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Laboratory of Molecular Growth Regulation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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The colony-forming potential of ICSBP-/- progenitor cells
was grossly abnormal, as they gave rise to a disproportionately high
number of granulocyte colonies and many fewer macrophage colonies.
IFN- inhibited colony formation, while promoting macrophage
maturation in ICSBP+/+ cells. In contrast, the effects of
IFN- were completely absent in ICSBP-/- progenitors.
By retrovirus transduction we tested whether reintroduction of ICSBP
restores a normal colony-forming potential in -/- progenitor cells.
The wild-type ICSBP, but not transcriptionally defective mutants,
corrected abnormal colony formation by increasing macrophage colonies
and decreasing granulocyte colonies. Taken together, ICSBP plays a
critical role in myeloid cell development by controlling lineage
selection and is indispensable for IFN--dependent modulation of
progenitor cell maturation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and C/EBP
, are both central to granulocyte
development. Genetic ablation of the C/EBP gene results in a
complete block in granulocyte formation, but not monocyte formation
(7). The disruption of the C/EBP gene prevents
functional maturation of granulocytes, but not granulocyte formation
per se (8).
IFN consensus sequence binding protein
(ICSBP),5 recently
renamed IFN regulatory factor-8 (IRF-8; hereafter ICSBP) is a member of
IRF family that is involved in IFN-/IFN-
-dependent transcription
(9). Unlike many other IRF members, ICSBP is expressed
only in hemopoietic cells and is induced by IFN- (10, 11). Analyses of ICSBP-/- mice revealed
a previously unsuspected role for ICSBP in myeloid cell development;
there is a striking increase in granulocytes in various lymphoid organs
in -/- mice, and as a result, -/- mice develop, with 100%
penetrance, a syndrome similar to chronic myelogenous leukemia
(12). Furthermore, ICSBP-/-
macrophages are functionally defective. They do not produce sufficient
amounts of reactive oxygen intermediates upon microbial challenges and
fail to express IL-12, making these mice highly susceptible to
infections (13, 14, 15, 16, 17, 18). Scheller et al. (19)
provided evidence that these abnormalities are traced to a defect in
myeloid progenitor cells. Also, by studying myeloid clones established
from ICSBP-/- bone marrow, we have recently
shown that ICSBP directs their differentiation into macrophages in
vitro (20). Additionally, recent reports from several
laboratories documenting the involvement of ICSBP in the pathogenesis
of chronic myelogenous leukemia further strengthen the possible role
for ICSBP in myeloid cell development (21, 22, 23, 24).
Despite the growing evidence that ICSBP has a pivotal role in the development of myeloid progenitor cells, a critical question as to whether normal progenitor cells express ICSBP has remained unresolved. With this uncertainty there remain two possible mechanisms by which ICSBP could contribute to myeloid cell development. If ICSBP expression is absent in the progenitor cells, its role in regulating the development must be indirect, presumably mediated through regulation of the cytokine milieu. However, if ICSBP is expressed in progenitor cells, it would support a more direct, intrinsic role for ICSBP in controlling myeloid cell development. Furthermore, if the latter proves to be the case, it would be possible for us to further assess the function of ICSBP by reintroducing the gene into -/- progenitor cells.
In addition to these issues we have been interested in the effect of
IFN- on myeloid cell development. IFN- is an immunoregulatory
cytokine produced by activated T cells and other cells in response to
various stimuli (25). IFN- elicits vital effects on
myeloid cells; it stimulates maturation of the monocyte-macrophage
lineage and activates macrophage functions by enhancing various
immunological activities (25). The contribution of IFN-
to hemopoiesis, however, may be complex. Although IFN- is generally
thought to suppress hemopoietic cell growth with an implication for
aplastic anemia (26, 27), its effects on early progenitor
cells appear to be mixed (28).
Here we show that ICSBP is expressed in normal bone marrow progenitor
cells and that the colony-forming potential of -/- progenitor cells
is severely distorted. Furthermore, although IFN- promoted
macrophage maturation in +/+ cells, it was unable to exert these
effects on -/- cells. Finally, reintroduction of an ICSBP retrovirus
vector into -/- progenitor cells fully restored the colony-forming
potential of +/+ cells. These results indicate that ICSBP contributes
to myeloid cell development by an intrinsic mechanism.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Experiments were performed with 6- to 10-wk-old homozygous ICSBP-/- and ICSBP+/+ mice on a C57BL/6 background housed under specific pathogen-free conditions. All animal work conformed to the guidelines of the animal care and use committee of National Institute of Child Health and Human Development.
Cytokines and cell cultures
Recombinant murine GM-CSF, stem cell factor (SCF), and M-CSF
were purchased from BioSource International (Camarillo, CA), and G-CSF,
IL-3, and IL-6 were obtained from PeproTech (Rocky Hill, NJ). Murine
IFN- was provided by Genentech (South San Francisco, CA). Murine
cells were cultured in RPMI 1640 (Quality Biologicals, Gaithersburg,
MD) supplemented with 2 mM L-glutamine, 100 U/ml
penicillin, and 100 µg/ml streptomycin (Life Technologies,
Gaithersburg, MD) with 20% FBS (Atlantic Biologicals, Norcross, GA).
BOSC23 obtained through American Type Culture Collection (Manassas, VA)
were maintained in DMEM (Quality Biologicals) containing 2 mM
L-glutamine, 10% FBS, and penicillin/streptomycin
(29).
Antibodies
FITC-conjugated Abs against Mac-1 (CD11b, M1/70), Gr-1 (Ly6-G), c-Kit (CD117, 2B8), PE-conjugated anti-Sca-1 (Ly-6A/E, E13-161.7), and B220 (CD45R, RA3-6B2) were purchased from BD PharMingen (San Diego, CA). Anti-F4/80 (Ly-71) Ab was obtained from Caltag Laboratories (Burlingame, CA).
Cell preparations
Lineage marker-negative (Lin-) cells were
prepared from bone marrow cell suspensions using the Enrichment of
Murine Hemopoietic Progenitor Cells (StemCell Technologies, Vancouver,
Canada). Briefly, lineage marker-positive cells were depleted with a
biotinylated Ab mixture for CD5 (Ly-1), CD45R (B220), CD11b (Mac-1),
Gr-1, TER 119, and 7/4, followed by immunomagnetic depletion on a
StemSep column. Sca-1+ or
c-Kit+ cells (30, 31) were purified
from bone marrow cells based on the MACS system (Miltenyi Biotec,
Auburn, CA) using Sca-1 MultiSort Kit and anti-FITC MicroBeads,
respectively. Gr-1+ granulocytes
(32) and F4/80+ macrophages
(33) were purified from spleen cell suspensions with
anti-FITC MicroBeads. Granulocytes were collected from the pellets
following density gradient centrifugation of cells on Lympholyte-M
(Cedarlane Laboratories, Hornby, Ontario, Canada). The yields of
Lin- cells are summarized in Table I
. Although the average numbers of
unfractionated bone marrow cells were 2-fold greater in
ICSBP-/- mice than in
ICSBP+/+ animals, there was no significant
difference in the yield of Lin- cells between
-/- and +/+ mice. B cell-enriched preparations were obtained from
spleen suspensions by density gradient centrifugation on
Lympholyte-M.
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Cells were mixed in Methocult M3100 (StemCell Technologies), 30% FBS, 1% BSA (Sigma-Aldrich, St. Louis, MO), 10-4 M 2-ME, and the indicated cytokines. The evaluation of colony formation was performed on days 7–9. Colonies containing 50 or more cells were scored. Colonies were classified microscopically into three types (CFU-M, CFU-G, and CFU-GM) according to the size, cell distribution patterns, and morphology as previously described (34, 35). Statistical analysis was performed with Student’s t test. Colonies placed on cytospin slides (Thermo Shandon, Pittsburgh, PA) were routinely inspected for morphological verification of colony types.
Retroviral transduction
Retrovirus vectors harboring the wild type and mutants have been previously described (20). A vector harboring ICSBP/enhanced green fluorescence protein (EGFP)-producing chimeric ICSBP fused to green fluorescence protein (GFP) at the C terminus was constructed as follows: open reading frames of ICSBP or K79E (20) with the stop codon removed were inserted into pEGFPN3 (Clontech Laboratories, Palo Alto, CA) to construct ICSBP/EGFP and K79E/EGFP, which were then transferred into pMSCV-puro (Clontech Laboratories). Transfection of these viruses was conducted as previously described (36) with some modifications (20). BOSC23 ecotropic retroviral packaging cells (29) were transiently transfected with retroviral vectors using Lipofectamine 2000 (Life Technologies). Retroviral supernatants were collected at 48 h. Virus titers measured on 32D cells were >1 x 106 infectious particles/ml. Before viral transduction, Lin- bone marrow cells (5 x 105/ml) were incubated in 24-well plates for 1 day in the presence of 6 ng/ml IL-3, 10 ng/ml IL-6, and 50 ng/ml SCF. Cells were transduced by two spinoculations (first, 2500 rpm for 1 h at 33°C; second, 2500 rpm for 45 min at 33°C) in a retrovirus supernatant supplemented with 4 µg/ml polybrene for 2 days. Twenty-four hours after spinoculation, cells were washed twice and plated in methylcellulose medium or cultured in suspension. Transduced cells were selected by 0.5 µg/ml puromycin, which produced no colonies from nontransduced cells, or were identified by the expressing of GFP.
Flow cytometric analysis
For detection of surface markers, cells were blocked with
anti-mouse FcR Ab (CD16/CD32. 2.4G2; BD PharMingen), followed by
incubation with either specific Abs or isotype controls at 4°C for 30
min. Flow cytometry was performed on FACSCalibur, and data were
analyzed using the FlowJo (Tree Star) or CellQuest software (BD
Biosciences, Mountain View, CA).
Semiquantitative RT-PCR
This was performed for ICSBP, -actin, the M-CSF receptor gene
(c-fms), scavenger receptor (SR), FcRI, the G-CSF
receptor gene (G-CSFR), C/EBP, C/EBP, and PU.1 transcripts as
previously described (20). Fifty nanograms of cDNA was
used for reaction of SR, FcRI, C/EBP, C/EBP, and PU.1; 5 ng
cDNA was used for ICSBP, c-fms, and G-CSFR; and 1 ng cDNA
was used for -actin. Reactions were performed at 94°C for 30
s, at 60°C for 30 s, and at 72°C for 45 s for 30 cycles
in the presence of 0.2 mM dNTPs, 2.5 µM MgCl2,
and 500 nM 5' and 3' primers. RT-PCR products were quantified by NIH
Image software and were normalized by -actin.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Growing evidence indicates that ICSBP plays a role in myeloid cell
development (12, 19, 20). However, it has not been clear
whether ICSBP is indeed expressed in hemopoietic progenitor cells. To
study this question, Lin- cells were isolated
from ICSBP+/+ bone marrow and tested for ICSBP
transcript expression by semiquantitative RT-PCR.
Lin- cells contain both primitive and mature
progenitors that can differentiate into cells of every hemopoietic
lineage, including the myeloid lineage (30, 37, 38). As
shown in Fig. 1A, ICSBP
transcripts were detected in Lin- progenitor
cells. Levels of the transcripts in the progenitor cells were about
half those in splenic macrophages and B cell-enriched splenocytes, both
known to express high levels of ICSBP (10). The transcript
levels in the progenitor cells were much higher than those in splenic
granulocytes. Interestingly, ICSBP expression in the progenitor cells
was increased by 
2.5-fold upon IFN- treatment, similar to other
cells (Fig. 1A). To further confirm ICSBP expression in
progenitor cells, Sca-1+ cells and
c-Kit+ cells were isolated separately and tested
for the transcripts. As depicted in Fig. 1B, the transcripts
were expressed in these cells at similar levels as
Lin- cells. These results indicate that ICSBP is
expressed in hemopoietic progenitor cells, and the expression is
augmented by IFN-.
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inhibition in ICSBP-/- Lin-
progenitor cells
To assess whether +/+ and -/- progenitors can properly give rise
to the cells of the macrophage and granulocyte lineages,
methylcellulose-based colony forming assays were performed with
Lin- cells. Lin-
progenitor cells rather than total bone marrow cells (19)
were chosen for this test, because we wanted to gain accurate
information on the developmental potential of immature cells. The
recovery of Lin- cell is summarized in Table I
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which indicated that ICSBP+/+ and
ICSBP-/- mice had a similar number of
progenitor cells in bone marrow, enabling us to compare their
colony-forming potential. Lin- cells were
allowed to form colonies in the presence of SCF (39) plus
three separate growth factors, M-CSF, GM-CSF, and G-CSF, for 7–9 days.
Colonies were classified into three distinct types, granulocyte,
macrophage, and granulocyte/macrophage colonies according to the
standard morphological criteria (34). As shown in Fig. 2, several features of colony formation
were strikingly different between +/+ and -/- cells. First, the
number of total colonies produced from ICSBP-/-
cells was greater than that from ICSBP+/+ cells
in all three growth factors. In the presence of M-CSF,
ICSBP-/- cells generated 60% more colonies
than ICSBP+/+ cells. The difference was less
pronounced in the presence of G-CSF and GM-CSF. Second,
ICSBP-/- cells gave rise to a
disproportionately large number of granulocyte colonies relative to
ICSBP+/+ cells in all three growth factor
conditions tested. Conversely, macrophage colonies produced from -/-
progenitor were many fewer than those from +/+ cells. This was most
noticeable in the presence of M-CSF, where macrophage colonies were
<5% of the total -/- colonies, but >30% of the +/+ colonies were
macrophages (Table II).
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on colony formation,
since this cytokine enhanced ICSBP expression in
Lin- progenitor cells (Fig. 1A).
IFN- has been shown to inhibit bone marrow stem cell growth and
colony-forming ability (26, 27). As shown in Fig. 2, IFN- treatment caused markedly different effects on +/+ and -/-
cells. IFN- inhibited colony formation in +/+ cells in the presence
of M-CSF and G-CSF. This inhibition was mostly due to the inhibition of
GM colonies and, to a lesser degree, that of granulocyte colonies (see
Table II also). In contrast, colony formation was not inhibited in
-/- cells by IFN-. Rather, IFN- markedly increased the total
number of colonies from -/- cells, resulting in at least twice as
many colonies as those from +/+ colonies. This was due to a large
increase in the number of granulocyte colonies formed from -/- cells
after IFN- addition. Granulocyte colonies formed in the presence of
IFN- were, in general, smaller and more tightly aggregated than
those formed in its absence both in +/+ and -/- cells. These results
indicate that the colony-forming potential of -/- progenitor cells is
markedly different from that of the normal progenitor cells, and that
in the absence of ICSBP, IFN- fails to inhibit colony formation. Impaired macrophage development in ICSBP-/- progenitors
It was remarkable that ICSBP-/- progenitor
cells gave rise to few macrophage colonies (<5% of total colonies)
even in the presence of M-CSF, where macrophage colonies generated by
+/+ progenitors were >33% of all colonies (Table II). To further
investigate progenitor differentiation in the presence of M-CSF, cells
were cultured under the same conditions as described above, but in
suspension without methylcellulose for 7 days. Within a few days
following culture, many +/+ cells adhered to plastic plates, and the
adherence was intensified by IFN-. In contrast, few
ICSBP-/- cells adhered to plastic, even in the
presence of IFN-. Wright-Giemsa staining of cytospin slides shown in
Fig. 3A revealed that
following 7 days of culture, 60% of ICSBP+/+
cells were macrophages in morphology, while the majority of
ICSBP-/- cells were typical granulocytes.
IFN- facilitated morphological maturation of macrophages in +/+
cells, but not in -/- cells. Upon IFN- treatment, -/- cells
transformed into large cells with abnormal morphology. Flow cytometric
analysis of day 7 cells shown in Fig. 3B indicate that
50% of +/+ cells expressed F4/80, a marker specific for macrophages
(33). In the presence of IFN-, F4/80-positive cells
were increased to 75%. In contrast, only 5% of -/- cells expressed
F4/80, and this was at a low level. Even in the presence of IFN-,
F4/80-positive cells were <15% in the -/- cell culture. In
addition, we tested the expression of Mac-1, a myeloid cell marker
normally expressed at higher levels in macrophages than granulocytes
(40). Most +/+ cells expressed high levels of Mac-1, but
the levels in -/- cells were, on the average, 3- to 4-fold lower
than those in +/+ cells. These results are consistent with the data in
Fig. 2 and indicate that ICSBP-/- progenitor
cells tend to generate more granulocytes and fewer macrophages than +/+
cells in the presence of M-CSF.
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To assess gene expression patterns in +/+ and -/- progenitor
cells developing in the presence of M-CSF, semiquantitative RT-PCR was
conducted for the FcRI and SR genes that are specifically expressed
in macrophages (41, 42). In addition, the G-CSFR gene; the
C/EBP and C/EBP genes, encoding transcription factors involved in
granulocytic differentiation (7, 8, 43); and
c-fms, known to stimulate the growth and differentiation of
monocyte-macrophages (44) were tested. The expression of
PU.1, a transcription factor critical for myeloid cell development that
interacts with ICSBP (4, 6, 22, 45, 46), was also
examined. The results are shown in Fig. 4A, where expression levels of
each transcript are compared with those in
ICSBP+/+ cells on day 0. Expression of
c-fms was detected in -/- cells at levels slightly higher
than or comparable to those in +/+ cells throughout the culture period
(see Discussion). G-CSFR transcripts were expressed in
ICSBP-/- progenitor cells at higher levels than
in +/+ cells on days 0 and 2, although the levels became comparable to
those in +/+ cells upon subsequent periods of culture. Expression
patterns for C/EBP, C/EBP, and PU.1 transcripts were similar, in
that they were expressed at higher levels in -/- cells than in +/+
cells in early stages, but the levels became comparable in later
stages. Fig. 4B shows RT-PCR analysis of macrophage-specific
genes, FcRI and SR, tested in the presence or the absence of
IFN-. FcRI was expressed constitutively in +/+ cells on day 7,
and the expression levels were augmented by IFN-. However, in -/-
cells this transcript was not expressed constitutively and was only
induced following IFN- treatment. SR transcripts were constitutively
expressed on day 2 in +/+ cells, but not in -/- cells, and were only
weakly expressed on day 7. These results indicate that the expression
of genes linked to granulocyte development is higher in -/- cells
than in +/+ cells, and conversely, the expression of genes linked to
macrophage development is lower in -/- cells.
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To assess whether reintroduction of ICSBP reverses the distorted
colony-forming potential of -/- cells,
ICSBP-/- Lin- cells were
transduced with ICSBP retroviruses and tested for colony-forming
ability. The wild-type ICSBP and three mutant ICSBP constructs were
tested (Fig. 5A for a diagram
of mutants) (20). ICSBP binds two target DNA elements: the
ISRE, a target sequence of all IRF family proteins, and the EICE, a
composite element to which ICSBP binds by interacting with PU.1
(45). Binding of ICSBP to both elements is dependent on
the intact DNA binding domain in the N terminus (DBD in Fig. 5A) and the IRF association domain (IAD) in the C-terminal
region (20, 45). Mutant 1–390 is a truncation lacking the
C-terminal region, but retaining the IAD (45). This mutant
is capable of binding to the both elements and is still functional
(20). Mutant 1–356, on the other hand, lacks a portion of
the IAD, does not bind to either element, and is without functional
activity. K79E, carrying a point mutation in the DNA binding domain,
similarly lacks DNA binding as well as transcriptional activities
(20). Whereas untransduced cells produced no colonies in
the presence of puromycin, cells transduced with retroviruses all
produced many colonies. The efficiency of viral transduction was judged
to be high, since the number of colonies produced in the presence of
puromycin ranged from 73 to 98% of the colonies generated in its
absence (not shown). Fig. 5B and Table III show the results of colony forming
assays performed with virus-transduced cells in the presence of M-CSF,
G-CSF, and GM-CSF. Cells transduced with the control vector gave rise
to a disproportionately large number of granulocyte colonies and few
macrophage colonies, similar to untransduced cells in Fig. 2. In
contrast, cells transduced with the wild-type ICSBP yielded a
dramatically higher number of macrophage colonies, concomitant with
many fewer granulocyte colonies. The changes were especially evident
with colony formation in M-CSF and GM-CSF where the number of
macrophage colonies was increased by 4-fold, while that of
granulocyte colonies was decreased by 4- to 10-fold upon ICSBP
transduction. Even in the presence of G-CSF, ICSBP transduction led to
an 45% reduction in granulocyte colonies. In all cultures the total
number of colonies was reduced in ICSBP-transduced cells relative to
that in cells transduced by the control vector. Among the mutants, only
1–390 rescued macrophage colony formation. Other ICSBP mutants, 1–356
and K79E lacking DNA binding activity and inactive in transcription,
did not rescue macrophage colony formation, did not inhibit granulocyte
colony formation, and did not reduce total colony numbers. Taken
together, simple reintroduction of ICSBP corrected the distorted
colony-forming potential of -/- cells, indicating that ICSBP has an
intrinsic role in lineage selection during myeloid cell development.
These results also indicate that the absence of ICSBP does not
irreversibly fix the developmental fate of progenitor cells.
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The morphology and surface makers were examined for
ICSBP-transduced cells cultured in suspension for 7 days with M-CSF. As
shown in Fig. 6A, cells
transduced with the control vector differentiated mainly into
granulocytes, similar to untransduced cells. In contrast, 60% of
ICSBP-transduced cells differentiated into macrophages, accompanied by
a reduction in granulocytes. Consistent with the colony forming assay
data in Fig. 5, cells transduced with the mutant 1–390 differentiated
into mostly macrophages. On the other hand, mutants 1–356 and K79E
produced mainly granulocytes, similar to the control vector. The
results of flow cytometric analysis shown in Fig. 6B
confirmed the morphological findings. While F4/80 expression remained
negative in most cells transduced with the control vector, 55% of
ICSBP-transduced cells expressed the marker at high levels. Similarly,
the expression of Mac-1, which was low in control cells, was increased
in ICSBP-transduced cells. Levels of F4/80 and Mac-1 expressed in the
cells transduced with mutant 1–390 were similar to those transduced by
the wild-type ICSBP. In contrast, there was no increase in the
expression of these markers when cells were transduced with mutants
1–356 or K79E. The results of semiquantitative RT-PCR performed with
cells transduced with ICSBP and cultured as described above are shown
in Fig. 6C. Macrophage-specific genes, FcRI and SR, were
both markedly induced in cells transduced with ICSBP, but not control
vector. The expression of c-fms was, however, not
significantly altered upon ICSBP transduction. Conversely, transcripts
for C/EBP and G-CSFR were reduced in ICSBP-transduced cells. The
patterns seen by the mutant 1–390 was similar to those of the
wild-type ICSBP, whereas mutants 1–365 and K79E displayed patterns
similar to those of untransduced cells. These results indicate that
ICSBP stimulates genes expressed along the macrophage lineage and
represses those along the granulocyte lineage.
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. ICSBP-/- Lin- cells
were transduced with these retroviruses and cultured for 7 days in the
presence of M-CSF as described above. Flow cytometric analysis was
performed to detect GFP signals and macrophage markers F4/80 and Mac-1.
In these assays cells transduced with retroviruses were mixed with
untransduced cells, providing an internal negative control. As shown in
Fig. 6D (left panel), cells transduced
with the vector for GFP only could be distinguished from untransduced
cells, since the former exhibited positive GFP signals, while the
latter did not. Both populations showed low F4/80 and Mac-1 expression,
indicating that few macrophages were generated when transduced with GFP
only. In contrast, when cells were transduced with ICSBP/EGFP vector
(Fig. 6D, middle panel), the cell population that
exhibited positive GFP signals, hence ICSBP, exhibited high levels of
F4/80 and Mac1, much higher than those seen by cells with GFP only. On
the other hand, cells transduced with the mutant K79E/EGFP, although
exhibiting GFP signals at levels comparable with or greater than those
of the wild-type ICSBP/EGFP, did not show a significant increase in the
expression of F4/80 or Mac-1 (Fig. 6D, right
panel). These results show that the ICSBP-GFP fusion protein is
expressed in the progenitor cells upon viral transduction, and that
only the wild-type ICSBP, but not the K79E mutant, enhances F4/80 and
Mac-1 expression and stimulates macrophage development. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. In line with our findings, a recent report
with purified stem cells indicates that ICSBP expression begins early
in hemopoiesis (46). These results pointed to the
possibility that ICSBP has a direct, intrinsic role in the regulation
of myeloid cell development, rather than acting through an indirect
mechanism such as regulation of cytokine environment. Impaired lineage commitment in ICSBP-/- progenitor cells
The absence of ICSBP severely distorted the colony-forming
potential of Lin- cells, as evidenced by the
disproportionately high number of granulocyte colonies generated from
-/- progenitor cells, coinciding with a marked decrease in macrophage
colonies. The distorted colony formation was seen in the presence of
all three growth factors tested, although the reduction in macrophage
colony formation was most pronounced in the presence of M-CSF. Thus,
ICSBP-/- progenitor cells are deficient in the
development of the macrophage lineage. This deficiency is apparently
compensated by the increase in the granulocyte colony formation.
Consistent with the impaired commitment to the macrophage lineage,
cells developed from -/- progenitor cells in the presence of M-CSF
expressed much lower levels of SR and FcRI and only later in
culture. Conversely, genes linked to granulocyte differentiation,
C/EBP and C/EBP, were expressed at higher levels in -/- cells,
indicating that the loss of ICSBP leads to preferential stimulation of
granulocyte lineage and genes associated with the lineage. The
expression of c-fms, encoding the receptor for M-CSF in
ICSBP-/- cells was comparable to or
higher than that in +/+ cells throughout the culture period, despite
the fact that -/- cells were deficient in forming macrophage colonies
in response to M-CSF. These results may have been unexpected, in light
of the fact that c-fms confers responsiveness to M-CSF and,
as such, stimulates the growth and maturation of cells in the
monocyte-macrophage lineage (44). These results may
imply that ICSBP does not directly control c-fms expression,
a possibility consistent with the observation that retrovirus
transduction of ICSBP did not significantly change c-fms
expression in -/- cells (Fig. 6C). It is possible that
ICSBP regulates a signaling event associated with the binding of M-CSF
to the receptor or may affect a pathway downstream from ligand-receptor
binding. Alternatively, however, it is possible that c-fms
expression varies among different bone marrow cells, and ICSBP may
contribute to the regulation of c-fms expression in some,
but not all, cells. Supporting this possibility, Scheller et al.
(19) reported that cells expressing M-CSFR were fewer in
-/- than +/+ bone marrow and that -/- cells are hyporesponsive to
M-CSF.
Requirement of ICSBP for IFN--mediated promotion of macrophage
development
IFN- elicited noticeable effects on colony formation by
ICSBP+/+ cells: it led to a reduction in total
colony numbers, facilitated macrophage maturation, and stimulated the
expression of SR and FcRI genes. These effects suggested that
IFN- can significantly alter the course of myeloid cell development
by preferentially reinforcing the development of macrophage lineage.
Our data on IFN-’s effects on +/+ cells are in line with previous
studies showing that IFN- stimulates macrophage differentiation in
normal bone marrow cells (47) and in established myeloid
cell lines (48). In addition, IFN- has been shown to
inhibit the growth of bone marrow stem cells in various culture
conditions (27). On the other hand, regulation of
hemopoietic cell growth by IFN- appears to be complex and may be
dependent on the developmental stages of cells, since other groups
found that IFN- inhibits colony growth by normal bone marrow
cells, but not by 5-fluorouracil-treated cells (28).
Strikingly, the above-described effects of IFN- were completely
absent in ICSBP-/- cells; instead of inhibiting
colony formation, IFN- markedly increased the total numbers of
colonies derived from -/- progenitors, which was attributed primarily
to a large increase in the number of granulocyte colonies. Moreover,
IFN- did not promote macrophage maturation in -/- cells, as judged
by morphology and the lack of F4/80 induction. Furthermore, IFN-
only weakly stimulated SR and FcRI genes in -/- cells. Our data
indicate that ICSBP is indispensable for the elicitation of the effects
of IFN- on progenitor cells.
Intrinsic role of ICSBP
The distorted colony formation seen by -/- progenitor cells was
fully corrected upon ICSBP-retrovirus transduction, resulting in a
marked increase in macrophage colony formation and a sharp decrease in
granulocyte colony formation. This was associated with the
normalization of total colony numbers and the ratio of macrophage to
granulocyte colonies and with the restoration of cellular morphology
and gene expression patterns. Importantly, these changes were seen only
with the wild-type ICSBP and mutant 1–390 that retained DNA binding
and transcriptional activities. Other mutants inactive in these
activities were unable to correct any aspects of abnormal colony
formation associated with the absence of ICSBP. In the present study
the expression of ICSBP protein in virus-transduced cells was verified
by employing ICSBP-GFP fusions (Fig. 6D). The visualization
of ICSBP-GFP allowed us to exclude the remote possibility that the
rescue was due to an indirect mechanism, rather than to ICSBP protein
expression. Taken together, these results provide compelling evidence
that ICSBP intrinsically regulates the lineage fate of progenitor
cells, presumably by regulating the transcription of various target
genes.
It is interesting to note that the formation of granulocyte colonies
and that of macrophage colonies was reciprocal in many criteria
examined. Namely, when the former was increased, the latter was
decreased and vice versa in our system. This reciprocity suggests that
the macrophage and granulocyte lineages are inseparably coupled, and
the development of one lineage is associated with the repression of the
other. Supporting the coupling of two lineages, we previously observed
that ectopic ICSBP expression stimulates macrophage differentiation,
but inhibits granulocyte differentiation (20).
Radmoska et al. (43) demonstrated another example for a
possible lineage coupling by showing that ectopic expression of
C/EBP promoted granulocytic differentiation, while it blocked
monocyte differentiation in U937 cells. In this context it is
interesting to note that the expression of C/EBP and C/EBP is
high in granulocytes and low in macrophages. Conversely, ICSBP
expression is high in macrophages and very low in granulocytes (see
Fig. 1A). These data suggest that expression levels of key
transcription factors, such as C/EBP and ICSBP, could influence the
process of lineage selection and strengthen the idea that increased
ICSBP expression favors the selection of macrophage lineage (see Fig. 7 for model).
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reinforces this
process by enhancing ICSBP expression.
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Acknowledgments |
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Footnotes |
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2 H.T. and T.N.-I. contributed equally to this work.
3 Present address: Division of Cell Processing, Institute of Medical Science, University of Tokyo, Tokyo, Japan.
4 Address correspondence and reprint requests to Dr. Keiko Ozato, Laboratory of Molecular Growth Regulation, National Institute of Child Health and Human Development, National Institutes of Health, Building 6, Room 2A01, Bethesda, MD 20892-2753. E-mail address: ozatok{at}mail.nih.gov
5 Abbreviations used in this paper: ICSBP, IFN consensus sequence binding protein; CSFR, CSF receptor; GFP, green fluorescence protein; IRF, IFN regulatory factor; IAD, IRF association domain; Lin-, lineage marker negative; SCF, stem cell factor; EGFP, enhanced GFP; SR, scavenger receptor.
Received for publication February 27, 2002. Accepted for publication May 16, 2002.
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