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Blood, Vol. 91 No. 10 (May 15), 1998:
pp. 3773-3783
The Effects of Colony-Stimulating Factor-1 on the Distribution of
Mononuclear Phagocytes in the Developing Osteopetrotic Mouse
By
Philip Roth,
Melissa G. Dominguez, and
E. Richard Stanley
From the Division of Neonatology, Department of Pediatrics and
Department of Developmental and Molecular Biology, Albert Einstein
College of Medicine, Bronx, NY.
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ABSTRACT |
Colony-stimulating factor-1 (CSF-1), the primary regulator of
mononuclear phagocyte (M ) production, exists as either a circulating or cell surface, membrane-spanning molecule. To establish
transplacental transfer of maternal CSF-1, gestational day-17 mothers
were injected intravenously with 125I-mouse CSF-1 or human
rCSF-1, and the 125I-cpm or human CSF-1 concentrations were
measured in fetal tissue, placenta, and fetal/maternal sera.
Biologically active CSF-1 crossed the placenta and peaked in fetal
tissue, placenta, and serum 10 minutes after injection. The role of
CSF-1 in perinatal M development was examined by studying the
CSF-1-deficient osteopetrotic
(csfmop/csfmop) mouse. Fetal/neonatal
mice, derived from matings of either +/csfmop
females with csfmop/csfmop males or the
reciprocal pairings, were genotyped and tissue M identified and
quantified. In the presence of circulating maternal CSF-1
(+/csfmop mother), M development in
csfmop/csfmop liver was essentially
complete at birth relative to +/csfmop
littermates, but significantly reduced in spleen, kidney, and lung. In
the absence of circulating maternal CSF-1
(csfmop/csfmop mother), M numbers at
birth were reduced in csfmop/csfmop
liver relative to the offspring of +/csfmop
mothers, but were similar in spleen, kidney, and lung. We conclude that
CSF-1 is required for the perinatal development of most M in these
tissues. Compensation for total absence of local CSF-1 production by
circulating, maternal CSF-1 is tissue-specific and most prominent in
liver, the first fetal organ perfused by placental blood. However,
because some M developed in the complete absence of CSF-1, other
factors must also be involved in the regulation of macrophage
development.
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INTRODUCTION |
COLONY-STIMULATING factor-1 (CSF-1) is a
hematopoietic growth factor that regulates the survival, proliferation,
and differentiation of the mononuclear phagocyte (reviewed in Pixley
and Stanley1). Whereas endothelial cells lining blood
vessels appear to be the primary source of circulating CSF-1 in vivo
(P. Roth and E.R. Stanley, unpublished observations),
numerous other cell types, including fibroblasts, monocytes, bone
marrow stromal cells, osteoblasts, thymic epithelial cells,
keratinocytes, astrocytes, myoblasts, mesothelial cells, liver
parenchymal cells, and epithelial cells of the pregnant uterus, have
been shown to produce this growth factor (reviewed in
Stanley2). Isolation and sequencing of both human and
murine CSF-1 cDNA clones3-10 coupled with studies of CSF-1
biosynthesis and expression have shown that CSF-1 exists in several
biologically active forms, including a long-lived (t1/2 = 7 hours)
membrane-spanning, cell surface glycoprotein, a secreted glycoprotein,
and a secreted chondroitin sulfate-containing
proteoglycan.11 The proteoglycan form may be localized to
specific types of extracellular matrix.11,12 The existence
of diverse forms of CSF-1 makes it well suited to exert its effects
both locally as well as at distant sites via the circulation. Some of
the major biological properties that have been attributed to CSF-1
include the regulation of mononuclear phagocyte production in vivo,
fetal osteoclast development, regulation of cells in the female
reproductive tract during pregnancy (reviewed in Stanley2
and Pollard and Stanley13), and regulation of testicular
macrophages involved in male reproductive functions.14,15
In addition, CSF-1 seems to preferentially regulate the development of
tissue macrophages, which are involved in tissue remodelling and
organogenesis during fetal and neonatal life.16 Whereas
CSF-1 appears to play an important role in mononuclear phagocyte
development, there are no data regarding its ability to cross the
placenta during fetal life. While the passage of IgG from mother to
fetus is the most striking and has been the most extensively studied,
other proteins may be transferred to a much more limited
extent.17 In particular, granulocyte colony-stimulating factor (G-CSF), another hematopoietic growth factor, has been shown to
cross the placenta after maternal administration and induce
myelopoietic activity in the fetal rat.18
A useful model for the study of the biologic role of CSF-1 is the
osteopetrotic (csfmop/csfmop) mutant
mouse, which is deficient in bone resorption due to a paucity of bone
osteoclasts19; has markedly reduced bone marrow
macrophages, blood monocytes, and serosal macrophages20-22;
displays defects in both male14,15 and female fertility
(reviewed in Pollard and Stanley13 and Pollard et
al23); and exhibits abnormalities in neural
processing.24 These abnormalities are the result of a null
mutation in the CSF-1 gene caused by a single thymidine insertion in
exon 4 leading to a frame-shift at base pair 262 with translational
termination 21 bp downstream.25 The resultant 63 amino acid
truncated translation product is totally devoid of biological
activity.21,25 The
csfmop/csfmop mouse has been extremely
helpful in defining mononuclear phagocyte populations that are
dependent on CSF-1 for their development. Administration of CSF-1 to
newborn csfmop/csfmop mice cured their
osteopetrosis and substantially corrected the deficiencies in
osteoclasts, bone marrow cellularity, and blood monocytes, although
having little effect on resident macrophages at serosal
surfaces.16,26-28 These observations suggest that
dependence on circulating CSF-1 for development is limited to specific
macrophage populations. In fact, a detailed analysis of the postnatal
development of various tissue macrophage populations in
csfmop/csfmop mice and their normal
littermates indicates that distinct populations may be subject to
complete dependence on, partial dependence on, and complete
independence of CSF-1 for their development and
maintenance.16 In general, macrophages in tissues, which
undergo significant remodelling and morphogenesis during the perinatal
period, seemed most dependent on CSF-1 and failed to develop in
csfmop/csfmop mice. These cells are
thought to act like osteoclasts as scavengers or produce trophic
factors in these tissues, such as the dermis and synovial membranes,
which are hypoplastic and hypotrophic in mutant
csfmop/csfmop mice,16 and
testes, in which the function of closely associated Leydig cells is
affected in csfmop/csfmop
mice.14,15 In contrast, the appearance of macrophages,
which are important in immunologic and inflammatory responses, in the epidermis (Langerhans cells), thymus, and lymph node is independent of
CSF-1.16,29-31 Because
csfmop/csfmop mice are totally devoid
of CSF-1, they are well suited to study the role of this growth factor
in the appearance of macrophages during perinatal development.
In the present study, we demonstrate that maternal CSF-1 crosses the
placenta and that CSF-1 is required for the perinatal development of
most macrophages in liver, spleen, kidney, and lung. We also show that
circulating maternal CSF-1 can compensate for the absence of
embryonic/fetal CSF-1 in csfmop/csfmop
fetuses and regulate normal macrophage development in liver but not
spleen, kidney, or lung.
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MATERIALS AND METHODS |
Animals.
C57BL/6 mice were obtained from the National Cancer Institute and
housed in the animal care facility of the Albert Einstein College of
Medicine according to established guidelines. Osteopetrotic csfmop/csfmop mice on a (C57BL/6 × C3HeB/FeJLe) F1 background (originally obtained from Jackson Laboratories, Bar Harbor, ME) and littermate controls were
bred and maintained in isolated units of the Albert Einstein College of
Medicine animal care facility as described previously.16,23 These latter mice were fed ad libitum with powdered chow and infant milk formula (Enfamil; Mead-Johnson, Evansville, IN). Animals were bred
and females were checked daily for the appearance of vaginal copulation
plugs. Gestational age was determined by counting days from the
appearance of plugs, with the day of appearance of the plug designated
as day 1. Mice were studied on gestational days 13 through 18 as well
as on postnatal days 1 through 7. Blood obtained at the time of death
was collected in capillary tubes and centrifuged, and the serum was
further handled as described below. Tissues obtained at the time of
death were handled as described below in the different experiments.
Identification of csfmop/csfmop
and +/csfmop offspring.
Matings were performed between +/csfmop females and
csfmop/csfmop males as well as the
reciprocal pairing of csfmop/csfmop
females with +/csfmop males. Distinction of
csfmop/csfmop and
+/csfmop offspring was accomplished by
radioimmunoassay (RIA) and/or polymerase chain reaction (PCR).
When RIA was used, tails from fetal and neonatal mice were removed
before tissue fixation for immunohistochemistry, homogenized, and
heated at 56°C for 30 minutes. CSF-1 levels in tail extracts were
measured by an RIA that detects biologically active mouse growth
factor32 and the presence or absence of CSF-1 was used to
distinguish +/csfmop and
csfmop/csfmop mice, respectively.
Alternatively, PCR was used to amplify the segment of exon 4 containing
the op mutation in DNA purified from the tails of fetal and neonatal
mice, as previously described.14,33 35S-dATP-labeled PCR products were run on sequencing gels,
which were dried and subjected to autoradiography. The wild-type
fragment is 59 bp in length, compared with the mutant fragment, which
is 60 bp in length due to the single thymidine insertion. Consequently, +/csfmop heterozygotes display both bands.
Transplacental transfer of CSF-1.
Pregnant C57BL/6 female mice on day 17 of gestation were injected
intravenously with approximately 1 × 106
cpm 125I-labeled CSF-1 in 0.1 mL physiologic saline. Pure
mouse L-cell CSF-1 was radioiodinated to high specific activity (~3 × 105 cpm/ng protein) and confirmed to be greater
than 95% immunoprecipitable, as previously described.34
Animals were killed 5 and 10 minutes after injection, and
125I-cpm in fetal and placental tissues was determined. In
addition, immunoprecipitable 125I-CSF-1 cpm were measured
in serum using an antibody that detects only biologically active growth
factor, correcting for counts precipitated by preimmune serum, as
previously described.34 In separate experiments, day-17
pregnant mice were injected intravenously with 12 µg of human
recombinant CSF-1, which is active on mouse CSF-1
receptors35 and was a generous gift from Chiron Corp
(Emeryville, CA), in 0.1 mL physiologic saline. Animals were killed at
1 minute, 10 minutes, 1 hour, 2 hours, or 24 hours, respectively, after CSF-1 injections and both sera and tissues were stored at
 80°C before analysis. CSF-1 was extracted from tissues by
homogenization and heating at 56°C for 30 minutes, as previously
described.32 Human CSF-1 levels were measured in tissue
extracts and sera by an RIA that detects biologically active growth
factor at concentrations  120 pg/mL, does not cross-react with mouse
CSF-1, and has interassay and intra-assay variabilities of
approximately 10%.36 CSF-1 concentrations in tissue
extracts were converted to picograms per milligram of tissue by
multiplying concentrations (in picograms per milliliter) by the volume
of RIA extraction buffer added per milligram of wet tissue weight
(milliliter per milligram).
Tissue macrophage immunohistochemistry.
Whole fetuses (13 to 18 days of gestation) and tissues excised from
neonatal (days 1 through 7)
csfmop/csfmop and
+/csfmop mice were fixed for 6 hours in
periodate-lysine-2% paraformaldehyde-0.05% glutaraldehyde, pH 7.4 (PLPG).37-39 Tissues were then dehydrated and embedded in
polyester wax. Sections of 5 µm were cut and air-dried on
gelatin-coated slides. Immunostaining was performed with the macrophage-specific rat monoclonal antibody F4/80,37 as
previously described,16 using the indirect
peroxidase-conjugated streptavidin procedure.22,38 Rat
gamma globulin (5 µg/mL) was used as a control.
At each age, at least two csfmop/csfmop
and two normal mice were examined. F4/80-positive cells were counted
under high power (40×) light microscopy with the aid of a video
monitor (Sony Trinitron, Sony Corp, Tokyo, Japan) by
scoring at least 5 to 10 fields (0.03 mm2/field) and were
expressed in cells per square millimeter. Cell densities were averages
derived from at least two animals and standard deviations for multiple
counts (n > 5) were less than 10% of the means.
Statistical analysis.
Data are expressed as the means ± standard error of the mean.
Comparisons were made using the Student's t-test and
nonparametric Tukey analysis for multiple comparisons, where
appropriate.40
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RESULTS |
Transplacental transfer of CSF-1.
To determine whether CSF-1 is transferred across the placenta, trace
amounts of 125I-CSF-1 labeled to high specific activity
were administered intravenously to pregnant female mice on day 17 of
gestation. Results of a representative experiment demonstrating
125I-cpm transferred to whole fetuses and placentas are
shown in Table 1. At 10 minutes
postinjection, approximately 5% of the injected cpm was transferred
across the placenta to the 10 fetuses in this particular experiment.
Determination of immunoprecipitable counts (due to intact
125I-CSF-1 only) showed that approximately 30% of
fetus-associated cpm were due to intact growth factor (data not shown).
This observation is not surprising in view of the fact that greater
than 80% of an intravenously injected dose of 125I-CSF-1
is normally taken up and rapidly degraded by the maternal liver,
leaving a relatively smaller amount available for transplacental transfer.34
To confirm that biologically active CSF-1 was, in fact, transferred
across the placenta, day-17 gestation pregnant female mice were
injected intravenously with human recombinant CSF-1, which also
interacts with mouse CSF-1 receptors.35 The mice were
killed at various times after injection and growth factor levels were
measured in placental and fetal tissues by a radioimmunoassay that
detects only biologically active growth factor and does not cross-react
with mouse CSF-1.36 As shown in the representative experiment in Fig 1A, biologically active
CSF-1 was already detectable at 1 minute, peaked at 10 minutes, and was
still detectable at 24 hours after injection in both fetal and
placental issues. Placental CSF-1 concentrations on a wet tissue weight
basis exceeded those in the fetus from 1 to 120 minutes by 7.4- to
13.2-fold. Interestingly, the amount of human CSF-1 transferred to each
fetus corresponds closely to the amount predicted from
125I-CSF-1 experiments. Because each fetus weighs
approximately 550 mg (Roth and Stanley, unpublished data),
the peak human CSF-1 concentration of 100 pg/mg fetal tissue
corresponds to 55 ng/fetus, which equals 0.46% of the injected dose of
12 µg. Although virtually identical to the 0.5% of the injected dose
predicted by the 125I-CSF-1 experiments (Table 1), it
represents a fourfold increase in biologically active (immunoreactive)
CSF-1, which is not surprising considering the rapid saturation of
maternal clearance and degradation of CSF-1 by the liver at elevated
circulating CSF-1 concentrations. Examination of pooled sera (Fig 1B)
again showed detectable CSF-1 at 1 minute, peak values at 10 minutes,
and levels that could still be detected at 24 hours in both maternal
and fetal sera. Although following similar kinetics, large differences
were apparent between circulating levels in the maternal and fetal
animals in this representative and other similar experiments.
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| Fig 1.
Transplacental transfer of human recombinant CSF-1.
Gestational day-17 pregnant mice were injected intravenously with human recombinant CSF-1 and killed at the indicated times postinjection, and
human CSF-1 levels were measured by a specific RIA in (A) whole fetuses
and placentas, with at least five of each measured at each time point
in this one of several representative experiments, and (B) fetal sera
pooled from individual litters at each time point and the corresponding
maternal serum, with the data representing one of several experiments.
Where indicated, placental tissue CSF-1 concentrations differed from
those measured in fetal tissues at #P = .06, $P < .01, and *P < .001, respectively. Multiple
comparisons over time showed differences, where indicated, of
aP < .025, bP < .005, and
cP < .001, respectively, versus 1 minute and
dP < .001 versus 10 minutes.
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Appearance of F4/80-positive macrophages in perinatal tissues.
To assess the role of circulating CSF-1 in the development and
maintenance of F4/80-positive macrophages, several representative tissues were studied in csfmop/csfmop
mice and their normal +/csfmop littermates born to
both phenotypically normal +/csfmop females (with
normal levels of circulating CSF-1 available for transplacental
passage) and csfmop/csfmop mothers
(with no circulating CSF-1).
Liver.
In animals born to +/csfmop mothers, F4/80-positive
cells were present at the earliest time points studied
(Fig 2A) in both csfmop/csfmop and
+/csfmop offspring. Whereas the densities of
F4/80-positive cells were somewhat diminished in
csfmop/csfmop mice compared with their
+/csfmop littermates during fetal life (gestational
days 13 and 15), there was no difference shortly after birth (postnatal
day 1). However, during the first postnatal week, F4/80-positive liver
macrophages continue to increase in the +/csfmop
mice, in contrast to the csfmop/csfmop
mice, in which they have plateaued.
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| Fig 2.
Appearance of F4/80-positive cells in fetal and neonatal
liver. Tissues were stained for F4/80, a macrophage-specific marker, as
described in the Materials and Methods and positive cells were quantified. Results are expressed as the mean ± SEM for
+/csfmop ( ) and
csfmop/csfmop ( ) offspring after
matings of (A) +/csfmop female × csfmop/csfmop male and (B)
csfmop/csfmop female × +/csfmop male mice, respectively. Where indicated,
+/csfmop differed from
csfmop/csfmop at *P < .001. Results of multiple comparisons over time, where indicated, showed
aP < .01 and bP < .001 versus day13; cP < .05, dP < .025, eP < .01, and fP < .001 versus day 14; gP < .01 and
hP < .001 versus day 15; iP < .005 and jP < .001 versus day 18;
and kP < .005 and lP < .001 versus postnatal day 1, respectively.
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As was the case for the offspring of +/csfmop
females, F4/80-positive cells were present at the earliest time points
studied in both csfmop/csfmop and
+/csfmop mice born to
csfmop/csfmop mothers (Fig 2B). Although
csfmop/csfmop mice had diminished liver
macrophage densities compared with their +/csfmop
littermates during fetal life, they also had reduced numbers shortly
after birth (postnatal day 1), in contrast to the situation observed
for the offspring of +/csfmop females (Fig 2A).
Once again, the density of F4/80-positive cells in
+/csfmop mice increased over the first week of life;
but, surprisingly, the density of these cells on postnatal day 7 in
csfmop/csfmop mice relative to that
observed in +/csfmop littermates was 70%, which
was very similar to the relationship observed in the offspring of
+/csfmop females. Interestingly,
+/csfmop fetal mice growing in
csfmop/csfmop mothers appeared to have
higher densities of F4/80-positive cells between gestational days 13 and 15 than those growing in +/csfmop mothers.
Despite the differences described above, the histologic appearance of
F4/80-positive cells in the livers of
csfmop/csfmop and
+/csfmop mice did not depend on whether they were
the offspring of +/csfmop or
csfmop/csfmop mothers (data not shown).
In both +/csfmop and
csfmop/csfmop neonatal livers,
F4/80-positive cells were dendritic and intimately associated with
islands of hematopoietic cells
(Fig 3a and b). However,
there was an obvious reduction in the density of F4/80-positive cells
in the neonatal csfmop/csfmop liver
(Fig 3b).
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| Fig 3.
F4/80-positive cells in neonatal
tissues. Neonatal tissues (postnatal day 7) were stained for the
macrophage marker, F4/80, as described in Fig 2, and counterstained
with hematoxylin in liver (a and b), spleen (c and d), kidney (e and
f), and lung (g and h) of +/csfmop (a, c, e, and
g) and csfmop/csfmop (b, d, f, and h)
newborn mice born to +/csfmop mothers. Original
magnifications are ×110 (a through f) and ×230 (g and h),
respectively. Bar = 50 µm.
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Spleen.
Although F4/80-positive cells were detectable in the spleens of both
csfmop/csfmop and
+/csfmop mice during fetal life, they were present
at extremely low levels (Fig 4). In the
+/csfmop offspring of +/csfmop
mothers, splenic F4/80-positive macrophages steadily increased in
density over the first week of life. In
csfmop/csfmop offspring, although
positive cells were present in substantial numbers, they were
significantly lower in density compared with +/csfmop littermates at birth (postnatal day 1) and
during the first week of life (Fig 4A). Despite the absence of
transplacental transfer of CSF-1, similar densities of F4/80-positive
cells were evident at birth and during the first postnatal week in
+/csfmop born to
csfmop/csfmop females (Fig 4B) as in
those born to phenotypically normal +/csfmop
females (Fig 4A). Furthermore, although F4/80-positive cells were
present in significantly lower numbers in
csfmop/csfmop mice born to
csfmop/csfmop mothers compared with
+/csfmop littermates (Fig 4B), they nevertheless
were present in similar numbers to those seen in
csfmop/csfmop born to
+/csfmop females.
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| Fig 4.
Appearance of F4/80-positive cells in fetal and neonatal
spleen. Tissues were stained, positive cells were quantified, and results are expressed for +/csfmop () and
csfmop/csfmop () offspring after
matings of (A) +/csfmop female × csfmop/csfmop male and (B)
csfmop/csfmop female × +/csfmop male mice as in Fig 2. Where indicated,
+/csfmop differed from
csfmop/csfmop at #P < .01 and
*P < .001, respectively. Results of multiple comparisons over
time, where indicated, showed aP < .025, bP < .01, and cP < .001 versus day13; dP = .08, eP < .05, fP < .025, gP < .01, hP < .005, and iP < .001 versus day 15; jP < .01 and
kP < .001 versus day 18; lP < .05, mP < .025, and nP < .001 versus postnatal day 1; and oP < .005 versus
postnatal day 4, respectively.
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On histological examination, differences were evident between
csfmop/csfmop and
+/csfmop fetal/neonatal mouse spleens independent
of the maternal genotype (Fig 3c and d and data not shown). Splenic
F4/80-positive cells were located exclusively in the red pulp and were
both stellate and rounded in +/csfmop mice (Fig
3c). In addition to the reduction in density, F4/80-positive cells in
csfmop/csfmop spleen were predominantly
rounded cells (Fig 3d).
Kidney.
During fetal life, there were minimal to no F4/80-positive cells in the
kidneys of +/csfmop mice born to either
csfmop/csfmop or +/csfmop
mothers (Fig 5). However, postnatally, the
densities were higher at birth and continued to increase over the first
week of life in these same mice (Fig 5). In
contrast, there was a virtual absence of F4/80-positive cells in
kidneys of csfmop/csfmop mice born to
both +/csfmop (Fig 5A) and
csfmop/csfmop (Fig 5B) mothers.
Consequently, there were many more F4/80-positive macrophages in
+/csfmop compared with
csfmop/csfmop kidneys throughout early
postnatal development (Fig 5).
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| Fig 5.
Appearance of F4/80-positive cells in fetal and neonatal
kidney. Tissues were stained, positive cells were quantified, and results are expressed for +/csfmop () and
csfmop/csfmop () offspring after
matings of (A) +/csfmop female × csfmop/csfmop male and (B)
csfmop/csfmop female × +/csfmop male mice as in Fig 2. Where indicated,
+/csfmop differed from
csfmop/csfmop at #P < .01 and
*P < .001, respectively. Results of multiple comparisons over
time, where indicated, showed aP = .09, bP < .05, and cP < .001 versus day14; dP < .025, eP < .005, and fP < .001 versus day 15;
gP < .05, hP < .025, and
iP < .001 versus day 18; jP < .05, and kP < .001 verus postnatal day 1; and
lP < .005 versus postnatal day 4, respectively.
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| Fig 6.
Appearance of F4/80-positive cells in fetal and neonatal
lung. Tissues were stained, positive cells were quantified, and results are expressed for +/csfmop () and
csfmop/csfmop () after following
matings of (A) +/csfmop female × csfmop/csfmop male and (B)
csfmop/csfmop female × +/csfmop male mice as in Fig 2. Because of technical
difficulties, Fig 6b includes only data through postnatal
day 1. Where indicated, +/csfmop differed from
csfmop/csfmop at *P < .001. Results of multiple comparisons over time, where indicated, showed aP = .07 and
bP < .001 versus day13;
cP < .025 versus day 14; dP < .005 and eP < .001 versus day 15;
fP < .05 and gP < .001 verus day 18; and hP < .025 versus postnatal day
4, respectively.
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Histologic examination showed no obvious effect of maternal
genotype on morphology of renal F4/80-positive macrophages, which for
the most part are located in the renal medulla and primarily line
collecting tubules in +/csfmop mice (Fig 3e). In
csfmop/csfmop mice, they are virtually
absent (Fig 3f).
Lung.
Similar to the kidney, there was a virtual absence of F4/80-positive
cells in the lung of csfmop/csfmop mice
born to either +/csfmop (Fig 6A) or
csfmop/csfmop (Fig 6B) mothers. In
contrast, F4/80-positive cells were present during fetal life in
+/csfmop mice born to either maternal genotype (Fig
6A and B). Whereas there was a clear increase in lung macrophages
during the first postnatal week in +/csfmop
offspring (Fig 6A), technical difficulties in preserving lung macrophages during the fixation process in
csfmop/csfmop offspring beyond 1 day of
age made it impossible to draw any conclusions regarding later time
points (Fig 6B). Nevertheless, the differences between mutant mice and
their normal littermates were readily detectable shortly after birth.
Examination of microscopic sections showed that, in developing
csfmop/csfmop lungs (Fig 3h), there
were marked reductions in both interstitial and alveolar macrophages,
which were present in the corresponding developing
+/csfmop tissue (Fig 3g).
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DISCUSSION |
Growth factors that are capable of crossing the placenta during
gestation, as we have demonstrated for CSF-1 in this report, have the
potential to exert significant effects on developing fetal cell
populations. Homologous CSF-1, radiolabeled to high specific activity
and administered to pregnant mice in trace quantities that do not
disturb steady-state concentrations, is detectable in fetal tissues and
serum shortly after injection. The presence of radioactivity in fetal
tissues and serum associated not only with intact but also degraded
CSF-1 is consistent with previous data,34 in which we
demonstrated that a large proportion of injected labeled CSF-1 is taken
up rapidly by the liver, in the present case that of the pregnant
mother, and rapidly degraded in that organ and released back into the
bloodstream. Furthermore, use of pharmacologic doses of heterologous
(human) CSF-1, which is capable of interacting with mouse CSF-1
receptors35 and is detectable by an RIA that specifically
detects biologically active human CSF-1 and not mouse
CSF-1,36 confirms the transplacental passage of this
hematopoietic growth factor, which can then potentially interact with
CSF-1 receptor bearing fetal cells. These data are similar to those
reported for G-CSF, which is detectable in the fetal circulation within
minutes after maternal administration.18 Furthermore, as in
the case of CSF-1 in this report, fetal levels of G-CSF were far lower
than maternal levels. Nevertheless, significant effects were still
observed on fetal myelopoiesis.18
The csfmop/csfmop mouse is well suited
for the study of the requirements of different tissues for circulating
versus locally produced CSF-1 for the establishment of mononuclear
phagocyte populations.16,28 Although the presence of
biologically active concentrations of both the proteoglycan and
glycoprotein forms of CSF-1 in the circulation11 and of
biologically active cell surface-associated CSF-1
glycoprotein41 have been described in normal animals, none
of these species can be detected in
csfmop/csfmop mice. Previous data on
the postnatal administration of exogenous CSF-1 to
csfmop/csfmop mice suggest that the
response of mononuclear phagocytes to circulating growth factor is
tissue specific, with complete restoration in tissues such as liver,
spleen, and kidney; partial restoration in gastrointestinal tissues,
bladder, salivary glands, and dermis; and no effect in muscle,
synovium, and tendon.16 Thus, although in some tissues
regulation of mononuclear phagocyte production may be mediated by
circulating CSF-1, in others, it may require growth factor locally, as
either the cell surface form or the sequestered proteoglycan.
In the current report, differences between +/csfmop
and csfmop/csfmop animals in the
appearance of F4/80-positive mononuclear phagocytes suggest that CSF-1
is responsible for the perinatal development of a very large proportion
of mononuclear phagocytes in the liver, spleen, kidney, and lung.
Furthermore, the ability of maternally derived circulating CSF-1 to
compensate for the total absence of local CSF-1 production during fetal
and early neonatal life is tissue specific. This phenomenon is most
striking in the liver, in which the densities of F4/80-positive cells
are equivalent shortly after birth in +/csfmop and
csfmop/csfmop mice born to
+/csfmop mothers, but not in similar littermate
pairs born to csfmop/csfmop mothers.
Thus, circulating CSF-1, which is present in normal concentrations in
phenotypically normal +/csfmop females, appears to
be capable of crossing the placenta and promoting the normal appearance
of hepatic mononuclear phagocytes in their csfmop/csfmop offspring. This
observation is not so surprising, because nearly 80% of hepatic blood
flow in the fetus consists of oxygen-enriched blood derived directly
from the umbilical vein42 (reviewed in Polin and
Fox43). Therefore, mononuclear phagocyte progenitors in the
fetal liver can be expected to be exposed to higher concentrations of
maternally derived circulating CSF-1 than any other tissue.
In contrast to the liver, splenic F4/80-positive cells were reduced by
similar proportions in csfmop/csfmop
compared with +/csfmop littermates, regardless of
whether they were born to csfmop/csfmop
or +/csfmop mothers, reflecting the failure of
transplacental CSF-1 to significantly alter F4/80-positive cell
density. Whereas the subnormal but substantial development of
mononuclear phagocytes in the spleen is consistent with the partial
ability of circulating CSF-1 to restore this population,16
the near total absence of F4/80-positive cells in the developing
perinatal csfmop/csfmop kidney and lung
intimate that local CSF-1 production is required for their development.
Unlike postnatal restoration of circulating CSF-1 concentrations
through exogenous administration, which readily increased the densities
of splenic and kidney F4/80-positive cells to normal
levels,16 circulating maternal CSF-1 was unable to effect
similar results. Unlike the liver, blood flow to the spleen in utero is
derived from the descending aorta distal to the ductus arteriosus and
thus consists of a mixture of relatively enriched umbilical vein/ductus
venosus and depleted fetal venous return blood.43
Consequently, the spleen may be exposed to CSF-1 concentrations substantially lower than those achieved in the experiments described by
Cecchini et al.16 In fact, those investigators demonstrate a requirement for the establishment of supraphysiologic concentrations of growth factor to fully restore the
csfmop/csfmop spleen. However, this
requirement was not the case in the kidney, which was extremely
responsive to restoration of its mononuclear phagocyte populations by
lower doses of exogenous CSF-1 in their experiments.16 The
apparent discrepancy between this observation and the failure of
maternally derived circulating CSF-1 to promote macrophage development
in this tissue during fetal life may actually be attributed to
alterations in renal blood flow that occur postnatally. During fetal
life, not only does the kidney receive its blood supply via the
postductal descending aorta, but it also is characterized by markedly
elevated vascular resistance. As a result, the kidney receives
approximately 2% of cardiac output in utero compared with the 15% to
18% it receives postnatally.43 Thus, under these conditions, perinatal kidney macrophages appear to depend on local production of CSF-1 for their development. Similar to the kidney, the
lung receives a far smaller proportion of cardiac output during fetal
life (5% to 10%) compared with postnatal life. Whereas no data exist
regarding the responsiveness of lung mononuclear phagocytes to
postnatal administration of exogenous CSF-1, our data clearly indicate
that, in the absence of local CSF-1 production, the available maternally derived circulating CSF-1 fails to foster the development of
either interstitial or alveolar macrophages in this tissue. Finally,
with the emergence of the fetal mononuclear phagocyte population in the
liver, it would be expected that these cells would play an increasing
role in clearance of growth factor from the circulation as it makes its
first pass through the fetal circulation, thereby reducing the amount
of growth factor available to other fetal tissues, including the
spleen, kidney, and lung.
Interestingly, our data on the density of F4/80-positive mononuclear
phagocytes in the liver, kidney, and spleen shortly after birth
(postnatal day 1) agree very well with those of Cecchini et
al16 (postnatal day 2). Based on a combination of our data and that of those investigators, it is apparent that, in these tissues,
the density of mononuclear phagocytes peaks at 1 week of age (current
report) and that, by 2 weeks,16 it has already begun to
decrease.
Although the current data point to the importance of CSF-1 in
mononuclear phagocyte development, they also suggest that other factors
are involved. For example, the data on both hepatic and splenic
mononuclear phagocytes, demonstrating the appearance of substantial
numbers of cells despite the total absence of CSF-1, are compatible
with the involvement of other hematopoietic growth factors or a growth
factor-independent pathway for mononuclear phagocyte development.
However, from the experiments described here, it is impossible to
determine whether such pathways are compensatory, depending on factors
other than CSF-1 that operate exclusively in its absence, or normal, in
that they operate even when CSF-1 is available in
+/csfmop mice. The degree to which such pathways
are operative may be responsible in part for the increased density of
F4/80-positive cells in fetal liver of +/csfmop
mice developing in csfmop/csfmop
mothers relative to those developing in +/csfmop
mothers as well as the increase in F4/80-positive cells seen in
postnatal livers of csfmop/csfmop mice
born to csfmop/csfmop mothers allowing
them to achieve a similar percentage of positive cells relative to
+/csfmop littermates at 1 week of age. Previous
data suggest that this cell compartment develops under the influence of
granulocyte-macrophage colony-stimulating factor (GM-CSF) and
interleukin-3 (IL-3) in addition to CSF-1.44 Although
exogenous GM-CSF corrects many of the macrophage deficiencies in
csfmop/csfmop mice, it is still unable
to induce osteoclast production and thereby correct the underlying
osteopetrosis.45 Furthermore, mice that are doubly
deficient in GM-CSF and CSF-1 still have substantial numbers of
mononuclear phagocytes, suggesting that a significant contribution to
production of these cells may derive from other factors, such as IL-3
and IL-2/IL-4, the inducible production of which is elevated in
csfmop/csfmop mice.21,46
Thus, it appears that mononuclear phagocyte production will occur to
the greatest degree in the presence of CSF-1 even in the absence of
both GM-CSF and IL-3 activity, as demonstrated in mice with inactive
receptors for GM-CSF and IL-5 (due to a mutation in their common
 C chain) and with a targeted inactivating mutation in
the IL-3 gene, which have normal numbers of mononuclear phagocytes.47 However, when CSF-1 is absent, even when
GM-CSF and IL-3 are both present, as is the case in the
csfmop/csfmop mouse, mononuclear
phagocyte production occurs, but to a much reduced level. Finally, when
IL-3 alone of the three mononuclear phagocyte growth factors is
present, as in the case of the mouse doubly deficient for CSF-1 and
GM-CSF, development of this cell compartment occurs to even a lesser
degree46 and possibly under the additional influence of as
yet undetermined additional factors. It is possible that each factor is
capable of stimulating a particular subpopulation of target cells and
may take on different relative importance in the face of deficiencies
of the other respective mononuclear phagocyte growth factors. Specific
in situ data may be helpful in elucidating the contributions of these
other potential factors in the absence of local CSF-1 expression or the
total absence of all CSF-1 expression, respectively.
| |
FOOTNOTES |
Submitted September 8, 1997;
accepted January 12, 1998.
Supported by National Institutes of Health Grant No. CA32551, by a
grant from the Lucille P. Markey Charitable Trust (to E.R.S.), and by
Albert Einstein Core Cancer Grant No. P30-CA13330.
Address reprint requests to Philip Roth, MD, PhD, Division of
Neonatology-4 East, Staten Island University Hospital, 475 Seaview Ave,
Staten Island, NY 10305.
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.
| |
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Abstract
Introduction
Abstract