of 15 pg/mL (100 nM). The addition of IGF-I
Marrow mononuclearcells were depleted of T- and B-lym- and IGF-II to cultures containing (YIR-3was delayedfor 2 h.
phocytes, monocytes, and natural killer cells by immunomag-
netic bead depletion (Dynabeads, Dynal, Oslo, Norway). The
Statistics
cellswere incubated for 30 min at 4 C with a cocktail of MY4
(Coulter, Hileah, FL), B4 (Coulter), OKT3 (Beckton Dickin- Statistical analyses were performed using the Student’s t
son, Sunnyville, CA) and NKHl (Coulter). Monoclonal anti- test or a one-way analysis of variance (ANOVA). Significance
bodies,washedtwice, and reincubatedwith Dynabeads(M-450, was defined as P < 0.05.
coatedwith antimouseimmunoglobulin), at 20 beads/cell.Dyn-
abead-bound(accessory)cells were depleted by attachment’to Results
a magnet.
Enhancement of erythroid colony formation by IGF-I
Enrichment of hemopoietic progenitors and IGF-II

This wasperformedby positive immunomagneticbeadselec- Biosynthetic IGF-I and IGF-II induced similar dose-
tion using monoclonal antibodies (ICH,) directed against the related growth-enhancing effects on human marrow
HPCA-1 membraneglycoprotein (27). Accessory cell-depleted CFU-E and BFU-E in serum-free cultures (Fig. 1). A
marrow cellswere incubated with ICH3, followed by immunom- significant increase in CFU-E (Fig. 1A) was detected at
agnetic bead separation at 0.025 bead/cell. Dynabead-bound 0.2 ng/mL IGF-I and IGF-II (P < 0.05, by ANOVA).
cells were attached to a magnet, the nonbound cells were Maximal enhancement of CFU-E by IGF-I (234 + 43%
discarded,and the bound cells (0.3 + 0.1% of initial mononu-
clear cells) were cultured. Microscopic examination of ICH3- 700, I
positive cells revealed0.5-3 beads/cell. s

1 600-
Colony assays
=v)
Marrow cells were cultured under serum-freeculture condi- ,‘: 500-
tions in modified Dulbecco’smedium(28) (Gibco, Grand Island, 0
NY), supplemented with 10 mg/mL bovine serum albumin
(Sigma, St. Louis, MO), 4 X 10m6M iron-saturated human A 400-
;a.
transferrin (Behringwerke, Darmstadt, Germany), 1 x 10m7M
w
selenite (Merck, Darmstadt, Germany), 5 x 10m5 M mercapto- 1 300
ethanol (Merck), 1 X 10m3 g/L nucleosides(Sigma), and 1.5 X z!
0
10m5 M eachof linoleic acid and cholesterol(Sigma),using 0.9%
methylcellulose(Methocel A4M, premium grade, Dow Chemi- 200 ,
0 0.2 0.6 2 6 20 CO
cal, St. Louis, MO) asviscoussupport. One-milliliter triplicate
cultureswere incubated in plastic petri dishes(Falcon, Cokeys- A IGF concentration (ng.ml-‘)
ville, MD) at 37 C in a humidified atmosphereof 7.5% CO, in
air. CFU-E werestimulatedwith 0.5 U/mL recombinant human
erythropoietin (rhEP0; 2.7 X lo5 U/mg protein; Boehrlnger-
Mannheim GmbH). BFU-E were stimulated with 2.5 ng/mL
recombinant human granulocyte-macrophagecolony-stimulat-
ing factor (rhGM-CSF; 1 X 10’ U/mg protein; Amgen, Thou-
sand Oaks, CA) and 1 U/mL rhEP0. IGF-I or IGF-II were
addedat the initiation of culture. Coloniesof 8-64 (CFU-E) or
>200 (BFU-E) hemoglobin-synthesizingcells were scored on
days 7 and 14, respectively.

Biosynthetic IGFs
Biosynthetic (recombinant) human IGF-I (lU/60 ng) and 5&Y
IGF-II (Kabi, Stockholm, Sweden) were more than 90% pure. 0 0.2 0.6 2 6 20 60
They were dissolvedand maintained at -20 C in culture me- B IGF concentration (ng.ml-‘)
dium containing 0.1% BSA. FIG. 1. Effects of IGF-I and IGF-II on the growth of human marrow
CFU-E and BFU-E. Cultures were supplemented with 0.2-60 ng/mL
Anti-IGF-I receptor monoclonal antibodies IGF-I (0) or IGF-II (0). CFU-E (A) were stimulated with 0.5 U/mL
rhEP0. BFU-E (B) were stimulated with 1 U/mL rhEP0 plus 2.5 ng/
An anti-IGF-I receptor monoclonal antibody, aIR-3 (26) mL rhGM-CSF. Results represent the mean f SEM of four independent
(provided by Prof. J. J. Van Wyk, University of North Carolina, experiments, performed in duplicate.

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 ERYTHROID-POTENTIATING EFFECTS OF IGF-I AND IGF-II 449

of control CFU-E frequency; P < 0.001) and IGF-II (189 induced by both peptides.
f 24%; P < 0.001) was found at 2 ng/mL of both IGFs As shown in Table 2, IGF-I and IGF-II were also
(P < 0.005, by ANOVA). similarly capable of erythroid colony enhancement in
Significant enhancement of BFU-E growth (Fig. 1B) cultures of highly enriched hemopoietic progenitors at
was detected at 0.6 ng/mL IGF-I and IGF-II (P < 0.05, both IGF concentrations tested. The magnitude of CFU-
by ANOVA), with maximal enhancement by IGF-I (186 E and BFU-E enrichment by the immunomagnetic bead
f 6%; P < 0.01) and IGF-II (176 f 17%; P < 0.001) method employed by us (14- and 32-fold, respectively)
detected at 2 and 6 ng/mL, respectively (P < 0.005, by was similar to that obtained by cell-sorting with MYlo
ANOVA). Enhancement of BFU-E was reflected not only antibodies (31) directed against the same membrane
in colony numbers, but the colonies also appeared some- marker as ICH3 (27).
what larger in size. The CFU-E and BFU-E numbers
detected in the presence of either IGF did not differ Effect of IGF-I receptor antibody on IGF-II-induced
significantly from each other. colony enhancement
The role of the type I receptor in the erythroid-en-
Interactions of IGF-I or IGF-II with erythropoietin and
hancing effects of IGF-II was evaluated with a monoclo-
GM-CSF
nal antibody (cuIR-3) that blocks the hormone-binding
The mode of action of IGF-I and IGF-II in relation to domain of that receptor (26). As shown in Table 3, (rIR-
erythropoietin was evaluated by assaying the effect of 3 completely abrogated the effect of IGF-II on CFU-E
both IGFs on CFU-E growth at various doses of rhEP0. and BFU-E at all concentrations tested. &R-3 also
Both IGFs similarly enhanced the growth of CFU-E at blocked the effect of IGF-I, while CFU-E and BFU-E
both limiting and saturating concentrations of rhEP0 growth in cultures with aIR-3 alone remained un-
(Fig. 2A), without altering the progenitor cell respon- changed.
siveness to this regulator (Fig. 2B). Neither peptide
stimulated any colony formation in the absence of Discussion
rhEP0. A similar phenomenon was found in cultures of Primarily regulated by erythropoietin (32, 33), mam-
BFU-E at various concentrations of rhGM-CSF (Fig. 3), malian erythropoiesis is also modulated by various hor-
the latter regulator required for the initial stages of their mones and growth peptides (34). The latter include IGF-
growth (29, 30). I and IGF-II, whose erythroid-enhancing effects have
been independently studied with purified native (22, 25)
Role of accessorycells in colony enhancement by IGF-I
or recombinant (21,23,24) molecules. Aimed at compar-
and IGF-II
ing the effects of biosynthetic IGF-I and IGF-II on
It was recently shown that the effect of IGF-I on human erythropoiesis, our study provides strong evi-
human CFU-E is not mediated by marrow accessory cells dence for a common mechanism of action for both IGFs
(23). The effects of IGF-I and IGF-II were further com- in the enhancement of erythroid progenitor cell growth.
pared in the absence of marrow accessory cells. As shown A strikingly similar dose-dependent magnitude of
in Table 1, accessory cell depletion led to a 2- to 3-fold erythroid colony enhancement was induced by biosyn-
enrichment of CFU-E and BFU-E, but did not alter the thetic IGF-I and IGF-II in serum-free cultures of human
similar dose-related magnitude of colony enhancement marrow CFU-E and BFU-E. The effect of IGF-II was

0°1
90
80
FIG. 2. Effects of IGF-I and IGF-II on
erythropoietin response of human mar- 70
row CFU-E. A, Absolute colony fre- 60
quency. B, Percentage of maximum col-
onies. Cultures stimulated with 0.1-2.5
U/mL rhEP0 (0) were supplemented
with 6 ng/mL IGF-I (0) or IGF-II (0).
Results represent the mean + SEM of
three independent experiments, per-
formed in duplicate.

0 0.1 1 10
Erythropoietin (U/ml) Erythropoletin (U/ml)

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450 MERCHAV ET AL. JCE & M. 1992
Vol74.NoZ

500- A
g 450-
&T
FIG. 3. Effects of IGF-I and IGF-II on 11
GM-CSF response of human marrow
BFU-E. A, Absolute colony frequency.
B, Percentage of maximum colonies. Ac- f
cessory cell-depleted marrow cells stim-
ulated with 0.75-5 ng/mL rhGM-CSF
(0) were supplemented with 6 ng/mL
IGF-I (0) or IGF-II (0). Results repre-
sent the mean f SEM of triplicate cul-
tures.
,
0.75 1.25 2.5 5.0
GM-CSF (rig/ml) GM-CSF (&ml>

TABLE 1. Effects of IGF-I and IGF-II on human marrow CFU-E and TABLE 2. Effects of IGF-I and IGF-II on human marrow CFU-E and
BFU-E in accessory cell-depleted serum-free cultures BFU-E in cultures of highly enriched haemopoietic progenitors

Cont. CFU-E” BFU-E” Cont. CFU-E” BFU-E”

(w/ml) (4
IGF-I IGF-II IGF-I IGF-II IGF-I IGF-II IGF-I IGF-II
ml)
0 567 f 88 196 + 23
0 2800 + 392 1925 + 203
0.2 974 + 215b 881 f 166 299 + 46 210 + 17 6 3850 + 462’ 4350 f 478’ 2675 f 232’ 2725 f 327
0.6 1065 f 177 964 f 92* 354 f 46’ 334 + 30’ 20 4500 f 474d 5100 f 510’ 2850 f 313’ 3200 + 330d
2.0 1135 f 122 1051 f 63 366 & 41 361 f 45
6.0 llOOf84 1036 + 60 371 f 45 359 + 43 Accessory cell-depleted marrow cells were incubated with ICH,
20.0 1097 f 66 1087 + 120 364 f 45 364 f 51 monoclonal antibodies, followed by positive selection with immuno-
60.0 1129 + 98 1044 f 78 355 + 44 358 + 37 magnetic beads. Cells were cultured at 4 x 103/mL in the presence of
0.5 U/mL rhEP0 (CFU-E) or 1 U/mL rhEP0 plus 2.5 ng/mL rhGM-
Marrow mononuclear cells were depleted of T- and B-lymphocytes, CSF (BFU-E). Cultures were supplemented with 10% fetal calf serum.
monocytes and natural killer cells by immunomagnetic bead separation. The frequency of CFU-E and BFU-E in the initial marrow mononuclear
Accessory cell-depleted marrow cells were cultured at 5 x lO’/ml in the cell suspension was 203 f 38/2 X lo5 and 56 f 6/l X lo5 cells,
presence of 0.5 U/mL rhEP0 (CFU-E) or 1 U/mL rhEP0 plus 2.5 ng/ respectively. Results represent the mean f SEM of three independent
mL rhGM-CSF (BFU-E). Results represent the mean + SEM of three experiments, performed in triplicate.
independent experiments, performed in triplicate. Significance was ’ CFU-E per 2 x lo5 and BFU-E per 1 X lo5 cells, respectively.
determined by ANOVA. * P < 0.05.
a CFU-E per 2 x 10’ and BFU-E per 1 x lo5 cells, respectively. c P c 0.02.
bP < 0.05. d P < 0.01.
c P < 0.01. e P c 0.001.

completely abrogated by aIR-3 antibodies, indicating

that the erythroid-promoting effects of this peptide are TABLE 3. Abrogation of IGF-II-induced enhancement of CFU-E and
BFU-E growth by an anti-IGF-I receptor antibody
mediated by the type I receptor.
Previous studies have shown a strong correlation be- CFU-E BFU-E
tween the affinity of IGF-II (relative to IGF-I) to the Stimulus
-aIR-3 +cuIR-3 -czIR-3 +aIR-3
type I receptor and its relative mitogenic potency in vitro
(13-16). While receptor binding studies for both IGFs on IGF-II (6 rig/ml) 433 + 43 289 + 26 131 k 11 80 + 9
IGF-II (20 rig/ml) 445 f 46 290 + 28 128 + 11 84 2 9
CFU-E and BFU-E are limited by the highly reduced IGF-II (60 rig/ml) 418 f 40 278 f 28 129 + 13 83 + 9
frequency of these cells in human marrow and by the
inability to distinguish them from coisolated IGF-re- IGF-I (6 rig/ml) 456 + 44 287 f 25 114 f 13 82 f 9
sponsive myeloid progenitors (27,35) (Merchav, S., Tat- IGF-I (20 rig/ml) 485 + 45 277 + 26 130 f 17 8528
sky, I., Lake, M., Shotter, A., submitted), the similar IGF-I (60 rig/ml) 457 + 46 280 + 25 139 + 17 83 f 8
erythroid-promoting effects we detected for both IGFs None 278 f 35 270 f 26 79 -c 9 78 + 8
agree well with their reported similar affinities for type
Results represent the mean f SEM of three (CFU-E) and two (BFU-
I receptors on unfractionated (17) and young (reticulo- E) independent experiments, performed in duplicate.
@e-enriched) (18) human erythrocytes. Although IGF- a (uIR-3 was added at a final concentration of 15 Fg/mL (100 nM).
II was previously shown to stimulate human K562 ery- The addition of IGF-I and IGF-II to cultures was delayed for 2 h.

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 ERYTHROID-POTENTIATING EFFECTS OF IGF-I AND IGF-II 451

throleukemic cells via the IGF-II receptor (20, 36), the 3. Bell GI, Merryweather JP, Sanchez-Pescador R, et al. Sequence of
a cDNA clone encoding human proinsulin-like growth factor II.
unique cell line described in these studies did not express Nature. 1984;310:775-7.
IGF-I receptors at all, in contrast to other existing lab- 4. D’Ercole AJ, Stiles AD, Underwood LE. Tissue concentrations of
oratory variants (19). The inability of IGF-I to stimulate somatomedin C: further evidence for multiple sites of synthesis
and paracrine or autocrine mechanisms of action. Proc Nat1 Acad
K-562 proliferation via the IGF-II receptor (37) agrees Sci USA. 1984;81:935-9.
well with the complete abrogation of IGF-I-induced 5. Zapf J, Froesch VR. Insulin-like growth factors/somatomedins:
erythroid colony growth by (uIR-3. The possibility that structure, secretion, biological actions and physiological roles.
Horm Res. 1986;24:121-30.
IGF-II may act indirectly via IGF-I production by mar- 6. Van Wyk, JJ. The somatomedins: biological actions and physio-
row accessory and/or progenitor cells is hardly unlikely, logic control mechanisms. In: Li CH, edy Hormonal proteins and
since IGF-II was also effective in cultures of accessory- neDtides. New York: Academic Press: 1984:12:82-120.
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than unseparated marrow cells. The slight (but insignif- 8. Morgan DO, Edman JC, Standring DN, et al. insulin-like growth
factor II receptor as a multifunctional binding protein. Nature.
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ing enriched progenitors with 20 us. 6 ng/mL IGF-I or 9. MacDonald RG, Pfeffer SR, Coussens L, et al. A single receptor
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inhibitors) may interfere with IGF-progenitor cell inter- over at the hormone receptor-exploring its role in human disease.
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452 MERCHAV ET AL. JCE & M .1992
Vol74.No2

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