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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 10 (May 15), 1998:
pp. 3702-3710
Myeloid Development Is Selectively Disrupted in PU.1 Null Mice
By
Karen L. Anderson,
Kent A. Smith,
Kris Conners,
Scott R. McKercher,
Richard A. Maki, and
Bruce E. Torbett
From The Burnham Institute, La Jolla, CA; and the Department of
Immunology, The Scripps Research Institute, La Jolla, CA.
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ABSTRACT |
The ets family transcription factor PU.1 is expressed in
monocytes/macrophages, neutrophils, mast cells, B cells, and early erythroblasts, but not in T cells. We have recently shown that PU.1
gene disruption results in mice with no detectable
monocytes/macrophages and B cells but T-cell development is retained.
Although neutrophil development occurred in these mice, it was delayed
and markedly reduced. We now proceed to demonstrate that PU.1 null
hematopoietic cells fail to proliferate or form colonies in response to
macrophage colony-stimulating factor (M-CSF), granulocyte CSF (G-CSF),
and granulocyte/macrophage CSF (GM-CSF). In contrast, PU.1 null cells did proliferate and form colonies in response to interleukin-3 (IL-3),
although the response was reduced as compared with control littermates.
Compared with control cells, PU.1 null cells had minimal expression of
G- and GM-CSF receptors and no detectable M-CSF receptors. The size of
individual myeloid colonies produced from PU.1 null primitive and
committed myeloid progenitors in the presence of IL-3, IL-6, and stem
cell factor (SCF) were reduced compared with controls. Under these
conditions, PU.1 null progenitors produced neutrophils but not
monocytes/macrophages. These observations suggest that PU.1 gene
disruption induces additional cell-autonomous effects that are
independent of the alterations in myeloid growth factor receptor
expression. Our results demonstrate that PU.1 gene disruption affects a
number of developmentally regulated hematopoietic processes that can,
at least in part, explain the changes in myeloid development and
reduction in myeloid and neutrophil expansion observed in PU.1 null
mice.
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INTRODUCTION |
MATURE BLOOD CELLS are continually
replenished throughout life. This requires establishment of a pool of
pluripotent stem cells early in embryogenesis from which various
hematopoietic lineages are derived by a tightly regulated developmental
process. One level of regulation of this process occurs via
transcription factors that direct the expression of genes controlling
commitment and/or differentiation. This pattern of
lineage-specific gene expression is complex, most likely requiring
interactions between lineage-specific and broadly expressed
transcription factors. These critical transcriptional events result in
the expression of various cytokine receptors, adhesion molecules, and
other key cellular proteins in pluripotent cells, thus providing the
means for individual cells to survive, proliferate, and
differentiate.1-3
PU.1 is a member of the ets family of transcription
factors4,5 recognizing a purine-rich DNA sequence
containing the core sequence 5 -GGAA/T-3.6 Expression of
PU.1 is limited to hematopoietic cells, including primitive
CD34+ cells, macrophages, B lymphocytes, neutrophils, mast
cells, and early erythroblasts.7-11 In vitro studies
suggest that PU.1 regulates the activity of a number of myeloid- and
lymphoid-specific promoters and enhancers.12 Recent
evidence also suggests that promoters for the genes encoding receptors
for macrophage colony-stimulating factor (M-CSF),13
granulocyte/macrophage CSF (GM-CSF),14 and granulocyte CSF
(G-CSF)15 are regulated by PU.1. CSF receptors have been
proposed to be critical for survival, proliferation, and terminal
maturation of myeloid cells.16,17
We18 and others19 have shown that the loss of
myeloid lineages in PU.1 gene-disrupted mice implicates PU.1 in the
regulation of myeloid development. Fetal18,19 or
newborn18 PU.1 null mice have no detectable
monocytes/macrophages or neutrophils. However, within 2 to 3 days after
birth, neutrophils, as defined by multisegmented nuclei, expression of
Gr-1, and chloroacetate esterase (CAE) staining, can be detected within
the liver, bone marrow, and spleen.18 Although these cells
express CD18, they fail to express CD11b. In contrast to neutrophils,
monocytes/macrophages18 and osteoclasts20 have
not been detected in older PU.1 null mice. This loss of normal
myelopoiesis in PU.1 gene-disrupted mice could be due to a
cell-autonomous defect. That is, the loss of PU.1 in myeloid
progenitors may directly affect development, producing the observed
multiple myeloid lineage defects. Alternatively, PU.1 gene disruption
may alter the hematopoietic microenvironment, for example, by
loss/dysfunction of stroma, resulting in the absence or reduction of
requisite signals for developmental progression of myeloid progenitors.
This is not without precedent, given the absence of osteoclasts in the
PU.1 null mouse and the subsequent failure of bone marrow cavity
formation.20 These changes caused by PU.1 gene disruption
are not mutually exclusive, and each could contribute to the myeloid
defects observed.
To investigate cell-autonomous defects produced by the loss of PU.1, we
assessed hematopoietic cell responses to M-CSF, G-CSF, GM-CSF, and
interleukin-3 (IL-3) or stem cell factor (SCF), IL-3, and IL-6 in
vitro. We show that PU.1 null hematopoietic cells do not respond to M-,
G-, and GM-CSF. Concomitant with the loss of response to these
cytokines, M-CSF receptors were not detected and minimal levels of G-
and GM-CSF receptors were detected on fresh or cultured PU.1 null
cells. When PU.1 null progenitor cells were assessed in clonogenic
assays using only SCF, IL-3, and IL-6, neutrophils were produced,
whereas monocytes/macrophages were absent. These results convincingly
demonstrate that PU.1 gene disruption affects a number of
developmentally regulated hematopoietic events that can, at least in
part, explain the alterations in myeloid development observed in PU.1
null mice. Thus, PU.1 appears pivotal in regulating myelopoiesis. PU.1
null mice should be useful for studying mechanisms controlling lineage
determination during myelopoiesis.
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MATERIALS AND METHODS |
Mice.
C57BL/6x129 PU.1 gene-disrupted mice were produced as previously
reported.18 PU.1 gene-disrupted hemizygous mice, F6
generation, were bred to produce PU.1 null homozygous neonates.
Homozygous neonates were identified as PU.1 null mice by the absence of
neutrophils/monocytes in blood. Tissue obtained from the tail was used
to confirm the genotype as previously reported.18 PU.1 null
neonates all developed septicemia within 24 hours and died by 48 hours
if not placed on enrofloxacin treatment (2.5 mg/kg/d). PU.1 null mice
treated in this manner have survived up to 20 days.
Isolation of hematopoietic cells.
Livers or spleens of the mice were aseptically removed, and a
single-cell suspension was generated. For bone marrow, femurs were
removed, stripped of soft tissue, and then crushed to release cells
within the marrow cavity. When required, red blood cells were lysed
with a 0.15-mol/L solution of ammonium chloride. For some studies,
low-density mononuclear cells were isolated by purification over a
density gradient (Histopaque 1083; Sigma, St Louis, MO) per the
manufacturer's instructions.
Cell proliferation.
Proliferation was assessed using an MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide)-based
colorimetric assay (Cell Proliferation Kit I; Boehringer, Mannheim,
Germany) as directed. Mononuclear cells (104 to
105 cells per well) from control and PU.1 null neonate
liver and spleen were grown in a 96-well plate in triplicate under the
following conditions: Iscove's media with 20% fetal calf serum
(medium) with no added factors (as a negative control); IL-3 only at
0.01%, 0.1%, and 1.0% conditioned media (from X63 cells; a kind gift from F. Melchers); and GM-CSF or G-CSF (R&D Systems, Minneapolis, MN)
only at 0.1, 1.0, and 10.0 ng/mL for 4 days at 37°C and 5% CO2. M-CSF was used at 10% to 30% L cell-conditioned
media or 5,000 U/mL rmM-CSF (a kind gift from D. Hume). Cell viability was assessed on the basis of trypan blue exclusion in a duplicate plate.
Enzyme histochemistry and immunohistochemistry.
Methods for CAE cytochemical staining21 and immunostaining
of cytospin slides for F4/80,22 M-CSF receptor/c-fms, and
Gr-123 were as previously described.18
Flow cytometric analysis.
Isolated hematopoietic cells were either directly analyzed or cultured
for 2 to 3 weeks in medium plus 1% IL-3-conditioned media, 10 ng/mL
G-CSF, and 10 ng/mL GM-CSF before flow cytometric analysis. Protocols
for staining the various cell populations for flow cytometric analyses
were as previously described.18 To determine cell surface
G- and GM-CSF receptor expression, we used commercially available
phycoerythrin (PE)-labeled cytokines (Fluorokines; R&D Systems) with
PE-streptavidin as a control, as directed by R&D Systems. Flow
cytometric analysis used a Becton Dickinson FACScan with Cell Quest
acquisition and analysis software (Becton Dickinson, Franklin Lakes,
NJ).
Colony-forming assays.
Assays for hematopoietic progenitor cells were performed as described
previously24 with some modification. For generation of
committed progenitor colonies and high-proliferative potential colony-forming cells (HPP-CFC), low-density mononuclear cells were
seeded at 5 × 103/mL in commercially available
methylcellulose media containing SCF, IL-3, IL-6, and erythropoietin
(MethoCult GF M3434; Stem Cell Technologies, Vancouver, BC, Canada).
This combination has been shown to be sufficient for generation of both
HPP-CFC and committed progenitor colonies.25 Assays were
performed in triplicate at 1 mL/35-mm2 petri dish.
Single-factor colony assays used either 1% IL-3-conditioned media
(from X63 cells; gift from F. Melchers) or 300 U/mL rmIL-3 (gift from
D. Hume), 10 ng/mL rmGM-CSF (R&D Systems), 10 ng/mL rhG-CSF (R&D
Systems), or 30% L929 cell-conditioned media (as a source of M-CSF).
Cultures were maintained at 37°C in a humidified atmosphere of 5%
CO2 in air. Colonies were scored as clusters containing
more than 50 cells. HPP-CFC were identified as tight colonies with a
diameter greater than 0.5 mm2 containing greater than
50,000 cells. Committed progenitor and single-factor colonies were
scored at 7 to 9 days and HPP-CFC at 21 days by in situ observation
with an inverted microscope. Colonies were routinely evaluated by
cytologic examination after aspiration of individual colonies with a
Gilson pipetman followed by cytospin preparation and Wright-Giemsa
(Sigma, St Louis, MO) staining to determine phenotype.
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RESULTS |
Impaired response of hematopoietic cells from PU.1 null mice to M-CSF,
G-CSF, or GM-CSF.
We have previously shown that PU.1 gene targeting results in the
absence of detectable monocyte/macrophage development, reduced and
delayed neutrophil generation, and generalized reduction of hematopoietic cell numbers in PU.1 null mice.18 Given these findings, the demonstration of PU.1 binding sites in the promoter region of M-, G-, and GM-CSF receptor genes,12 and the
possible role of PU.1 in regulating these receptors genes, we examined whether alterations in myeloid development in PU.1 null mice were a
consequence of the inability to respond to each of these cytokines.
The ability of neonatal PU.1 null (deficient) hematopoietic cells to
use M-, G-, GM-CSF or IL-3 was first assessed in clonogenic assays.
Cells removed from the bone marrow, spleen, or liver of PU.1 null
neonatal mice failed to produce colonies when cultured with M-, G-, or
GM-CSF, but generated colonies when cultured with IL-3. Although the
level of response to IL-3 varied in the studies (Table
1, and data not shown), at no time did we
observe M-, G-, or GM-CSF supporting colony generation from
PU.1-deficient hematopoietic cells. In contrast, cells from similar
hematopoietic compartments of control littermates generated colonies in
response to M-, G-, GM-CSF or IL-3.
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Table 1.
Hematopoietic Cells From PU.1 Null Mice Are Unable to
Generate Colonies in Response to GM-CSF, G-CSF, or M-CSF
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Next, the ability of neonatal PU.1-deficient liver cells to proliferate
in response to IL-3, G-CSF, or GM-CSF stimulation was assessed in
short-term assays. Proliferation in these cytokines was assessed at the
end of a 4-day culture. There was a readily detectable difference
between cells from control and PU.1 null mice: cells from PU.1 null
mice cultured with G- or GM-CSF did not proliferate above the baseline
values observed for cultures without addition of cytokines (Fig
1). M-CSF was also found not to support
proliferation of PU.1 null cells (data not shown). Although
hematopoietic cells from PU.1 null mice exhibited baseline values when
cultured with G- or GM-CSF, they proliferated above baseline values
when cultured with IL-3. These results were common to all experiments.
However, as shown in Fig 1 and in other experiments (data not shown),
hematopoietic cells from PU.1 null mice compared with control
littermates proliferated suboptimally in IL-3. To assess whether M-,
G-, or GM-CSF might function as PU.1 null cell survival factors rather
than proliferation factors, PU.1 null cells established in parallel to
proliferation assays were tested for viability at the end of these
short-term cultures by trypan blue exclusion. We could not detect an
increase in survival of PU.1 null cells in M-, G-, or GM-CSF as
compared with controls without cytokines (data not shown).
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| Fig 1.
Proliferation of PU.1 deficient cells was reduced in IL-3
and absent in G-CSF and GM-CSF as compared with control cells.
Mononuclear cells isolated from neonates were incubated in IL-3 (1%
SN), G-CSF (10 ng/mL), or GM-CSF (10 ng/mL) for 4 days. Proliferation
was measured by colorimetric assessment of MTT reduction and by
counting viable cells at the end of the culture period. Results for
cellular proliferation are presented as the mean ± SD of absorbance.
Similar results were obtained for spleen and bone marrow cells (not
shown). Note an approximately threefold reduced proliferation of PU.1 deficient cells ( ) in IL-3 compared with control ( ) and no
proliferation in G-CSF or GM-CSF detectable above the baseline (medium
only conditions).
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To determine if G-CSF- or GM-CSF-responsive cells were present at low
frequency and could be expanded in the absence of IL-3, freshly
isolated PU.1 null neonatal liver cells were plated in triplicate at
500,000, 50,000, and 5,000 cells/well in 1% IL-3-conditioned medium
(as positive controls) or in 20 ng/mL G- or GM-CSF. After 1 week, few
viable cells (< 1%) other than adherent cells remained in either
the G-CSF- or GM-CSF-containing wells, and at 3 weeks, only the cells
in IL-3-containing medium remained viable and continued to expand
(data not shown).
Our results demonstrate that PU.1 disruption imparts a cell-autonomous
defect in myeloid cells that manifests as an inability to use M-, G-,
or GM-CSF for proliferation and colony formation. Since survival and
proliferation were evaluated after 4 days, our studies cannot fully
eliminate the possibility that progenitors that use M-, G-, or GM-CSF
are present at very low frequency and survive for very brief periods in
the presence of these cytokines. Lastly, it should be noted that at the
population level, hematopoietic cells from PU.1-deficient liver,
spleen, and bone marrow did not respond as well to IL-3 as did
populations from control mice.
Hematopoietic cells from PU.1 null mice bound minimal amounts of
PE-labeled G-CSF and GM-CSF.
The lack of clonogenic growth and in vitro proliferation in response to
M-, G-, or GM-CSF by hematopoietic cells obtained from PU.1 null mice
could be due to the absence of cytokine receptors or the inability of
the receptors to respond to their respective growth factors. Given our
results, we next assessed whether these receptors were present. Both G-
and GM-CSF receptors have been reported to be present on early
progenitors and highly expressed during various stages of myeloid
development.16,17 Since low numbers of myeloid cells were
observed in liver, spleen, and bone marrow of PU.1 null
neonates18 (and data not shown), we reasoned that in vitro
expansion of hematopoietic cells from the liver for 1 or 2 weeks in
cultures containing IL-3, G-CSF, and GM-CSF should allow for sufficient
numbers of cells for receptor analyses. This combination of cytokines
normally allows for both expansion of myeloid cells and G- and GM-CSF
receptor expression. Furthermore, as we have shown, hematopoietic cells
from PU.1 null mice do not expand in either G- or GM-CSF alone.
Assessment of M-CSF receptors on PU.1 null cells is presented elsewhere
in this report.
Representative results from flow cytometric analyses for expression of
G- and GM-CSF receptors on neonatal PU.1 null and wild-type myeloid
cells after short-term culture are presented in Fig
2A. Cytokine-expanded PU.1 null cells were
found to bind reduced amounts of PE-labeled G-CSF (PE control mean
fluorescence, 4.2; mean fluorescence for PE G-CSF binding, 4.9) as
compared with binding of PE-labeled G-CSF to cytokine-expanded cells
from wild-type (PE control mean fluorescence, 3.2; mean fluorescence
for PE G-CSF binding, 17.1) littermates. As seen for G-CSF binding to
PU.1 null cells, PE-labeled GM-CSF binding to PU.1 null cells was also
reduced (PE control mean fluorescence, 4.2; mean fluorescence for PE
GM-CSF binding, 5.5) as compared with wild-type cells (PE control mean
fluorescence, 3.2; mean fluorescence for PE GM-CSF binding, 24.1).
These results demonstrate the absence of detectable PU.1 null cells
capable of binding high levels of PE-labeled G- and GM-CSF after
culture (note the right shoulder of both the G- and GM-CSF binding
curves merge with controls). Furthermore, the vast majority of cultured PU.1 null cells were incapable of binding detectable amounts of G- and
GM-CSF, as compared with cells from wild-type littermates.
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| Fig 2.
Binding of the PE-labeled myeloid growth factors G- and
GM-CSF to cultured PU.1 null cells was minimal. Cells were prepared directly from liver of control and PU.1 null neonatal mice and cultured
in the presence of IL-3, GM-CSF, and G-CSF for 9 days to expand
populations of Gr-1+ cells. Wild-type and PU.1 deficient
cells were harvested, live cells enriched over a density gradient and
then analyzed for the presence of receptors for G-CSF (G-CSFR), and for
GM-CSF (GM-CSFR) by the binding of PE-conjugated G-CSF or GM-CSF (A) or
for the neutrophil specific marker Gr-1 (B). (A) PE-control staining
( ); PE-conjugated G-CSF or GM-CSF binding (- - -). (B) Irrelevant control antibody staining (); specific anti-Gr-1 antibody staining (- - -).
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We next analyzed the PU.1 null and wild-type cultured cells, used for
G- and GM-CSF receptor assessment, for Gr-1 expression. Gr-1 has been
used as a marker of mouse granulocyte differentiation.21 Gr-1 expression is highest on terminally differentiated neutrophils, lower on myeloblasts, transient on differentiating monocytes and not
detectable on primitive progenitors.21,26 IL-3, G-CSF, and
GM-CSF all induce Gr-1, progenitor colony formation, and neutrophil production, whereas proliferation to these cytokines is inversely related to Gr-1 expression.21 Cultured PU.1 null cells were found to be 41% Gr-1+ (as well as CAE+, data not shown),
as compared with 67% Gr-1+ for wild-type cells (Fig 2B).
Cells expressing intermediate and high levels of Gr-1 are known to bind
G- and GM-CSF.21 We find that 43% of the Gr-1+
PU.1 null cells are intermediate to high as compared with 86% of the
Gr-1+ wild-type cells (channel values from 50 to 10,000).
These results suggest that sufficient PU.1 null myeloid cells are
present after culture that have the potential to express G- and GM-CSF
receptors.
Since culture conditions might alter G- and GM-CSF receptor expression
on neonatal PU.1 null cells we directly assessed fresh pooled bone
marrow and liver cells from 9-day-old PU.1 null (n = 2) and control
(n = 1) mice for binding of PE-labeled G- and GM-CSF. We have
previously demonstrated neutrophil development in older PU.1 null
mice18; therefore, differentiated myeloid cells should be
present for G- and GM-CSF receptor analysis. Binding of PE-labeled
G-CSF to fresh PU.1 null cells was extremely reduced (PE control mean
fluorescence, 5.1; mean fluorescence for PE G-CSF binding, 6.0), as
compared with the binding of PE-labeled G-CSF to cells from a wild-type mouse (PE control mean fluorescence, 5.5; mean fluorescence for PE
G-CSF binding, 21.9). Receptors for GM-CSF binding were also reduced on
fresh PU.1 null cells (PE control mean fluorescence, 5.1; mean
fluorescence for PE GM-CSF binding, 7.8), as compared to cells from a
wild type mouse (PE control mean fluorescence, 5.5; mean fluorescence
for PE GM-CSF binding, 31.5).
In summary, PE-labeled G- and GM-CSF binding to cells taken directly
from PU.1 null mice or after short-term culture was minimal, although
myeloid cells were present that should bind higher amounts of G- and
GM-CSF. Although our results do not allow us to quantify the number of
receptors remaining on cells after PU.1 gene disruption, the lack of G-
and GM-CSF-induced proliferation and colony formation is consistent
with too few receptors for normal function or the loss of normal
receptor function for the remaining receptors.
Hematopoietic cells from PU.1 null mice generate myeloid colonies
that are reduced in cell number and are lacking monocytes/macrophages.
We have demonstrated the delayed appearance of neutrophils, the absence
of monocytes/macrophages in vivo,18 and the inability of
myeloid cells from PU.1 null mice to use M-, G-, and GM-CSF in vitro
(Table 1 and Fig 1). We next investigated the effects of PU.1 gene
disruption on progenitor expansion and development in assays that do
not rely on M-, G-, or GM-CSF. For these studies, we used a
methylcellulose media containing SCF, IL-3, and IL-6 since this has
been shown to be sufficient for the assessment of colony-forming
progenitors.25 The number of total colonies produced from
the PU.1 deficient bone marrow was substantially lower, 53- to 86-fold,
compared with the number of colonies produced from the control bone
marrow (Table 2). Progenitor cells from PU.1 deficient liver were reduced threefold to 22-fold compared with
control liver.
Differences other than the total number of colonies between PU.1 null
and control mice were readily apparent. First, it was noted that
colonies produced from PU.1 deficient cells were reduced in cell
number. To confirm this observation colonies were aspirated from
selected clonogenic dishes and counted demonstrating that the average
number of myeloid cells present in colonies from PU.1 deficient liver
was approximately sixfold lower (Fig 3A).Secondly, it was clear that differences existed in the cell types
present in colonies produced from PU.1 null mice (Table
3). Based on morphology, it was apparent
that PU.1 deficient progenitor cells did not differentiate into
monocytes/macrophages. These observations were confirmed by the absence
of the macrophage-associated marker F4/80 and the M-CSF receptor after
immunohistochemical staining of cells generated from PU.1 null mice in
clonogenic assays (Fig 4A, D, and E).
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| Fig 3.
Hematopoietic colony size was reduced in 7-day CFC and
21-day HPP-CFC colonies established from livers of neonatal PU.1 null mice. Individual colonies were aspirated and counted from plates established in triplicate at 5,000 input cells per plate (see Table 2).
Results are presented as the mean ± SD of the number of cells per
colony. Note that the average PU.1 deficient CFC colony size () at 7 to 10 days was approximately sixfold reduced compared to control ()
littermates (A). PU.1 deficient HPP-CFC () were reduced in size
3.5-fold compared with control () littermates (B). Colony size = number of cells per colony. These colony sizes were obtained from CFC
and HPP-CFC shown in experiment 2, Table 2, and are representative of
colony sizes from other experiments.
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Table 3.
Neutrophils But Not Monocytes/Macrophages Are Present in
Clonogenic Colonies From the Liver of Neonatal PU.1 Null Mice
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| Fig 4.
CAE+ and Gr-1+ cells
(neutrophils) but not F4/80+ or M-CSF receptor
(c-fms)+ cells (monocyte/macrophages) developed in vitro
from PU.1 deficient progenitors when cultured in SCF, IL-3, and IL-6.
(A) Wright-Giemsa-stained cytospins of representative multilineage
colonies from control and PU.1 null individuals revealed a conspicuous
absence of macrophages in PU.1 deficient colonies. However, neutrophils
and megakaryocytes were evident in both panels (1,150×). (B) The
neutrophil enzyme CAE, demonstrated by a pink reaction product, was
evident in PU.1 deficient and control polymorphonuclear cells. Note the
larger, nonstaining macrophage in the control individual (1,150×).
(C) Both PU.1 deficient and control polymorphonuclear cells expressed the cell surface marker Gr-1 which was demonstrated by immunoperoxidase staining. Detection was with diaminobenzidine (DAB) which yields an
orange-brown reaction product (1,150×). (D) M-CSF receptor immunostaining revealed no M-CSF receptor-positive cells in PU.1 deficient colonies. Cell debris was present that stained
nonspecifically with DAB in PU.1 null cultures (1,150×). (E)
Immunocytochemical staining for the macrophage marker F4/80 revealed no
positive cells in PU.1 deficient colonies, whereas many orange-staining F4/80+ cells were found in control colonies (1,150×).
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In contrast to colonies derived from cells of PU.1 null mice,
macrophages were identified in 63% and 87% of all control colonies assessed in two separate experiments (Table 3 and Fig 4; Control; A and
D). Neutrophils as identified by morphologic criteria (Fig 4, PU.1
null; A), CAE staining (Fig 4, PU.1 null; B), and Gr-1 immunostaining
(Fig 4, PU.1 null; C) were present in 91% and 100% of all PU.1 null
colonies compared with 58% and 93% of all control colonies tested in
two separate experiments. Mast cells were present in 59% and 28% of
all PU.1 null colonies as compared with 87% and 33% of all control
colonies, and megakaryocytes were found in 23% and 14% of PU.1 null
and 30% and 25% of all control colonies in these experiments (Table
3).
These in vitro results revealed that disruption of the PU.1 gene
results in an intrinsic defect in committed myeloid progenitor cells
that precludes monocyte/macrophage development from progenitors. The
inability to detect M-CSF receptors on cells produced in clonogenic assays provides an explanation for the loss of M-CSF response demonstrated in Table 1. Given that SCF, IL-3, and IL-6 were used in
the clonogenic assays it is most likely that absence of monocyte/macrophage development is independent of the loss of hematopoietic cell response to M-, G-, or GM-CSF. Finally, these results also confirm that the effect of PU.1 gene disruption is selective: PU.1 appears not to be absolutely necessary for mast cell or
neutrophil development from myeloid progenitors, but is essential for
monocyte/macrophage generation (Table 3).
Neonatal bone marrow and liver of PU.1 null mice have fewer HPP-CFC.
To determine whether PU.1 gene disruption only targeted more committed
progenitors (Table 2) and selected myeloid lineages (Table 3), such as
monocytes/macrophages, we next assessed whether myeloid HPP-CFC cells
were affected by PU.1 gene disruption. HPP-CFC have been proposed as
candidates for a primitive myeloid progenitor cell with some stem cell
properties.28 These cells are capable of giving rise to
CFU-S, marrow repopulating cells, erythroid and megakaryocyte
reconstituting cells in lethally irradiated mice.29 In in
vitro assays, these cells form large colonies, greater than 0.5 mm2 and greater than 50,000 cells.30 As for the
CFC assessment we relied on methylcellulose media containing the growth
factors SCF, IL-3, and IL-6. It must be noted that SCF, IL-3, and IL-6 have been shown to give rise to HPP-CFC, although the frequencies of
colonies produced per number of input cells were not as numerous as
when M-CSF and/or GM-CSF were included.28 Scoring
of colony production from bone marrow at 21 days of culture revealed
28- to 93-fold fewer HPP-CFC from PU.1 null mice than found in control cultures (Table 2). The number of HPP-CFC colonies from PU.1 deficient
liver was found to be threefold to ninefold less than control cultures
(Table 2). As was the case for committed progenitors, counts of cells
per HPP-CFC colony from the liver revealed lower numbers of cells from
PU.1 null mice, as compared with control littermates (Fig 3B).
In summary, a reduced number of HPP-CFC-derived colonies were obtained
from PU.1 deficient hematopoietic cells as compared with control
hematopoietic cells. The reduced number of cells in HPP-CFC colonies is
consistent with a cell-autonomous defect limiting colony expansion of
primitive myeloid progenitor cells as the result of PU.1 gene
disruption. Taken together the CFC and HPP-CFC assays demonstrate that
disruption of PU.1 results in an intrinsic defect in committed and
primitive myeloid cells in mice that reduces the expansion of primitive
and committed myeloid progenitors and disrupts development to
monocytes/macrophages.
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DISCUSSION |
In this study, we investigated the mechanisms responsible for myeloid
disruption in PU.1 null mice. Our previous studies demonstrated that
PU.1 gene disruption results in the delay of neutrophil development and
the absence of monocytes/macrophages18 and
osteoclasts.20 Lymphopoiesis was also affected with the
loss of B cells, but not T cells. Other lineages, such as
megakaryocytes and erythrocytes, were minimally affected.18
Our initial studies suggested that PU.1 gene targeting imposed
lineage-specific alterations in hematopoietic development, rather than
ablation of more primitive progenitors that give rise to myeloid or
lymphoid lineages. In our current study, we provide evidence that
disruption of PU.1 results in the loss of M-, G-, and GM-CSF-mediated
proliferation and development. Concomitant with this loss M-CSF
receptors were undetectable, whereas G- and GM-CSF receptor expression
was substantially reduced. Independent of the loss of M-, G-, and
GM-CSF receptor function, PU.1 deficient myeloid progenitor cells have
additional defects that alter differentiation and expansion. Our
findings demonstrate that PU.1 is not essential for myeloid or
neutrophil commitment, but is required for optimal myeloid expansion
and necessary for the development of monocytes/macrophages.
It is apparent from our studies that PU.1 is required for normal
expression of M-, G-, and GM-CSF receptors. These results are not
surprising particularly given that PU.1 cooperates with other
transcription factors, such as C/EBP and AML1, to regulate the
promoters of the myeloid growth factor receptors for M-, G-, and GM-CSF
and other myeloid specific genes.12 Although hematopoietic cells from PU.1 null mice do not respond to G- or GM-CSF in vitro, a
low level of granulopoiesis still occurs in these mice. A number of
mechanisms have been proposed as to how G-CSF regulates granulopoiesis in vivo; these include stimulation of primitive progenitors,
proliferation of granulocyte progenitors and induction of granulocyte
maturation. However, the in vivo importance of these mechanisms is not
at all clear.31 The hypothesis that G-CSF receptor
engagement17 is required for differentiation of neutrophils
from progenitors is controversial. Evidence from the G-CSF
ligand-null,33 G-CSF receptor-null mice,31 and
G- and GM-CSF cytokine deficient mice33 demonstrates that
cytokines other than G- or GM-CSF allow bone marrow granulopoiesis, but
it appears that G-CSF might be required for normal neutrophil numbers
in the periphery.31 Results obtained from PU.1 null mice,
both in vivo and in vitro, would also argue that granulopoiesis occurs
in the absence of detectable G- and GM-CSF response. The presence of
neutrophils in PU.1 null mice is consistent with a variation of the
proposed stochastic developmental model,34,35 where IL-3,
possibly IL-6, or other cytokines provide survival signals to
developing neutrophils thereby allowing intrinsic developmental
programs to commence. That cytokine/cytokine receptor interaction
provides a survival signal, allowing cells at a specific stage to
exploit intrinsic or external signals, has been demonstrated for
monocytes/macrophages,26 T cells,36 and B
cells37 as well. Although developing PU.1 null neutrophils
appear to be morphologically normal in having segmented nuclei,
CAE+ expression and intermediate expression of Gr-1 (Fig
4), we found that colony expansion is limited and terminal
differentiation and functional maturity does not occur (K.L. Anderson
et al, submitted). What still remains to be determined is
whether reduced expansion, loss of functional maturity, and reduced
Gr-1 expression are due to the postulated role of
G-CSF17,38 or other cytokines such as GM-CSF31
or are the result of additional defects induced by the absence of PU.1.
Studies are under way to address these issues by restoring the
expression of G-, GM-CSF receptors or PU.1 directly in PU.1 null
progenitor cells.
The absence of monocytes/macrophages in PU.1 null mice would not be
predicted from the studies of M-CSF, G-CSF, and GM-CSF cytokine null
mice, which develop monocytes/macrophages, albeit at reduced
levels.32,33 Recent studies have provided further support
that M-CSF functions as a survival factor, rather than as a
differentiation factor, for monocytes.26 Given the clear lack of M-, G-, and GM-CSF response in hematopoietic and myeloid cells
from PU.1 deficient mice, and the absence of a normal bone marrow
microenvironment, the question of whether IL-3 or cytokines other than
M-, G-, and GM-CSF are limiting in vivo and thus not supporting
monocyte/macrophage development must be considered. However, we show
that SCF, IL-3, and IL-6 allow for HPP-CFC generation and neutrophil
development, thus arguing for at least intact IL-3 signaling in PU.1
null cells. However, the loss of PU.1 function cannot be supplanted by
these cytokines to rescue monocyte/macrophage development in vitro.
Based on our results and published reports demonstrating in vitro (and
in myeloid growth factor null mice) that other cytokines in the absence
of M-, G-, and GM-CSF are sufficient for low levels of
monocyte/macrophage generation, we propose that PU.1, in addition to
regulating M-, G-, and GM-CSF receptor expression, is necessary for
intrinsic programs required for monocyte/macrophage survival or
differentiation. Alternatively, the loss of M- and GM-CSF receptors is
not equivalent to loss of their respective cytokines during myeloid
development, so that monocyte commitment occurs in the PU.1 null mouse,
but in the absence of either M- or GM-CSF receptor expression committed
cells do not survive.
Whether PU.1 disruption causes intrinsic defects in primitive myeloid
cells beyond the loss of a M-, G-, or GM-CSF mediated response is
difficult to ascertain in this study. PU.1 deficient hematopoietic
cells generated HPP-CFC in the presence of SCF, IL-3, and IL-6, but the
frequency of HPP-CFC per hematopoietic compartment (liver or bone
marrow) and the number of cells per colony was reduced as compared with
age-matched controls. A recent study has suggested the existence of at
least four stages in the mouse HPP-CFC hierarchy, from pro-HPP-CFC to
HPP-CFC-3, where HPP-CFC are positioned within the hierarchy based on
in vitro cell expansion to combinations of cytokines.28 The
most primitive HPP-CFC utilizes M-CSF and/or GM-CSF with SCF,
IL-1, IL-3, and/or IL-6 in various combinations for maximum
colony expansion.28 The decreased number of HPP-CFC from
PU.1 deficient mice could be the result of a more generalized in vivo
disruption of primitive progenitors that give rise to pro-HPP-CFC.
Alternatively, but not mutually exclusive, is the possibility that the
reduced HPP-CFC frequency is due to a diminished survival or expansion
of selected HPP-CFC progenitors in vivo as the result of the loss of
normal expression of M-CSF or GM-CSF receptors. Although a role for
PU.1 in hematopoietic progenitor cells is supported by a recent study in which constitutive expression of PU.1 resulted in enhanced size and
numbers of colonies in IL-3, G-CSF, or GM-CSF, the mechanism is
unknown.39 Since our studies do not distinguish as to what stage the HPP-CFC belong, we cannot conclude precisely as to where the
lack of PU.1 affects early myeloid development. Studies are under way
to isolate primitive lin Sca-1+ populations
from age-matched PU.1 null and control littermates to address these
issues on a per cell basis, both in vitro and in vivo.
The role of PU.1 in hematopoietic development has also been studied in
an independently derived PU.1 gene-targeted mouse.19 A
comparison of the PU.1 null mouse discussed here and that generated by
Scott et al19 reveals similarities but also some major
differences. One difference is that the PU.1 null mice reported here
are born alive, whereas in the studies by Scott et al19 all
mice die in utero by day 18. A second major difference that exists is
the block of all myeloid and lymphoid development in the mouse
developed by Scott et al.19 Recent studies by Scott et
al40 show that PU.1 gene disrupted mouse fetal liver cells
failed to reconstitute the myeloid or lymphoid lineages or rescue
lethally irradiated mice. These results suggest that the absence of
PU.1 results in a cell-autonomous defect that disrupts primitive
hematopoietic stem cell commitment to myeloid and lymphoid lineages.
The molecular mechanism(s) contributing to the observed hematopoietic
defects in and the fetal death of the PU.1 null mice generated by Scott et al19 are still unexplained. Recently, we have proposed
possible explanations for the observed differences between the distinct PU.1 null phenotypes generated in these mice.18
We observe hematopoietic cell-autonomous defect(s) in the PU.1 null
mice described herein. Rather than a generalized block in all myeloid
development, lineage specific effects were observed. As shown here and
previously,18 monocyte/macrophage development is disrupted,
but commitment and development along the neutrophil lineage occurs. At
least part of the mechanism for the abnormal myeloid development in
PU.1 null mice appears to be the loss of normal M-, G-, and GM-CSF
receptor expression during myeloid development. In addition to the loss
of M-, G-, and GM-CSF receptor regulation there appears to be an
independent requirement for PU.1 expression during monocyte/macrophage
development and possibly during HPP-CFC expansion. It is tempting to
speculate that PU.1 plays a role beyond simply regulating growth factor
receptor expression during myelopoiesis. Finally, we would like to
propose that the cumulative effect of altered or the absent expression
of PU.1-regulated genes, a group which is likely to include genes other
than those already known, contributes to the observed PU.1 null
phenotype.
| |
FOOTNOTES |
Submitted September 29, 1997;
accepted December 31, 1997.
Supported by National Institutes of Health Grants No. DK49886 (B.E.T.)
and AI30656 (R.A.M.).
Address reprint requests to Bruce E. Torbett, PhD, Department of
Immunology, IMM-7, The Scripps Research Institute, 10550 N Torrey Pines
Rd, La Jolla, CA 92037.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors gratefully acknowledge the technical assistance of Michelle
Butler and Kari Carver and the secretarial assistance of Bonnie Towle.
We would like to thank Laura Crisa, Toñi Ortiz, Greg Henkel,
Deborah Vestal, and Donald Mosier for critically reading the
manuscript. This is publication IMM 10705 from The Scripps Research
Institute.
| |
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Abstract
Introduction
Abstract