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Blood, Vol. 94 No. 3 (August 1), 1999:
pp. 950-958
Differential Effects of Human Granulocyte Colony-Stimulating Factor
(hG-CSF) and Thrombopoietin on Megakaryopoiesis and Platelet Function
in hG-CSF Receptor-Transgenic Mice
By
Feng-Chun Yang,
Kohichiro Tsuji,
Atsushi Oda,
Yasuhiro Ebihara,
Ming-jiang Xu,
Azusa Kaneko,
Sachiyo Hanada,
Tetsuo Mitsui,
Akira Kikuchi,
Atsushi Manabe,
Sumiko Watanabe,
Yasuo Ikeda, and
Tatsutoshi Nakahata
From the Department of Clinical Oncology and the Department of
Molecular and Developmental Biology, The Institute of Medical Science,
The University of Tokyo; and the Department of Internal Medicine, Keio
University, Tokyo, Japan.
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ABSTRACT |
Granulocyte-colony stimulating factor (G-CSF) has been found to act
on the neutrophilic lineage. We recently showed that human G-CSF
(hG-CSF) has effects similar to early-acting cytokines such as
interleukin-3 (IL-3) in the development of multipotential hematopoietic progenitors in transgenic (Tg) mice expressing receptors (R) for hG-CSF. In the present study, we examined the effects of hG-CSF on more
mature hematopoietic cells committed to megakaryocytic lineage in these
Tg mice. The administration of hG-CSF to the Tg mice increased the
numbers of both platelets in peripheral blood and megakaryocytes in the
spleen, indicating that hG-CSF stimulates megakaryopoiesis in the Tg
mice in vivo. The stimulatory effect of hG-CSF was also supported by
the results of studies in vitro. hG-CSF supported megakaryocyte colony
formation in a dose-dependent fashion in clonal cultures of bone marrow
cells derived from the Tg mice. Direct effects of hG-CSF on
megakaryocytic progenitors in the Tg mice were confirmed by culture of
single-cell sorted from bone marrow cells. hG-CSF showed a stronger
effect on maturation of megakaryocytes in the Tg mice than that of IL-3 alone, but weaker than that of TPO alone. In addition, hG-CSF induced
phosphorylation of STAT3 but not Jak2 or STAT5, while TPO induced
phosphorylation of both. In contrast to TPO, hG-CSF did not enhance
ADP-induced aggregation. Thus, hG-CSF has a wide variety of functions
in megakaryopoiesis of hG-CSFR-Tg mice, as compared with other
megakaryopoietic cytokines, but the activity of hG-CSF in
megakaryocytes and platelets does not stand up to a comparison with
that of TPO. Specific signals may be required for the full maturation
and activation of platelets.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE PROLIFERATION, differentiation and
maturation of hematopoietic cells are regulated by a number of
cytokines.1 These molecules exert biological functions
through specific receptors expressed on the surface of target
cells.2 Binding of the cytokines to the receptors induces
phosphorylation of a variety of cellular proteins, including a group of
signal transducers and activators of transcription (STAT) proteins,
involved in intracellular signal transduction.3 Although it
was earlier postulated that each cytokine has specific functions,
recent studies have shown that the specific activity of each cytokine
depends on the cellular context in which the receptor is expressed.
Dubart et al4 reported that erythropoietin (EPO) supported
the formation of multilineage colonies comprising not only erythrocytes
but also granulocytes, macrophages and megakaryocytes from
multipotential progenitor cells transduced with the EPO receptor cDNA.
We have also shown that human granulocyte-macrophage colony-stimulating
factor (hGM-CSF) stimulated development of multiple lineages of cells,
such as erythrocytic and megakaryocytic cells in addition to myelocytic cells in transgenic (Tg) mice expressing hGM-CSF receptors
(R).5 Thus, cytokines specific for erythropoiesis or
myelopoiesis can support megakaryopoiesis to some extent if their
receptors are expressed on megakaryocytic cells. Whether intracellular
signals emanating from ectopic receptors can activate and otherwise
mimic the biological effects on megakaryopoiesis in a fashion identical to that of natural receptors for megakaryopoietic cytokines has remained unclear.
Megakaryopoiesis is a multistage developmental process governed by a
series of megakaryopoietic cytokines.6,7 Some, such as
interleukin-3 (IL-3), called megakaryopoietic colony-stimulating factors, support proliferation of megakaryocytic progenitors and differentiation into immature megakaryocytes. In contrast, some such as
IL-6, IL-11, leukemia inhibitory factor, and oncostatin M, which are
known as Mk-potentiators, affect the maturation of megakaryocytes.
Thrombopoietin (TPO) has been found to be a major physiological
regulator of megakaryopoiesis. TPO stimulates both growth of
megakaryocytic progenitors and megakaryocyte maturation.8 TPO also enhances human platelet aggregation, and induces tyrosine phosphorylation of Jak2, STAT3, and STAT5.9
Recently, we generated Tg mice expressing receptors for human
granulocyte colony-stimulating factor (hG-CSFR), a myelopoietic growth
factor.10 A 3-kb fragment of G-CSFR cDNA was inserted into
the EcoRI site of a pLG1 expression vector that is under control of mouse major histocompatibility L-locus gene promoter, resulting in ubiquitous expression of the transgene. hG-CSF induces colony formation that includes multiple lineages when incubated with
bone marrow (BM) from those mice.10 In the present study, we scrutinized effects of hG-CSF on megakaryopoiesis and thrombopoiesis in the Tg mice in vivo and in vitro and compared these effects with
cytokines known to stimulate megakaryopoiesis and thrombopoiesis. In
addition, we examined activities of hG-CSF and TPO on aggregation and
STAT phosphorylation in platelets of the Tg mice. The biological, signaling, and functional effects of hG-CSF on megakaryopoiesis of the
Tg mouse differ from those of any other megakaryopoietic cytokines,
including TPO.
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MATERIALS AND METHODS |
Mice.
Tg mice constitutively expressing high-affinity hG-CSFR were generated
in our laboratory, as described.10 The Tg mice and their
normal littermates were 8 weeks old, of both sexes, and their
backgrounds were C3H/HeN and (C57BL/6 × C3H/HeN) F1. These mice
were maintained in an environmentally controlled clean room with
12-hour light-dark cycles under specific pathogen-free conditions in
micro-isolator cages. The mice were not used in more than two protocols, and were not repeatedly bled. Injection schedules of hG-CSF
were according to the modified method described
elsewhere.11 Briefly, the Tg mice and normal littermates
were injected intraperitoneally with 0.2 mL of phosphate-buffered
saline (PBS) containing 1% of C3H/HeN mice serum (serum/PBS) or 500 µg/kg of hG-CSF twice a day at 12-hour intervals for 7 consecutive
days at 9:00 AM and 9:00 PM, and peripheral
blood (PB), BM and spleen cells were harvested 12 hours after the last injection.
Cytokines and antibodies.
Recombinant hG-CSF and hTPO were kindly provided by Kirin Brewery
(Tokyo, Japan). Recombinant mouse IL-3 (mIL-3) and recombinant hIL-6
were provided by Amgen (Thousand Oaks, CA), and Tosoh Co (Kanagawa,
Japan), respectively. Fluorescein isothiocyanate (FITC)-conjugated hamster 1C2 antibody that immunoreacts with mouse platelets and megakaryocytes is a gift from Dr Junichiro Fujimoto12
(National Children's Medical Research Center, Tokyo, Japan).
Allophycocyanin (APC)-conjugated anti-c-Kit antibody (ACK-2) was a
generous gift from Dr Shin-Ichi Nishikawa (Kyoto University, Kyoto,
Japan). Biotin-conjugated monoclonal antibodies (MoAbs) specific for
CD45R (B220, RA3-6B2), Gr-1 (Ly-6G, RB6-8C5), CD4 (L3T4, RM4-5), CD8a (Ly-2, 53-6.7), TR119 (TER119), Mac-1 (CD11b, M1/70), phycoerythrin (PE)-conjugated anti-mouse Sca-1 (Ly-6A/E, E13-161.7), APC-conjugated rat IgG2b, rat anti-mouse CD32/CD16 (Fc II/III receptor, 2.4G2), and
mouse anti-STAT5A and B polyclonal antibodies (pAb) were purchased from
Pharmingen (San Diego, CA). An anti-Jak2 pAb and an antiphosphotyrosine MoAb (4G10) were from Upstate Biotechnology (Lake Placid, NY). PE-conjugated streptavidin, rat IgG2a, texas red (TR)-conjugated streptavidin, and R-phycoerythrin-cyanine 5 (RPE-Cy5)-conjugated streptavidin were purchased from Becton Dickinson Immunocytometry Systems (San Jose, CA), Cedarlane Laboratories, Ltd (Ontario, Canada),
Life Technologies, Inc (Gaithersburg, MD), and DAKO (Glostrup, Denmark), respectively. Anti-STAT3 pAb was from Santa Cruz (Santa Cruz, CA).
Cell and platelet preparations.
Mice were anesthetized with ether, and killed by rapid cervical
dislocation. BM cells were flushed from femurs and tibiae into a
minimum essential medium ( -MEM; Flow Laboratories, Rockville, MD)
with 2% fetal bovine serum (FBS; Hyclone, Logan, UT) using 21-gauge
needles. Then cells were passed through a 70-µm nylon cell strainer
(no. 2350; Becton Dickinson Labware, Franklin Lakes, NJ). BM
mononuclear cells (MNC) were prepared using a density gradient
centrifugation method. Cells were diluted with PBS, layered over
Lympholyte-M (Cosmo Bio, Tokyo, Japan), and centrifuged for 30 minutes
at 1,500 rpm at room temperature. Interface cells were collected and
washed twice with PBS. For platelets preparation, mice were
anesthetized with ether, and blood was drawn from superior vena cava
into 40 mmol/L D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone
(PPACK; Sigma, St Louis, MO), and gently mixed. Platelet-rich plasma
(PRP) was prepared by centrifuging the whole blood at 200g for
20 minutes and aspirating PRP. PRP was incubated with aspirin (2 mmol/L) for 30 minutes at room temperature. To prepare washed platelets, prostaglandin E1 (1 mmol/L) was added from a stock solution
in absolute ethanol (1 mmol/L). The PRP was spun at 800g to
form a soft platelet pellet. The pellet was resuspended in 1 mL of a
modified HEPES-Tyrode buffer (129 mmol/L NaCl, 8.9 mmol/L NaHCO3, 0.8 mmol/L KH2PO4, 0.8 mmol/L MgCl2, 5.6 mmol/L dextrose, and 10 mmol/L HEPES, pH
7.4) also containing apyrase (2 U/mL) and washed twice with PBS.
Platelets were resuspended in the same buffer at a concentration of 3 to 9 × 108 cells/mL with apyrase (2 U/mL) containing 1 mmol/L CaCl2 at 37°C.
Clonal cell culture.
Clonal cell culture was performed in triplicate, as described
previously.13-15 Briefly, 1 mL of culture mixture
containing 2.5 × 104 BM cells, -MEM, 1.2%
methylcellulose (Shinetsu Chemical, Tokyo, Japan), 30% FBS, 1%
deionized fraction V bovine serum albumin (BSA; Sigma),
10 4 mol/L mercaptoethanol (Eastman Organic Chemicals,
Rochester, NY), and varying concentrations of hG-CSF, 20 ng/mL of hTPO,
10 ng/mL of mIL-3, or 100 ng/mL of hIL-6 was plated in each 35-mm suspension culture dish (no. 171099; Nunc, Inc, Naperville, IL) followed by incubation at 37°C in a humidified atmosphere flushed with 5% CO2 in air. Colony types were determined on days 7 and 14 of incubation by in situ observation according to documented criteria14,16,17 using an inverted microscope. To assess
the accuracy of in situ identification of the colonies, individual colonies were lifted using an Eppendorf micropipette under direct microscopic visualization, spread on glass slides using a
cytocentrifuge (Cytospin 2; Shandon Inc, Pittsburgh, PA), then stained
with May-Grünwald-Giemsa (Muto Chemica, Tokyo, Japan) or
acetylcholine esterase (AChE) for megakaryocytes.18 Except
for megakaryocyte colonies, cell aggregates consisting of more than 50 cells were scored as colonies. Megakaryocyte colonies were scored as
such when they had four or more megakaryocytes.19,20
Abbreviations for the colony types are as follows: Mk, megakaryocyte
colonies; M-Mix, mixed hematopoietic colonies containing
megakaryocytes; GM, granulocyte and/or macrophage colonies; others,
other colonies including mast and blast cell colonies.
Clone-sorting and single cell culture.
Clone-sorting of lineage
(Lin)c-Kit+Sca-1+ and
Linc-Kit+Sca-1 cells from BM
cells of (C57BL/6 × C3H/HeN) F1 Tg mice was performed using a modification of a previously described technique.21 Briefly, BMMNC were enriched by negative selection with
streptavidin-conjugated beads (PerSeptive Biosystems, Framingham, MA),
using a cocktail of biotin-conjugated MoAb specific for CD45R/B220,
Gr-1, CD4, CD8, TR119, and Mac-1. After incubation with rat anti-mouse
CD32/CD16 to avoid nonspecific antibody binding, the lineage
marker-negative cells were stained with PE-conjugated Sca-1 and
APC-conjugated anti-c-Kit antibodies. The cells were then washed twice
with PBS and incubated with TR-conjugated streptavidin. The negative
controls were stained with PE-conjugated rat IgG2a, APC-conjugated rat IgG2b, or only TR-conjugated streptavidin. Individual
Linc-Kit+Sca-1+ and
Linc-Kit+Sca-1 cells were
sorted into each well of 96-well flat-bottomed plates (no. 163320, Nunc) with a FACSVantage equipped with an automatic cell deposition
unit (ACDU; Becton Dickinson). The clone-sorted cells were then
cultured with 200 µL culture medium containing 30% FBS, 1%
deionized fraction V BSA, 104 mol/L mercaptoethanol, and
with the addition of 20 ng/mL of hG-CSF, 100 ng/mL of hIL-6, 20 ng/mL
of hTPO, or 10 ng/mL of mIL-3. On days 5 and 8 of culture, colony
formations were observed and classified using of an inverted microscope.
Determination of size of megakaryocytes in megakaryocyte colonies.
BM cells (2.5 × 104) were cultured in methycellulose
medium containing 20 ng/mL of hG-CSF, 20 ng/mL of hTPO, or 10 ng/mL of mIL-3. After 5 days culture, Mk colonies were lifted from
methylcellulose medium, pooled, and washed with -MEM. The cells were
cytospun and stained with AChE. The diameter of each megakaryocyte
identified by AChE-staining was measured using a microscope equipped
with an ocular micrometer. The mean of two perpendicular diameters was
calculated.17
Peripheral blood cell counts.
Mice were anesthetized with ether, and PB was taken from superior vena
cava using a 1-mL syringe with a 21-gauge needle. After the collection,
PB was quickly mixed with 2 mg EDTA to avoid aggregation. The numbers
of red blood cells (RBCs), white blood cells (WBCs), platelets, and the
hemoglobin concentration (Hb) were counted using a hemacytometer
(Sysmex K1000; Sysmex, Kobe, Japan). Smear preparations were made on
glass slides and stained with May-Grünwald-Giemsa solution.
Histological examination of spleens.
Spleens of the Tg mice and normal littermates treated with hG-CSF or
serum/PBS were taken for histological examination. After fixation with 10% formalin solution, the sections were
stained with hematoxylin-eosin.
Examination of hG-CSFR expression on platelets.
Flow cytometry analysis was performed by a modified method previously
described.10 Briefly, PRP was incubated with PE-conjugated hG-CSF (R & D Systems, Inc, Minneapolis, MN) or FITC-conjugated 1C2
MoAb for 60 minutes at room temperature, diluted with staining buffer
(PBS containing 2% FBS and 0.1% sodium azide), then analyzed using a
FACScan (Becton Dickinson). PE-conjugated streptavidin or
FITC-conjugated goat anti-hamster IgG (Cedarlane, Ontario, Canada) was
used as a control, respectively.
Measurement of platelet aggregation.
Platelet aggregation using PPACK (final concentration: 40 mmol/L)-PRP
was measured using an aggregometer (Hema Tracer TM Model 601; Niko Bio
Science, Tokyo, Japan) with continuous stirring (1,000 rpm), as
previously described.22,23
Immunoprecipitation.
Platelet stimulation was terminated by adding an equal amount of lysis
buffer (15 mmol/L HEPES, 150 mmol/L NaCl, 1 mmol/L phenylmethylsulfonyl
fluoride [PMSF], 10 mmol/L EGTA, 1 mmol/L sodium orthovanadate, 0.8 µg/mL leupeptin, 2% Triton X-100 [vol/wt], pH 7.4). After 20 minutes of incubation on ice, the lysates were centrifuged at
10,000g for 20 minutes at 4°C. The supernatant (from 3 to
9 × 108 platelets) was removed and incubated with
preimmune serum and Protein A-Sepharose (Transduction Laboratories,
Lexington, KY) (40 µL of 50% slurry) for 1 hour. After preclearing,
the desired pAb were added followed by incubation for 2 to 3 hours on
ice. Protein A-Sepharose (40 µL of 50% slurry) was then added and
the preparation was incubated for several hours. The immune complexes were washed three times with 1 mL of cold washing buffer (15 mmol/L HEPES, 150 mmol/L NaCl, 1 mmol/L PMSF, 10 mmol/L EGTA, 1 mmol/L sodium
orthovanadate, 0.8 µg/mL leupeptin, 1% Triton X-100 [vol/wt], pH
7.4) and then resuspended in Laemmli's sample buffer (10% glycerol, 1% sodium dodecyl sulfate (SDS), 5% 2-mercaptoethanol, 50 mmol/L Tris-HCl, pH 6.8). After boiling at 95°C for 5 minutes,
one-dimensional SDS-electrophoresis was performed on 10% or 7.5% to
15% polyacrylamide gels. Separated proteins were electrophoretically
transferred from the gels onto polyvinylidene difluoride
(PVDF) membranes in buffer containing Tris (25 mmol/L),
glycine (192 mmol/L) and 20% methanol at 0.2 A for 12 hours at room
temperature. To block residual protein binding sites,
membranes were incubated in TBST (TBS: Tris-buffered-saline, 10 mmol/L
Tris, 150 mmol/L NaCl, pH 7.6 with 0.1% Tween 20) with 10% chicken
egg albumin. The blots were washed with TBST and incubated overnight
with primary antibodies at a final concentration of 2 µL/mL (for
STAT5 A and B) or 2 µg/mL (for STAT3) in TBST. The primary antibody
was removed and the blots were washed four times in TBST and incubated
with horseradish peroxidase-conjugated second antibody diluted 1:5,000
in TBST. Blots were then washed four times with TBST. Antibody
reactions were detected with chemiluminescence, according to the
manufacturer's instructions.
Statistical analysis.
The data are expressed as the mean ± SD of triplicate plates in
colony formation. Student's t-test was used to determine the statistical significance.
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RESULTS |
hG-CSF administration to hG-CSFR-Tg mice.
The peripheral blood WBC, RBC, Hb and platelet counts of the Tg mice
were similar to those of normal littermates. In vivo effects of hG-CSF
on megakaryopoiesis in the Tg mice and normal littermates were examined
after administration of hG-CSF (1,000 µg/kg/d) for 7 consecutive
days. After the administration of hG-CSF or serum/PBS, both the Tg mice
and normal littermates looked healthy. The number of PB RBCs and the Hb
level showed no remarkable changes on day 8, and the number of WBCs
increased to similar levels in both the Tg mice and normal littermates
(Table 1). The most marked difference was
in the number of platelets; while a 1.4-fold increase of platelets was
seen in the Tg mice treated with hG-CSF, a decrease in platelet count
was seen in normal littermates.
After the injection of hG-CSF, a significant splenomegaly was observed
in all the mice. The spleen weight increased 5-fold in the Tg mice and
normal littermates injected with hG-CSF (431.2 ± 76.8 and
421.4 ± 75.6 mg, respectively) compared to mice injected with
serum/PBS (75.9 ± 11.4 and 79.9 ± 11.3 mg, respectively). The
numbers of spleen cells increased in a parallel with spleen weights in
the Tg mice and normal littermates (3.6 ± 0.8 and
3.5 ± 0.9 × 108, respectively, v serum/PBS
1.0 ± 0.2 and 1.1 ± 0.1 × 108, respectively).
Histological analysis showed that the spleens of the Tg mice contained
an increased number of megakaryocytes compared with normal littermates
(Fig 1). The megakaryocytes in spleens of
the Tg mice included various stages of maturation ranging from small
immature to huge mature megakaryocytes. These results indicate that
hG-CSF simulates megakaryopoiesis and thrombopoiesis in the Tg mice in
vivo.
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| Fig 1.
Photographs of spleens of a littermate (A) and a Tg mouse
(B) injected with hG-CSF. Note increased numbers of megakaryocytes in
the spleen of the Tg mouse (original magnification ×100).
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Effects of hG-CSF on megakaryocyte colony formation in the Tg mice.
To clarify effects of hG-CSF on megakaryopoiesis in the Tg mice, we
used methylcellulose clonogenic assay of BM cells derived from Tg mice
and normal littermates. hG-CSF significantly stimulated Mk and M-Mix
colonies from BM cells of the Tg mice but not from littermates (Table
2). In the Tg mice, hG-CSF supported a
larger number of Mk and M-Mix colonies than mIL-3, hTPO, or hIL-6.
mIL-3, hTPO, or hIL-6 showed the same effects on Mk and M-Mix colonies in both the Tg mice and their normal littermates. While a maximal effect on Mk colony formation was seen with 20 ng/mL of hG-CSF in the
Tg mice (data not shown), no Mk colonies were observed at
concentrations of up to 100 ng/mL in the case of littermates. To assess
the accuracy of in situ identification of Mk colonies (Fig
2A), cytospin preparations of individual
colonies were stained with AChE. All Mk colonies consisted of
AChE-positive megakaryocytes (Fig 2B).
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| Fig 2.
(A) In situ appearance of a representative Mk colony
induced by 20 ng/mL of hG-CSF from BM cells of hG-CSFR-Tg mice
(original magnification ×200). (B) AChE-positive megakaryocytes in
the Mk colony (original magnification ×400).
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Colony formation from clone-sorted
Linc-Kit+Sca-1+/
cells.
In the Tg mice, accessory cells expressing hG-CSFR may also be
responsive to hG-CSF and produce various cytokines which in turn induce
growth of megakaryopoietic progenitors. To exclude this possibility, we
sorted Linc-Kit+Sca-1+ and
Linc-Kit+Sca-1 cells, which
have been shown to reflect murine primitive hematopoietic progenitors
and more mature populations, respectively,24 from BM cells
of the Tg mice and normal littermates. Because it was reported that
only some of primitive hematopoietic cells expressed Sca-1 antigen in
C3H/HeN,25 we used BM cells of (C57BL/6 × C3H/HeN) F1 backgrounds as the source of sorted cells. The
single-sorted cells were cultured with hG-CSF, or three
megakaryopoietic cytokines, hIL-6, mIL-3, and hTPO, and the colony
formation was compared (Table 3). hIL-6,
mIL-3, and hTPO had the same effects on Mk colony formation on sorted
cells derived from the Tg mice and normal littermates. In both, hIL-6
supported no or only a few Mk colonies, mIL-3 supported Mk colonies
from both Linc-Kit+Sca-1+ and
Linc-Kit+Sca-1 cells, and
hTPO supported Mk colonies from only
Linc-Kit+Sca-1 cells.
Conversely, hG-CSF supported Mk colonies from both fractions (Sca-1+/Sca-1) of Tg mice, whereas Mk
colonies were never observed in response to G-CSF in normal
littermates. The number of Mk colonies induced by hG-CSF was larger
than that by mIL-3 in the Tg mice. These results confirmed the direct
effect of hG-CSF on megakaryocytic progenitors of Tg mice and further
indicate that hG-CSF has differential effects on Mk colony formation
compared with those other megakaryopoietic cytokines.
Effect of hG-CSF on megakaryocyte maturation.
We next examined the effect of hG-CSF on megakaryocyte maturation.
Because of the simultaneous presence of a large number of myelocytic
cells stimulated by hG-CSF in the culture system, it was not feasible
to analyze the ploidy of megakaryocytes by flow cytometry. Therefore,
the size of megakaryocytes was used to evaluate megakaryocyte
maturation, since the size achieved by each megakaryocyte is
proportional to its ploidy.26 On day 5 of methylcellulose
culture of BM cells from the Tg mice or normal littermates, Mk colonies
were pooled from 120 plates containing hG-CSF, 120 plates containing
mIL-3, and 150 plates containing hTPO, then processed for measurement
of the size of constituent megakaryocytes. As shown in Fig
3, hG-CSF induced larger megakaryocytes compared to mIL-3, although the size was smaller than that stimulated with hTPO (P < .001). Therefore, it appears that hG-CSF
stimulates not only the proliferation of megakaryocytic progenitors but
also the maturation of megakaryocytes in the Tg mice.
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| Fig 3.
Effects of mIL-3, hG-CSF, and hTPO on megakaryocyte size
in Mk colonies. Three independent experiments were analyzed to generate
this figure. The horizontal bars in each group represent the mean
perpendicular diameters of megakaryocytes in Mk colonies. The numbers
of measured megakaryocytes are presented in parentheses. The
megakaryocytes from Mk colonies supported by hG-CSF were significantly
larger than those in case of mIL-3, but smaller than those in case of
hTPO (P < .001, for both).
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Effect of hG-CSF on platelet aggregation.
We examined the expression of hG-CSFR on platelets using PE-conjugated
hG-CSF. Platelets in PRP from both the Tg mice and normal littermates
were confirmed by staining with 1C2, an antibody reacting with mouse
platelets (Fig 4A). As shown in Fig 4B,
hG-CSF bound to the platelets of the Tg mice, but not normal
littermates, indicating the expression of hG-CSFR on platelets of the
Tg mice. We next compared the effect of hG-CSF with that of hTPO on
platelet aggregation induced by ADP in the Tg mice, since we earlier
reported that hTPO enhances human platelet functions.23 As
shown in Fig 5, the addition of hG-CSF to
PPACK PRP did not enhance the aggregation of murine platelets induced
by ADP, while hTPO enhanced this aggregation, as was observed in human
platelets. Phosphorylation of STAT3 and STAT5 (Fig
6) showed that hTPO stimulated tyrosine
phosphorylation of STAT3, STAT5, and Jak2 in platelets from both the Tg
mice and normal littermates, which are consistent with our previous
data on human platelets. In the Tg mice, however, STAT3 but not STAT5 was modestly phosphorylated in platelets stimulated by hG-CSF, while
hG-CSF did not induce tyrosine phosphorylation of either STAT3 or STAT5 in littermates. Thus, platelets of the Tg mice express
functional hG-CSFR but hG-CSFR-mediated signals and their biological
effects differ from those of the c-Mpl, the TPO receptor, in the Tg
mouse platelets.
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| Fig 4.
The expression of hG-CSFR on platelets of the Tg mice
(the unshaded areas represent isotype control, the shaded areas
represent specific staining). (A) Platelets gated in R1 of the Tg mice
were stained with 1C2, an anti-mouse platelet antibody. The same
finding was obtained with platelets of normal littermates. (B)
PE-conjugated hG-CSF bound to platelets of the Tg mice, but
not littermates.
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| Fig 5.
hTPO and hG-CSF were diluted in autologous platelet-poor
plasma (PPP, final concentration 10 µg/mL). PRP from Tg mice was
diluted with PPP to adjust the platelet concentration at 2.0 × 108/mL. hTPO or hG-CSF was added 5 minutes before adding
ADP (1 mmol/L).
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| Fig 6.
(A) Tyrosine phosphorylation of STAT3. Platelets were
lysed by the addition of an equal amount of a buffer containing 2%
Triton X-100 before or 5 minutes after stimulation with hTPO (100 ng/mL) or hG-CSF for 5 minutes. STAT3 was immunoprecipitated from 6.0 × 108 platelets from littermate mice or 2.0 × 108 platelets from Tg mice. Immune complexes were
resuspended in SDS-sample buffer and divided into two. Proteins were
separated by 15 to 7.5% SDS-PAGE and transferred onto PVDF membranes.
One immunoblot was probed with anti-phosphotyrosine antibodies and
bands were visualized by chemiluminescence (top). The other blot was
probed for STAT3 (bottom). The arrows indicate the bands of interest.
(B) Tyrosine phosphorylation of STAT5. The same as in (A) except the
combination of STAT5A and B antisera were used instead of STAT3
antisera. (C) Tyrosine phosphorylation of Jak2. The same as in (B)
except the combination of an anti-Jak2 polyclonal antibody was used
instead of STAT5 antisera.
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| |
DISCUSSION |
Accumulating data were shown that G-CSF specifically regulates the
proliferation and differentiation of myelocytic progenitor cells.27 However, the ectopic expression of the G-CSFR in
hematopoietic progenitor cells has also been shown to allow for
G-CSF-dependent multilineage differentiation.28 We also
demonstrated that hG-CSF supports the development of various types of
hematopoietic progenitors, including Mast, Mix, and Mk other than GM
progenitors in hG-CSFR-Tg mice.10 These reports indicate
that hG-CSF can stimulate megakaryopoiesis if hG-CSFR is expressed on
cells of megakaryocytic lineage.
In the present study, we extended these observations to determine more
precisely the effects of hG-CSF on megakaryopoiesis. Evidence for the
stimulatory effect of hG-CSF on megakaryopoiesis in the Tg mice was
supported by findings in vivo and in vitro. The administration of
hG-CSF resulted in an increase in platelets in PB and megakaryocytes in
spleen of the Tg mice, indicating that hG-CSF stimulates
megakaryopoiesis and thrombopoiesis in vivo. Because mouse and human
G-CSF have been shown to crossreact with the receptors of both
species,10,29,30 the normal platelet count in uninjected Tg
mice indicates that the concentrations of endogenous mouse G-CSF are
not sufficient to affect megakaryocytopoiesis in the Tg mice in vivo.
In vitro studies showed the effect of hG-CSF on megakaryopoiesis in
even more detail. The effect of hG-CSF on megakaryocytic progenitors of
the Tg mice was confirmed in a dose-response study of hG-CSF and
single-cell cultures of sorted hematopoietic progenitors. The results
clearly show that the proliferation of megakaryocytic progenitors is
directly triggered by hG-CSF, without the influence of accessory cells.
hG-CSF also stimulated the enlargement of megakaryocytes, reflecting
megakaryocyte maturation, in the Tg mice. Thus, hG-CSF showed a wide
variety of functions in megakaryopoiesis of the Tg mice.
In the single-cell culture of sorted hematopoietic progenitors, hIL-6,
mIL-3, and hTPO had different effects on Mk colony formation. hIL-6, an
Mk potentiator, had no activity related to Mk colony formation, and
mIL-3, an Mk-CSF, supported Mk colony formation in case of both
Linc-Kit+Sca-1+ and
Linc-Kit+Sca-1 cells. hTPO
supported Mk colony formation for only
Linc-Kit+Sca-1 cells,
suggesting that colony-forming activity of hTPO predominantly acts on
relatively mature megakaryocytic progenitors. Effects of hG-CSF on Mk
colony formation differed from effects of these three megakaryopoietic
cytokines in the Tg mice. hG-CSF induced Mk colony formation in the
cases of both Linc-Kit+Sca-1+
and Linc-Kit+Sca-1 cells of
the Tg mice, but the number of Mk colonies derived from Linc-Kit+Sca-1+ cells exceeds
that seen with mIL-3. Thus, hG-CSF, as a single factor, is the most
potent stimulator of Mk colony formation, although the combination of
mIL-3 and hTPO supported almost the same number of Mk colonies from
Linc-Kit+Sca-1+ cells (data not
shown). Differences in activities between hG-CSF and other
megakaryopoietic cytokines were also observed in case of megakaryocyte
maturation. hG-CSF showed greater activity than mIL-3 in enlarging
megakaryocytes, albeit the activity being weaker than that of hTPO.
Because Linc-Kit+Sca-1+ cells
are only about 10% of
Linc-Kit+Sca-1 cells in BM
(data not shown), it can be estimated that the majority of
megakaryocytes cultured from BM cells by hG-CSF are derived from
Linc-Kit+Sca-1 cells.
Therefore, the difference in size of megakaryocytes supported by hG-CSF
and hTPO is unlikely to be caused by the immaturity of the target
progenitor population by hG-CSF.
The distinct activities of hG-CSF on megakaryocytic development may be
explained by differences in the distribution between hG-CSFR and each
cytokine receptor. The present study suggests that IL-6R is not
expressed on murine megakaryocytic progenitors, and IL-3R is
predominantly expressed on early stage of cells in murine
megakaryopoiesis, mainly megakaryocytic progenitors. Although TPO acts
on all stages of megakaryocytic cells, our observation shows that c-Mpl
may be predominantly expressed at the late stage of cells. There is
also a possibility that these receptors are present but not functional
at certain stages. On the other hand, it is conceivable that hG-CSFR is
expressed throughout the development of megakaryocytic cells in the Tg
mice, so that hG-CSF has a wide variety of functions in the Tg mouse megakaryopoiesis.
Alternatively, hG-CSFR on megakaryocytic cells may activate different
signals from those by natural receptors for megakaryopoietic cytokines,
resulting in different effects on the Tg mouse megakaryopoiesis. Platelets are an excellent model system for investigating signaling induced by TPO. Platelets from patients with thrombocytopenia with
absent radii have been shown to have reduced responses to exogenous
TPO.31 In platelets of these patients, exogenous TPO did
not induce protein tyrosine phosphorylation or prime platelet aggregation. Accordingly, we examined TPO- and G-CSF-induced proximal signaling in murine platelets. Our studies show for the first time
presence of Jak2, STAT3 and STAT5 in murine platelets and the inducible
tyrosine phosphorylation following stimulation by TPO. We earlier found
that these signaling molecules were tyrosine phosphorylated in
hTPO-stimulated platelets.9,32 Coexpression of hG-CSFR and
c-Mpl on platelets of the Tg mice was shown by the phosphorylation of
STAT3 in platelets stimulated by hG-CSF and hTPO, although the degrees
of tyrosine phosphorylation was much less in G-CSF-stimulated
platelets than in TPO-stimulated platelets. However, neither Jak2 nor
STAT5 was phosphorylated in Tg mouse platelets stimulated by hG-CSF.
Interestingly, Parganas et al33 have shown G-CSF signaling
was still functional in Jak2-deficient mice; the hG-CSFR may be able to
activate different kinases.
The difference in activities between hG-CSF and hTPO was also observed
in platelet aggregation studies. hTPO but not hG-CSF enhanced the
platelet aggregation induced by ADP. Because STAT3 was tyrosine
phosphorylated by hG-CSF stimulation, there is a possibility that the
intensity of signal activated by hG-CSF could explain the lack of
response of Tg mouse platelets to hG-CSF. Drachman et al34
found that TPO induced tyrosine phosphorylation of Jak2, STAT3, and
STAT5 in murine mature megakaryocytes. In contrast, G-CSF may not be
able to induce tyrosine phosphorylation of signaling molecules like
Jak2 and STAT5 in mature megakaryocytes from the Tg mice. G-CSF, as a
result, may have only weak biological effects on the late stage of
megakaryocyte maturation. Shimoda et al35 reported that
human platelets possessed functional G-CSFR, and G-CSF augmented
ADP-induced aggregation. However, other workers could not confirm the
costimulatory effects of G-CSF on human platelets,36 and
the presence or absence of functional G-CSFRs on human platelets has
remained controversial. If human platelets do express G-CSFR,
mechanisms of aggregation between human and murine platelets may differ.
We previously reported that hG-CSF, as well as early acting cytokines
such as IL-3, stimulated the proliferation of multipotential progenitor
cells of the Tg mice, but had no effect on their commitment to specific
hematopoietic lineages.10 Similar results have been reported from studies using murine hematopoietic cells expressing transgenes of receptors for mouse macrophage colony-stimulating factor, EPO receptor, c-Mpl, hGM-CSFR, or mIL-5R chain-Tg
mice.4,5,37-39 We propose that cytokines
function as proliferation-promoting factors in multipotential
progenitors and that cellular differentiation is determined by an
intrinsic program. Although intracellular signal-transducing pathways
of these transreceptors in murine multipotential progenitors are
unknown, there may be common signals for mitosis. In the present study,
hG-CSF had a wide variety of effects on Tg mouse megakaryopoiesis, but
the activities cannot be compared with those of TPO with regard to
megakaryocytes and platelets of the Tg mice. We suggest that specific
intracellular signals may be required for full maturation of
megakaryocytic progenitors committed to megakaryocytes and for
activation of functions in platelets.
| |
ACKNOWLEDGMENT |
We thank Dr. David A. Williams for critical reading of the manuscript,
M. Ohara for comments on the manuscript, and I. Hirose and K. Sudo for
excellent technical assistance.
| |
FOOTNOTES |
Submitted August 19, 1998; accepted April 6, 1999.
Supported in part by the Japan Society for the promotion of Science.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Tatsutoshi Nakahata, MD, DM Sci, Department
of Clinical Oncology, The Institute of Medical Science, The University
of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan; e-mail:
nakahata{at}ims.u-tokyo.ac.jp.
| |
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ABSTRACT
INTRODUCTION