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Blood, Vol. 93 No. 8 (April 15), 1999:
pp. 2525-2532
Soluble Interleukin-6 (IL-6) Receptor With IL-6 Stimulates
Megakaryopoiesis From Human CD34+ Cells Through
Glycoprotein (gp)130 Signaling
By
Xingwei Sui,
Kohichiro Tsuji,
Yasuhiro Ebihara,
Ryuhei Tanaka,
Kenji Muraoka,
Makoto Yoshida,
Kaoru Yamada,
Kiyoshi Yasukawa,
Tetsuya Taga,
Tadamitsu Kishimoto, and
Tatsutoshi Nakahata
From the Department of Clinical Oncology, The Institute of Medical
Science, The University of Tokyo, Tokyo, Japan; Biotechnology Research
Laboratory, Tosoh Co, Ayase, Kanagawa, Japan; and the Institute for
Molecular and Cellular Biology and Department of Medicine III, Osaka
University Medical School, Suita, Osaka, Japan.
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ABSTRACT |
We have recently shown that stimulation of glycoprotein (gp) 130, the membrane-anchored signal transducing receptor component of IL-6, by
a complex of human soluble interleukin-6 receptor (sIL-6R) and IL-6
(sIL-6R/IL-6), potently stimulates the ex vivo expansion as well as
erythropoiesis of human stem/progenitor cells in the presence of stem
cell factor (SCF). Here we show that sIL-6R dose-dependently enhanced
the generation of megakaryocytes (Mks) (IIbIIIa-positive cells) from
human CD34+ cells in serum-free suspension culture
supplemented with IL-6 and SCF. The sIL-6R/IL-6 complex also
synergistically acted with IL-3 and thrombopoietin (TPO) on the
generation of Mks from CD34+ cells, whereas the synergy
of IL-6 alone with TPO was barely detectable. Accordingly, the addition
of sIL-6R to the combination of SCF + IL-6 also supported a
substantial number of Mk colonies from CD34+ cells in
serum-free methylcellulose culture, whereas SCF + IL-6 in the absence
of sIL-6R rarely induced Mk colonies. The addition of monoclonal
antibodies against gp130 to the suspension and clonal cultures
completely abrogated the megakaryopoiesis induced by sIL-6R/IL-6 in the
presence of SCF, whereas an anti-TPO antibody did not, indicating that
the observed megakaryopoiesis by sIL-6R/IL-6 is a response to gp130
signaling and independent of TPO. Furthermore, human
CD34+ cells were subfractionated into two populations of
IL-6R-negative (CD34+ IL-6R ) and
IL-6R-positive (CD34+ IL-6R+) cells by
fluorescence-activated cell sorting. The CD34+
IL-6R cells produced a number of Mks as well as Mk
colonies in cultures supplemented with sIL-6R/IL-6 or TPO in the
presence of SCF. In contrast, CD34+ IL-6R+
cells generated much less Mks and lacked Mk colony forming activity under the same conditions. Collectively, the present results indicate that most of the human Mk progenitors do not express IL-6R, and that
sIL-6R confers the responsiveness of human Mk progenitors to IL-6.
Together with the presence of functional sIL-6R in human serum and
relative unresponsiveness of human Mk progenitors to IL-6 in vitro,
current results suggest that the role of IL-6 may be mainly mediated by
sIL-6R, and that the gp130 signaling initiated by the sIL-6R/ IL-6
complex is involved in human megakaryopoiesis in vivo.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
HEMATOPOIESIS IS A HIGHLY complex process
mediated by cytokines, by which blood cells of various lineages are
produced from a small population of stem cells. The development of
megakaryocytes (Mks) from their stem/progenitor cells is one of the
least understood aspects of hematopoiesis. The recent cloning of
thrombopoietin (TPO), the ligand for c-Mpl receptor, and the findings
that TPO acts through c-Mpl to promote proliferation and maturation of Mk progenitors, have provided impetus for the study of Mk
biology.1-4 Extensive data published recently support that
TPO acts as the primary regulator of Mk development and platelet
production in vivo.5 However, the facts that although Mpl
knock-out mice are thrombocytopenic, they do not suffer a bleeding
diathesis,6,7 and that transcription factor
NF-E2-deficient mice developed absolute thrombocytopenia independent
of action of TPO,8 suggest that additional factors act in
vivo to promote Mk development and platelet production.
In recent years, three well-characterized cytokines, interleukin-6
(IL-6), IL-11, and leukemia-inhibitory factor (LIF) have been shown to
have various effects on megakaryocyte development in vitro and in
vivo.9-12 These three cytokines together with ciliary
neurotrophic factor (CNTF), oncostatin M (OSM), and recently cloned
cardiotrophin-1 (CT-1), form a subset of cytokines with structural and
functional similarities that share glycoprotein (gp)130, a 130 kD
transmembrane glycoprotein with a large intracytoplasmic domain, as
their signal transducing receptor component.13,14 gp130 has
been shown to be ubiquitously expressed in various tissue and organs,
whereas the ligand-specific receptor components ( -chain) display a
more limited expression.14,15 Our recent studies have shown
that gp130 was expressed in almost all human CD34+ cells,
and that a complex of sIL-6R/IL-6, but not IL-6 or sIL-6R alone, can
activate this glycoprotein and transduce the functional signals that
stimulate the hematopoietic progenitor expansion as well as
erythropoiesis in the presence of stem cell factor (SCF) in vitro, thus
suggesting a novel role of gp130 signaling in human
hematopoiesis.16-18
To examine the potential role of sIL-6R with IL-6 through gp130
signaling in human megakaryopoiesis in vitro, we have examined the
effect of a complex of sIL-6R/IL-6 on Mk production and colony formation from purified human CD34+ cells in serum-free
suspension and methylcellulose cultures. In the present study, we show
that sIL-6R confers the IL-6 responsiveness of human Mk progenitors,
and that a complex of sIL-6R/IL-6, but not sIL-6R or IL-6 alone,
significantly stimulates the proliferation and differentiation of human
Mk progenitors through gp130 signaling in the presence of SCF.
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MATERIALS AND METHODS |
Cell preparation.
Human umbilical cord blood (CB) samples, collected according to
institutional guidance, were obtained during normal full-term deliveries. Mononuclear cells (MNCs) were separated by Ficoll-paque (Pharmacia LKB, Uppsala, Sweden) density-gradient centrifugation after
depletion of phagocytes with Silica (IBL, Fujioka, Japan).
Receptor, cytokines, and antibodies.
Recombinant human IL-6 and sIL-6R were prepared as
described.19 Recombinant human SCF was kindly provided by
Amgen Biologicals (Thousand Oaks, CA). Recombinant human TPO, IL-3 and
EPO were generously provided by Kirin Brewery (Tokyo, Japan). All the
cytokines were pure recombinant molecules and were used at
concentrations that induced optimal response in methylcellulose culture
of human hematopoietic cells. These concentrations are 100 ng/mL of
SCF, 4 U/mL of TPO, and 200 U/mL of IL-3. Preparation of antihuman gp130 monoclonal antibodies (MoAbs) (GPX7, GPX22 and GPZ35) and anti-IL-6R MoAb (MT26) has been described.20,21 The three
anti-gp130 MoAbs recognize different epitopes on gp130 and have been
shown to inhibit IL-6-mediated biological response through inhibition of the IL-6-induced association of gp130 and IL-6
receptors.22 The rabbit antihuman TPO neutralizing
polyclonal antibody was kindly provided by Kirin Brewery. The
anti-IL-6R MoAb was labeled with biotin (Pierce Chemical Co, Rockford,
IL) according to conventional methods. Mouse fluorescein isothiocyanate
(FITC)-labeled monoclonal immunoglobulin (Ig)G1 antibody specific for
CD34(anti-HPCA-2), FITC- and biotin-labeled irrelevant IgG1 MoAb, and
SA-PE (phycoerythrin-conjugated streptavidin) were provided by Becton
Dickinson (San Jose, CA).
Purification of CD34+ cells.
Human CB MNCs were resuspended at 3-5 × 107/mL in
phosphate-buffered saline (PBS) and mixed with Dynabeads M-450 CD34
(Dynal AS, Oslo, Norway), with a bead to cell ratio of 1:1. The
cell-bead suspension was resuspended and incubated at 4°C for 30 minutes with gentle rotation. After incubation, the cell-bead volume
was expanded and placed in a DYNAL Magnetic Particle Concentrator (MPC)
to collect the Dynabeads M-450 CD 34/rosetted cells. The rosetted cells
were incubated with DETACHaBEAD CD34 (Dynal) at room temperature for 45 minutes to detach the Dynabeads M-450 CD34 from the positively selected
cells. The released cells (CD34+), collected by placing the
tube in MPC, were further evaluated by flow cytometric analysis and
colony assay. Eighty-five percent to 95% of the cells separated were
CD34+ by flow cytometric analysis.
Flow cytometry and cell sorting.
Cell sorting based on the CD34 and IL-6R markers was performed with
MNCs. Cells were first incubated with biotin-labeled IL-6R MoAb in ice
for 30 minutes. After washing, the cells were then incubated
simultaneously with FITC-anti-CD34 MoAb and SA-PE for another 30 minutes. After two washings, the cells were suspended in -medium
(Flow Laboratories, Rockville, MD) at concentrations of 5 to 10 × 105/mL and separated by cell sorting. Cells were sorted on
a FACS Vantage flow cytometer (Becton Dickinson). A morphologic gate including about 25% of the events and all the CD34+ cells
was determined on two-parameter histograms (side scatter and forward
scatter). Compensation for two-color labeled samples was set up with
single-stained samples. Positivity or negativity for IL-6R antigen
among CD34+ cells was determined using control cells
labeled with the biotin-PE and FITC-labeled irrelevant IgG1 MoAb. Cells
were sorted into CD34+ IL-6R and
CD34+ IL-6R+ fractions. Purity of sorted
populations as verified by reanalysis was greater than 95%.
Suspension culture.
Purified CD34+ cells were incubated in serum-free
suspension culture as we recently described.16,17 One mL of
culture mixture containing 2000 CD34+ cells, -medium,
2% pure bovine serum albumin (BSA) (Sigma, St Louis, MO), 10 µg/mL
of insulin (Sigma), 200 µg/mL of transferrin (Sigma), 0.01mmol/L
2-mercaptoethanol (Eastman Organic Chemicals, Rochester,
NY), 40 µg/mL of low-density lipoprotein (Sigma), and different
combinations of cytokines was incubated in 24-well tissue plates (Nunc,
Kamstrup, Denmark) at 37°C in a humidified atmosphere flushed with
5% CO2, 5% O2, and 90% N2. At
weekly intervals, cultures were demidepopulated by removal of half the
culture volume, which was then replaced by newly prepared medium with
the same combinations of cytokines. Cells in the collected medium were
washed, counted, cytocentrifuged, and stained. Total Mks generated at
various time points in the suspension culture were calculated based on
the proportion of the IIbIIIa+ cells in cytospin
preparations and the total cell number induced by each combination. For
blocking studies, anti-gp130 MoAbs, anti-TPO Ab, and their control IgG
were added at the beginning of the culture.
Clonal culture.
CD34+ cells were incubated in triplicate at concentrations
of 500 cells/mL in serum-free methylcellulose culture as previously reported with minor modification.23-25 One mL of culture
mixture containing cells, -medium, 0.9% methylcellulose (Shinetsu
Chemical, Tokyo, Japan), 2% pure-BSA (Sigma), 300 µg/mL of human
transferrin, 160 µg/mL of soybean lecithin (Sigma), 96 µg/mL of
cholesterol (Nacalai Tesque, Kyoto, Japan), 10 µg/mL of insulin, 0.05 mmol/L 2-mercaptoethanol and various combinations of cytokines with or without sIL-6R, was plated in each 35-mm Lux standard nontissue culture
dish and incubated at 37°C in a humidified atmosphere flushed with
5% CO2 in air.
The pure Mk colonies were divided into two subtypes according to their
size: colony forming unit-megakaryocyte (CFU-Mk)-derived colonies and
burst forming unit-megakaryocyte (BFU-Mk)-derived colonies.
CFU-Mk-derived colonies were scored as such when they had 4 to 50 Mks,
whereas BFU-Mk-derived colonies were scored when they had more than 50 Mks. Megakaryocyte-mixed colonies including granulocyte-macrophage-megakaryocyte (GMM), erythroid-megakaryocyte (EM), and granulocyte-erythroid-macrophage-megakaryocyte (GEMM) colonies, were scored according to the criteria reported
previously.23-25 The Mks in colonies were determined by
observation on an inverted microscope, and were typically large cells
that had nongranular, translucent cytoplasm and highly refractile cell
membranes. To assess the accuracy of the in situ identification,
individual colonies were lifted with an Eppendorf micropipette under
direct microscopic visualization, spread on glass slides using a
cytocentrifuge (Cytospin II; Shandon Southern, Sewickley, PA), and
stained for morphological examination.
Cytochemical and immunological staining.
Cytocentrifuge preparations from suspension culture and methylcellulose
culture were stained for the observation of cellular morphology.
Staining with May-Grünwald-Giemsa was performed by conventional
method. Immunostaining with the alkaline phosphatase antialkaline
phosphatase (APAAP) method using MoAbs of anti-gpIIbIIIa was performed
as described previously.26 Briefly, cytocentrifuged samples
were fixed with buffered formalin-acetone at 4°C, washed with Tris
buffer saline (Wako, Osaka, Japan), and preincubated with normal rabbit
serum to saturate the Fc receptors on the cell surface. After washing,
the samples were successively incubated with mouse MoAbs and rabbit
antimouse IgG Ab (Medical and Biological Laboratories, Nagoya, Japan),
and then reacted with calf intestinal alkaline phosphatase-mouse
monoclonal antialkaline phosphatase complex (Dako, Osaka, Japan).
Alkaline phosphatase activity was detected with naphthol AS-TR
phosphate sodium salt (Sigma) and fast red TR salt (Sigma) in pH 7.6, 40 mmol/L barbital buffer (Wako) containing levamisole (Sigma) to
inhibit nonspecific alkaline phophatase activity. Positive cells were
stained with reddish granules.
Statistical analysis.
For the statistical comparison in scoring the numbers of Mks, and that
of various Mk colonies, Student's t-test was applied. The
significant level was set to 0.05.
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RESULTS |
Soluble IL-6R stimulates Mk production from human
CD34+ cells in serum-free suspension culture
dose-dependently in the presence of IL-6 and SCF.
To determine the effect of sIL-6R on human Mk progenitors, we first
examined its effect on the Mk generation from human CB CD34+ cells in the presence of 100 ng/mL of IL-6 and SCF in
serum-free culture. The culture was kept up to 1 week and the generated
Mks were detected by immunostaining the cytospin preparations with MoAb
of anti-IIbIIIa. As shown in Fig 1, the
addition of sIL-6R in the presence of IL-6 significantly enhanced Mk
generation in a dose-dependent manner. The increase in the number of
Mks was detectable at concentrations of sIL-6R as low as 10 ng/mL, and reached a plateau at 200 ng/mL. In the absence of sIL-6R, IL-6 also
induced the generation of Mks, but to much less extent. The same
results were also obtained when CD34+ cells purified from
human bone marrow MNC were used. These results clearly indicate that
sIL-6R is functional and capable of stimulating Mk generation in the
presence of IL-6 and SCF, and that sIL-6R at 200 ng/mL appears to be
the optimal concentration for the generation of Mks in serum-free
suspension culture in the presence of SCF and IL-6.
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| Fig 1.
Effect of sIL-6R on Mk generation from human
CD34+ cells in suspension culture in the presence of 100 ng/mL of IL-6 and SCF. 2,000 CB CD34+ cells were
initiated in the culture and results were examined at day 7. Total Mks
generated at each concentration of sIL-6R were calculated based on the
proportion of IIbIIIa+ cells on cytocentrifuge
preparations and total cell number. Results are obtained from three
separate experiments. Standard deviations are represented by error
bars.
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Effects of sIL-6R and IL-6 in combination with various cytokines on
the Mk generation in suspension culture.
To examine the sIL-6R and IL-6-mediated Mk generation in more detail,
we performed CD34+ cell suspension culture supplemented
with sIL-6R, and IL-6 in combination with various cytokines, over a
period of 2 weeks with weekly analysis of the generated cells. The
total number of Mks generated at each time point by various
combinations of cytokines is shown in Table
1. Soluble IL-6R and IL-6 alone or in combination failed to support
cell growth and no Mks were detected in their cultures. A combination
of IL-6 and SCF induced a small number of Mks, but a striking increase
in Mks was observed when sIL-6R was added to the combination. The
addition of sIL-6R to the combination of SCF + IL-6 enhanced the Mk
generation about 13- and 45-fold at days 7 and 14, respectively, with
the maximal generation of Mks at day 14. A typical Mk induced by the
three factors in suspension culture and positively immunostained with
anti-IIbIIIa MoAb, is shown in Fig 2A. The
addition of sIL-6R to the combination of IL-3 + IL-6, also
significantly enhanced the production of Mks, but to a lesser extent
than that to the combination with SCF. The addition of sIL-6R to the
combination of TPO + IL-6 increased Mk numbers about 2- and 5-fold at
days 7 and 14, respectively, although no significant synergistic effect
was observed between IL-6 and TPO. Thus, while having no effects alone,
the complex of IL-6/sIL-6R potently stimulated the Mk generation from
human CD34+ cells in combination with either SCF, IL-3, or
TPO in suspension culture with the most pronounced synergy, in terms of
the fold increase of Mks after addition of sIL-6R, observed in
combination with SCF at day 14. Norol et al27 recently
showed that a combination of TPO with either IL-3 or SCF appeared to be
the optimal combination in the Mk production from adult peripheral
blood and bone marrow CD34+ cells. In the present study,
our results also showed that these combinations exert synergistic
action on the Mk production from CB CD34+ cells. A
combination of TPO + SCF is more potent than that of TPO + IL-3, and
addition of both sIL-6R and IL-6 but not IL-6 alone to the combination
of TPO + SCF greatly enhanced Mk production (Table 1).
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| Fig 2.
Development of Mk generation from CB CD34+
cells by sIL-6R, IL-6, and SCF. (A) A typical Mk generated in
serum-free suspension culture was positively immunostained with
anti-IIbIIIa MoAb by APAAP technique. Magnification: ×1000. (B) A
representative Mk colony derived from CD34+ cells in
serum-free methylcellulose culture (original magnification ×100).
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The effect of sIL-6R/IL-6 in combination with other cytokines on human
Mk generation was also tested. No synergies were found between the
complex of sIL-6R/IL-6 with granulocyte colony-stimulating factor
(G-CSF), macrophage colony-stimulating factor (M-CSF), fibroblast
growth factor (FGF), hepatocyte growth factor (HGF), IL-1 ,
transforming growth factor- (TGF-), insulin-like growth factor-1
(IGF-1), macrophage inflammatory protein-1 (MIP-1), or tumor necrosis
factor (TNF), in the presence or absence of SCF (data not shown). The
effects of other members of the IL-6 family (gp130-stimulatory
cytokines) on megakaryopoiesis were also tested. IL-11 and LIF had
comparable effects to IL-6 in the presence of SCF on the generation of
Mks, whereas no Mks were observed in the cultures supplemented with
either OSM or CNTF in the presence or absence of SCF. The addition of
IL-11, LIF, CNTF, and OSM alone or in combination to the culture
supplemented with sIL-6R, IL-6, and SCF, did not affect the
megakaryopoiesis induced by the three factors (data not shown),
indicating that the observed effect on Mk generation through gp130
stimulation was provided specifically by a complex of IL-6/sIL-6R.
Mk colony formation by sIL-6R and IL-6.
It is likely that the stimulatory effects of sIL-6R and IL-6 on Mk
generation in suspension culture were provided by the proliferation and
differentiation of Mk progenitors. To examine this possibility, methylcellulose clonal assay of CD34+ cells was performed
(Table 2). Although SCF alone or in
combination with IL-6 stimulated no or few Mk colonies from the human
CD34+ cells, the addition of sIL-6R to the combination of
SCF and IL-6 significantly enhanced Mk colony formation. The Mk
colonies induced by the three factors usually consisted of 10 to 300 Mks. A large Mk colony containing about 250 cells induced by sIL-6R,
IL-6 and SCF is shown in Fig 2B. The nature of the Mks was confirmed by immunostaining of cytospin preparations with MoAb against IIbIIIa. In
addition to a number of pure Mk colonies, the combination of sIL-6R,
IL-6, and SCF also induced a large number of megakaryocyte-mixed colonies, in which the cells of other lineages such as granulocyte, macrophage, and/or erythroid cells, in addition to a number of Mks,
were also present (GMM, GEMM, or EM), suggesting a complex of
IL-6/sIL-6R in the presence of SCF acts on Mk progenitors as well as
more primitive progenitors to promote their proliferation and
differentiation. In accordance with the finding obtained in suspension
culture, the addition of sIL-6R also enhanced the Mk colony formation
induced by IL-3 and IL-6. Although no significant synergistic effect
was observed between IL-6 and TPO, the addition of sIL-6R to the
combination of IL-6 + TPO did increase the size and number of Mk
colonies. A larger number of BFU-Mk-derived colonies developed in the
culture supplemented with a combination of sIL-6R, IL-6, and TPO than
in that with TPO alone. In the presence of SCF, IL-6/sIL-6R in
combination with TPO induced the largest number of megakaryocyte-mixed
colonies, most of which were GEMM, in addition to a number of pure Mk
colonies, suggesting that the IL-6/sIL-6R complex and TPO, apart from
their effects on Mk progenitors, also synergistically act on more
primitive progenitors (CFU-GEMM) in the presence of SCF.
sIL-6R and IL-6 stimulate megakaryopoiesis independent of the action
of TPO.
To verify the involvement of membrane-anchored gp130 in the
sIL-6R/IL-6-mediated megakaryopoiesis and to exclude the possibility that the observed megakaryopoiesis by sIL-6R/IL-6 was mediated by TPO,
we performed blocking studies using MoAbs against gp130 as well as
neutralizing Ab against TPO. The addition of anti-gp130 MoAbs to the
culture dose-dependently inhibited the Mk production supplemented with
a combination of sIL-6R, IL-6, and SCF although no effects of control
IgG were detectable, whereas anti-TPO neutralizing Ab had no effect on
the Mk generation in the same condition
(Fig 3A). By contrast, although anti-TPO Ab
efficiently inhibited the TPO-dependent megakaryopoiesis, anti-gp130
MoAbs failed to affect the Mk generation induced by TPO and SCF (Fig
3B). Similar results were also observed in the clonal assay, in which
the anti-gp130 MoAbs, but not TPO Ab, abrogated the Mk colonies induced
by sIL-6R, IL-6, and SCF (Fig 4A,B). These
results clearly indicated that the observed megakaryopoiesis mediated
by a sIL-6R/IL-6 complex was provided by the interaction of sIL-6R/IL-6
with membrane-anchored gp130 and was independent of the action of TPO.
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| Fig 3.
Effects of various concentrations of anti-gp130 MoAbs
( ) and TPO-neutralizing Ab ( ) on the generation of Mks from
CD34+ cells stimulated by a complex of IL-6/sIL-6R (A),
or TPO (B) in serum-free suspension culture in the presence of SCF. The
data from isotype control IgG for anti-TPO Ab ( ) and anti-gp130 MoAb
( ) were also presented. 2,000 CD34+ cells were
cultured in suspension condition and the antibodies were added at the
beginning of the culture. Total Mks was calculated based on the
proportion of IIbIIIa+ cells on the cytospin preparation
and total cell number at day 10 of culture. The absolute number of Mks
produced in the wells without Abs were presented (*) and estimated as
control data. Data indicate the ratio of the total Mks in each well
treated with antibodies or control IgG to those from control, and are
expressed as percent (%) of the control.
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| Fig 4.
Effects of anti-gp130 MoAbs (A) and TPO-neutralizing Ab
(B) on the Mk clonal growth from CD34+ cells supported by
a complex of IL-6/sIL-6R in serum-free methylcellulose culture in the
presence of SCF. 500 CD34+ cells purified from cord blood
were initiated and various concentrations of Abs were added at the
beginning of the culture. Mk colonies including CFU-Mk, BFU-Mk, MK-Mix
were scored at day 11. The number of Mk colonies indicates mean ± SD
from triplicate cultures.
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Most of the human Mk progenitors express little or no IL-6R, and
sIL-6R can confer their responsiveness to IL-6.
The effects of sIL-6R/IL-6 on human megakaryopoiesis together with the
results of blocking studies by anti-gp130 MoAbs suggest that most of
the human Mk progenitors may not express IL-6R but do express the
signal transducer gp130. Indeed, our recent studies, using FACS
analysis, have shown that almost all of the CD34+ cells
express gp130 whereas 50% to 70% of the CD34+ cells do
not express IL-6R.18 To examine the different effects of
sIL-6R and IL-6 on the generation of Mks from IL-6R-positive and
negative CD34+ cells, we performed suspension culture of
CD34+ IL-6R+ and CD34+
IL-6R cells sorted by FACS
(Fig 5A). In cultures of CD34+
IL-6R cells, the addition of sIL-6R to the
combination of IL-6 and SCF dramatically increased the total number of
Mks, whereas CD34+ IL-6R+ cells failed to do so
under the same conditions. Moreover, although TPO alone was capable of
stimulating CD34+ IL-6R cells to
generate a number of Mks, Mks generated from CD34+
IL-6R+ cells by TPO were hardly detectable (Fig 5B).
Similar results were obtained in the methylcellulose assay, in which
CD34+ IL-6R but not CD34+
IL-6R+ cells gave rise to a number of pure Mk and
megakaryocyte-mixed colonies (data not shown). Collectively, these
findings indicate that most Mk progenitors do not express IL-6R, and
that sIL-6R confers the IL-6 responsiveness of human Mk progenitors on
which gp130 but not IL-6R is expressed.
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| Fig 5.
Different effects of IL-6/sIL-6R on the generation of Mks
from CD34+ IL-6R and CD34+
IL-6R+ cells. (A) Selection of CD34+
IL-6R and CD34+ IL-6R+ cells
from human umbilical cord blood MNCs by flow cytometry. R2:
CD34+ IL-6R+, R3: CD34+
IL-6R cells. (B) Megakaryocyte generation from the two
populations of the CD34+ cells. 2,000 CD34+
IL-6R cells and CD34+ IL-6R+
cells sorted by FACS were initiated in serum-free culture,
respectively. The total number of Mks generated by each combination was
determined at day 10. Results are from one representative experiment.
Similar results were obtained in three additional experiments.
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DISCUSSION |
Hematopoietic cytokines control numerous aspects of hematopoiesis
through binding to specific receptors on the surface of target cells.
Most cytokine receptors in the hematopoietic system consist of a
multichain complex, a ligand-binding chain and a signal-transducing
chain, the latter of which is usually used in common by several
receptor complexes. gp130, a membrane-anchored 130 kD glycoprotein with
large transmembrane and intracellular domains, originally identified as
the signal transducing receptor component of IL-6, is shared in common
by the receptor complexes of IL-11, LIF, CNTF, OSM, and
CT-1.14,28,29 Subsequent cloning of the cDNA encoding
murine gp130 showed the ubiquitous expression of gp130 in every murine
organ, including heart, spleen, kidney, lung, liver, and brain, whereas
the expression of various ligand-binding chains displayed a more
limited distribution. In the human hematopoietic system, information on
what cytokine receptors are normally expressed on stem/progenitor cells
remains incomplete. The potential role of gp130, which can be initiated
by a complex of IL-6/sIL-6R, in normal human cells remains largely
unknown. In our recent study a complex of IL-6/sIL-6R through gp130
signaling in the presence of SCF, was found to stimulate expansion of
human primitive progenitor cells and erythropoiesis from human
stem/progenitor cells in vitro. In the present study, we showed that
sIL-6R with IL-6, but not IL-6 or sIL-6R alone, significantly
stimulates the proliferation and differentiation of human Mk
progenitors through gp130 signaling in the presence of SCF. A recent
study showed that the production of murine Mks in response to SCF,
IL-6, and IL-11 alone or in combination was eliminated by neutralizing
the biological activity of TPO, suggesting that the effects of SCF,
IL-6, and IL-11 on megakaryocytes were indirect and mediated by
TPO.30 However, this may not be the case with human
hematopoietic progenitor cells when SCF, IL-6, and sIL-6R are employed,
as shown in the present study. The results of the blocking experiments
by TPO-neutralizing Ab and anti-gp130 MoAbs clearly indicate that the
observed effect of sIL-6R/IL-6 on human megakaryopoiesis is provided
directly by gp130 signaling in the presence of SCF independent of TPO. Thus, the present study may provide new information as to the mechanisms that control the development of human megakaryocytes.
The stimulatory effect of sIL-6R on the megakaryopoiesis in the
presence of IL-6 and SCF, and the lack of this effect in the culture
without sIL-6R, are reminiscent of our recent findings that sIL-6R
confers IL-6 responsiveness to human hematopoietic primitive and
erythroid progenitors.16,17 Our flow cytometric analysis of
the expression of gp130 and IL-6R on CD34+ cells has
provided a plausible explanation for this observation.18 The distinct responsiveness of CD34+
IL-6R and CD34+ IL-6R+ cells
to IL-6/sIL-6R and TPO as shown in the present study, clearly indicates
that most of the Mk progenitors are included in the IL-6R populations, and that the activation of the
signal pathway of gp130 in these Mk progenitors can only be achieved by
a complex of IL-6/sIL-6R, but not by IL-6 alone. Because it is
conceivable that most of the Mk progenitors express both gp130 and
c-Mpl, the activation of either signal pathway can contribute to
megakaryopoiesis. The activation of gp130 (homodimerization), induced
by a complex IL-6/IL-6R, is believed to result in the juxtaposition of
the cytoplasmic regions that appear to initiate a downstream signaling cascade such as RAS/MAPK and JAK2/STAT1,3 leading to cellular response.14 Interestingly, recent studies showed that TPO
also induces activation of JAK2, STAT1, STAT3, and STAT5 through c-Mpl signaling,31-33 suggesting that the ability to activate
JAK/STAT pathway may underline the effects common to c-Mpl signaling by TPO and the gp130 signaling by IL-6/sIL-6R.
It is interesting to note that gp130 signaling plays a role in both
erythropoiesis17 and megakaryopoiesis in the presence of
SCF. In recent years, several lines of evidence support a common origin
of erythroid and Mk lineages. For example, erythroleukemia cell lines
express markers of Mk differentiation, and erythroid and megakaryocytic
cells display a number of common surface markers and transcription
factors including GATA-1, NF-E2, and Tal/SCL.34-36 EPO and
TPO are structurally related growth factors. EPO possesses megakaryopoietic activity although as a physiological regulator of
erythropoiesis.23,37 Similarly, recent studies also show that TPO enhances proliferation of erythroid
progenitors.38,39 The present study together with our
previous one,17 suggests that both erythroid and
megakaryocyte progenitors express gp130 and respond to gp130 signaling
induced by IL-6/sIL-6R, adding further evidence for the hypothesis that
the progenitors within the two lineages respond to overlapping signals.
Recently cloned c-Mpl ligand (TPO) has been shown to be the primary
regulator of megakaryocyte development in vivo. However, the failure of
the Mpl (TPO receptor) knock-out to absolutely eliminate marrow
megakaryocytes or circulating platelets argues that alternative routes
to platelet production exist.6,7 Our results suggest that
the three factors of sIL-6R, IL-6, and SCF may contribute to human
TPO-independent megakaryopoiesis in vivo. This hypothesis is supported
by the following: (1) Soluble IL-6R, IL-6, and SCF are detectable in
human serum, and the half-maximal effect of sIL-6R observed in the
present study was 50 ng/mL, which is within the physiological
range.40-43 In fact, a serum concentration of sIL-6R at
approximately 50 ng/mL in MRL/lpr mice was reported to mediate IL-6
signal in IL-6R gp130+
cells.44 Given the dissociation constant of IL-6 and sIL-6R at approximately 109 mol/L, it is assumed that the
IL-6 will be almost completely complexed with sIL-6R in
serum.45 (2) gp130 and IL-6-knock-out mice have decreased
number of Mk progenitors and megakaryocytes.46,47 (3)
SCF-deficient mice have a pronounced abnormality of
megakaryocytes.48,49 (4) IL-6-sIL-6R double transgeneic
mice have extramedullary expansion of hematopoietic progenitor cells
and a strong increase of peripheral platelets and other blood
cells.50 Taken together, the current results suggest that
although TPO is the principal regulator in the development of Mk, gp130
and c-kit signaling may play a role in megakaryopoiesis in vivo. The
TPO-independent megakaryopoiesis and platelet production observed in
Mpl knock-out mice might be provided, at least in part, by the combined
signals through the gp130 and c-kit.
| |
FOOTNOTES |
Submitted August 12, 1998; accepted December 1, 1998.
Supported by grants from Ministry of Education, Sciences, Sports, and
Culture, Japan.
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, Japan; e-mail:
nakahata{at}ims.u-tokyo.ac.jp.
| |
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ABSTRACT
INTRODUCTION