1. Healthcare

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Effects of the Stem Cell Factor, c-kit Ligand, on Human Megakaryocytic Cells
By Hava Avraham, Edouard Vannier, Sally Cowley, Shuxian Jiang, Sheri Chi, Charles A. Dinarello, Krisztina M. Zsebo, and
Jerome E. Groopman
The kit ligand (KL), also termed stem cell factor (SCF), is a
recently discovered hematopoietic growth factor that augments response of early progenitor cells t o other growth
factors and supports proliferation of continuous mast cell
lines. Histological studies suggest that the receptor for
SCF/KL, the c-kit proto-oncogene product, is present in bone
marrow megakaryocytes. We studied the effects of SCF/KL
on immortalized human megakaryocytic cell lines (CMK,
CMK6, and CMK11-5) and on isolated human marrow megakaryocytes. Human SCF/KL alone or in combination with the
hematopoietic growth factors, interleukin-3 (IL-3). granulocyte-macrophage colony-stimulating factor (GM-CSF), and
IL-6, stimulated proliferation of these megakaryocytic cell
lines. SCF/KL treatment did not alter expression of gplb,
gpllb/llla, LFA-1, ICAM-1, or GMP-140 in CMKcells. No effect
on ploidy was observed. Furthermore, human SCF/KL induced expression of IL-la, IL-lp, IL-2, and IL-6 in CMK cells. In
a fibrin clot system, SCF/KL modestly potentiated megakaryocyte colony formation when added alone t o cultures
containing CD34’, DR’ bone marrow cells. Addition of SCF/KL
with IL-3 or GM-CSF t o these cultures resulted in a more
marked increase in colony number. Modest stimulatory effects were observed when SCFfKL was added t o more
mature marrow megakaryocytic cells. SCF/KL may directly
affect megakaryocytopoiesis, as well as secondarily modulate hematopoiesis through induction of cytokines in target
cells.
o 1992b y The American Society of Hematology.
A
vided by Amgen (Thousand Oaks, CA). IL-1p was provided by Dr
C. Dinarello (Tufts University, Boston, MA). Plateau doses of each
factor were determined from dose-response curves and used for
culture at the following concentrations: SCFKL, 100 ng/mL; IL-6,
10 ngimL; IL-lp, 10 ngimL; IL-3, 10 ngimL; GM-CSF, 200
ng/mL; and G-CSF, 500 ng/mL.
Monoclonal antibodies. Monoclonal antibodies for gpIb and
gpIIbiIIIa, as well as other monoclonal antibodies for glycophorin
A, CDla, CD3, CD4, CD8, CD9, CD1.5, CD16, CD33, CD34, and
HLA Class I, were supplied by Becton Dickinson (Mountain View,
CA). Monoclonal antibodies TS1122 and TSlIl8 recognize the
LFA-la subunit and LFA-1f3subunit, respectively, and were kindly
provided by Dr T.A. Springer (Center for Blood Research,
Harvard Medical School, Boston, MA).’ Monoclonal antibody
RR1/1, which recognizes ICAM-1 (CD54):was also provided by
Dr T.A. Springer. Polyclonal antibodies against GMP-140 were
kindly provided by Dr Bruce Furie (Tufts University Medical
School).
Cell culture. Three different clones of the immortalized parent
megakaryocytic cell line CMK (termed CMK6, CMK11-5, and
CMK cells) were grown in RPMI 1640 medium with 10% fetal calf
serum (FCS) or with 7.5% platelet-poor plasma supplemented with
2 mmoliL L-glutamine, and 50 kg/mL penicillin and streptomycin
(GIBCO Laboratories, Grand Island, NY), as described.“’.”
The immortalized Dami megakaryocytic cell line’’ and the
immortalized CHRF megakaryocytic cell line’3 were kindly provided by Dr S.M. Greenberg (Brigham and Women’s Hospital,
Boston, MA) and Dr L. Zon (Children’s Hospital, Boston, MA),
HEMATOPOIETIC growth factor derived from mesenchymal cells that supports the proliferation of early
hematopoietic progenitors and of permanent mast cell lines
was recently identified.’” The cell surface receptor for this
hematopoietic growth factor has been identified as the
product of the c-kit proto-oncogene. The growth factor has
been termed kit ligand (KL), stem cell factor (SCF), and
mast cell growth factor (MGF), based on its in vitro effects
with respect to growth stimulation and receptor binding.
Recent studies in non-human primates demonstrate that
recombinant human SCF/KL is biologically active and
augments production of cells of myeloid and erythroid
lineages: The absolute number of megakaryocytes in the
bone marrow also increased after SCF/KL administration.
A modest increase in circulating platelet number was
observed in these initial studies. Histologic studies of
normal human bone marrow using specific antibodies to the
c-kit protein have suggested expression of this surface
structure on immature blast-like cells and on megakaryocytes.*
We have studied the effects of human SCFIKL. on several
permanent human megakaryocytic cell lines to provide a
model of its modulation of growth and function in cells of
this lineage.5 SCF/KL significantly increased the proliferation of immortalized human megakaryocytic cell lines in
vitro. SCF/KL also induced gene expression for cytokines.
These studies using the CMK cell lines as a model prompted
us to investigate the effects of SCF/KL alone and in
combination with other hematopoietic growth factors on
human megakaryocytopoiesis. Using a serum-depleted fibrin clot assay system, we observed proliferative effects of
SCF/KL on the CD34+DR’ human bone marrow subpopulation, which is enriched for the colony-forming unit
megakaryocyte (CFU-MK)6 and on megakaryocytes isolated by immunomagnetic beads’ using anti-human glycoprotein gpIIb/IIIa monoclonal antibody.
MATERIALS AND METHODS
Growth factors. Human SCFiKL was cloned and expressed in
Escherichia coli and purified as previously described.” Human
SCF/KL, human interleukin-3 (IL-3), human granulocyte-macrophage colony-stimulating factor (GM-CSF), human IL-6, and
human granulocyte colony-stimulating factor (G-CSF) were proBlood, Vol79, No 2 (January 151,1992:pp 365-371
From the Division of HematologyiOncology, New England Deaconess Hospital, Harvard Medical School, Boston, MA; the Division of
GeographicMedicine and Infectious Diseases, Tufts University School
of Medicine, Boston, MA; and Amgen, lnc, Thousand Oaks, CA.
Submitted March 15, 1991; accepted September 19, 1991.
Supported in part by Grants No. HL33774, HL42112, HL46668,
A129847, and AI-15614from the National Institutes of Health, and by
the Department of Defense Grant No. DAMD 17-90-C-0106.
Address reprint requests to Jerome E. Groopman, MD, Division of
HematologyiOncology, New England Deaconess Hospital, 110 Francis St, 4A, Boston, M A 02215.
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 I 8 U.S.C. section 1734 solely to
indicate this fact.
0 1992 by The American Society of Hematology.
0006-49711921 7902-0025$3.00/0
365
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366
respectively. The Dami cell line was grown in Iscove's modified
Dulbecco's medium (IMDM) containing 10% horse serum. The
CHRF cell line was grown in Fisher's medium containing 25%
horse serum.
Bone marrow preparation and fractionation of marrow cells. Ten
milliliters of bone marrow aspirate was collected from one site of
the sternal bone or posterior iliac crest of normal volunteers who
had given informed consent. Cells were collected into 20-mL
plastic syringes containing 1/10 vol of acid-citrate-dextrose
(ACDIA) and EDTA to a final concentration of 2.5 mmoliL. One
volume of marrow aspirate was mixed with 1 vol of MK medium,
which consisted of Ca2'-MgZ' free phosphate-buffered saline
(PBS) containing 13.6 mmoliL sodium citrate, 1mmoliL theophylline, 1% bovine serum albumin (BSA, fraction V; Sigma Chemical,
St Louis, MO, and 11 mmoliL glucose, adjusted to pH 7.3 and an
osmolarity of 290 mOsmiL. The cell suspension was layered over
two different kinds of Percoll(5 mL; Pharmacia, Uppsala, Sweden)
at densities of 1.020 g/mL and 1.050 g/mL containing 13.6 mmoliL
sodium citrate. The 1.020 Percoll density cut was helpful in
preventing aggregate formation, and most morphologically identifiable megakaryocytes were contained in the fraction heavier than
1.020 g/mL and lighter than 1.050 g/mL. After centrifugation at
400 x g for 20 minutes, the upper medium layer was removed, and
the cells were collected from the interface ( < 1.050 g/mL). These
cells were washed twice with MK medium and adjusted to a
concentration of 1 to 5 x 106/mLwith MK medium.
Isolation of megakaiyocytes. The light-density ( < 1.050 gimL)
cells were mixed with 10 ILLof monoclonal antibody directed to the
human platelet glycoprotein (gp)IIb/IIIa complex and incubated
for 1 hour at 4°C. After washing completely with MK medium, 20
KLof 10 times diluted immunomagnetic beads (4 x lo7beadsiml)
coated with anti-mouse IgG antibody (Dynabeads M-450, Dynal,
Oslo, Norway) were added to the cell suspension in 1mL. The cells
rosetted with immunomagnetic beads were collected with a Dynal
magnetic particle concentrator (Dynal MPC l),washed three times
with MK medium, and resuspended with 10% FCS in RPMI 1640
medium.
Serum-depleted assay system. Megakaryocyte progenitor cells
were isolated and separated into a nonadherent, low-density,
T-cell-depleted (NALDT), marrow subpopulation.6 The NALDT
were further enriched for megakaryocyte progenitors (CFU-MK)
by using monoclonal antibodies for anti-CD34 and anti-HLA-DR.
The NALDT- marrow cells consistently contained greater than
94% CD34 and HLA-DR-positive cells. Approximately, 5 X lo3
CD34+, DR+ were assayed for their ability to produce CFU-MK
derived colonies in a serum-depleted fibrin clot culture system as
described.I4Various concentrations of SCF/KL, alone or in combination with other hematopoietic growth factors, were added and
the cultures were incubated for 12 to 14 days at 37°C in a 100%
humidified atmosphere of 5% CO, in air.
Viability and identification of megakaryocytes. Viability of separated cells was assessed by trypan blue exclusion. Megakaryocytes
were identified by Wright's staining and flow cytometric expression
of gpIIbiIIIa and gpIb. The maturation stages of the megakaryocytes were classified by cell size, nuclear morphology, and cytoplasmic staining.'
Flow cytometiy. For flow cytometry, 5 x lo5cells were exposed
to monoclonal antibodies (4"C, 30 minutes), washed three times,
and followed by fluorescein-conjugated goat anti-mouse IgG (Boehringer Mannheim Biochemicals, Indianapolis, IN) (150 dilution in
Hanks balanced salt solution [HBSS] with 0.1% BSA, at 4°C for 20
minutes) and fixed in 1% paraformaldehyde in PBS.
Immunojuorescentstainingfor GMP-140. The CMK cells grown
on glass coverslips were fixed in PBS containing 3.7% formaldehyde for 20 minutes and permeabilized for 15 minutes with 0.5%
AVRAHAM ET AL
Triton X-100 in the same buffer. Antibodies to GMP-140 (50
KgimL) were added for 30 minutes at 4"C, washed three times, and
followed by fluorescein-conjugated goat anti-rabbit IgG (Boehringer-Mannheim) (1:50 dilution in HBSS with 0.1% BSA, at 4°C
for 20 minutes). The coverslips were mounted in Geevatol and
photographed through a Zeiss Axioscope microscope (Carl Zeiss,
Germany) equipped with a fluorescein filter set.
Preparation ofphorbol-l2-myristaie-I
3-acetate (PU4)-treated CMK
cells. PMA (Sigma) was dissolved in dimethylsulfoxide (DMSO)
and stored at 80°C.Just before use, PMA was diluted in the RPMI
culture medium. Cells were incubated with PMA at a concentration of 50 ngimL or 100 ng/mL at 37°C in a 5% CO, humidified
atmosphere for 3,6,24, or 48 hours as indicated.
Flow cytometric measurement of DNA content of CMK cells.
CMK cells were seeded at 2 x lo5 cells and fed again after 2 days.
Cells were harvested after 5 days and the nuclei were isolated,
stained with propidium iodide, and analyzed on a Becton Dickinson FACS Analyzer as previously described.I2 Freshly prepared
lymphocytes were used to mark the position of the 2N cells.
Proliferation assays. Cell proliferation and viability was assessed by ['Hlthymidine incorporation and by trypan blue exclusion
(0.4% trypan blue stain in 0.85% saline; GlBCO Laboratories).
Dilutions of growth factors were made in 96-well flat-bottom tissue
culture plates (Costar, Cambridge, MA) in the appropriate growth
medium containing either 1% platelet-poor plasma or 1% FCS in a
final volume of 50 pL. For [3H]thymidine incorporation assays,
cells were seeded at time zero (50 KL vol) and the plates were
incubated at 37°C in a humidified atmosphere of 5.5% CO, for 48
hours. Cells were pulsed with 0.5 Ci per well of [-'H]thymidine (25
Aimmol; DuPont-New England Nuclear, Boston, MA) and incubated for an additional 5 hours. Samples were harvested onto glass
fiber filters and counted by liquid scintillation spectrometry. The
concentrations of cytokines added at the initiation of the cultures
were SCFIKL, 100 ng/mL; IL-3,10 ng/mL; GM-CSF, 200 ng/mL;
IL-6,10 ng/mL; IL-lp, 0.5 ng/mL; and G-CSF, 500 ng/mL.
RNA isolation and Northern blot analysis. Total RNA from
CMK cells was extracted by the guanidine isothiocyanate procedure, followed by ultracentrifugation through a CsCl cushion.15For
each sample, 20 pg of total RNA was electrophoresed in a 1%
agarose, 6% formaldehyde gel, and blotted to an S & S Nytran
membrane (Schleicher & Schuell, Keene, NH). The blot was
prehybridized as described' for 6 hours and then hybridized
overnight at 42°C with radiolabeled probe (1 to 3 x lo6cpm/mL).
The blot was washed as described' and exposed to Kodak XAR-5
film (Eastman Kodak, Rochester, NY) with intensifying screens at
-70°C.
DNA probes. Expression of the c-kit gene was assessed by
Northern blot analysis using a 1,250-bp fragment of human c-kit
gene subcloned in phc-kit 171 (ATCC 59492).
Cytokine analysis. Total cellular RNA was extracted from the
CMK cell lines, as indicated above, with or without SCFiKL
treatment (100 ng/mL). Total RNA was run on a 1.2% formaldehyde agarose gel and the intact RNA was visualized by ethidium
bromide staining. Reverse transcription (RT) of RNA was performed using 2 pg of total RNA from each sample and polymerase
chain reaction (PCR) assays for each primer set was set up
according to Clontech's Cytokine Mapping PRI-MATE methods
(Clontech Laboratories, Palo Alto, CA). The following human
primer sets were used: IL-la (816 bp); IL-1p (811 bp); IL-2 (453
bp); GM-CSF (420 bp); tumor necrosis factor-a (TNF-a) (691 bp);
p-actin primer set (584 bp, or 300 bp, or 100 bp).
The products for each set were analyzed on a 2% agarose gel
(BRL, Bethesda, MD). The amplified DNA bands were visualized
with a UV transilluminator. Internal reaction standards for PCR
controls were performed for each set of primers, which included
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367
SCF/KL, EFFECTS ON MEGAKARYOCYTIC CELLS
RNA with or without primers: primers without RNA: primers with
target DNA provided by the PCR kit (Cetus-Perkin Elmer,
Emeryville, CA) as positive control; and mRNAwith actin primers.
All the primer stocks and total mRNA preparations were analyzed
to exclude contamination by cellular DNA. These studies for
cytokine expression provide qualitative information about the
presence or absence of mRNA for cytokines.
First-strand cDNA synthesis. First-strand DNA was synthesized
at 37°C for 1 hour in a final volume of 10 p L with oligo-dT as
primer: 4.5 p L RNA in DEPC-dH,O. 2.0 pL 5 x buffer (250
mmol/LTris-HCI, pH 83,375 mmol/L KCI, 50 mmol/L dithiothreitol, 15 mmol/L MgCI,, and 250 pglmL actinomycin D), 0.5 p L
RNAsin (40 UlpL, Promega, Madison, WI), 1.0 p L dNTP (dATP,
dCTP, dGTP, dTTP mix, 10 mmol/L each: Pharmacia, Piscataway,
NJ), 1.0 FL oligo-dT, 1.0 p L Moloney murine leukemia virus
(MMLV) RT (200 UlmL, Boehringer-Mannheim. Chicago, IL).
PCR. Eighty microliters of PCR mix was added to 10 pL of
first-strand cDNA. PCR mix contains 53.5 p L sterile water, 10 p L
I0x reaction buffer, 16 p L dNTP mix (each at 1.25 mmol/L), and
0.5 p L (2.5 U) of the Thennus aquaticits thermostable DNA
polymerase (Cetus-Perkin Elmer). PCR reaction buffer ( 1 0 ~ )
contains 500 mmollL KCI, 100 mmol/L 0.1% gelatin. Five microliters of each primer (final primer concentration, 1 FmollL) was
added and the mixture was then subjected to PCR amplification
using the Perkin-Elmer thermal cycler set for 40 cycles. The
temperatures used for PCR were denature 94°C. 1 minute: primer
anneal 5ST, 2 minutes: primer extension 72"C, 3 minutes. Normally. I-minute ramp times were used between these temperatures. Ethidium bromide-stained 2% agarose gels were used to
separate PCR fragments.
c-kit analysis hv PCR. Total RNA from CMK cells was used as a
template for reverse transcription and PCR assays for c-kit primer
set was set up as described above. The primers were provided by
Amgen.' The expected size of the c-kit cDNA synthesized, using
the c-kit primers, was 143 bp, and was verified by hybridization
using the c-kit probe.
Cytokine assays. For cytokine assays, CMK cells were grown in
RPMI 1640containing 1% platelet-poor plasma at a density of 5 x
10"lmL. Cell suspensions were centrifuged for 10 minutes at 1,200
rpm. Supernatants and cell pellets were assayed for cytokine
production by specific radioimmunoassays (RIAs) as described for
TNF-a,16IL-lp,'7IL-la," and GM-CSF."The sensitivities (defined
as 95% binding) of RIAs for TNF-a, IL-Ip, IL-la. IL-6, and
GM-CSF were 42 13,32 f 3,28 f 7.22 2 2, and 25 4 pg/mL,
respectively.''' In addition, cytokine assays were performed on
CMK cells stimulated with SCF/KL (100 ng/mL), GM-CSF (200
ng/mL), G-CSF (500 ng/mL), IL-6 (10 nglmL), IL-3 (10 ng/mL).
IL-Ip (10 ng/mL), or combinations of these cytokines for 48 hours.
Supernatants and cell pellets were assayed for cytokine production.
Statistical ana!vsis. The results were expressed as the mean 2
SEM of data obtained from two or more experiments performed in
duplicate or triplicate. Statistical significance was determined using
the Student's t-test.
*
IIIa, which is a structure specifically expressed on megakaryocytcs and platelets, was expressed in 71% of the less
mature CMK6 cells, approximately 86% of CMK cells, and
94% of more mature CMKll-5 cells.
Effect of SCFIKL on proliferation of immortalized CMK
cells. The CMK megakaryocytic cell lines were analyzed
for their proliferative response to various concentrations of
exogenous SCF/KL. As shown in Fig 1, all the megakaryocytic ccll lines exhibitcd a proliferative response to SCF/KL
in a dose-dependent fashion as asscssed by counting the
number of viable cells. The CMK cell lines showed higher
proliferation rates in rcsponsc to SCF/KL as comparcd
with the Dami and CHRF cell lines. Maximum growth
stimulation was seen at a SCF/KL concentration of 10
ng/mL in evcry ccll line tested. However, the maximum
growth reached by CMK ccll lines after 48 hours was higher
compared with the maximum growth reached by Dami or
CHRF cclls.
Prolifcration of CMK cclls was augmcntcd by combinations of human SCF/KL with the hematopoietic growth
factors IL-3, GM-CSF, and IL-6 (Fig 2). These proliferation assays were initially performed at submaximal concentrations of each growth factor and then at optimal combinations of these growth factors as described in the Methods.
We did not observe any pronounced enhancement when
suboptimal concentrations of these cytokines were used.
The combination of human SCF/KL with GM-CSF, IL-3,
or IL-6 resulted in higher levels of stimulation of CMK cell
growth, but the effects were not fully additive.
Effects of human SCFIKL on CMK cellfrtnction. Human
SCF/KL treatment over a range of concentrations (10
ng/mL, 50 ng/mL, or 100 ng/mL) did not alter CMK cell
surface expression of gpIb, gpIIb/IIIa, LFA-1, or ICAM-1,
and did not induce expression of GMP-140 (data not
shown). No effect on CMK ploidy was observed (data not
shown).
Effects of human SCFIKL on cytokine expression in CMK
cells. The effects of SCF/KL on the gene expression for
cytokines in CMK cells were studied using PCR analysis.
0 CMK-6
0 CMK
t
Z
2 CMK11-5
..
IDAM1
ICHRF
RESULTS
Expression of surface antigens by CMK cell lines. To
explore the effects of purified recombinant human SCF/KL
on human megakaryocytes, experiments were performed
using the three immortalized CMK clones (CMK6, CMK,
and CMKll-5), as wcll as the Dami and CHRF human ccll
lines. The characterization of Dami cells" and CHRF cells"
has been previously reported. For the three CMK clones,
lymphoid surface antigens wcrc uniformly absent, as were
those of granulocytes, monocytcs, and macrophages. gpllb/
0
1
5
fi..
[
100
SCF/KL ( n g / m l )
Fig 1. Effect of SCF/KL on the prolieration of human megakaryocytic cell lines. Cells (2 x lo5cells/mL) were cultured for 48 hours in
the presence of SCF/KL (1 to 100 ng/mL). Cell numbers were
determinedby counting viable cells by trypan blue exclusion. Data are
mean
SEM of pooled data from three separate experiments
performed in duplicate. (0)Statisticallysignificant as compared with
the control culture (P < .OS)
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AVRAHAM ET AL
368
11-6
GMCSF
Cylokines
Fig 2. Effect of SCF/KL on proliferation of CMK cells in the
presence of IL-3 or GM-CSF. CMK cells (6 x 10'/mL) were cultured
with SCF/KL (100 ng/mL), IL-3 (10 ng/mL). GM-CSF (200 ng/mL), or
combinations of these factors as indicated in Materials and Methods.
Cell numbers were determined by trypan blue exclusion. Data are
reported as mean -t SEM of triplicate cultures. ( 0 )Statistically
significant as compared with the control culture (P < .05).
Total RNA from CMK cells was used as a template for RT
reactions. PCR mapping was performed using specific
primers for each cytokine or cytokine receptor. These
studies provided a readout of mRNAs being produced by
CMK cells at a given point in time for the presence or
absence of the examined cytokines. A number of cytokine
genes were induced by treatment of CMK cells with human
SCF/KL (Fig 3). There appeared to be differences in the
IL1-p
IL1-a
'A B C
' A B C
DI
I L-6
'A B C
kinetics of expression of different genes with SCF/KL
treatment. CMK cells were found to constitutively express
moderate levels of mRNA for TNF-a and IL-1p (Fig 3). No
induction of GM-CSF expression was observed after stimulation. Cytokine expression was detected after 6 hours of
SCF/KL stimulation, which included IL-la, IL-2, and IL-6.
This repertoire of cytokine mRNA expression was also
present after 24 hours of SCF/KL treatment (Fig 3). All
PCR standard controls were negative. DNA-positive controls with each primer set were positive, as well as the
p-actin primers.
Effect of human SCFIIU on cytokine production in CMK
cells. Studies for cell-associated or secreted GM-CSF,
TNF-a, IL-2, or IL-la by RIA showed that none of these
cytokines was readily detectable in cultures of the unstimulatcd CMK cells (data not shown). Cell-associated IL-1p
protein was detected (Fig 4) in unstimulated CMK cells, as
well as after 3 hours and 24 hours of SCF/KL treatment. By
48 hours of SCF/KL treatment, the level of IL-lp was
decreased. Low levels of TNF-a and IL-la were detected as
secreted and cell-associated forms. RIA for IL-2 showed
very low levels of secreted and cell-associated protein after
SCF/KL treatment (data not shown). Although SCF/KL
had a modest effect on IL-2 production by CMK cells,
combinations of SCF/KL with GM-CSF or IL-3 markedly
increased the level of secreted and cell-associated IL-2
protein (data not shown).
D'
._' A
9
2322
D
GM-CSF
B C D'
I L-2
E F '
' A B C
TNF-a
A B C
D'
Actin
D
IG H I
J'
-
-
564
4204
--
500
300
+loo
Fig 3. Mapping of CMK cells using amplimer sets for cytokines at 6 and 24 hours poststimulation with SCF/KL. Total RNA from CMK cells
(10 x 10') or bone marrow megakaryocytes (10' cells) was prepared and mapped as described in Materials and Methods for the following
cytokines: IL-la, IL-1s. IL-2, IL-6, GM-CSF, TNF-a, and pactin. Lanes: A, RNAfrom untreated CMK cells; B, 6 hours poststimulation with SCF/KL;
C, 24 hours poststimulation with SCF/KL; D, DNA-positive control with each specific set primers; E, 6 hours poststimulation of isolated
megakaryocytes by immunomagnetic beads; F, 24 hours poststimulation of isolated megakaryocytes by immunomagnetic beads; G. pactin
primers with RNA from untreated CMK cells; H, pactin primers with RNA from SCF/KL stimulated CMK cells for 6 hours; I, pactin primers with
RNA from SCF/ KL stimulated CMK cells for 24 hours; J, p-actin primers with RNA for SCF/KL stimulated megakaryocytes for 6 hours.
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SCF/KL, EFFECTS ON MEGAKARYOCYTIC CELLS
IL-10
TNF-a
IL-6
IL-la
369
GM-CSF
5.L
0'
2 CELL-ASSOCIATED
9 1
-SECRETED
r
r
-
-
-
-
r
r
1
24 h
I1
0.5
Fig 4. Production of TNF-re, IL-1s. IL-lu, IL-6, and GM-CSF by CMK
cells for 3, 24, and 48 hours. CMK cells were stimulated by SCF/KL
(100ngImL) or bySCF/KL(lOOng/mL) and PMA(lOng/mL)for3,24,
or 48 hours and assayed for cytokine production in supernatants and
cell pellets. ( 3 )
SCF; (C)
PMA; (B) SCF PMA.
+
Effect of SCFIKL. on proliferation of primary human megakaryocytes. The direct effect of human SCF/KL on CMK
cell proliferation suggested that the SCF/KL receptor
would be present on the surface of primary human megakaryocytes. Constitutive expression of the 5.5-kb specific
transcript of the c-kit gene was observed on isolated human
megakaryocytes either by Northern blot analysis or by PCR
analysis (Fig 5). We thus investigated the effects of this
growth factor on megakaryocyte progenitors and mature
megakaryocytes.
The effects of addition(s) of SCF/KL, IL-3, GM-CSF,
and IL-6, either alone or in combination, on CFU-MKderived colony formation are shown in Table 1. CFU-MKderived colonies appeared in the presence of SCF/KL,
IL-3, and GM-CSF, but not IL-6 or G-CSF (P < .05). IL-3
significantly promoted megakaryocyte colony formation
compared with either GM-CSF or SCF/KL. The addition
of IL-3 plus SCF/KL or GM-CSF plus SCF/KL to cultures
containing CD34+, DR' cells resulted in an increase in
CFU-MK-derived colony formation over that stimulated by
IL-3, GM-CSF, or SCF/KL alone (Table 1). This increase
was additive, because the combination of the two cytokines
approximated the sum of effects of the addition of each
cytokine alone. IL-6 in combination with SCF/KL resulted
in an increased number of CFU-MK colonies.
The effects of SCF/KL alone or in combination with
IL-3, GM-CSF, and IL-6 on the proliferation of marrow
megakaryocytes isolated by immunomagnetic beads are
summarized in Table 2, using the ['Hlthymidine uptake
method. Both IL-3 and GM-CSF stimulated ['Hlthymidine
incorporation of these isolated megakaryocytes. A modest
but not statistically significant increase in ['Hlthymidine
incorporation was observed after treatment with SCF/KL.
The addition of IL-3, GM-CSF, or IL-6 to SCF/KL resulted
in an increasc in ['Hlthymidine incorporation, although the
increase was not fully additive compared with the sum of
the effects of each cytokine.
DISCUSSION
Human SCF/KL has a broad range of activities in
vitro. 1.3.: I Alone, the growth factor has modest effects on
primitive progenitor cells,-'' but in combination with lateracting hematopoietic growth factors, such as GM-CSF, it
acts to markedly increase the number and size of myeloid
colonies in vitro. In combination with erythropoietin, human SCF/KL significantly stimulates erythropoiesis. Our
studies demonstrate that immortalized cell lines with
megakaryocytic properties, as well as human megakaryo-
A
+&
' G
5.5 kb Fig 5. Northern blot analysis of c-kif transcript.
Total mRNA was prepared and analyzed as described
in the Methods. Twenty micrograms of total RNA
was analyzed. Cells used were CMK-6 (lane l),
CMK
(lane 2). CMK11-5 (lane 3) and PMA (10 ng/mL)stimulated CMK for 24 hours (lane 4). Arrows indicate
the positions of 18s and 28s ribosomal RNAs. (A)
Hybridization with c-kit probe (after 5 days of exposure). (6)Ethidium bromide staining of the Northern
blot. (C) Mapping of CMK cells and isolated megakaryocytes by PCR using primer pair and detector probe
for e-kit as described in Materials and Methods.
ma
B
28S-
PCR
Analysis
Southern
Blot
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370
AVRAHAM ET AL
Table 1. Effects of the Addition of Recombinant Cytokines on
Megakaryocyte Colony Formation
Addition io Culture
None
SCF/KL (10 ng/mL)
SCF/KL (25 ng/mL)
SCF/KL (50 ng/mL)
SCF/KL (100 ng/mL)
IL-3 (10 ng/mL)
GM-CSF (200 ng/mL)
IL-6 (10 ng/mL)
G-CSF (500 ng/mL)
IL-3 (10 ng/mL) + SCF/KL (100 ng/mL)
GM-CSF (200 ng/mL) SCF/KL (100 ng/mL)
IL-6 (10 ng/mL) SCF/KL (100 ng/mL)
G-CSF (500 ng/mL) SCF/KL (100 ng/mL)
+
+
+
Table 2. Effects of SCF/KL on the Proliferationof Megakaryocytes
Factor
L3H]Thymidine
Incorporation
IcDm 2 SEM)
Medium
SCF/KL (10 ng/mL)
SCF/KL (50 ng/mL)
SCF/KL (100 ng/mL)
IL-3 (10 ng/mL)
GM-CSF (200 ng/mL)
IL-6 (10 ng/mL)
IL-3 (10 ng/mL) SCF/KL (100 ng/mL)
GM-CSF (200 ng/mL) + SCF/KL (100 ng/mL)
IL-6 (10 ng/mL) SCF/KL (100 ng/mL)
5,630 ? 258
5,810 f 45
7,231 2 120
8,761 f 225
14,687 2 236
14,053 f 631
7,220 f 189
19,103 2 250*t
18,992 f 389*t
10,775 ? 225
Mean Colonies z S E M
0.0 f 0.0
0.0 ? 0.0
2.0 f 0.1
3.6 ? 0.2
4.5 f 0.5
10.9 ? 0.8
5.6 f 0.6
0.3 ? 0.1
0.5 ? 0.1
16.3 ? 2.0*t§
10.5 2 1.3**§
6.3 f 0.6*T
4.2 f 0.3*1I
+
+
CD34'. DR' cells were plated at 5 x 103/mL.Results are expressed as
mean number of megakaryocyte colonies 2 1 SEM taken from three
separate studies performed in duplicate.
*Statistically significant as compared with the control culture (P < .05).
tStatistically greater than IL-3 alone (P < .05).
*Statistically greater than GM-CSF alone (P < .05).
§Statistically greater than SCF/KL alone (P < .05).
TStatistically greater than IL-6 alone (P < .05).
llStatistically greater than G-CSF alone (P < .05).
cytes, express the c-kit gene product and directly respond to
human SCFIKL. The role of SCF/KL in promoting human
megakaryocyte colony formation was studied, using a serumdepleted fibrin clot assay system. SCF/KL showed modest
stimulatory effects on megakaryocyte colony formation
when added alone to cultures containing CD34', DR' bone
marrow cells. When SCF/KL was added to these cultures in
combination with GM-CSF, IL-3, or IL-6, a greater increase in CFU-MK-derived colony formation was observed. However, when SCF/KL was added to megakaryocytes isolated by immunobeads using anti-gpIIb/IIIa
monoclonal antibodies, its effect on proliferation was modest. When SCF/KL was added to megakaryocyte cultures in
combination with IL-3 or GM-CSF, a nearly additive
proliferative effect was observed. The augmented increase
in CFU-MK-derived colony formation associated with the
addition of SCFIKL and IL-3 or SCF/KL and GM-CSF was
paralleled by the effects seen on permanent cell lines with
megakaryocytic properties.
CMK cells have previously been reported to produce a
variety of cytokines whose genes can be induced by agents
such as PMA in vitro.6 Human SCFiKL treatment of CMK
cells led to increased expression of the genes for several
cytokines, including IL-1 species and IL-6. Human SCFIKL
may thereby promote early hematopoiesis by increasing
proliferation via activation of the c-kit receptor on progenitor cells, as well as by inducing production of certain
cytokines by mature hematopoietic cells, such as megakaryocytes. We observed induction of IL-1p in marrow mega-
SCF/KL at the indicated concentration was added to the cells
(105/mL) for 48 hours in a final volume of 0.1 mL followed by 5-hour
pulse with [3Hlthymidine. Megakaryocytes were isolated by immunomagnetic beads. Data are mean f SEM for triplicate cultures in each
experiment after substraction of the medium (control). P < .05. These
results are comparable with those seen in three other similar experiments.
*Statistically significant as compared with the control culture (P < .05).
tStatistically greater than SCF/KL alone (P < .05).
karyocytes following SCFIKL treatment (Fig 3). Megakaryocyte-derived cytokines such as IL-1 could amplify the effects
of SCFIKL on early progenitors. Further studies on human
SCF/KL induction of cytokines in responsive cell types,
such as mast cells and blasts, will be of interest in this
regard.
Several hematopoietic growth factors, including GMCSF, IL-3, and IL-6, have previously been reported to
augment megakaryocyte proliferation and maturation in
vitro.6,22-25Our study suggests that these growth factors may
act in combination with SCF/KL to further augment human
megakaryocyte progenitor cell growth. It will be particularly
interesting to extend these in vitro observations to in vivo
studies in non-human primates and, ultimately, in humans.
Such trials should determine which combinations of growth
factors result in stimulation of megakaryocytopoiesis under
physiological conditions and after radiation therapy or
chemotherapy, which suppress bone marrow activity.
Human SCF/KL is a broadly acting hematopoietic growth
factor that augments in vitro, and under certain in vivo
conditions, myelopoiesis, erythropoiesis, and lymphopoiesis. Based on our in vitro observations of direct effects of
SCF/KL on cell lines with megakaryocytic properties,
megakaryocyte progenitors, and isolated primary marrow
megakaryocytes, the augmentation of megakaryocyte number observed after invivo treatment of primates4may reflect
direct proliferation of these cells in response to treatment
with human SCFIKL. Such effects on megakaryocytopoiesis may ultimately be most evident when human SCF/KL is
used in combination with other hematopoietic growth
factors.
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1992 79: 365-371
Effects of the stem cell factor, c-kit ligand, on human megakaryocytic
cells
H Avraham, E Vannier, S Cowley, SX Jiang, S Chi, CA Dinarello, KM Zsebo and JE Groopman
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