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Blood, Vol. 93 No. 9 (May 1), 1999:
pp. 2849-2858
Commitment to the Monocytic Lineage Occurs in the Absence of the
Transcription Factor PU.1
By
Gregory W. Henkel,
Scott R. McKercher,
Pieter J.M. Leenen, and
Richard A. Maki
From The Burnham Institute, La Jolla, CA, and Department of
Immunology, Erasmus University, Rotterdam, Netherlands.
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ABSTRACT |
Mice homozygous for the disruption of the PU.1 (Spi-1) gene do not
produce mature macrophages. In determining the role of PU.1 in
macrophage differentiation, the present study investigated whether or
not there was commitment to the monocytic lineage in the absence of
PU.1. Early PU.1 / myeloid colonies were generated from neonate
liver under conditions that promote primarily macrophage and
granulocyte/macrophage colonies. These PU.1/ colonies were found
to contain cells with monocytic characteristics as determined by
nonspecific esterase stain and the use of monoclonal antibodies that
recognize early monocyte precursors, including Moma-2, ER-MP12, ER-MP20, and ER-MP58. In addition, early myeloid cells could be grown
from PU.1/ fetal liver cultures in the presence of
granulocyte-macrophage colony-stimulating factor (GM-CSF). Similar to
the PU.1 null colonies, the GM-CSF-dependent cells also possessed
early monocytic characteristics, including the ability to phagocytize
latex beads. The ability of PU.1/ progenitors to commit to the
monocytic lineage was also verified in vivo by flow cytometry and
cytochemical analysis of primary neonate liver cells. The combined data
shows that PU.1 is absolutely required for macrophage development after
commitment to this lineage.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
DIFFERENT DEVELOPMENTAL stages, from
progenitors to mature cells, have been defined during the process of
monocyte/macrophage differentiation. Nevertheless, the critical
molecular switches necessary to orchestrate monocyte/macrophage
differentiation are poorly understood. One important regulator required
for monocyte/macrophage development is the transcription factor PU.1
(Spi-1). In PU.1/ mice, mature F4/80 positive macrophages are not
observed in various tissues known to contain these
cells.1-3 The use of in vitro colony-forming assays further
showed that no distinctive macrophage colonies are generated from yolk
sac, fetal liver, neonate liver, or bone marrow cells from PU.1 null
animals.4-7 Likewise, PU.1/ embryonic stem cells,
when induced to differentiate along the macrophage lineage, do not
produce mature F4/80 positive cells.4,8 It is not clear how
PU.1 is involved in the regulation of macrophage development. The
defect in macrophage differentiation may occur before commitment, or it
may occur after commitment at an early point in monocyte maturation.
Therefore, determining where the block in monocyte/macrophage
development occurs is an important step in discovering the function of
PU.1 in the differentiation of this lineage.
Although mature macrophages are not detected in PU.1/ animals, it
has never been shown if early monocytic precursors exist in PU.1/
mice. The earliest cell described in the monocytic lineage is the
monoblast, which on division gives rise to the promonocyte.9 Promonocytes are the direct precursors to
monocytes. Low F4/80 expression is detectable as early as the
promonocyte stage and increases as the cells mature.9-11
Other surface markers found on early macrophage precursors include
receptors for immunoglobulin G (IgG) and complement.12 Characteristic enzyme activity associated with the monocytic lineage, such as nonspecific esterase and lysozyme, can be detected as early as
the monoblast stage of development.13 Monoblasts and promonocytes also show moderate peroxidase activity that diminishes as
the cells mature.13 These early macrophage precursors also engage in pinocytosis and phagocytosis, although less actively relative
to mature macrophages.13
Recently, several monoclonal antibodies, including ER-MP12, ER-MP20,
and ER-MP58, have been shown to recognize antigens associated with
early precursor cells of the monocyte/macrophage
lineage.14,15 ER-MP12hi/ER-MP20 ,
ER-MP12+/ER-MP20+, and
ER-MP12/ER-MP20hi population of cells in
bone marrow contain macrophage colony or cluster-forming
progenitors.16 In addition, these three populations represent distinct stages of increasing monocyte maturation as cells
develop from ER-MP12hi only to ER-MP20hi.
Mature macrophages no longer express ER-MP12 or ER-MP20.16 High ER-MP58 expression is found on the majority of M-CSF-responsive monocyte/macrophage precursors in the bone marrow, but diminishes as
the cells mature toward macrophages.15,17
Another monoclonal antibody, MOMA-2, recognizes both mature subsets of
macrophages and bone marrow monocyte/macrophage
precursors.18 The antigen recognized by this antibody is
primarily intracellular, although some level of surface expression has
been shown. Moma-2 is reported not to react with neutrophils and
lymphocytes.18 In contrast, various levels of ER-MP58
expression can be found on the majority of cells in normal bone
marrow.14 Similarly, both ER-MP12 and ER-MP20 recognize
other hematopoietic cell types, including lymphocytes and
granulocytes.16,19 ER-MP12 has also been shown to be a
marker on multiprogenitor cells and costains with other stem cell
markers, including Sca-1 and c-Kit.20 The ER-MP12 antibody
has recently been found to recognize the CD31 antigen (PECAM-1),
whereas ER-MP20 recognizes the Ly-6C antigen.21,22 Although
ER-MP12, ER-MP20, and ER-MP58 recognize other cells, the use of these
antibodies together with Moma-2 should be valuable for identifying
early monocytes.
In this report, we demonstrate using monoclonal antibodies, Moma-2,
ER-MP12, ER-MP20, and ER-MP58, along with cytochemical and functional
assays, that PU.1/ hematopoietic progenitors commit to the
monocytic lineage, but are blocked in their ability to become mature macrophages.
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MATERIALS AND METHODS |
Mice.
C57BL/6 × 129Sv PU.1 targeted mice were generated as previously
reported.2 PU.1 null fetal and neonatal mice were produced by mating PU.1 targeted heterozygous or PU.1 null, normal bone marrow-reconstituted adults. Genotyping of pups was accomplished by
genomic DNA polymerase chain reaction (PCR).2
Antibodies.
Monoclonal antibodies: ER-MP12 (MP12), ER-MP20 (MP20), and ER-MP58
(MP58)14; Moma-2 (Harlan Bioproducts, Indianapolis, IN); F4/80 (C1:A3-1; Biosource Int'l, Camarillo, CA); CD11b, CD18, CD16/32,
c-Kit, Sca-1, Gr-1 (PharMingen, San Diego, CA). Secondary antibodies:
anti-Rat Ig2a fluorescein isothiocyanate (FITC) and anti-Rat Ig2a
Biotin (PharMingen); and Goat (Fab')2 anti-Rat IgG-PE (Biosource Int'l). Polyclonal antibodies: Rabbit
anti-granulocyte-macrophage colony-stimulating factor (GM-CSF)
receptor  and control Rabbit IgG (Santa Cruz Biotechnology, Santa
Cruz, CA).
Colony assays.
Normal and PU.1 null livers were removed from neonatal mice and
passaged through a nylon mesh screen followed by a 25-gauge needle. Red
blood cells were lysed with a 0.15 mol/L solution of ammonium chloride.
Cells were plated in 1% methylcellulose containing media with 15%
heat inactivated fetal bovine serum (FBS), 50 ng/mL stem cell factor
(SCF; R&D Systems, Minneapolis, MN), 2 ng/mL recombinant GM-CSF (R&D
Systems), 100 U/mL recombinant interleukin-3 (IL-3), and 5,000 U/mL
human recombinant macrophage colony-stimulating factor (M-CSF). (Both
the IL-3 and M-CSF were kindly provided by David Hume, Queensland
University, Australia.) Colonies were scored after 7 days in culture.
Enzyme histochemistry.
Cells for enzyme histochemical analysis were spun onto slides with a
cytocentrifuge and fixed in a mixture of Citrate Acid Solution (Sigma,
St Louis, MO), acetone, and formaldehyde (CAF). To identify nonspecific
esterase (NSE) activity, a commercially available kit for NSE staining
(Sigma) was used. The NSE substrate in this kit was -naphthyl
acetate. 3-amino-9 ethylcarbazole was used as a substrate for
peroxidase staining.23
Immunohistochemical stains.
Slides were fixed in CAF and incubated with primary antibodies. To
visualize cells recognized by the antibodies, immunoperoxidase staining
was performed with the Vectastain Elite ABC and Vector VIP
substrate kit (Vector Labs, Burlingame, CA). Cells were counterstained with Vector Methyl Green (Vector Labs).
Reverse transcriptase-PCR analysis.
RNA from normal and PU.1 null colonies was isolated using Trizol
according to the manufacturer's protocol (GIBCO/BRL, Gaithersburg, MD). Two micrograms of total RNA was used to generate single strand cDNA. PCR conditions were performed as previously described, except the
magnesium concentration was increased to 3 mmol/L final
concentration.8 The PCR primers for PU.1 and GM-CSF
receptor have been reported.8 The primers for these
genes amplify across introns to control for genomic contamination.
Fetal liver cultures.
A single cell suspension of normal or PU.1 null E17-E18 fetal liver
cells was plated in T25 flasks in Iscove's media with 20% FBS and 2.5 ng/mL GM-CSF. PU.1/ myeloid cells growing from these cultures
were established away from the fetal liver stroma and sustained as a
suspension cell culture with 1.25 ng/mL GM-CSF. Clones were generated
by limiting dilution in 96 well plates.
Phagocytic assay.
GM-dependent PU.1/ cells were plated in media with 1.25 ng/mL
GM-CSF and 3.6 × 109 particles/mL of 0.2 µm
carboxylated fluorescent latex beads (Molecular Probe, Eugene OR).
After 1 or 4 hours, cells were washed with fluorescence-activated cell
sorter (FACS) buffer (phosphate-buffered saline [PBS] with 2% FBS
and 0.1% azide), resuspended in PBS, layered over FBS, and pelleted
through the serum. Cells were fixed in 0.8% paraformaldehyde for 5 minutes on ice, washed, and resuspended in FACS buffer for analysis. In
addition, slides were made for microscopic examination.
Flow cytometry.
After a single cell suspension of neonate or fetal liver was prepared,
the cells were incubated with DNase I at 100 U/mL final concentration
for 30 minutes at 37°C. Cells were washed in FACS buffer and
prestained in 50 µL of FACS buffer with Fc Block (PharMingen) (CD
16/32) for 15 minutes before the addition of the appropriate monoclonal
antibodies. Cell surface fluorescence was determined using
FITC and r-phycoerythrin (PE) directly conjugated antibodies. For
unconjugated primary antibodies, a biotinylated, FITC- or PE-conjugated secondary antibody was used. A 1:500 dilution of Streptavidin-Cy-Chrome (PharMingen) was used in the triple
staining experiments. Appropriate isotype controls were also included. Events were collected from samples using a Becton Dickinson FACScan (Becton Dickinson, San Jose, CA), and analysis was performed using Cell
Quest (Becton Dickinson).
Cell sorting.
Normal and PU.1 null liver cells were incubated with MP12, followed by
anti-Ig2a-biotin and Streptavidin-PE (PharMingen). Moma-2
FITC was added next, followed by anti-FITC Microbeads (Miltenyi Biotec,
Auburn, CA). The cells were washed, resuspended in FACS buffer, and run
over a steel wool column surrounded by a magnet (Miltenyi Biotec). The
nonmagnetic fraction was collected into one tube. The cells retained in
the steel wool were recovered by removing the magnet and washing the
cells with FACS buffer. The enrichment of the magnetic fraction of
ER-MP12+Moma-2+ cells was verified by flow cytometry.
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RESULTS |
Cells from PU.1 null myeloid colonies show features of monocytic
precursors.
It has recently been reported by our laboratory and others that
neutrophil colonies can be generated from progenitors isolated from
PU.1/ neonatal and fetal liver.6,7 The production of
neutrophil colonies is consistent with the presence of neutrophils detected in PU.1/ mice.2 The identification of
granulocyte colony-forming cells (G-CFC) from PU.1/ liver
suggests that either the earlier granulocyte/macrophage (GM) progenitor
also exists or some alternative pathway bypassing the GM stage of
development results in the production of neutrophil colonies. If GM
progenitors exist in the PU.1/ mice, then there may also be
commitment down the monocytic lineage. To determine if PU.1/
progenitors can commit to the monocytic lineage, colony-forming (CFU)
assays were set up with cells from normal and PU.1 null neonate liver
under myeloid growth factor conditions that included SCF, IL-3, GM-CSF, and M-CSF.
These growth factor conditions were found to induce primarily
macrophage and GM colonies from normal progenitors (Table
1). In addition, the GM colonies contained
a higher proportion of macrophages than neutrophils. No erythroid
colonies were detected on the CFU plates. Cells isolated from PU.1 null
neonate liver also formed colonies under these same conditions. In
agreement with our previously published results, the number of
PU.1/ CFU cells from neonate liver (Table 1) was significantly
reduced compared with normal livers.6 Individual
PU.1/ colonies were isolated and stained by Wright-Giemsa.
In some cases, neutrophil-only colonies were identified; however, most
of the colonies examined contained immature mononuclear cells or a
mixture of mononuclear cells and some neutrophils (Fig
1A). No erythroid or typical macrophage colonies were detected on the PU.1/ CFU plates. Also shown in the
same figure, a normal macrophage colony stained with Wright-Giemsa (Sigma) illustrates the morphology of both mature and immature macrophages. The PU.1 null mononuclear cells showed a similar morphology to that of normal early macrophage precursors, including in
some cases, the presence of vacuoles. However, early granulocytic cells
also have a similar morphology.
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| Fig 1.
Characterization of CFU-derived early PU.1/ myeloid
cells. Normal and PU.1/ neonate liver cells were grown in
methylcellulose CFU plates for 7 days with 50 ng/mL stem cell factor
(SCF), 100 U/mL IL-3, 2.5 ng/mL GM-CSF, and 5,000 U/mL M-CSF. (A)
Individual PU.1/ myeloid colonies were harvested and
stained with Wright-Giemsa stain. / in the upper right-hand
corner of the panels indicates PU.1 null colonies. A normal macrophage
colony is shown in the lower right-hand panel. (B) Pooled normal or
PU.1/ colonies were examined for NSE activity using  -naphthyl
acetate as the substrate. The dark staining cells show an NSE-positive
reaction. (C) Pooled normal and PU.1/ colonies were tested for
Moma-2 expression. Biotinylated secondary antibody and avidin
peroxidase were used to identify Moma-2-expressing cells. Purple
staining cells indicate Moma-2 expression. Moma-2 negative neutrophils
(N) are indicated in the normal panel.
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To determine if these early PU.1 null myeloid cells were monocytic,
normal and PU.1/ colonies were isolated and stained for NSE
activity. NSE-positive normal macrophages, as indicated by the dark
stain, are shown in Fig 1B. Similarly, some of the early PU.1/
myeloid cells also showed NSE activity. Neutrophils from normal and
PU.1 null CFU plates were negative for NSE activity. This is in
agreement with what has been reported, that granulocyte esterase
activity can be distinguished from esterase activity found in cells of
the monocyte lineage.24,25 However, weak activity in
granulocytes has been observed when the pH in the NSE reaction goes
above 7.0 and the incubation time is prolonged.25 Although
this was not a problem with the conditions used in this study, we
decided to verify that the NSE-positive reaction was monocyte-specific.
Sodium fluoride (NaF) specifically inhibits NSE activity found in cells
of the monocytic lineage, whereas the esterase activity in granulocytes
is NaF resistant.25 The addition of NaF to the NSE reaction
inhibited the appearance of dark staining cells on both normal and
PU.1/ slides, confirming that the esterase activity measured in
the PU.1/ cells was monocyte derived (data not shown).
The PU.1/ cells in these colonies clearly represent an early
stage in myeloid development. Many early and late myeloid markers are
shared by monocytic and granulocytic cells. Because Moma-2 recognizes
cells of the monocyte lineage, we used this monoclonal antibody to
stain pooled myeloid colonies from normal or PU.1/ CFU plates.
Normal macrophages and early precursors were positive for Moma-2,
whereas neutrophils were weakly stained or completely Moma-2 negative
(Fig 1C). Most of the PU.1 null early myeloid cells were also Moma-2
positive. However, between 10% and 20% of the cells were either
weakly positive or did not stain with Moma-2. Additional monoclonal
antibodies recognizing antigens found on monocytic precursors,
including ER-MP12, ER-MP20, and ER-MP58, were used to stain cytospins
of cells pooled from PU.1/ myeloid colonies. As shown in Table
2, the majority of the cells were ER-MP12-
and ER-MP58-positive, whereas 30% to 50% of the cells were
ER-MP20-positive. The presence of these markers on the majority of the
cells from the PU.1/ colonies shows that these cells are blocked
at a very early stage in development. This is further shown by the
detection of c-Kit on most of these cells. We also stained the cells
with an antibody that recognizes the Gr-1 (Ly-6G) antigen found on
early and late granulocytes.26 In addition, Gr-1 is
expressed transiently on cells of the monocytic lineage.26
Ten to twenty percent of the PU.1/ cells were Gr-1 positive. Less
than 50% of the Gr-1-positive cells were strongly positive for this
antigen, whereas the majority of the cells were weakly positive. High
levels of Gr-1 expression are reported to be found on neutrophils,
whereas low to moderate levels of Gr-1 are found on myeloblasts and
cells of the monocytic lineage.26 In agreement with what
has been reported, there were no PU.1/ cells expressing the late
monocytic F4/80 marker, although F4/80-positive cells were detected on
slides of normal cells. In addition, except for a rare one or two
weakly positive cells, CD11b protein could not be detected in the
PU.1/ colony-derived myeloid cells, but was readily detected on
normal myeloid cells.
The presence of cells in PU.1 null colonies with early monocyte
morphology, NSE activity, and early precursor antigens, including Moma-2, supports the hypothesis that PU.1/ progenitors commit to
the monocytic lineage.
PU.1/ monocytic precursors can be expanded from fetal
liver with GM-CSF.
We have reported minimal levels of GM-CSF receptor (GMR) on PU.1/
cells, either from short-term neonate liver cultures or freshly
isolated cells from bone marrow and liver of 9-day-old PU.1 null
animals.6 In contrast, no c-fms positive cells were detected in that study. GM-CSF has been shown to be
important for sustaining multipotent progenitors as well as regulating
myeloid development.27 Therefore, we stained slides of
normal and PU.1 null colonies with an antibody to GMR and found greater
than 50% of normal and PU.1 null cells were positive for GMR (Fig
2A). Control antibody did not stain either
normal or PU.1/ cells. As further evidence for specificity, we
observed that anti-GMR did not stain an IL-7-dependent B-cell line
generated from normal C57BL-6 mice (data not shown). RNA was isolated
from both normal and PU.1 null myeloid cells and examined for GMR-
gene expression by reverse transcriptase-PCR (RT-PCR). GMR- message
was detected in both normal and PU.1 null samples (Fig
2B). To verify that normal cells had not contaminated the PU.1/
CFU plates, we also looked for PU.1 message. Normal myeloid colonies
expressed PU.1 message, but no message was detected in PU.1/
colonies.
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| Fig 2.
(A) Normal and PU.1/ myeloid colonies contain cells
that express GM-CSF receptor (GMR). A 1:10 dilution of an affinity
purified rabbit polyclonal antibody to a cytoplasmic portion of GMR was
used to stain pooled normal or PU.1/ colonies. The same
concentration of preimmune rabbit control antibody was also used. (B)
The expression of GMR was verified by reverse transcriptase-PCR
(RT-PCR). Normal (N) and PU.1/ colonies were harvested, and total
RNA was extracted. Two micrograms of total RNA was used for
cDNA synthesis and PCR. Expression of both GMR and PU.1
was examined. PCR conditions: 94°C 1", 60°C 1", and 72°C 2", for
40 cycles. A no-cDNA control (C) was included in this assay.
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We showed that progenitors from normal neonate liver were able to form
myeloid colonies in the presence of GM-CSF alone, whereas PU.1/
progenitors did not form any colonies.6 In addition, PU.1
null cells from neonate liver were not able to proliferate in the
presence of GM-CSF alone.6 Therefore, although PU.1/ cells expressing GMR can be detected from these CFU assays with mixed
growth factors, no measurable response to GM-CSF has been shown.
Because the number of PU.1/ progenitors were reduced relative to
normal mice, the frequency of GM-CSF-responsive PU.1 null progenitors
may be very low and might not be detected in either a CFU or
proliferative assay. To maximize the input of GM-CSF-responsive
progenitors, we generated single-cell suspensions of whole fetal liver
from PU.1 null animals and cultured the entire mix in the presence of
GM-CSF. We chose the fetal liver, because at this stage of development,
this organ is the primary site of hematopoiesis and would likely be a
better source of early myeloid progenitors compared with neonate
liver.28 At birth, the bone marrow becomes the predominate
site of hematopoiesis, while liver hematopoiesis begins to
wane.28 In PU.1/ neonates, bone marrow formation is
deficient and contains a minimal number of cells.3 After 7 to 10 days, normal fetal liver cultures in the presence of GM-CSF
primarily contained macrophages. In contrast to the CFU and
proliferation assays, a population of early myeloid cells grew out of
the PU.1 null fetal liver cultures in the presence of GM-CSF alone.
These PU.1/ early myeloid cells were found to be
GM-CSF-dependent, and a stable culture was established away from the
fetal liver stroma and has been passaged over several months.
The generation of GM-CSF-dependent PU.1/ cells from fetal liver
cultures was repeated three times. In addition, fifteen PU.1/
GM-CSF-dependent clones have been generated. One of the GM-CSF-dependent clones (GM-pu12798-4 or GM-pu4) was further
characterized. GM-pu4 cells were analyzed by flow cytometry for the
expression of various myeloid markers. These cells coexpressed markers
found on early monocytic precursors, including Moma-2, ER-MP12,
ER-MP20, and ER-MP58 (Fig 3). As previously
mentioned, monocytic precursors express Fc receptors for IgG. A
monoclonal antibody that recognizes both CD16 (Fc III) and CD32
(FcRII) positively stained GM-pu4 cells. GM-pu4 cells did not
express CD11b or F4/80, but did express low levels of Gr-1. GM-pu4
cells also expressed CD18 and low levels of c-Kit but were Sca-1
negative.
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| Fig 3.
Antigen marker analysis of a GM-dependent PU.1/
myeloid cell line. PU.1/ fetal liver cultures were set up with
GM-CSF. GM-dependent myeloid cells that grew from these cultures were
cloned. One of the clones, GM-pu4, was analyzed by flow cytometry for
the expression of various markers. Cells were incubated with either
unconjugated (ER-MP12, ER-MP20, and ER-MP58), FITC-conjugated (Moma-2,
Gr-1, CD11b, F4/80, c-Kit, and Sca-1), or PE-conjugated (CD18 and
CD16/32) antibodies. FITC- or PE-conjugated secondary antibody was used
to detect ER-MP12, ER-MP20, or ER-MP58. The light tracings on the
histogram plots show background staining using isotype control
antibodies.
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Although many of the markers idenitified on the GM-pu4 cells can also
be found on cells of the granulocytic lineage, the expression of Moma-2
indicated that these cells were of the monocytic lineage. To verify
that GM-pu4 cells were of the monocytic lineage, they were tested for
NSE activity. These cells were found to be NSE positive and sensitive
to NaF treatment (Fig 4A). Another feature of early monocytic precursors is that they display phagocytic capabilities. GM-pu4 cells were incubated for 1 or 4 hours with fluorescent latex beads to determine if they were capable of
phagocytosis. Cells were harvested and analyzed by flow cytometry.
After 1 hour, the majority of cells had engulfed some latex beads, and
by 4 hours, there were very few cells remaining that had not taken up
some of the beads (Fig 4B). As a control for nonspecific sticking of
the latex beads to the cells, the beads were added to the cells for 5 minutes and then washed. More than 95% of the cells were unstained
(data not shown). In addition, approximately 90% of a myeloblast cell
line, M1, was unstained after a 90-minute incubation with the latex
beads, demonstrating that not all cells will phagocytize the latex
beads (data not shown). The engulfment of the latex beads by GM-pu4
cells was verified visually using a fluorescent microscope. There was
some variability in the number of particles phagocytosed by the cells,
which was consistent with the broad FL-1 peak shown by the flow
cytometric analysis.
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| Fig 4.
(A) GM-pu4 was found to be NSE-positive. The GM-pu4 clone
was tested for NSE activity. The dark staining cells are NSE-positive.
The activity was verified as being monocyte-specific by the ability of
NaF to inhibit the reaction. (B) GM-pu4 cells have phagocytic activity.
GM-pu4 cells were incubated with latex beads for 1 or 4 hours. The
engulfment of beads was evaluated by flow cytometry and with a
fluorescent microscope. The clear tracing on the histogram plot are
cells that did not receive beads. For visualization, cells were spun
onto slides and stained with Wright-Giemsa. The figure shows the same
two cells viewed with bright field or UV.
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These results show that the GM-CSF-dependent cells possess monocytic
characteristics similar to the early PU.1/ myeloid cells
characterized in the CFU assay.
Flow cytometric analysis of primary normal and PU.1 null liver cells
using antibodies to monocytic precursor antigens.
The data presented thus far show that under in vitro culturing
conditions, PU.1/ cells with monocytic characteristics are present. We next wanted to determine whether or not similar cells exist
in vivo. A single cell suspension of normal and PU.1 null neonate liver
cells was double stained with Moma-2 and either ER-MP12 or ER-MP20
monoclonal antibodies. After staining, the liver cells were analyzed by
flow cytometry (Fig 5A). Normal liver showed both ER-MP12 and ER-MP20 single positive populations. In contrast, the PU.1/ liver contained few single positive ER-MP12 or ER-MP20 cells. The scarcity of ER-MP12 and ER-MP20 single positive cells is consistent with the published data showing that the PU.1 null
mice are deficient in lymphocytes and granulocytes.1,2 On
the other hand, both normal and PU.1/ livers did contain a
comparable proportion of ER-MP12+Moma-2+ and
ER-MP20+Moma-2+ double positive cells.
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| Fig 5.
Monocytic precursors are found in the PU.1 null liver.
Cells from normal and PU.1/ neonate livers were triple stained
with Moma-2FITC and either unconjugated ER-MP12 (MP12) or
ER-MP20 (MP20) monoclonal antibodies, along with F4/80PE,
CD11bPE, or Gr-1PE-conjugated antibodies. MP12
or MP20 were stained with a biotinylated isotype-specific (IgG2a)
secondary antibody followed by Streptavidin Cy-Chrome (A) Moma-2 versus
MP12 or MP20 FACS data of normal and PU.1/ liver cells
demonstrate single positive, double positive, and unstained cells. The
quadrants were determined based on background staining of cells with
isotype control antibodies. The percentage of each positive staining
population is indicated in the corners of the quadrants. (B) Histogram
plots of F4/80, Mac-1, or Gr-1 cells found in gated normal and
PU.1/ MP12+Moma-2+ population. The
rectangular box in (A) was the gate used to analyze the double positive
populations.
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Further characterization indicated that the population of normal and
PU.1/ double positive cells was different. In these experiments,
normal and PU.1 null liver cells were triple stained with Moma-2 and
ER-MP12, along with either F4/80, CD11b, or Gr-1 antibodies (Fig 5B).
Most of the cells in the normal ER-MP12+Moma-2+
populations were positive for the monocyte-specific marker F4/80, and
the myeloid markers CD11b and Gr-1. F4/80, CD11b, and Gr-1 staining was
broad from mostly low to intermediate, whereas a few cells expressed
high levels of CD11b or Gr-1. The coexpression of Gr-1 with F4/80 in
the ER-MP12+Moma-2+ population is consistent
with Gr-1 detection on early monocytes.26 The Gr-1 bright
neutrophils were located in the ER-MP20 single positive population.
Less than 30% of the ER-MP12+Moma-2+
population were found to be F4/80, CD11b, or Gr-1 negative, using a
gate to subtract out cells expressing these late markers. Therefore,
the double positive population consists of monocytic precursors at
various stages of development. Similar results were obtained when we
analyzed the normal ER-MP20+Moma-2+ population
(data not shown). In contrast, no significant F4/80 or Gr-1 expression
was detected in the PU.1/ ER-MP12+Moma-2+
population, although a small shift in the Gr-1 histogram plot relative
to background levels indicates that some cells express low levels of
this antigen. In contrast to our in vitro results, a small percentage
of PU.1/ cells in this population did express low to intermediate
levels of CD11b. Similar results were found using day 17 to 18 fetal
liver from both normal and PU.1/ animals.
To determine if the PU.1/ double staining population contained
cells with monocytic characteristics, we used antibodies with magnetic
beads to isolate the ER-MP12+Moma-2+ population
from both normal and PU.1 null livers (see Materials and Methods). The
sorted cells were examined for NSE activity. As shown in Fig 6 (see
page 2855), both normal and PU.1/
ER-MP12+Moma-2+ populations included cells with
NSE activity and were NaF-sensitive. It has been reported that
monocytic precursors are also positive for peroxidase activity.
Therefore, both the normal and PU.1/ double staining populations
were analyzed for peroxidase activity and found to contain
peroxidase-positive cells (Fig 6).
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| Fig 6.
Cells showing monocytic enzyme characteristics are found
in both the normal and PU.1/ double staining populations. The
MP12+Moma-2+ population from normal and
PU.1/ livers was isolated using magnetically labeled antibodies
(see Materials and Methods). Flow cytometry data show the enrichment of
the double positive cells from (A) normal and (B) PU.1/ livers.
Cytochemical stains of normal or PU.1/
MP12+Moma-2+ cells for NSE (black stain)
and peroxidase (PO; red stain) activity are also presented.
|
|
The combined data from the flow cytometric and cytochemical analysis
shows that a low percentage of monocytic precursors are produced in the
PU.1 null mice.
| |
DISCUSSION |
Earlier reports have repeatedly shown that mature macrophages are not
produced in the absence of PU.1. However, it had not been shown where
in the process of monocyte/macrophage differentiation PU.1 was
required. In this report, we show that commitment to the monocytic
lineage takes place without PU.1, but development beyond the stage of
early monocytic precursors is blocked. Several criteria were used to
identify PU.1/ cells committed to the monocytic lineage,
including morphology, expression of Moma-2, ER-MP12, ER-MP20, and
ER-MP58, the presence of peroxidase, NaF-sensitive NSE activity and
phagocytic function.
Initially, the existence of PU.1/ monocytic precursors was shown
under in vitro culturing conditions in either CFU assays or fetal liver
cultures. These in vitro results were confirmed by demonstrating the
presence of primary NSE and peroxidase-positive cells expressing Moma-2
and ER-MP12 in PU.1 null liver. Likewise, we showed the presence of
cells coexpressing these early markers and later markers, including
CD11b and/or low Gr-1. It has been noted that the early markers ER-MP12
and ER-MP20 can be found on cells of other hematopoietic
lineages.14,15 However, in agreement with what has been
reported for Moma-2,18 the coexpression of this marker with
ER-MP12 and ER-MP20 can be used to specifically identify cells of the
monocyte lineage. This was supported by the triple color flow
cytometric analysis, which showed that the bulk of the cells in the
normal double positive populations expressed late monocytic markers
F4/80 and CD11b, as well as variable levels of Gr-1.
The expression pattern of ER-MP12 and ER-MP20 has been shown to
delineate stages of monocyte maturation.16 Cells expressing high levels of ER-MP12 alone were defined as early monocyte precursors, including GM and macrophage (M) progenitors. As monocyte precursors matured, ER-MP12 expression decreased, and ER-MP20 expression increased. By the monocyte stage of development, cells expressed ER-MP20 but not ER-MP12. High ER-MP58 expression was also found at the
early GM and M stage of development and remained constant until cells
mature to macrophages, at which time the expression diminished.17 The late monocytic marker CD11b was first
detected on 40% of the cells found in the population expressing both
ER-MP12 and ER-MP20, and on all cells in the ER-MP20-only (monocytes) population.16 Therefore, an early stage of monocyte
precursors before late marker expression does occur. Consistent with
these results, our analysis of the primary normal double positive
population from neonate liver did show a small percentage of cells that
expressed only Moma-2 with ER-MP12 and/or ER-MP20. We would argue that
the early PU.1/ monocytic precursors detected in vitro and in
vivo represent normal stages in mononuclear phagocyte development. The
majority of PU.1/ monocytic precursors detected were at the
pre-late marker stage of development, expressing only Moma-2, ER-MP12,
ER-MP58, and ER-MP20. However, some PU.1/ monocytic precursors
did mature to the late marker stage and expressed CD11b and/or low
levels of Gr-1. The ability of PU.1/ monocytic precursors to
mature to the late marker stage could also account for the detection of
rare F4/80 positive cells in older PU.1 null animals.2
The lack of complete macrophage development in the PU.1 null animal may
be caused by an inability to respond to cytokines known to regulate
monocyte/macrophage development. Cytokines such as M-CSF, GM-CSF, and
IL-3 have been shown to regulate macrophage development.27
Although no M-CSF response has been shown in cells from the PU.1/
mouse, PU.1/ progenitors have been shown to respond to
IL-36 and now to GM-CSF. Not only do the PU.1/
progenitors respond to GM-CSF, but the cells that grow out from the
PU.1/ fetal liver culture were characterized as belonging to the
monocytic lineage. Interestingly, GM-CSF-dependent cells could not be
derived from PU.1/ neonate liver cultures (data not shown). It is
unclear as to why there is a change in response to GM-CSF from
PU.1/ fetal liver cultures compared with neonate liver cultures.
As already mentioned, one explanation could be that the number of progenitors in the liver expressing GMR is extremely low at birth in
the PU.1/ animal. An alternative explanation, although not mutually exclusive, may be that the stroma in the fetal liver cultures
provide additional secreted or surface-bound factors that prime PU.1
null progenitors to respond to GM-CSF and proliferate. In support of
this theory, a study was published in which the investigators
found that adult bone marrow and fetal liver stroma provided a better
environment to maintain GM-CFC compared with neonate
liver.29 This is consistent with both the adult bone marrow
and fetal liver being active sites of hematopoiesis, whereas neonate
liver has low hematopoietic activity.28
It is possible that the inability of IL-3 and GM-CSF to enable
complete macrophage maturation in the PU.1 null mice is that PU.1 is
required to receive signals initiated by these cytokines through their
receptors. After receiving IL-3- or GM-CSF-induced signals, PU.1 may
modulate the expression of genes required for monocyte/macrophage
development. We have shown that PU.1 is important for mediating M-CSF-
and GM-CSF-induced proliferation of bone marrow-derived
macrophages.30 However, it is clear from studies on mice
lacking M-CSF or both M-CSF and GM-CSF, that macrophage development
proceeds in the absence of these growth factors, although in some
tissues the level of production is diminished.31,32 Similarly, mice deficient in both the common  c chain used by the
IL-3 and GM-CSF receptor and the IL-3 ligand were able to generate
monocytes.33 Therefore, PU.1 likely regulates
monocyte/macrophage development independent of growth factor
receptor-mediated signaling.
| |
ACKNOWLEDGMENT |
The authors thank Dr David Hume for the IL-3 and M-CSF. They also thank
Drs Bruce Torbett, Karen Anderson, and Melody Clark for their critical
review of this manuscript.
| |
FOOTNOTES |
Submitted September 14, 1998; accepted December 11, 1998.
G.W.H. and S.R.M. contributed equally to this study.
Supported by National Institutes of Health Grant No. AI30656.
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 Richard A. Maki, PhD, The Burnham
Institute, 10901 N Torrey Pines Rd, La Jolla, CA 92037.
| |
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ABSTRACT
INTRODUCTION