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Blood, Vol. 91 No. 8 (April 15), 1998:
pp. 2745-2752
Cytokine Production and Function in c-mpl-Deficient Mice: No
Physiologic Role for Interleukin-3 in Residual Megakaryocyte and
Platelet Production
By
Timothy Gainsford,
Andrew W. Roberts,
Shinya Kimura,
Donald Metcalf,
Glenn Dranoff,
Richard C. Mulligan,
C. Glenn Begley,
Lorraine Robb, and
Warren S. Alexander
From The Walter and Eliza Hall Institute for Medical Research, PO
Royal Melbourne Hospital, Victoria, Australia; the Dana Farber Cancer
Institute, Boston; and the Howard Hughes Medical Institute, Harvard
University School of Medicine, Boston, MA.
 |
ABSTRACT |
Mice lacking thrombopoietin (TPO), or its receptor c-Mpl, display
defective megakaryocyte and platelet development and deficiencies in
progenitor cells of multiple hematopoietic lineages. The contribution of alternative cytokines to thrombopoiesis in the absence of TPO signalling was examined in mpl / mice.
Analysis of serum and organ-conditioned media showed no evidence of a
compensatory overproduction of megakaryocytopoietic cytokines. However,
consistent with a potential role in vivo, when injected into
mpl/ mice, interleukin-6 (IL-6) and
leukemia inhibitory factor (LIF) retained the capacity to elevate
megakaryocytes and their progenitors in hematopoietic tissues and
increase circulating platelet numbers. However, double mutant mice bred
to carry genetic defects both in c-Mpl and IL-3 or the alpha chain of
the IL-3 receptor, displayed no greater deficiencies in megakaryocytes
or platelets than mpl-deficient animals, suggesting absence of
a physiologic role for IL-3 in the residual megakaryocytopoiesis and
platelet production in these mice.
| |
INTRODUCTION |
THE PRODUCTION OF platelets, the small
anuclear cells shed into the circulation by mature megakaryocytes,
plays an important role in blood clotting and hemostasis. A number of
hematopoietic growth factors have been implicated in
megakaryocytopoiesis, including interleukin-3 (IL-3),
granulocyte-macrophage colony-stimulating factor (GM-CSF),
erythropoietin (EPO), and stem cell factor (SCF), as well as the
cytokines that use the gp130 receptor signalling chain (IL-6, IL-11,
leukemia inhibitory factor [LIF] and oncostatin-M [OSM]).1 However, recently it has become clear that
thrombopoietin (TPO) is the major physiologic regulator of this
process. Isolated and cloned on the basis of interaction with its
receptor c-Mpl, a member of the hematopoietin receptor family, TPO is
transcribed predominantly in the liver, kidney, and smooth muscle and
is secreted as a glycoprotein, the circulating concentration of which
is regulated by megakaryocyte and platelet mass.2 As a
single agent, TPO is a specific inducer of the proliferation of
progenitor cells committed to megakaryocyte production. In semisolid
culture assays, TPO stimulates the formation of small colonies of
mature megakaryocytes from bone marrow or spleen cells, and this
activity is augmented by the addition of other factors particularly
IL-3 and SCF.3-6 TPO also stimulates megakaryocyte
maturation, promoting expansion of cell size, increased DNA ploidy, and
the cytoplasmic reorganization that typically precedes platelet
release.3,5-9 Indeed, culture systems have been devised in
which TPO can support complete in vitro development of primitive
CD34+ hematopoietic progenitor cells to mature,
platelet-shedding megakaryocytes.8
In vivo, administration of TPO elevates the number of circulating
platelets up to 10-fold and stimulates production of mature megakaryocytes and their progenitors in hematopoietic
tissues.6,10-14 Although other cytokines, particularly
IL-3, IL-6, IL-11, and LIF, share these thrombopoietic
properties,15-18 at maximal concentrations their effects
are significantly less potent than those of TPO. The essential
physiologic roles of TPO have been established in mice genetically
manipulated to lack the cytokine or its receptor, c-Mpl. The
hematopoietic organs of TPO/ and
mpl/ mice produce only 5% to 10% of
the normal number of megakaryocytes, most of which are relatively
immature. Consequently, the mice are thrombocytopenic and display a
platelet deficiency of similar magnitude.19-21 In addition
to deficiencies in megakaryocyte progenitor cells,
c-mpl/ mice also display reduced
numbers of immature cells committed to all other hematopoietic
lineages.20 This phenotype is shared with TPO-deficient
mice22 and suggests that signalling through c-Mpl may also
play a critical role in regulation of the hematopoietic stem cell
compartment.
Despite the severe thrombocytopenia that characterizes
c-mpl-deficient animals, residual platelet production in
mpl/ mice is sufficient to prevent
hemorrhage and allows ostensibly normal development and adult life. To
examine the contribution of alternative stimuli to the residual
megakaryocytopoiesis in mice lacking TPO signalling, we have examined
cytokine production and activity in mpl-deficient animals.
Although we found no evidence of elevation of non-TPO
megakaryocytopoietic cytokines in mpl-deficient mice, injection
of IL-6 or LIF stimulated megakaryocyte and platelet production in
mpl/ mice to a similar extent to that
observed in normal animals, suggesting alternative megakaryocytopoietic
stimuli can function in the absence of TPO signalling. However, IL-3
appeared not to contribute significantly to megakaryocytopoiesis
because double mutant mice deficient in c-Mpl and IL-3 or its receptor
alpha chain (IL-3R ) displayed no greater deficiencies in platelets, megakaryocytes, or their progenitors than is evident in
mpl/ mice.
| |
MATERIALS AND METHODS |
Mice.
NZB mice bearing mutant IL-3R genes
(Il3ran),23 c-mpl-deficient
mice,20 and mice lacking a functional IL-3
gene24 have been described previously. Mice genetically
defective for both c-Mpl and IL-3R were generated by breeding
mpl/ and NZB mice to yield F1
offspring (mpl+/- Il3ra+/n),
which were subsequently interbred. The peripheral blood platelet counts
of 98 male F2 mice were measured at 6 weeks of age and 27 thrombocytopenic (mpl/) mice were
identified and analyzed. The IL-3R genotype of these mice was then
determined using the polymerase chain reaction method previously
described.23 As mpl/ mice
were of mixed C57Bl/6 and 129/Sv genetic background, and IL-3/ mice were a mixture of C57Bl/6, 129/Sv,
and Balb/c, in the mpl/
IL-3/ intercross experiments, wild-type
controls included a combination of data from mice of each of these
strains. All mice were housed in a conventional animal facility and
analyzed at between 2 and 4 months of age.
Cytokines.
Murine GM-CSF, IL-3, IL-5, and TPO were produced in purified
recombinant form and kindly provided by Dr N. Nicola (The Walter and
Eliza Hall Institute of Medical Research, Melbourne). Recombinant murine IL-6 was a kind gift of Dr R. Simpson (Joint Protein Structure Laboratory, The Walter and Eliza Hall Institute of Medical
Research and The Ludwig Institute for Cancer Research, Melbourne).
Recombinant human G-CSF and recombinant murine LIF were kindly provided
by Amgen (Thousand Oaks, CA) and AMRAD (Boronia, Australia),
respectively, and recombinant human IL-11 was purchased from R&D
Systems (Minneapolis, MN).
Cytokine bioassays.
Organs from male mpl/ mice or their
wild-type littermates were collected, fragmented with scissors, and
incubated in 2 mL of serum-free Dulbecco's modified Eagle's medium
(DMEM) in a fully humidified atmosphere of 10% CO2 in air.
Supernatants conditioned by each tissue were collected after 4 days,
filter sterilized, and stored at 4°C for analysis. Cytokine
concentrations were determined by bioassay as
described25,26 using parental Ba/F3 cells for IL-3
(detection limit, 20 pg/mL) or Ba/F3 cells transfected with the
specific receptors for IL-6 (detection limit, 100 pg/mL), IL-11
(detection limit, 100 pg/mL), LIF (detection limit, 200 pg/mL), G-CSF
(detection limit, 400 pg/mL), or TPO (detection limit, 100 pg/mL). As
no organ-conditioned medium contained IL-3, FDC-P1 cells,
which respond to IL-3 or GM-CSF, provided a specific bioassay for
GM-CSF (detection limit, 100 pg/mL). Microwell assays were performed in
60-well microtiter dishes (Lux, Nashville, TN) as
described.25 Briefly, 200 cells in 10 µL DMEM containing 10% newborn calf serum (NCS) were added to duplicate 5 µL volumes of
serially diluted organ conditioned medium. The numbers of viable cells
in each well were determined by manual inspection under phase-contrast
microscopy after incubation for 2 days at 37°C in a fully
humidified atmosphere of 10% CO2 in air. Where cell counts
exceeded 200 per well, the culture was scored as containing >200
cells. Each assay included a titration of a known concentration of the
relevant purified recombinant cytokine, which was used as a standard
for calculating the amount of that cytokine present in each conditioned
medium. As previously described,25 the media conditioned by
four organs, brain, salivary gland, kidney, and liver, contain
components toxic to the assay cells. Because mixing experiments
indicated that the toxic effects were eliminated at dilutions greater
than 1:8, the limits of detection for cytokines in these conditioned
media were eightfold higher than stated. Similarly, as serum samples
were initially diluted fourfold, the limit of detection for cytokines
in serum was fourfold higher than stated.
Cytokine injections.
Groups of weight-matched male mpl/
mice or wild-type littermates were injected twice daily subcutaneously
with 0.2 mL of cytokine solution or dilution medium (saline
supplemented with 5% NCS). Mice injected with IL-6 (2 µg/day) and
LIF (1 µg/day) were analyzed after 7 and 8 days, respectively.
Hematologic and progenitor cell analysis.
Peripheral blood was collected by retro-orbital bleeding and diluted
into 3% acetic acid containing methylene blue (white cells) or 1%
ammonium oxalate (platelets) for manual cell counts using hemocytometer
chambers and standard microscopy. Megakaryocytes were enumerated by
microscopic examination of hematoxylin and eosin-stained histologic
sections of sternal bone marrow and spleen, which had been cut to
standard thicknesses of 1 and 2 µm, respectively. The clonal culture
of hematopoietic progenitor cells was performed in 1 mL cultures of 2.5 × 104 (bone marrow) or 105 (spleen) cells
in 0.3% agar in Iscove's modified Dulbecco's medium (IMDM)
supplemented with 20% fetal calf serum (FCS), 10 ng/mL murine IL-3,
100 ng/mL murine SCF, and 4 U/mL human EPO as previously described.18 Single stimulus cultures included cytokines at the following final concentrations: murine TPO, 100 ng/mL; IL-5, 103 U/mL; IL-6, 100 ng/mL; murine GM-CSF, 10 ng/mL; human
G-CSF, 10 ng/mL; murine M-CSF 10 ng/mL. Agar cultures were fixed and sequentially stained for acetylcholinesterase, Luxol fast Blue, and
hematoxylin, and the composition of each colony was determined at
100-fold to 400-fold magnifications.
| |
RESULTS |
Cytokine production in mpl/ mice.
The concentration of cytokines in the serum or in media conditioned by
the organs of adult mpl/ mice was
examined using factor-dependent cells ectopically expressing specific
cytokine receptors (see Materials and Methods). Consistent with
previous reports,19 the level of serum TPO, which was
undetectable in wild-type mice, was elevated in
mpl/ animals (640 ± 0 pg/mL, n = 4). Recent studies suggest that lack of platelet-mediated clearance of
TPO is the predominant mechanism accounting for the elevated TPO levels
in mpl-deficient animals.27 We were unable to
determine whether changes in production also contribute, as TPO
production was not sufficient to be detected in conditioned medium from
any organs of normal or mpl/ mice. To
investigate whether maintenance of the residual thrombopoiesis in mice
lacking TPO signalling may be due to increased production of
alternative cytokines with megakaryocytopoietic activities, we also
examined production of IL-3, IL-6, IL-11, LIF, and GM-CSF in
mpl/ mice. None of these regulators
was detected in the serum of wild-type or mpl-deficient animals
(Table 1). Similarly, IL-3 was undetectable in all conditioned media examined from mice of both genotypes. As
previously observed,25 LIF, IL-6, and GM-CSF were detected in a wide range of organ-conditioned media from normal mice, in particular that of the lungs and muscle, and no significant differences in the concentrations of these cytokines was observed in analysis of
mpl/ animals (Table 1). Similarly, no
difference in the organ distribution or level of IL-11 production,
which was evident at lower concentrations and from a more restricted
range of tissues, was observed between mpl-deficient and
wild-type littermates (Table 1). Together, these data suggest that of
the megakaryocytopoietic cytokines under study, dramatic elevation in
production does not contribute to the mechanisms by which
mpl/ mice maintain residual platelet
levels.
In vivo activity of megakaryocytopoietic cytokines in
mpl/ mice.
To investigate whether alternative cytokines are capable of stimulating
megakaryocytopoiesis in the absence of TPO signalling, the response of
mpl/ mice to daily administration of
IL-6 or LIF was compared with that of normal animals. Presumably
reflecting their thrombocytopenic state, at analysis, several
mpl/ mice displayed evidence of
subcutaneous hemorrhage at the injection site and two LIF-injected
animals that exhibited hematocrit values below 40 were excluded from
analysis.
Consistent with previous reports,16 IL-6 injections for 6 days in normal mice elevated spleen weight and induced a 1.6-fold increase in platelet numbers. Similar findings were observed in mpl/ mice, with the platelet count
increasing by approximately twofold (Table
2). In mice of both genotypes, the increase in platelet counts was
accompanied by significant increases in megakaryocytes and
megakaryocyte progenitor cells, particularly in the spleen, but also in
the bone marrow (Table 2). Little alteration in the hematocrit or white
blood cell count were observed in IL-6-injected mice of either
genotype (Table 2).
Similarly, LIF administration for 7 days also increased the number of
circulating platelets in both normal and
mpl/ mice
(Table 3). The magnitude of the increase,
1.6-fold, was consistent with that observed in previous studies of LIF
activity in normal animals.17 In contrast to IL-6-treated
mice, little if any significant elevation in the number of mature
megakaryocytes was observed in LIF-injected
mpl/ mice. No significant elevation
in megakaryocyte progenitor cells was observed in either wild-type or
mpl/ mice receiving LIF (Table 3).
Thrombopoiesis in mice deficient in both c-Mpl and the IL-3R
chain.
To determine whether IL-3 plays an essential role in the residual
megakaryocytopoiesis observed in the absence of TPO signalling, mice
deficient in both c-Mpl and the IL-3R chain were generated. NZB mice
are homozygous for a mutation in the IL-3R chain gene (Il3ra), and their cells are markedly hyporesponsive to IL-3 in vitro23 (see Table 4). A total
of 98 F2 mice from an intercross between
mpl/ and NZB mice were generated.
Twenty-seven (27.5%) of these animals were thrombocytopenic and were
genotyped as mpl/. As expected with
normal Mendelian segregation of alleles, of the 27 mpl-deficient animals, seven (26%) were also homozygous for
the mutant IL-3R chain (Il3ran/n). Although one
of the seven mpl/
Il3ran/n mice died before analysis of an
undetermined cause at 3 weeks of age, the mutant IL-3R gene appeared
to have no significant impact on the survival of
mpl/ mice. Moreover, the
mpl/ Il3ran/n
animals were no more thrombocytopenic than
mpl/ Il3ra+/+ or
mpl/ Il3ra+/n
mice (Table 4) suggesting that IL-3 does not contribute significantly to the maintenance of residual platelet numbers in
mpl/ mice. As expected, cells from
mice homozygous for the Il3ran allele displayed
only a residual response to IL-3, while normal colony numbers were
observed in response to GM-CSF (Table 4).
Hematopoiesis in mice deficient in both c-Mpl and IL-3.
Given that some residual IL-3 responsiveness may still exist in NZB
mice (Table 4), mice created through gene targeting to definitively
lack IL-324 were bred with mpl-deficient animals for analysis of hematopoiesis in mpl/
IL-3/ double mutant offspring. Genotyping of
276 weanlings from matings of
mpl+/-IL-3+/- parents showed normal
ratios of offspring of each of the expected genotypes
(Table 5). In addition, no adult lethality
was observed, indicating that the loss of IL-3 had no significant
effect on the survival of mpl/ mice.
Peripheral blood analysis showed that, like the
mpl/ Il3ran/n
mice, mpl/
IL-3/ animals displayed the thrombocytopenia
typical of mpl/ mice. However, the
superimposed lack of IL-3 did not reduce the platelet count further, or
was any thrombocytopenia observed in mice lacking IL-3 alone
(Table 6). Similarly, the reduction in megakaryocytes evident in histologic sections of
mpl/ mice was not exacerbated in
mpl/ IL-3/
animals and megakaryocyte numbers were normal in
IL-3/ mice (Table 6). Both
IL-3/ and
mpl/ IL-3/
mice also displayed normal hematocrits and total white blood cell
counts, as well as levels of circulating lymphocytes, monocytes, neutrophils, and eosinophils that were within the normal range (Table
6). The number of peritoneal cells, the cellularity of the femoral bone
marrow and spleen, and the distribution of morphologically recognizable
precursor cells in these populations was not significantly different in
mutant mice of all genotypes from that in normal controls (Table 6).
Moreover, flow cytometric analysis of cells from bone marrow, spleen
and thymus, using antibodies directed against a range of T lymphoid, B
lymphoid, myeloid, and erythroid markers,20 showed no
perturbations in mutant animals of any genotype (data not shown).
To determine whether IL-3 plays a physiologic role in earlier stages of
hematopoiesis, progenitor cells from mutant animals were assayed in
semisolid agar cultures containing a combination of SCF, IL-3, and EPO,
which stimulates a broad range of erythroid and myeloid
colonies.20 The total numbers of hematopoietic progenitor cells were normal in IL-3/ mice and the low
levels evident in mpl/
animals20 were not further lowered in
mpl/ IL-3/
mice (Table 7). The enumeration of
individual colony types also indicated that loss of IL-3 did not alter
the numbers of progenitor cells committed to specific hematopoietic
lineages, including megakaryocyte progenitors, either in normal or
mpl/ mice (Table 7). As expected, in
cultures stimulated solely by TPO, no colony formation was evident with
the cells from mpl/ or
mpl/ IL-3/
mice, while equal numbers of small megakaryocyte colonies developed from wild-type and IL-3/ bone marrow cells
(wild-type: 9 ± 4; IL-3/: 8 ± 5 colonies per 2.5 × 105 cells plated, n = 3).
Interestingly, despite development in the absence of IL-3, normal
numbers of IL-3-responsive cells had developed in
IL-3/ bone marrow (wild-type: 88 ± 28;
IL-3/: 71 ± 30 colonies per 2.5 × 105 cells plated, n = 3) and no further reduction from that
already evident in mpl/ mice was
evident in the mpl/
IL-3/ double mutants
(mpl/: 29 ± 9;
mpl/ IL-3/:
33 ± 12 colonies per 2.5 × 105 cells plated, n = 3). In addition, colony formation in response to stimulation by single
cytokines (IL-5, IL-6, GM-CSF, G-CSF, M-CSF, or SCF) was similar
between bone marrow cells from mpl/
and mpl/
IL-3/ mice, as well as between
IL-3/ and wild-type animals (data not shown).
Consistent with these observations of bone marrow progenitor cells, the
lack of IL-3 also failed to alter the numbers of erythroid, mixed
erythroid, or myeloid colony-forming cells in the spleens of these
animals (data not shown). Together, these analyses suggest that even in the absence of signalling from the major regulator of
megakaryocytopoiesis, TPO, IL-3 does not contribute to the residual
megakaryocyte and platelet production in
mpl/ mice. Moreover, these data
confirm and extend previous observations24 to suggest that
IL-3 has no essential physiologic role in the maintenance of other
mature hematopoietic cells or their committed progenitor cells, in
otherwise normal animals or in mpl/
mice.
| |
DISCUSSION |
The dominant role of TPO in the regulation of platelet production has
been convincingly demonstrated by the severe thrombocytopenia evident
in mice lacking this regulator or its receptor.19-21
However, as TPO/ and
mpl/ mice retain the capacity to
produce sufficient platelets to prevent bleeding, an important
contribution to thrombopoiesis in vivo is likely to be made by other
cytokines with megakaryocytopoietic potential. Candidate cytokines
include IL-6, IL-11, IL-3, and LIF, which significantly elevate
platelet numbers when injected into animals.15-18 Although
gene targeting studies have shown that in the presence of TPO, the loss
of such cytokines does not significantly affect platelet
levels,28-31 subtle actions of these factors may exist and
be shown or amplified in mice lacking the dominant TPO signalling
system. We initially investigated this possibility by examining
mpl/ mice for evidence of elevated
production of known megakaryocytopoietic factors. Although we found, as
previously described,19 that serum TPO was elevated, we did
not detect biologically active IL-6, IL-11, IL-3, LIF, or GM-CSF in the
circulation of mice of either genotype. A measure of production of
these cytokines from individual tissue sources was obtained by
analyzing organ conditioned medium. As evident in a previous
survey,25 factors were produced by multiple tissues of
normal adult mice, in particular the lungs, muscle, thymus, and bone
shaft. However, no significant differences in the organ distribution or
level of cytokine production were observed in analyses of media
conditioned by the organs of mpl/
mice (Table 1). Thus, dramatic elevation in production of alternative megakaryocytopoietic cytokines does not appear to be a major mechanism by which mice lacking an intact TPO signalling system maintain residual
platelet production. An interesting characteristic of mpl/ mice is that despite producing
reduced numbers of progenitors cells for essentially all hematopoietic
lineages, deficits in mature blood cells are restricted to
platelets.19,20 This observation complements previous
studies, including those of cytokine administration32 and
other mutant mouse models of hematopoiesis,33 in which
alterations in the number of committed progenitor cells are not
necessarily reflected by the numbers of circulating blood cells. In the
case of the granulocyte lineage, for example, we also found no evidence that elevated cytokine production provides a compensatory mechanism: the concentrations and spectra of organs producing G-CSF (data not
shown) and GM-CSF (Table 1) were not significantly different between
normal and mpl-deficient mice.
To determine the in vivo megakaryocytopoietic potential of alternative
cytokines in the absence of TPO signalling, control and
mpl/ mice were injected with LIF or
IL-6. Previous studies with these cytokines in normal mice showed a
capacity to elevate platelet numbers up to twofold.16,17
Our studies confirmed these observations and demonstrated that LIF and
IL-6 retain this activity in mpl/
mice. Although the platelet numbers in LIF- or IL-6-injected mpl/ mice did not recover to normal
levels, reflecting the reduced numbers of megakaryocytes and their
progenitors available for stimulation, the magnitude of the platelet
increase was at least as significant in mpl-deficient mice as
in the wild-type controls (IL-6: mpl+/+ 1.6-fold,
mpl/ 2.2-fold; LIF:
mpl+/+ 1.7-fold,
mpl/ 1.6 fold, Table 2).
Our results confirm previous studies demonstrating the thrombopoietic
potential of IL-6, and also of IL-11 and SCF, in
mpl/ and
TPO/ mice22 and further
demonstrate the potency of LIF in the absence of TPO signalling. These
observations suggest that, in vivo, such cytokines act substantially
independently of TPO, in contrast to their reported action in certain
in vitro assays.34 The elevation in platelet numbers in
mpl/ mice injected with IL-6 was also
accompanied by significant increases in the numbers of mature
megakaryocytes and their progenitors in hematopoietic organs, a
response consistent with that in wild-type animals (Table 2). Thus, our
data also extend previous observations to show that in vivo,
alternative cytokines cannot only elevate platelet numbers, but can
stimulate the full process of megakaryocytopoiesis in the absence of
TPO signalling.
These analyses provide important proof that alternative cytokines have
the capacity to stimulate megakaryocytopoiesis in the absence of TPO
signalling, but do not provide direct evidence that any of these
factors control the residual steady-state megakaryocyte and platelet
development in mpl/ mice. To further
address this issue, we are conducting genetic crosses between
mpl/ mice and other genetically
modified mice unable to produce alternative megakaryocytopoietic
cytokines or their receptors. As IL-3 is the most potent single
stimulus for megakaryocyte colony formation in vitro,1 we
initiated studies with mice deficient in either IL-3 or the IL-3R
chain. NZB mice have a naturally-occurring mutation in the IL-3R
chain gene preventing normal cell-surface expression of the receptor
and resulting in a markedly reduced response to IL-3.23
Interbreeding of mpl/ and NZB mice
yielded the expected number of mpl/
Il3ran/n double mutant offspring and platelet
numbers in these mice were equivalent to those in their
mpl/ Il3ra+/(+/n)
littermates (Table 4), suggesting little or no role for IL-3 in
mpl-deficient platelet development.
As our data and that of others23 suggested that some
residual IL-3 responsiveness may exist in NZB mice (Table 4), we
definitively addressed the role of IL-3 in normal and residual
mpl/ megakaryocytopoiesis using
animals genetically modified to lack this cytokine.24 The
numbers of circulating platelets, mature megakaryocytes, and
megakaryocyte progenitor cells were normal in
IL-3/ mice (Tables 6 and 7). Moreover, even
in the absence of TPO signalling, lack of IL-3 did not exacerbate the
megakaryocyte deficiency or the thrombocytopenia of
c-mpl/ mice (Tables 6 and 7). In
previous studies, initial indications that IL-3 may not play a
prominent role in megakaryocytopoiesis came from the normal
megakaryocyte and platelet production evident in athymic nu/nu
mice, which lack T lymphocytes, considered a prominent source of
IL-3.35 Our data from genetically modified mice
specifically lacking the cytokine provide definitive evidence that,
despite its potent in vitro activity on megakaryocyte
proliferation1 and its ability to stimulate this lineage on
administration in vivo,15 physiologically IL-3 has no
significant essential role in megakaryocytopoiesis, or is it involved
in the residual megakaryocyte and platelet production in
mpl/ mice. Of broader significance,
IL-3/ mice displayed normal numbers of
hematopoietic progenitor cells in the bone marrow and spleen, as well
as their mature progeny in the circulation, marrow, spleen, and
peritoneal cavity24 (Tables 6 and 7). Similarly, the
reduction in committed progenitor cells characteristic of
mpl/ mice was not exacerbated in the
absence of IL-3. Thus, in addition to providing no evidence of a role
for IL-3 in megakaryocyte and platelet production, our studies confirm
previous analyses24 to suggest that IL-3 plays little
physiologic role in the development of blood cells of other lineages.
| |
FOOTNOTES |
Submitted May 5, 1997;
accepted November 24, 1997.
Supported by the National Health and Medical Research Council,
Canberra, the Anti-Cancer Council of Victoria, the Cooperative Research
Centre for Cellular Growth Factors, the National Institutes of Health,
Bethesda, Grant No. CA22556, and by support to S.K. by the Naito
Foundation.
Address reprint requests to Warren S. Alexander, PhD, The Walter and
Eliza Hall Institute for Medical Research, PO Royal Melbourne Hospital,
Victoria 3050, Australia.
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.
| |
ACKNOWLEDGMENT |
We thank Ladina DiRago and Sandra Mifsud for excellent technical
assistance and Jodie deWinter and Anne Chow for animal husbandry.
| |
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Abstract
Introduction
Abstract