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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 7 (October 1), 1999:
pp. 2310-2318
PU.1 and the Granulocyte- and Macrophage Colony-Stimulating Factor
Receptors Play Distinct Roles in Late-Stage Myeloid Cell
Differentiation
By
Karen L. Anderson,
Kent A. Smith,
Hugh Perkin,
Gary Hermanson,
Carol-Gay Anderson,
Douglas J. Jolly,
Richard A. Maki, and
Bruce E. Torbett
From The Burnham Institute, La Jolla, CA; the Department of
Immunology, The Scripps Research Institute, La Jolla, CA; Vical, Inc,
San Diego,CA; and Chiron Technologies Center for Gene Therapy, San
Diego, CA.
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ABSTRACT |
PU.1 is a hematopoietic cell-specific ets family transcription
factor. Gene disruption of PU.1 results in a cell autonomous defect in
hematopoietic progenitor cells that manifests as abnormal myeloid and
B-lymphoid development. Of the myeloid lineages, no mature macrophages
develop, and the neutrophils that develop are aberrantly and
incompletely matured. One of the documented abnormalities of PU.1 null
(deficient) hematopoietic cells is a failure to express receptors for
granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage (GM)-CSF, and M-CSF. To elucidate the roles of the myeloid growth factor receptors in myeloid cell differentiation, and to distinguish their role from that of PU.1, we have restored expression of the G- and
M-CSF receptors in PU.1-deficient cells using retroviral vectors. We
have similarly expressed PU.1 in these cells. Whereas expression of
growth factor receptors merely allows a PU.1-deficient cell line to
survive and grow in the relevant growth factor, expression of PU.1
enables the development of F4/80+,
Mac-1+/CD11b+ macrophages, expression of
gp91phox and generation of superoxide, and
expression of secondary granule genes for neutrophil collagenase and
gelatinase. These studies reinforce the idea that availability of PU.1
is crucial for normal myeloid development and clarify some of the
molecular events in developing neutrophils and macrophages that are
critically dependent on PU.1.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE ETS FAMILY transcription factor PU.1
is expressed exclusively in hematopoietic cells. Evidence from PU.1
gene-disrupted mice indicates a pivotal role for PU.1 in myeloid
lineage (as well as B-lymphocyte) development. To summarize the PU.1
null phenotype, leukocytes are absent at birth in these mice, but low numbers of neutrophils eventually develop by 2 to 3 days, and T
lymphocytes by 5 to 8 days, after birth. Mature monocytes/macrophages and B cells are not detectable in these mice at any age examined, up to
2 weeks.1 The defect in PU.1 null hematopoietic progenitors is cell autonomous and may be due, at least in part, to their failure
to express receptors for the myeloid growth factors granulocyte colony-stimulating factor (G-CSF), macrophage (M)-CSF, and
GM-CSF.2 Currently there is still debate as to the specific
role of these receptors in hematopoietic cell survival, proliferation,
and differentiation, and it is not clear whether the absence of
receptors can account for the hematopoietic deficits seen in the PU.1
null mouse. It is possible and indeed likely that nonexpression or
dysregulation of other key genes that are regulated by PU.1 contributes
to the abnormal myeloid development in these gene-disrupted mice.
Because PU.1-deficient cells fail to express myeloid growth factor
receptors, they represent a useful vehicle for elucidating the distinct
functions served by these receptors in hematopoietic cell expansion and
development. Therefore, we reintroduced PU.1, G-CSF receptor, and M-CSF
receptor into an established PU.1-deficient myeloid cell line,
503.3 We observed in PU.1 restored myeloid cells the
expression of genes and acquisition of functions associated with normal
terminal neutrophil maturation that were absent from PU.1-deficient
cells. In addition, restoration of PU.1 expression in these cells
enabled the development of F4/80+, Mac-1/CD11b+
cells (monocytes and macrophages), whereas previously only
Gr-1+, CAE+ cells (neutrophils) were
generated.3 Furthermore, PU.1 transduction restored the
ability of these cells to grow in the presence of G-and M-CSF. Unlike
cells transduced with a PU.1-expressing vector, those transduced with
the G-CSF receptor showed no detectable change in surface-marker
phenotype (other than expression of G-CSF receptor), gene expression,
or functional capacity to suggest terminal differentiation or
maturation of these cells beyond the point that we have described to
occur in PU.1 null neutrophils.3 Similarly, transduction
with the M-CSF receptor allowed the cells to use M-CSF for survival and
growth with no apparent progression of differentiation along the
monocyte/macrophage pathway.
These results support the concept of a permissive but not an inductive
or instructive role for growth factor receptors in myeloid cell
development. In contrast, PU.1 can regulate the expression of molecules
associated with cell differentiation. The results obtained from these
studies suggest that normal components of myeloid cells and neutrophils
can be restored and distinct differentiation pathways enabled even when
PU.1 is reintroduced at a relatively late stage of myeloid development,
as shown by the PU.1 null cell line that was used in these experiments.
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MATERIALS AND METHODS |
The PU.1 null cell line 503.
The myeloid cell line 503 was originally derived from PU.1 null
neonatal liver as described3 and was used for PU.1 and G-
and M-CSF receptor restoration studies. These cells are maintained in
Iscove's medium supplemented with 20% fetal bovine serum with 100 U/mL recombinant mouse (rm) interleukin-3 (IL-3), 5 ng/mL rmGM-CSF, 5 ng/mL rmG-CSF, and 5,000 U/mL recombinant human (rh) M-CSF. Previous
work has shown that the cells will only respond to IL-3,2,3
but they are maintained in all factors for experimental consistency and
comparison with normal cells.
Construction of retroviral plasmids.
Pfu polymerase (Stratagene, San Diego, CA) was used for high-fidelity
polymerase chain reaction (PCR) amplification of cDNAs encoding PU.1
and the G-CSF receptor. The complete coding region of PU.1 (nucleotides
132 to 1000, GenBank accession no. M32370) and the G-CSF receptor
coding region (nucleotides 180 to 2691, GenBank accession no. M58288)
was amplified from a mixed population of mouse bone marrow myeloid
cells obtained after activation in IL-3, G-, and GM-CSF. The 5'
and 3' amplimers introduced a NotI cloning site at each
end and the 5' primer also includes a Kozak consensus
translational initiation sequence.4 PCR fragments were
cloned into pCR-script (Stratagene) and verified by sequencing. cDNAs
were excised with NotI and cloned into the SrfI site of retroviral vector pBA8b-L1 (Chiron Corp, San Diego, CA). The plasmid pBA8b-L1 was constructed in a 3-fragment ligation using the following 3 fragments: (1) The NdeI-ClaI fragment from pBA-5b
(described in Patent Cooperative Treaty [PCT]
application no. WO9742338), containing the 3' long terminal
repeat (LTR) and the pUC18 backbone; (2) the
ClaI-HindIII fragment from pCI-PLAP (the cDNA encoding human Placental Alkaline Phosphatase
[PLAP]5 inserted into the pCI cytomegalovirus (CMV)
expression plasmid (Invitrogen Inc, San Diego, CA); and (3) the
HindIII-NdeI fragment from pBA-6bL1, containing the
5' LTR and the SV40 promoter. Plasmid pBA-6bL1 is based on pBA-6b
(described in example 10 of patent application WO 9742338), where the
HIVenv/rev-coding region was deleted via XhoI-ClaI
digestion and replaced with the L1 polylinker.
In these vectors, now termed pBA8b-L1.PU.1 (encoding PU.1) and
pBA8b-L1.GR (encoding the G-CSF receptor), the Moloney murine leukemia
virus LTR drives expression of the gene of interest and the SV40
promoter drives expression of PLAP. The M-CSF receptor expressing
vector pMZen (c-fms) was a gift from Dr Larry
Rohrschneider (University of Washington, Seattle, WA).6
Retroviral infection of PU.1-deficient cells.
The 293-based amphotropic packaging line 2A-LB (patent application WO
no. 9742338) was maintained in Dulbecco's modified Eagle's medium
(DMEM) plus 10% fetal calf serum (FCS) and 2 mmol/L L-glutamine. Vector-producing cell lines (VPCL) were made by cotransfecting the
plasmid pMLG-G encoding vesicular stomatitis virus glycoprotein (VSV-G)7 along with plasmids containing gene of interest
into 293 2-3 cells7 to generate transient VSV-G-pseudotyped
vectors that were then used to transduce the human 293-based
amphotropic packaging cell line, 2A-LB. The resulting pool of vector
producing cells were enriched for PLAP-expressing cells using anti-PLAP antibody (Sigma, St Louis, MO) and magnetic beads (Miltenyi Biotec Inc,
Auburn, CA). To produce virus-containing supernatants,
PLAP+ cells were grown to confluency, fresh medium was
added, and supernatants were collected 24 and 48 hours later. Viral
titers were determined by infection of HT1080 cells using a series of
dilutions of the vector in the presence of 8 µg/mL polybrene and
scoring the number of PLAP+ colonies 48 hours later.
Briefly, the infected HT1080 cells were fixed in phosphate-buffered
saline (PBS) containing 2% formaldehyde and 0.2% glutaraldehyde and
stained with the alkaline phosphatase substrate Fast Red TR/Napthol
AS-MX phosphate (Sigma) as directed by the manufacturer. Titer
(colony-forming units [CFU])/mL was calculated as [No. Red
Colonies/µL Vector Added] × Dilution Factor × 1,000. Typical supernatants used contained 0.5 to 1.0 × 106
CFU/mL.
To transduce 503 cells, the wells of a tissue-culture plate were first
coated with fibronectin (Sigma) by incubating with 10 µg/mL
fibronectin solution at 37°C for 2 hours. The fibronectin was
aspirated and 1 mL of viral supernatant was added.8,9 The
plate was further incubated for 0.5 to 1 hour at 37°C to allow adherence of viral particles to the fibronectin-coated surface. Cells
were then added in a minimal volume of media plus polybrene (final
concentration 6 to 8 µg/mL), and the plate was centrifuged at
1,000g for 3 hours at room temperature. Afterwards, cells were resuspended and allowed to recover for 24 hours in growth
factor-containing medium. The transduction protocol was repeated for a
total of 3 times.
Selection of transduced cells and flow cytometric analysis.
Forty-eight hours after the last transduction (5 days after
the first transduction), an aliquot of cells was removed and stained with an anti-human alkaline phosphatase (PLAP) antibody (Sigma) followed by a phycoerythrin-conjugated secondary donkey anti-mouse antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) to
determine transduction efficiency. Cells were then analyzed for
alkaline phosphatase expression by flow cytometry with a Becton Dickinson FACScan and data were analyzed with CellQuest (Becton Dickinson, Franklin Lakes, NJ). For assessment of Gr-1 and
Mac-1 expression, cells were incubated with Gr-1PE and
Mac-1/CD11bFITC at concentrations recommended by the
manufacturer (Pharmingen, San Diego, CA), and washed and analyzed as
previously described.1
To isolate PU.1 and G-CSF receptor transduced cells,
107 cells were incubated with 20 µg/mL of anti-PLAP
antibody (Sigma) for 30 minutes on ice. This step was followed by 3 washes, then goat anti-mouse antibody-conjugated magnetic beads were
added as per manufacturer's instructions (Miltenyi Biotec, Auburn,
CA). Positively stained cells were isolated using a VarioMacs magnetic
device as directed by the manufacturer (Miltenyi Biotec). Because the M-CSF receptor-expressing vector did not contain a selectable marker,
transduced cells were selected by their ability to survive and expand
in 5,000 U/mL rhM-CSF.
Isolation of RNA and reverse transcription (RT)-PCR analysis.
Total RNA was isolated and PCR was performed as described
previously.3 PCR primers used herein included the following
(listed 5'  3'):  actin 5'
GTGGGCCGCTCTAGGCACCAA, 3' CTCTTTGATGTCACGCACGTATTC; PU.1
(573-873) 5': TCTGATGGAGAAGCTGATGG, 3':
CTTGACTTTCTTCACCTCGC (these primers are located in exon 4 and
exon 5, respectively); c-fms 5': GCGATGTGTGAGCAATGGCAGT,
3': AGACCGTTTTGCGTAAGACCTG; murine neutrophil
collagenase 5': CACGATGGTTGCAGAGAAGC, 3':
TCTCCTCCAATACCTTGGCC; murine neutrophil gelatinase 5':
ACGGTTGGTACTGGAAGTTCC, 3': CCAACTTATCCAGACTCC- TGG;
gp91phox.3
Enzyme histochemistry and immunohistochemistry.
Methods for immunostaining of cytospin slides for F4/80 and CD11b were
as described.1 Sialoadhesin antibody was obtained from
Serotec Inc (Raleigh, NC) and used at 1:25 dilution.
Modified colony-forming assays.
Cells were seeded at 500 cells/mL in Methocult 3234 (Stem Cell
Technologies, Vancouver, BC, Canada) which was supplemented with either
10 ng/mL rmG-CSF (R&D Systems, Minneapolis, MN) or 5,000 U/mL rhM-CSF
(gift of Dr David Hume, University of Queensland, Brisbane,
Australia). Colony formation (50 or more cells) was scored
after 7 days.
Western blotting.
Detection of proteins by Western blot was performed as
described.3 Polyclonal anti-PU.1 and anti-G-CSF receptor
antibodies and secondary antibody were obtained from Santa Cruz
Biotechnology (Santa Cruz, CA).
Cytochrome c reduction assay for superoxide generation.
Cells were assayed for their ability to reduce cytochrome c exactly as
described.3
| |
RESULTS |
Expression of PU.1 in PU.1-deficient cells restores expression of G-
and M-CSF receptors and restores response to G- and M-CSF.
PU.1 regulation of the G- and M-CSF receptor promoters in transfection
assays has previously been reported,10,11 and we have shown
that PU.1-deficient cells fail to express receptors for G- and
M-CSF.2 PU.1-deficient hematopoietic cells will grow in the
presence of IL-3 but not in G-CSF or M-CSF, further confirming the
absence of selective receptor function.2 The PU.1 null
myeloid cell line 503 was originally derived from PU.1 null neonatal
liver as described.3 The phenotype of this cell line is
very similar to cells that can be expanded in short-term (<1 month)
cultures of PU.1 null neonatal liver. Most cells are CAE+
(90%) and CD18+ (80%), with approximately 40% to 60%
Gr-1+, and <3% CD11b+ 1,3 (and
data not shown). Our previous work has shown that these cells express
markers consistent with developing neutrophils, but fail to express
markers and functions of terminally differentiated neutrophils.3 503 cells exhibit a 10% frequency of colony
formation in semisolid media containing IL-3; thus, these cultures
consist of a progenitor-type cell in addition to more differentiated, neutrophil-like progeny. Therefore, 503 cells should be useful for the
investigation of PU.1-mediated growth factor regulation and myeloid differentiation.
We first tested whether retroviral delivery of PU.1 to PU.1-deficient
503 cells would restore G- and M-CSF receptor expression and function.
To assess whether restoration of either G-CSF receptor or M-CSF
receptor alone would convey the same biological potential as PU.1
expression, G-CSF- or M-CSF-expressing 503 lines were also produced
(called 503-GR and 503-MR, respectively). To deliver PU.1 and the G-CSF
receptor, the Moloney murine leukemia virus-based retroviral vector
pBA8b-L1 was used. In this vector, PU.1 (or G-CSF receptor) cDNA was
under the control of the viral LTR, and the cell-surface-expressed
selectable marker, human placental alkaline phosphatase (PLAP), was
driven by the SV40 promoter (see Materials and Methods). Delivery of
the M-CSF receptor used the pMZen(cfms) vector.6
After transduction of the 503 cell line with pBA8b-L1.PU.1,
PLAP+ cells were isolated as discussed in Material and
Methods. To confirm PU.1 expression, RNA was prepared from these cells,
treated with DNase, and subjected to RT-PCR. mRNA for PU.1 was detected in transduced samples (503-PU) but not in the parental 503 cells, or in
503 cells transduced with an M-CSF receptor-expressing retroviral vector (Fig 1A) or a G-CSF
receptor-expressing retroviral vector (data not shown). We then
analyzed whole-cell lysates for presence of PU.1 protein. Confirmation
that PU.1 protein expression was restored can be seen in Fig 1B. The
PU.1 protein level in 503-PU cells approached the level of normal mixed
myeloid cells, but was less than the B-cell line A20-2J.
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| Fig 1.
PU.1 is detectable in 503 cells transduced with a
PU.1-expressing retrovirus. PU.1-deficient 503 cells were transduced
with retroviral vectors containing the cDNA for PU.1, G-CSF receptor,
or M-CSF receptor. Cells that were positive for the selectable marker
PLAP (PU.1 and G-CSF receptor) or positive for growth in M-CSF (M-CSF
receptor) were isolated for analysis. (A) RT-PCR showed PU.1 mRNA in
normal macrophages (MAC) and PU.1-transduced 503 cells (503-PU), but
not in 503 or 503 transduced with M-CSF receptor (503-MR). Primers used
were intron-crossing, and controls for DNA amplification in the absence
of RT were also included and were negative (not shown). The far
righthand lane contains a 100-bp DNA ladder for size determination of
PCR products. (B) Whole-cell lysates were prepared and 50 µg (or 80 µg where indicated) of total cellular protein was separated by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After
blotting and probing with a polyclonal anti-PU.1 antibody, PU.1 protein
was visible in normal mixed myeloid cells, A20-2J B cells, and 503-PU
cells. As expected, nonhematopoietic HT1080 cells were negative for
PU.1 expression. The lane labeled MW contains protein size standards
(CruzMarker; Santa Cruz Biotechnology). MW, molecular weight.
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Unlike parental PU.1-deficient 503 cells, pBA8b-L1.PU.1-transduced 503 cells (503-PU) were shown to express M-CSF receptor mRNA
(Fig 2A). 503 cells that were transduced
with an M-CSF receptor-containing retroviral vector (503-MR) also
expressed mRNA for M-CSF receptor (Fig 2A). 503-PU cells also expressed
G-CSF receptor protein, as did 503 cells transduced with a G-CSF
receptor-expressing retroviral construct (503-GR) (Fig 2B). Parental
503 cells expressed no G-CSF receptor protein, consistent with our
previous results2 (Fig 2B). To test whether reintroduction
of PU.1 would also restore responsiveness to G- and M-CSF,
PLAP+ 503-PU were seeded into methylcellulose media
containing either G- or M-CSF. We have shown that cells derived from
PU.1 null neonatal liver will form colonies in semisolid media only in
the presence of IL-3, but not G- or M-CSF.2 Similarly,
parental PU.1-deficient 503 cells formed no colonies in either G- or
M-CSF; however, 2 different 503-derived PU.1 transduced clones produced
9.3 ± 3.5 and 20.7 ± 5.6 colonies in G-CSF, and 1.3 ± 0.6 and 4.0 ± 2.7 colonies in M-CSF, per 500 cells seeded in
methylcellulose (Table 1).
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| Fig 2.
Transduction of PU.1 into PU.1-deficient cells restores
G- and M-CSF receptor expression. (A) PU.1- and M-CSF receptor
transduced cells (503-PU, 503-MR) that were selected by PLAP expression
or growth in M-CSF, respectively, were analyzed by RT-PCR. M-CSF
receptor mRNA was evident in normal macrophages (MAC), 503-PU, and
503-MR. These primers were intron-crossing, and controls for DNA
amplification in the absence of RT that were also performed were
negative (not shown). The far lefthand lane contains a 100-bp DNA
ladder. (B) Whole-cell lysates were prepared and 25 µg of total
cellular protein was separated by SDS-PAGE. After blotting and probing
with polyclonal anti-G-CSF receptor antibody, G-CSF receptor protein
was detectable in G-CSF receptor- and PU.1-transduced 503 cells
(503-GR, 503-PU) and normal neutrophils, but not in 503 cells or the B
cell line A20-2J. The lane labeled MW contains protein size standards
(CruzMarker; Santa Cruz Biotechnology).
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Table 1.
Only PU.1-Transduced 503 Cell Lines and Not Parental
PU.1-Deficient 503 Cells Can Form Colonies in Response to G- and
M-CSF
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Thus, we have documented that retroviral delivery of PU.1 into 503 cells results in PU.1 expression and the restoration of expression of
functional G- and M-CSF receptors.
Gene expression consistent with neutrophil terminal differentiation
is seen after restoration of PU.1 expression to PU.1-deficient myeloid
cells.
The late stages of neutrophil differentiation are characterized by the
appearance of secondary or specific granule gene products. These genes
are believed to be coordinately regulated, and are transcribed in a
stage-specific manner at the myelocyte stage of
development.12-14 We have shown that the PU.1 null
neutrophil-like cells that develop, both in vivo and in vitro, do not
express detectable mRNA for secondary granule components.3
When PLAP+ 503-PU cells were examined using RT-PCR, we were
able to detect mRNA for the secondary granule genes neutrophil
collagenase and neutrophil gelatinase, whereas no mRNA was found in the
parental cell line (Fig 3) or in G-CSF
receptor-transduced 503 cells (503-GR) (Fig 3 and data not shown).
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| Fig 3.
503 cells transduced with PU.1 express markers of
neutrophil terminal differentiation. RNA was prepared from
PLAP+ PU.1-transduced 503 (503-PU) cells and analyzed by
RT-PCR. mRNA for secondary granule genes neutrophil collagenase (mNC)
and neutrophil gelatinase (mNG) is present in 503-PU as well as normal
neutrophils, but not in 503 or 503 transduced with G-CSF receptor
(503-GR). The far righthand lane of each gel contains a 100-bp DNA
ladder.
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PU.1 transduction restores gp91phox expression and
superoxide production in PU.1-deficient 503 cells.
We previously documented that PU.1 null myeloid cells failed to
transcribe the gp91phox gene, which encodes a major
subunit of the enzyme NADPH oxidase, and also failed to generate the
metabolite superoxide (O2 ) that is the
exclusive product of this enzyme.3 As shown in Fig 4A, 503-PU cells expressed
gp91phox mRNA, unlike the parental 503 cell line,
or 503-GR cells. To confirm the restoration of NADPH oxidase function,
we analyzed the cells for their ability to reduce cytochrome c in
response to PMA stimulation. Unlike 503 or 503-GR cells that produced
no detectable O2, 503-PU and normal
cells were able to increase their O2
production by roughly 74% and 64%, respectively, in the presence of
PMA (Fig 4B).
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| Fig 4.
Expression of gp91phox and associated
NADPH oxidase enzyme activity is restored by PU.1 transduction of 503 cells. (A) RT-PCR analysis of cells revealed
gp91phox mRNA in normal neutrophils and
PU.1-transduced 503 cells (503-PU), but not in 503 as previously
documented or in G-CSF receptor-transduced 503 (503-GR). The far
lefthand lane contains a 100-bp DNA ladder. (B) An assay for cytochrome
c reduction in response to PMA, which measures
O2 production, was performed to address the
functionality of the enzyme NADPH oxidase, of which
gp91phox is a principal subunit. Detectable
production of O2 was found in normal
neutrophils (NORMAL) and 503-PU, but not in 503 or 503-GR. SOD,
superoxide dismutase.
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G-CSF receptor expression enables PU.1-deficient myeloid cells to
survive and proliferate without further differentiation in G-CSF.
Our previous studies have shown that neutrophil development proceeds in
the absence of PU.1 and the accompanying lack of detectable G-CSF
receptor expression. The number of neutrophils that develop in vivo is
severely reduced, however, and the cells that develop in vivo and in
vitro are abnormal in their surface marker and functional
profile.1-3 As shown above, PU.1 restoration in 503 cells
leads to the expression of genes and functions associated with mature
neutrophils. To attempt to distinguish the contribution of the G-CSF
receptor to neutrophil development from the contribution of PU.1, we
transduced PU.1-deficient 503 cells with the retroviral vector
pBA8b-L1.GR as discussed above. PLAP+ cells were isolated
after transduction and shown to express G-CSF receptor protein (Fig
2A). By plating equal numbers of cells initially and counting live
versus dead cells on the basis of trypan blue exclusion over the course
of 7 days, we were able to document enhanced survival and growth in
G-CSF of PLAP+ G-CSF receptor-expressing 503 cells compared
to PLAP cells (Fig 5).
However, as shown in Figs 3 and 4, we detected neither mRNA for murine
neutrophil gelatinase nor superoxide production in 503-GR cells, which
represented no change from the parental PU.1 null cell line, even after
2 weeks of culture in G-CSF. Similarly, no change in the Gr-1 and
Mac-1/CD11b surface staining profile of G-CSF-cultured 503-GR cells
was detected (data not shown). Therefore, we conclude that G-CSF
receptor expression is not sufficient for terminal neutrophil
differentiation in the absence of PU.1. Finally, because the G-CSF
receptor-expressing retroviral vector was the same vector that was used
for PU.1 transduction, it is clear that the phenotypic changes produced
by PU.1 transduction were specific to PU.1 expression and not a
nonspecific effect of retroviral integration.
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| Fig 5.
503 cells transduced with the G-CSF receptor display
enhanced survival and proliferation in the presence of G-CSF compared
with parental 503 cells. After transduction with a G-CSF-expressing
retrovirus, an equal number of selectable marker PLAP+
( ) and PLAP ( ) 503 cells were plated in 10 ng/mL
G-CSF and live cell (on the basis of trypan blue exclusion) counts
monitored over 7 days. Note an almost 5-fold greater number of live
PLAP+ versus PLAP cells after 7 days in
G-CSF.
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Macrophage differentiation is partially restored by transduction with
a PU.1-expressing retrovirus.
As we have previously reported,3 cell line 503 was derived
from the liver of a PU.1 null neonate. Based on their expression of
Gr-1 and chloroacetate esterase, 503 cells appear to be of the
neutrophil lineage. No cells displaying specific monocyte or macrophage
characteristics such as F4/80, M-CSF receptor, scavenger receptor, or
mannose receptor expression have been detected in PU.1 null cell
cultures2,3 or in PU.1 null mice of any age examined.1 After transduction of 503 cells with PU.1,
however, Wright-Giemsa staining showed cells with typical mature
macrophage morphology (Fig 6A, compare 503 and 503-PU). Immunohistochemical staining demonstrated the expression
of F4/80 and sialoadhesin, two classic markers for this lineage (Fig 6B
and C). Flow cytometric analysis of the PU.1-transduced population
showed an overall increase in cell size (forward scatter), and both a
decrease in Gr-1 expression and an increase in Mac-1/CD11b expression
relative to the parental 503 PU.1 null cell line (data not shown). mRNA
for macrophage-specific genes including M-CSF receptor (Fig 2A),
mannose receptor, and IL-18 was detected in PU.1-transduced but not
PU.1-deficient cells (data not shown). Interestingly, scavenger
receptor mRNA was still absent from these cells. With the exception of
IL-18, the promoters of each of these genes have previously been
reported to be regulated by PU.1.11,15,16 Thus, by
retroviral delivery of PU.1 into the PU.1-deficient cell line 503, which previously expressed only markers of early myeloid or neutrophil
development, we have restored the ability of these cells to express
markers associated with mature monocyte and macrophage development.
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| Fig 6.
Macrophage development can be restored by PU.1
transduction of PU.1-deficient 503 cells. (A) PU.1 transduction of 503 cells (503-PU) restored the development of cells with typical
macrophage appearance as shown by Wright-Giemsa staining. (B)
Immuno-cytochemistry was used to show that 503-PU cells expressed the
macrophage marker F4/80, unlike the parental cells (503).
Positive-staining cells demonstrate an orange-brown reaction product.
Note the presence of F4/80+ 503-PU cells with lobate
nuclear morphology of monocytes. (C) An adherent subpopulation 503-PU
cells was also positive for the macrophage subset marker sialoadhesin
by immunocytochemical staining, whereas no positive cells were found in
parental 503 cultures.
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Restoration of M-CSF receptor expression alone permits survival and
proliferation, but not macrophage differentiation, of PU.1 null cells.
We have documented that hematopoietic cells deficient in PU.1 express
neither M-CSF receptor mRNA nor protein2 (Fig 2A). Recent
studies on an independently derived PU.1 null mouse indicated that some
macrophage characteristics could be restored when fetal progenitor
cells were transduced with PU.1.17 In addition, it has
recently been shown that monocyte/macrophage progenitors may be present
in our PU.1 null mouse model.18 To determine whether restoration of signaling through the M-CSF receptor could compensate for the absence of PU.1 and allow macrophage differentiation to proceed, we transduced an M-CSF receptor-expressing retrovirus into the
PU.1 null myeloid cell line 503. We found that M-CSF receptor
expression permitted the cells to use M-CSF for survival and growth,
but did not detectably alter the differentiation status of the cells.
In contrast to PU.1-complemented 503 cells, M-CSF receptor-restored 503 cells did not express F4/80 or Mac-1/CD11b (data not shown).
PU.1-deficient cells transduced with the M-CSF receptor did not express
mRNA for macrophage-associated genes such as IL-18 or mannose receptor
or the myeloid-associated gene gp91phox, which
represented no change from the parental cell line (data not shown).
| |
DISCUSSION |
We previously established that myeloid and lymphoid commitment occurs
in the absence of PU.1.1,2 Although myeloid commitment was
evident in PU.1 null mice, monocytes and macrophages were not produced
in vivo or in vitro.1,2 In contrast, neutrophils were
produced in the mouse relatively late in development (postnatally, in
fact) and remained extremely few in number. Their surface markers, gene
expression, and functional profiles were consistent with incompletely
or aberrantly matured neutrophils. Further studies documented that
PU.1-deficient neutrophils could be expanded in vitro in the presence
of IL-3, but not G- and/or GM-CSF.2 Therefore, at least
part of the observed developmental abnormality of PU.1 null myeloid
cells could be attributable to their failure to express receptors for
G-, GM-, and M-CSF.2 The importance of the CSF receptors in
normal production and expansion of myeloid lineage-restricted cells is
well accepted, although their specific roles are less clear. To
determine the contribution of G- and M-CSF receptors and PU.1 to the
expression of characteristics associated with late stages of
myeloid/neutrophil development, G- and M-CSF receptors and PU.1 were
introduced into the PU.1 null 503 myeloid cell line.
As reported previously, both primary PU.1-deficient myeloid cells and
the myeloid cell line 503 produce cells that express markers consistent
with developing neutrophils, but these cells fail to express markers
and functions of mature neutrophils or monocytes/macrophages.3 Restoration of the G-CSF receptor
in the 503 cell line did not allow these cells to progress detectably beyond the point of differentiation that they were able to achieve in
the absence of PU.1 and the concomitant absence of the effects of
G-CSF. It remains a possibility that disruption of PU.1 has resulted in
additional downstream effects that preclude normal G-CSF receptor
signal transmission or its effects. However, at least a part of the
G-CSF receptor pathway was intact, as indicated by the enhanced cell
survival and proliferation seen in the presence of G-CSF after receptor
restoration. In contrast, PU.1 restoration in 503 cells permitted
expression of mRNA for the NADPH oxidase component
gp91phox, which was absent from PU.1-deficient
neutrophils. Subsequently, the transduced cells were able to reduce
cytochrome c in vitro, which is an indication of superoxide generation
and restored enzyme activity. Furthermore, we were able to detect mRNA
in 503-PU cells for murine neutrophil collagenase and neutrophil
gelatinase, terminal maturation-associated secondary granule
genes12,13 that were not expressed in PU.1-deficient
neutrophils. Importantly, transduction of cell line 503 with PU.1
restored expression of the G-CSF receptor and the ability to survive
and proliferate in G-CSF, confirming an essential role for PU.1 in
G-CSF receptor expression as has been predicted.2,10
Table 2 summarizes the neutrophil
characteristics of 503 cells and transduced variants.
View this table:
[in this window]
[in a new window]
|
Table 2.
Expression of Certain Neutrophil Characteristics Is
Restored in PU.1-Transduced But Not in G-CSF Receptor-Transduced or
Parental PU.1-Deficient 503 Cells
|
|
Our earlier studies clearly established that neutrophil commitment and
some differentiation can take place in the absence of PU.1 and G-CSF
receptor signaling, although the resulting cells were not mature and
marginally functional. Although G-CSF and its receptor are believed to
play an essential role in neutrophil development and
function,19 their specific roles are not clear. For
example, mice with a targeted disruption of the G-CSF or G-CSF receptor
genes were still able to generate neutrophils, albeit in low
numbers.20,21 These results suggested that G-CSF-receptor signaling is not necessary for granulocyte lineage commitment or
differentiation, but is necessary for lineage expansion. These studies
also highlight the point that alternative developmental pathways exist
for neutrophils in vivo. Consistent with our original observations,
DeKoter et al17 recently described neutrophil development
in vitro in their PU.1 knockout system. Thus, these collective results
clearly indicate that neutrophil commitment and some development is
possible in the absence of PU.1. However, both our previous
studies2,3 and the results presented herein show that the
late stages of neutrophil development are critically dependent on the
presence of PU.1. In contrast to the results obtained in studies with
G-CSF or G-CSF receptor null mice, the molecular events allowing
neutrophil maturation cannot be circumvented by cytokine receptor
signaling in PU.1-deficient cells and PU.1 restoration is required
(Table 2). In addition, our current studies put a temporal context on
the effects of PU.1 expression; PU.1 reintroduction in later stages of
myeloid development can rescue phenotype and functions associated with
mature neutrophils.
When M-CSF receptor expression was restored in PU.1-deficient myeloid
cells, cell proliferation occurred, but no change in cell phenotype was
found that would suggest any progression of macrophage development.
These results, similar to what was observed for G-CSF receptor
reintroduction, indicated that receptor expression alone could not
compensate for the absence of PU.1. In contrast, expression of PU.1 in
the same cell line resulted in the development of F4/80+,
Mac-1/CD11b+, and large monocyte- and macrophage-like
cells. Consistent with previous data that implicated PU.1 in the
critical regulation of the M-CSF receptor,11 we found that
expression of the M-CSF receptor and response to M-CSF was restored by
PU.1 reintroduction. Macrophage characteristics of PU.1 null 503 cells
and 503 transduced with PU.1 or M-CSF receptor are summarized in
Table 3. Our findings of reintroducing
M-CSF receptor at a relatively late stage of myeloid cell development
are similar to those obtained from an independently derived PU.1 null
mouse model where pluripotent fetal lineage marker-negative
hematopoietic cells were targeted for M-CSF receptor delivery. After
transduction of PU.1 or M-CSF receptor into these PU.1 null
hematopoietic cells before myeloid commitment and/or possibly at very
early stages of myeloid commitment, these investigators documented
restoration of macrophage development only in the PU.1-transduced
cells.17 Thus, our studies show that PU.1 need not be
present for commitment of a myeloid lineage that retains the potential
for monocyte/macrophage development, but needs to be present for
monocyte/macrophage development.
View this table:
[in this window]
[in a new window]
|
Table 3.
Expression of Certain Macrophage Characteristics Is
Restored in PU.1-Transduced But Not in M-CSF Receptor-Transduced or
Parental PU.1-Deficient 503 Cells
|
|
M-CSF and its receptor have been reported to be crucial for macrophage
and osteoclast development.22,23 However, the naturally occurring mutant op/op mouse, which fails to produce measurable M-CSF, was still able to develop detectable macrophages and
osteoclasts.24,25 This mutant showed populations of
M-CSF-dependent and M-CSF-independent macrophages; the latter were
severely and persistently deficient and the former achieved almost
normal levels postnatally.26 No M-CSF receptor-deficient
mice have yet been described. The hematopoietic cells of the PU.1 null
mouse have been shown to lack M-CSF receptor mRNA or protein
expression, and no cells with distinct mature monocyte/macrophage
characteristics could be identified1,2 in vivo or in vitro.
Populations of Moma-2+ER-MP12+ and
Moma-2+ERMP-20+ cells have been detected in
fetal and neonatal PU.1 null mice18 that may reflect
commitment to the monocyte/macrophage lineage. In normal mice, most of
these cells were also F4/80+, CD11b+, and
Gr-1+.18 Thus, these populations could include
early common myeloid and neutrophil precursors as well as monocyte
precursor cells. Nevertheless, it is clear that monocyte/macrophage
differentiation is blocked early and cells are unable to proceed along
the macrophage developmental pathway as a result of the lack of PU.1.
One potential explanation to account for the lack of differentiation
after restoration of M-CSF receptor is that PU.1 must be present to
turn on a critical molecular switch (or switches) that enables
macrophage development in a common macrophage/neutrophil progenitor
cell. In this model, the choice of neutrophil development may hinge
upon the precise balance of PU.1 and other
neutrophil-differentiation-favoring factor(s), such as the
transcription factors C/EBP and C/EBP , members of the basic
leucine zipper class of transcription factors.27-29 C/EBP and C/EBP gene targeted mice both displayed disruptions in
neutrophil development. Loss of C/EBP resulted in markedly decreased
expression of IL-6 and G-CSF receptors,30 whereas the loss
of C/EBP in mice resulted in the generation of atypical neutrophils
(hyposegmented), that appeared to express mRNA for the G-CSF receptor,
but failed to mature and were not fully functional.31 Interestingly,25 ectopic overexpression of C/EBP in the
human leukemic cell line U937 promoted secondary granule gene
transcription, which suggests that C/EBP might have additional
effects in neutrophil development.32 A recent study by
Iwama et al33 suggests that C/EBP message level was
undetectable in whole fetal PU.1 null liver. This observation is
consistent with our previous demonstration that primitive myeloid
lineages were reduced and neutrophils and lymphoid cells were
undetectable from PU.1 null mice at birth.2 We have found
mRNA for both C/EBP and C/EBP in PU.1 null myeloid cells isolated
from older mice (K.L.A., unpublished data, April 1998);
however, further studies need to be performed to quantify levels of
mRNA and protein to determine if alterations are present.
Alternatively, molecular events regulating neutrophil versus
monocyte/macrophage differentiation in a shared progenitor may not be
reliant on PU.1. However, PU.1 appears to be absolutely necessary to
allow the monocyte/macrophage differentiation program to proceed in an
already-committed macrophage progenitor. Either model is partially
supported by the observation that in the absence of PU.1, neutrophils
become the principal myeloid lineage that is produced. In contrast to
monocytes/macrophages, the need for PU.1 in the neutrophil lineage
apparently becomes critical at a relatively late stage of development,
because the absence of PU.1 during neutrophil development manifests as
a failure of these cells to display characteristics of terminal
differentiation and functional maturity.3
The present study has illuminated certain myeloid developmental events
that are critically regulated by PU.1. These include expression of
genes common to both monocyte/macrophages and neutrophils, such as
CD11b and gp91phox, with the restoration of NADPH
oxidase function as shown by superoxide generation. Additionally,
lineage-specific programs such as restricted expression of G- and M-CSF
receptors, neutrophil expression of secondary granule genes, and the
monocyte/macrophage expression of F4/80, mannose receptor, and IL-18,
are dependent on the presence of PU.1. From these results, coupled with
prior observations made in the PU.1 null mouse,1-3 we
conclude that PU.1 is indispensable for macrophage differentiation,
neutrophil maturation, and myeloid cell proliferation via regulation of
CSF receptors. It is still not clear from the present studies whether
the developmental characteristics that have been restored in PU.1 null
cells by retroviral reintroduction of PU.1 are directly or indirectly
mediated by PU.1. Nevertheless, this work defines certain
differentiation and development-associated molecular events that are
regulated by PU.1 in myeloid cells. Characterization of a PU.1-rescued
mouse model will add relevance and further elucidate the role of PU.1
in the developing hematopoietic system.
| |
ACKNOWLEDGMENT |
We gratefully acknowledge the technical assistance of Giano Panzarella
and of the animal facility personnel at both the Burnham Institute and
The Scripps Research Institute, and the secretarial assistance of
Bonnie Towle. We also thank Drs Dan Tenen and Dan Link for helpful
discussions, and Dr Larry Rohrschneider for providing the
pMZen(cfms) vector and packaging line.
| |
FOOTNOTES |
Submitted April 6, 1999; accepted June 2, 1999.
Supported by National Institutes of Health (NIH) Grant No. DK49886
(B.E.T.). K.L.A. was supported by an NIH Training Grant [T328HLO7195-22]. This is publication 12368-IMM from The Scripps Research Institute.
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 Bruce E. Torbett, PhD, Department of
Immunology, IMM-7, The Scripps Research Institute, 10550 N Torrey Pines
Rd, La Jolla, CA 92037; e-mail: betorbet{at}scripps.edu.
| |
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A. D. Panopoulos, D. Bartos, L. Zhang, and S. S. Watowich
Control of Myeloid-specific Integrin alpha Mbeta 2 (CD11b/CD18) Expression by Cytokines Is Regulated by Stat3-dependent Activation of PU.1
J. Biol. Chem.,
May 17, 2002;
277(21):
19001 - 19007.
[Abstract]
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N. E. S. Sibinga, M. W. Feinberg, H. Yang, F. Werner, and M. K. Jain
Macrophage-restricted and Interferon gamma -inducible Expression of the Allograft Inflammatory Factor-1 Gene Requires Pu.1
J. Biol. Chem.,
May 3, 2002;
277(18):
16202 - 16210.
[Abstract]
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S. Akbarzadeh, A. C. Ward, D. O. M. McPhee, W. S. Alexander, G. J. Lieschke, and J. E. Layton
Tyrosine residues of the granulocyte colony-stimulating factor receptor transmit proliferation and differentiation signals in murine bone marrow cells
Blood,
February 1, 2002;
99(3):
879 - 887.
[Abstract]
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Y. Li, Y. Okuno, P. Zhang, H. S. Radomska, H.-m. Chen, H. Iwasaki, K. Akashi, M. J. Klemsz, S. R. McKercher, R. A. Maki, et al.
Regulation of the PU.1 gene by distal elements
Blood,
November 15, 2001;
98(10):
2958 - 2965.
[Abstract]
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R. N. Laribee and M. J. Klemsz
Loss of PU.1 Expression Following Inhibition of Histone Deacetylases
J. Immunol.,
November 1, 2001;
167(9):
5160 - 5166.
[Abstract]
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Z. Lian, L. Wang, S. Yamaga, W. Bonds, Y. Beazer-Barclay, Y. Kluger, M. Gerstein, P. E. Newburger, N. Berliner, and S. M. Weissman
Genomic and proteomic analysis of the myeloid differentiation program
Blood,
August 1, 2001;
98(3):
513 - 524.
[Abstract]
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P. Zhang, X. Zhang, A. Iwama, C. Yu, K. A. Smith, B. U. Mueller, S. Narravula, B. E. Torbett, S. H. Orkin, and D. G. Tenen
PU.1 inhibits GATA-1 function and erythroid differentiation by blocking GATA-1 DNA binding
Blood,
October 15, 2000;
96(8):
2641 - 2648.
[Abstract]
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K. R. Calvo, D. B. Sykes, M. Pasillas, and M. P. Kamps
Hoxa9 Immortalizes a Granulocyte-Macrophage Colony-Stimulating Factor-Dependent Promyelocyte Capable of Biphenotypic Differentiation to Neutrophils or Macrophages, Independent of Enforced Meis Expression
Mol. Cell. Biol.,
May 1, 2000;
20(9):
3274 - 3285.
[Abstract]
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W. B Strawn, R. H Dean, and C. M Ferrario
Novel mechanisms linking angiotensin II and early atherogenesis
Journal of Renin-Angiotensin-Aldosterone System,
March 1, 2000;
1(1):
11 - 17.
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S. Harendza, D. H. Lovett, and R. A. K. Stahl
The Hematopoietic Transcription Factor PU.1 Represses Gelatinase A Transcription in Glomerular Mesangial Cells
J. Biol. Chem.,
June 23, 2000;
275(26):
19552 - 19559.
[Abstract]
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K. L. Anderson, S. L. Nelson, H. B. Perkin, K. A. Smith, M. J. Klemsz, and B. E. Torbett
PU.1 Is a Lineage-specific Regulator of Tyrosine Phosphatase CD45
J. Biol. Chem.,
March 2, 2001;
276(10):
7637 - 7642.
[Abstract]
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TOP
ABSTRACT
INTRODUCTION