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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 11 (December 1), 1998:
pp. 4119-4127
Functional Analysis of Mature Hematopoietic Cells From Mice Lacking
the  c Chain of the Granulocyte-Macrophage Colony-Stimulating Factor
Receptor
By
C.L. Scott,
D.A. Hughes,
D. Cary,
N.A. Nicola,
C.G. Begley, and
L. Robb
From The Walter and Eliza Hall Institute of Medical Research, The
Cooperative Research Centre for Cellular Growth Factors, PO Royal
Melbourne Hospital, Victoria, Australia; Rotary Bone Marrow Research
Laboratories Factors, PO Royal Melbourne Hospital, Victoria, Australia;
and the Sir William Dunn School of Pathology, Oxford, UK.
 |
ABSTRACT |
Mice with a null mutation of the  c chain of the
granulocyte-macrophage colony-stimulating factor (GM-CSF),
interleukin-3 (IL-3), and IL-5 receptors (c-null mice) develop an
alveolar proteinosis-like lung disease. The pathogenesis of this
disease is uncertain and, although a defect in alveolar macrophage
function has been postulated, no previous analysis of mature
hematopoietic cells in mice with alveolar proteinosis has been
reported. Therefore, we undertook a functional analysis of the mature
hematopoietic cell compartment in c-null mice. In addition, we
reexamined the roles of the GM-CSF receptor  chain and the c
chain in signaling by GM-CSF. Neutrophils and macrophages from
c-null mice were capable of normal survival and phagocytosis in the
absence of stimulus and of similar levels of nitric oxide production in
response to interferon- and lipopolysaccharide. GM-CSF-mediated
augmentation of survival, phagocytosis, and hydrogen-ion production
were absent in neutrophils from c-null mice. Interestingly, we were
unable to show any ability of the GM-CSF receptor -chain alone to
mediate glucose transport in these cells. In keeping with the c-null mice lung pathology, examination of lavage fluid from the lungs of
c-null mice showed increased cellularity. This was caused by an
increase in the number of lymphocytes, neutrophils, and macrophages.
Large foamy cells in the lavage fluid from c-null mice were
identified as macrophages using immunohistochemistry. Functional
analysis showed that these c-null alveolar macrophages were capable
of phagocytosis but uptake of colloidal carbon and cellular adhesion
were reduced. In summary, mature hematopoietic cells with a null
mutation of the c receptor were unable to perform GM-CSF-mediated
hematopoietic cell functions including glucose transport, but responded
normally to a range of other ligands.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
THE HEMATOPOIETIC cytokines
granulocyte-macrophage colony-stimulating factor (GM-CSF),
interleukin-3 (IL-3), and IL-5 stimulate proliferation and
differentiation of hematopoietic progenitor cells. These cytokines also
play a role in mature hematopoietic cell functions, including mature
cell survival and phagocytosis.1 They act on target cells
via a receptor complex composed of an alpha () and a beta ()
chain. The subunit for each cytokine is unique and binds the ligand
with low affinity, while the chain converts the interaction with
ligand to one of high affinity and is required for intracellular
signaling. In both mice and humans a common subunit is used by
GM-CSF, IL-3, and IL-5 receptors and is known as the common beta chain
(c).2-4 In the mouse, an additional IL-3-specific chain exists, known as IL-3,5 which is used
in preference to c for signaling by IL-3.6
The requirement for the c chain in mediating the actions of GM-CSF
has been formally demonstrated by the creation of c-null mice,7,8 and mutational analysis of c has shown that
multiple different signaling pathways are initiated from distinct
regions of the cytoplasmic domain of the c receptor.9,10
In contrast to the c chain, the only signaling function so far
attributed to the GM-CSF receptor chain (GM-CSF R) alone is
GM-CSF-mediated glucose transport.11,12 This function was
shown in Xenopus oocytes transfected with the human GM-CSF R
and it was postulated that such chain-mediated involvement in
glucose transport may be associated with prolongation of cell survival.
Mice with a null mutation for the c chain (c-null)7,8
have normal baseline hematopoiesis except for a low basal circulating eosinophil level. The eosinopenia is similar to that observed in mice
with a null mutation of the IL-5 gene13 or the IL-5 receptor chain gene.14 Like mice with a null mutation
of the gene for GM-CSF,15,16 the c-null mice have lung
disease, the pathology of which is reminiscent of the human disease
pulmonary alveolar proteinosis (PAP). The alveolar spaces of the lungs
progressively accumulate surfactant and GM-CSF null mice have been
shown to have markedly reduced alveolar clearance and catabolism of
surfactant protein A (SP-A).17 This pulmonary lesion,
thought to be due at least in part to defective function of alveolar
macrophages,18 can be cured by bone marrow
transplantation19 and, in GM-CSF null mice, by expression
of a GM-CSF transgene in type II pneumocytes.20
To date, analysis of the functional activity of mature hematopoietic
cells in mice lacking the c receptor has been limited to an
examination of the resident and elicited peritoneal
populations.21 A more complete examination of mature
hematopoietic cell function in these mice is required to explore the
pathogenesis of the PAP-like disease.
In this study we used the c-null mice to reexamine the role of the
GM-CSF R in influencing cell survival. We also report experiments in
which we assessed the function of granulocytes and macrophages from
these mice using a variety of parameters including cell survival,
phagocytosis, cell adhesion, and nitric oxide production.
| |
MATERIALS AND METHODS |
Mice.
129/Sv c-null7 or wild-type (WT) control mice were used
for the majority of experiments. In some experiments C57BL/6 x 129/Sv
mice were used. No significant differences were seen comparing the two
strains. Mice were 6 to 12 weeks old for neutrophil function experiments and were 8 to 12 weeks old for peritoneal and alveolar macrophage studies. As indicated, selected experiments were performed on 2-week-old mice.
Cytokines.
Lyophilized recombinant human G-CSF (rhG-CSF; AMRAD
Melbourne, Australia) was dissolved in sterile water and diluted in
sterile normal saline for injection with 5% bovine calf serum (BCS;
Hyclone, Logan, UT). Recombinant murine (rm) GM-CSF was produced in
Escherichia coli or Saccharomyces cerevisiae and
purified by conventional chromatography (specific activity
108 U/mg). rmIL-3 was from Peprotech (Rocky Hill, NJ).
Interferon- (IFN-) was from Genzyme Corp (Cambridge, MA). E
coli lipopolysaccharide was from Sigma Chemical Co (St Louis, MO).
G-CSF-elicited neutrophil preparation.
Mice were injected with 2.5 µg of rhG-CSF twice daily at 8 AM and 7 PM for 5 days. All analyses were commenced at 9 AM on the morning
following the last evening injection. Mice were anesthetized and
retro-orbital plexus blood and axillary vessel blood was collected. White blood cell counts and differential cell counts were performed as
previously described.22 Blood obtained from the axilla was subjected to hypotonic lysis for 10 minutes followed by washing in
Dulbecco's modified Eagle's medium with 10% BCS (10% DME BCS). After this, 200 cell differential counts were performed on
cytocentrifuge preparations stained with May-Grünwald-Giemsa. The
purity of neutrophils in the preparations was similar for both WT and
c-null mice (WT 87% ± 4% n = 5 mice, c-null 83% ± 6%,
n = 5 mice). Radioiodination of rmGM-CSF, binding assays, and Scatchard
analysis were performed as described.23
Neutrophil survival assay.
Survival assays were performed by culturing neutrophils in 60-well Lux
5260 microtiter plates (Nunc, Naperville, IL) according to a method
previously described.24 Each well contained 10 µL 10%
DME BCS and 200 cells. Serial dilutions of 5 µL of
cytokine-containing preparations were added to quadruplicate
microwells before the addition of target cells. Cultures were
incubated at 37°C in a fully humidified atmosphere of 10%
CO2 in air. Cultures were examined in replicate wells at
intervals and cells that were highly refractile with a clearly defined
cell border were counted as viable using an inverted microscope at
200× magnification.
Neutrophil phagocytosis assay.
Cells were plated at a cell density of 105 cells/mL in
24-well plates (Falcon, Becton Dickinson, Lincoln Park,
NJ) in 10% DME BCS. 106 latex beads
conjugated with a fluorescein dye (Flouresbrite carboxy YG
microspheres; Polysciences Inc, Northampton, PA) were then added, with
50 µL of carrier with or without rmGM-CSF. After 6 hours of
incubation at 37°C cells were detached with 100 µL of 0.1 mol/L
EDTA. Cytocentrifugation of 200 µL of cells and beads was performed.
After staining with May-Grünwald-Giemsa, enumeration of beads per
neutrophil was performed for 200 consecutive neutrophils. The Weighted
Phagocytic Index (WPI)25 was calculated by multiplying the
number of neutrophils with 1, 2 to 3, 4, or  5 associated beads by 1, 2, 3, and 4, respectively, and dividing the total score by the number
of neutrophils examined.
Cytosensor analysis.
105 neutrophils were analyzed per point on the Cytosensor
Microphysiometer (Molecular Devices Corp, Sunnyvale, CA) according to a
method previously described.26 The extracellular
acidification rate (ECAR) was measured in microvolts per
second and normalized in running buffer (DME without
bicarbonate buffering, 0.1% bovine serum albumin [BSA], endotoxin
free) before exposure of the cells to ligand. The change in ECAR versus
time was documented over 1 to 2 hours after exposure to cytokine for 6 minutes.
Peritoneal and bronchoalveolar lavage.
Resident peritoneal cells were washed from the peritoneal cavities of
sacrificed mice by injecting 5 mL phosphate-buffered saline (PBS),
gently massaging the abdominal wall, then aspirating the lavage, first
with a syringe and 18-gauge needle, then with a glass pasteur pipette
inserted through the peritoneum, and again after exposure of the
peritoneal cavity. Alveolar cells were lavaged from murine
lungs after peritoneal lavage. The trachea was exposed transthoracically and a piece of fine bore peristaltic tubing inserted
to just above the carina and secured. The lungs were then lavaged with
10 × 1-mL aliquots of PBS with 0.5 mmol/L EDTA. The percentage of
macrophages was determined by staining with crystal violet (to examine
nuclear morphology) and cell counts were performed with eosin (to
assess cell viability). Cells were washed and suspended at
106 per mL in RPMI 1640 with 10 mmol/L HEPES pH 7.3, and
10% BCS (heat-inactivated at 56°C for 60 minutes). Cells, 2 × 104, were cytocentrifuged and stained with
May-Grünwald-Giemsa or stored at  70°C. Frozen cell
preparations were brought slowly to room temperature in a mixture of
acetone and methanol (50:50 vol/vol).
Immunohistochemistry.
Cytospin preparations were washed in PBS containing 0.1% vol/vol
Triton X-100 (BDH-Merck, Darmstadt, Germany) and treated with a solution of 2% normal rabbit serum, 2% normal mouse serum, and
2% BCS in PBS for 30 minutes. Cytospin preparations were incubated for
90 minutes in monoclonal antibody (MoAb), PBS, or isotype-matched control MoAb, and endogenous peroxidase activity was blocked as previously described.27 The cells were incubated with
primary peroxidase conjugated secondary antibody for 45 minutes,
washed, and then incubated with 0.5 mg/mL diaminobenzidine
(Polysciences Inc) and 0.024% H2O2 in 10 mmol/L imidazole, pH 7.4. Sections were counterstained in Mayers
hematoxylin and mounted using DePeX (BDH, Poole, Dorset, UK).
Antibodies.
The following MoAbs raised in rats and directed against mouse antigens
were used as hybridoma supernatants for immunohistochemistry: F4/8028 recognizes a 160-kD antigen of unknown
function; 5C629 was used to recognize the 2 integrin,
complement receptor type 3 (CR3); IC2 recognizes
sialoadhesin30; FA-1131 recognizes the major
wheat germ agglutinin-binding lectin of murine macrophages, macrosialin; 2F8 recognizes the murine macrophage scavenger receptor (mMSR)32; 8D2 recognizes the hyaluronan receptor
CD44/pgp-133; and TIB 120, which recognizes class II major
histocompatibility complex (MHC).34
Adhesion assays.
Cells were resuspended in RPMI 1640 with 10% BCS, plated at a density
of 3 × 105 macrophages per well in flat-bottom tissue
culture plastic (TCP) 96-well plates, and incubated with MoAb (5 µg/mL) and/or chelator (5 mmol/L EDTA) as previously
described.35 In some assays cells were plated in 10-µL
volumes in microtiter plates and adherent cells were enumerated under
phase-contrast microscopy, counting selected regions of a grid (average
number of cells per area) for a minimum of three independent wells.
Macrophage phagocytosis assays.
For colloidal carbon phagocytosis, cells were cultured at a density of
105 cells per well in flat-bottom TCP 96-well plates in
RPMI 1640 with 10% BCS and allowed to adhere for 90 minutes at
37°C. After one wash, 5 µL of 5% colloidal carbon (Pelikan Ink,
Gunther Wagner, Germany) in combination with MoAb and/or
chelator was added to test wells and incubated for 1 hour at 37°C.
After washing gently three times in PBS, cells were viewed by
phase-contrast microscopy and the percentage of phagocytic cells was
enumerated. For latex bead phagocytosis, cells were cultured at 2 × 104 cells per well in flat-bottom 96-well TCP and
adhered overnight at 37°C. Latex beads, 2 × 105,
were added and incubated for 12 hours at 37°C. After one gentle wash in PBS adherent cells were fixed using 100 µL 1% glutaraldehyde and the number of beads per cell determined for 100 consecutive cells
using phase-contrast microscopy. For sheep red blood cell (RBC)
phagocytosis, cells were cultured as for latex bead phagocytosis. Thirty microliters of sheep RBCs (prepared by incubation with or
without anti-sheep RBC serum and suspended at 2 × 107
cells/mL) was added and incubated for 2 hours (in selected experiments for 1/2, 1, or 2 hours) at either 4°C or 37°C.
After gentle washing of all wells and hypotonic lysis of selected
wells, adherent cells were fixed, the number of sheep RBCs per
macrophage enumerated for 100 consecutive macrophages, and the WPI
calculated as for neutrophil latex bead phagocytosis. For endocytosis
of acetylated low-density lipoprotein (AcLDL) labeled with
1,1 -dioctadecyl-1-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI; PerImmune, Inc, Rockville,
MD),32 cells were adhered overnight on glass
coverslips, washed three times, and incubated with 10 µg/mL DiI-AcLDL
for 30 minutes at 37°C. Coverslips were then washed three times for
5 minutes each with PBS and mounted on glass slides. Uptake was
detected by confocal microscopy using rhodamine excitation and emission
filters. Where phagocytosis studies involved c-null alveolar
macrophages, all test samples were washed very gently to minimize cell
loss caused by the poor adhesion of these cells to TCP.
Nitric oxide assays.
105 cells per well were plated in U-bottom 96-well TCP in
RPMI 1640 with 10% BCS and allowed to adhere for 3 hours at 37°C. After washing, medium containing saline with or without IFN- (102 U/mL final concentration) and lipopolysaccharide (LPS)
(0.1 µg/mL) was added and cells were incubated for 48 hours at
37°C. Culture supernatants were assayed for nitrite
content.36 Fifty microliters was reacted for 10 minutes at
room temperature with an equal volume of the colorimetric Griess
reagent [0.5% sulfanilimide and 0.05%N-(1-napthyl)ethylenediamine dihydrochloride in phosphoric acid]. The absorbance at 540 nm was
measured and the nitrite content was quantified by comparison with a
standard curve generated with NaNO2 in the range of 0 to 100 µmol/L. Nitric oxide (NO) production by resident peritoneal macrophages and peritoneal macrophages obtained 4 days after
intraperitoneal injection of 2 mL of 3% thioglycollate broth was
examined. NO produced in response to Listeria monocytogenes was
assessed as follows: peritoneal cells were cultured at a concentration
of 2 × 106 cells/mL in 24-well TCP (Nunc, Roskilde,
Denmark) in the presence or absence of 2 × 108
heat-killed Listeria organisms/mL for 24 hours.37
Culture supernatant was assayed for NO production as described above.
Glucose uptake assay.
Uptake of 2-deoxy-D-glucose (2-DOG) was performed as previously
described.11,38,39 Bone marrow cells were washed three times in a serum-free, glucose-free buffer (15 mmol/L HEPES/135 mmol/L
NaCl/5 mmol/L KCl/1.8 mmol/L CaCl2/0.8 mmol/L
MgCl2 pH 7.4). Cells, 2 × 106, were
incubated in 1-mL cultures with 5% serum/saline with or without
cytokine and 2-DOG (0.01 mmol/L final concentration; Sigma) for 50 minutes. 2-deoxy-D-(1,2-3H)glucose (3H-2-DOG, 1 µCi; Amersham, Buckinghamshire, UK) was then added to
each culture for exactly 10 minutes. Three 10-mL washes of ice-cold 5 mmol/L D-glucose were performed, followed by solubilization with 1%
sodium dodecyl sulfate (SDS) and addition to 2 mL of aqueous scintillant (Starscint; Packard, Groningen, The Netherlands). Incorporated radioactivity was then determined. Cytochalasin B or E (10 µmol/L final concentration; Sigma) were added to selected cultures 10 minutes before the addition of 3H-2-DOG.
| |
RESULTS |
Neutrophil function in c-null mice.
To obtain sufficient neutrophils for this study, WT and c-null mice
were injected with 2.5 µg rhG-CSF subcutaneously twice daily for 5 days. Both c-null and WT mice responded with a comparable leukocytosis (c-null 53.2 ± 1.2 × 106/mL
[mean ± SD] and WT 55.6 ± 5.7 × 106/mL, n = 3 to 4 mice per group and 82% and 81% neutrophilia, respectively) and
increase in circulating progenitor cells (c-null 7,700 ± 775 cells/mL, and WT 7,000 ± 556 cells/mL).22
There was also a similar increase in splenic weight, cellularity, and
number of splenic progenitor cells in both groups. As shown in
Fig 1, top panel, G-CSF-elicited blood
neutrophils from WT mice bound 125I-GM-CSF with a single
class of high-affinity binding (kd = 500 pmol/L, n = 1,700 receptors). Cells from c-null mice (Fig 1, bottom panel) bound
GM-CSF with only low affinity (kd 4.2 nmol/L, n = 2,100 receptors per
cell) as was previously reported for bone marrow cells from untreated
c-null mice.6,7 Thus, c-null mice responded normally
to administration of G-CSF and in vivo exposure to G-CSF did not alter
the GM-CSF binding characteristics of elicited cells.
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| Fig 1.
Binding of GM-CSF to G-CSF-elicited blood neutrophils
from c-null and WT mice. Cells were incubated with
125I-GM-CSF for 3 hours at 4°C and assayed for binding.
Data were corrected for nonspecific binding and are shown plotted in
the Scatchard coordinate system ( ). (Top) Neutrophils (87% pure)
from WT mice bound with a single class of high-affinity binding: kd = 500 pmol/L, n = 1,700 per cell. (Bottom) Neutrophils (83% pure) from
c-null mice bound GM-CSF only with low affinity: kd = 4.2 nmol/L,
n = 2,100 per cell.
|
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We examined the survival of murine G-CSF-elicited neutrophils in
microwell cultures. Survival of neutrophils from WT mice was prolonged
by the addition of GM-CSF, G-CSF, or IL-3
(Fig 2A and B; data for G-CSF not shown).
Neutrophils from c-null animals exhibited equivalent baseline
survival in saline and prolongation of survival in the presence of IL-3
and G-CSF but not GM-CSF. Increasing the concentration of GM-CSF did
not result in prolongation of survival of c-null neutrophils (Fig
2C). This was observed even when the concentration of GM-CSF was
increased up to 106 U/mL, equivalent to a calculated
receptor occupancy of 99% for the low-affinity GM-CSF R. For WT
neutrophils, the concentration of GM-CSF at which 50% neutrophil
survival was seen was 100 U/mL. This was higher than previously
observed for human neutrophils24 and may be caused by the
in vivo exposure to G-CSF. A plateau-effect in prolongation of cell
survival was seen at concentrations greater than 500 U/mL of GM-CSF for
the WT cells. Results obtained for incubation of cells in IL-3 were
similar for both WT and c-null cells, with an IL-3 dose-response
relationship demonstrable for both (data not shown). Thus, there was a
lack of survival response of c-null neutrophils to GM-CSF, while
responsiveness to IL-3 was unaltered compared with WT cells. No
evidence was seen for acceleration of cell death in the absence of c
(timepoints: 3 to 16 hours, n = 2 mice; >16 hours, n = 9 mice).
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| Fig 2.
The survival of G-CSF elicited peripheral blood
neutrophils (87% ± 2% pure) from WT () and c-null ( ) mice
in in vitro cultures containing (A) 3 × 104 U/mL mGM-CSF
(continuous line) or saline (broken line). (B) 4 × 103U/mL mIL-3 (continuous line) or saline (broken line).
Cells were placed in microtiter trays (200 cells per well) and the
number of viable cells was counted in four replicate wells at various
time points thereafter (hours). (C) Dose-response relationship for
neutrophils from WT () and c-null () mice in mGM-CSF, starting
concentration 3 × 104U/mL with fivefold dilutions.
Results are the means of four wells. Error bars represent SD. One of
six similar experiments.
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The ability of neutrophils from c-null and WT animals to phagocytose
latex beads was examined. With WT neutrophils, baseline phagocytic
activity increased after incubation in GM-CSF (fold change 3.9 ± 1.2, n = 9 mice, Fig 3). In the c-null
mice, baseline levels of neutrophil phagocytosis were normal. However,
no increase in phagocytic ability was seen in c-null neutrophils
incubated in GM-CSF (fold change 0.9 ± 0.2, n = 9 mice). The lack
of response in terms of phagocytic activity in c-null cells was also
evident with higher doses of GM-CSF (up to 50,000 U/mL, equivalent to a
calculated occupancy of 85% for the GM-CSF R; data not shown).
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| Fig 3.
Phagocytosis of latex beads by G-CSF-elicited peripheral
blood neutrophils from WT and c-null mice. 105
neutrophils were incubated in 1-mL cultures with 106 beads
and either carrier or mGM-CSF, 4,000 U/mL, for 6 hours. Cytospin
preparations were stained and 200 consecutive neutrophils were scored
for number of cell-associated beads. The WPI was derived for each mouse
by multiplying the number of neutrophils with 1, 2 to 3, 4, or 5
associated beads by 1, 2, 3, or 4, respectively, and dividing the total
score by the number of neutrophils examined. Results for two mice of
each genotype are shown and similar results were obtained in a further
seven mice.
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When cultured in vitro, cells excrete acidic metabolites into the
culture medium. The production of acid metabolites can be quantitated
and may increase in response to certain stimuli, such as exposure to
cytokines.40 Figure 4 shows the
ECAR after cytokine treatment of G-CSF-elicited neutrophils. GM-CSF
induced an increase in the ECAR of cells from WT animals, with maximal
levels achieved within 6 minutes. This gradually returned to baseline
over several hours. This increase was absent when cells from c-null
animals were exposed to GM-CSF. The lack of response was maintained at concentrations of GM-CSF up to 106 U/mL. However, both WT
and c-null cells responded to IL-3 to a comparable degree.
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| Fig 4.
Acidification responses of WT and c-null
G-CSF-elicited peripheral blood neutrophils in response to stimulation
with cytokine. The change in ECAR versus time following normalization
in running buffer (DME without bicarbonate buffering, 0.1% BSA,
endotoxin free) and exposure to cytokine for 6 minutes is shown.
Results for mIL-3, 102 U/mL (dashed line); WT (),
c-null#1 ( ), and c-null#2 ( ). Results for mGM-CSF
(continuous line) (102 U/mL, WT [] and 104
U/mL c-null animal #1 [ ] and c-null animal #2 [ ]) and
buffer alone (continuous line), WT (), c-null#1
(). The cycle time was 2 minutes, and the pump speed
was 120 µL/min with a pump-off time of 30 seconds. Three additional
separate experiments were performed with similar results using a
concentration of mGM-CSF in 0.1% BCS of 102 U/mL for both
WT and c-null mice with other conditions being unchanged.
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Bronchoalveolar lavage of c-null mice.
In WT mice the number of cells obtained by bronchoalveolar lavage was
0.23 ± 0.24 × 106 cells per animal (n = 40 mice).
Macrophages were the predominant cell type (76% ± 22%) with
lymphocytes (14% ± 6%) and neutrophils (10% ± 16%) also
present. The number of cells in lavage fluid from c-null animals was
greatly increased (15.26 ± 7.78 × 106 cells per
animal, n = 19 mice). Differential cell counts showed increased numbers
of neutrophils (28% ± 13%), lymphocytes (29% ± 9%), and
cells with typical macrophage morphology (27% ± 10%). In
addition, there were cells with an atypical macrophage-like morphology,
with increased amounts of foamy cytoplasm (16% ± 7%). Although
there was a decrease in the percentage of alveolar macrophages in
c-null mice, the absolute number of typical alveolar macrophages was
increased 20-fold (WT 0.18 × 106 per animal,
c-null 4.12 × 106). We also examined the
bronchoalveolar lavage fluid of 2-week-old WT and c-null mice to
determine if changes in the cellular content were present at this young
age. Again, neutrophils (56% ± 5%) were present and large
atypical cells were seen (8% ± 1%) in lavage fluid from c-null
mice. However, the cellular yield was not increased compared with WT
mice (c-null 0.27 ± 0.35 × 106 per animal, n = 6 mice; WT 0.31 ± 0.2 × 106, n = 8 mice).
Immunohistochemistry was performed to determine whether the large,
foamy cells present in lung lavage fluid were macrophages
(Table 1). Peritoneal macrophages from both
WT and c-null mice stained positively for the macrophage markers
F4/80, macrosialin, and murine macrophage scavenger receptor (mMSR). Alveolar cells with typical macrophage morphology from WT and c-null
animals were positive for the macrophage surface markers macrosialin,
sialoadhesin, and mMSR, and, as expected from previous reports, were
negative for F4/80 and CR3.41 The large foamy alveolar
cells were positive for macrophage markers (F4/80, macrosialin, sialoadhesin, and mMSR), the adhesion marker CR3 (2 integrin), and
for MHC class II, which when present on macrophages is a marker of
activation. Thus, the foamy cells from bronchoalveolar lavage showed
surface markers consistent with their being of macrophage origin.
Macrophage function in c-null mice.
Cellular adhesion is an important function of macrophages which allows
them to display site-preference and which facilitates endocytosis/phagocytosis. We assessed the adhesion of WT and c-null peritoneal and alveolar macrophages in the presence and absence of EDTA
and using the 2F8 antibody that blocks the function of mMSR. Peritoneal
macrophages from c and WT animals adhered normally to TCP. This
adhesion was mediated via both divalent cation-dependent mechanisms and
via mMSR (data not shown). Alveolar macrophages from WT animals
remained adherent to TCP in the absence of inhibitors. This adhesion
was substantially divalent cation-dependent. Alveolar macrophages from
c-null animals, however, displayed markedly reduced adhesion
(Fig 5A).
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| Fig 5.
Cellular adhesion and phagocytosis of three agents by WT
and c-null macrophages. (A) Alveolar macrophage adhesion (average
cell number per area, derived as shown in Materials and Methods) with
incubation in IgG2b isotype control antibody (IC5), chelator EDTA, 2F8
(MoAb to mMSR), or combination of EDTA plus 2F8. (B) Alveolar
macrophage endocytosis of colloidal carbon in presence or absence of
EDTA or 2F8. (C) Phagocytosis of latex beads. (D) Phagocytosis of
opsonized sheep RBCs. Mean results of 2 to 4 wells, incubated at
37°C. No significant phagocytosis was observed in duplicate assays
performed at 4°C. Error bars represent SD. One of three similar
experiments.
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We examined the phagocytic ability of WT and c-null peritoneal and
alveolar macrophages using four different agents: colloidal carbon
(alveolar macrophages only), opsonized sheep RBCs, latex beads, and
DiI-AcLDL. Adherent peritoneal macrophages from both WT and c-null
were able to phagocytose all agents tested (Fig 5C and D, data for
endocytosis of DiI-AcLDL not shown). Alveolar macrophage phagocytosis
assays were performed by modifying the washing procedure as described
in Materials and Methods. There was no significant difference in the
ability of adherent alveolar macrophages from WT and c-null mice to
take up latex beads or sheep RBCs (Fig 5C and D). However, there was a
reduction in the uptake of colloidal carbon (Fig 5B) by adherent
c-null alveolar macrophages. Neither the adhesion defect nor the
colloidal carbon uptake defect was altered by addition of IL-3 or G-CSF
to the assays (data not shown).
We also examined NO production by peritoneal macrophages. As shown in
Fig 6, NO production in response to IFN-
and LPS by resident peritoneal macrophages from c-null animals was
the same as for macrophages from WT animals (WT 28 ± 4 µmol/L
nitrite; c-null 32 ± 5, n = 2 mice). There was no consistent
difference between cells from WT and c-null mice in five
experiments, although considerable inter-experimental variability was
observed. NO production by thioglycollate-elicited peritoneal
macrophages from c-null animals was also similar to WT (WT 61 ± 4; c-null 57 ± 16, n = 2 mice, similar results in
three experiments). We then examined NO production in vitro by
peritoneal macrophages in response to heat killed L
monocytogenes and did not detect a difference between c-null and
WT macrophages (data not shown). We were not able to examine NO
production using c-null alveolar macrophages because of the adhesion
defect described above. In summary, in these assays the peritoneal
macrophages from c-null mice showed normal adhesion, phagocytic
ability, and nitric oxide production. In contrast, the c-null
pulmonary macrophages showed reduced cellular adhesion. However, the
macrophages that did adhere sufficiently to be examined were able to
phagocytose latex beads, opsonized sheep RBCs, and DiI-AcLDL normally,
but displayed a reduction in the ability to take up colloidal carbon.
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[in a new window]
| Fig 6.
Nitrite production by (A) resident peritoneal macrophages
and (B) thioglycollate-elicited peritoneal macrophages from WT and
c-null mice in response to saline or IFN-/LPS after 48 hours of
incubation. Mean of 4 to 5 wells. Error bars represent SD. Results
shown are for two mice per genotype. Five experiments performed with
similar results.
|
|
Glucose transport in bone marrow cells from c-null
mice.
It has been previously suggested that the GM-CSF R alone may mediate
signaling for glucose transport.11,12 Therefore, this
hypothesis was examined using c-null bone marrow cells which express
only the GM-CSF R. In normal WT bone marrow cells the fold increase
over control in uptake of 3H-2-DOG after incubation in
GM-CSF was 2.53 ± 1.13 (n = 14 mice, GM-CSF concentrations ranging
from 500 U/mL to 50,000 U/mL: Fig 7).
However, in bone marrow cells from c-null animals there was no
significant increase in glucose uptake after incubation in GM-CSF (fold
increase in 3H-2-DOG uptake 1.02 ± 0.30, n = 22 mice
examined). At the highest concentration of GM-CSF used, the calculated
receptor occupancy of the low-affinity receptor was 85%. In contrast,
a similar increase in uptake of 3H-2-DOG was seen after
incubation in IL-3 for both WT cells (fold change 1.85 ± 0.12, n = 4 mice) and c-null cells (2.00 ± 0.69, n = 8 mice). GM-CSF- or
IL-3-stimulated uptake of 3H-2-DOG was inhibited by
cytochalasin B, an inhibitor of facilitative glucose transport (average
percent inhibition 91% ± 6%, n = 11 samples) but not by the
inactive analogue, cytochalasin E (average percent inhibition 33% ± 20%, n = 2 samples). These data showed that the GM-CSF R
alone was insufficient to mediate GM-CSF signaling for increased
glucose transport in hematopoietic cells from mice lacking the c
chain.
View larger version (11K):
[in this window]
[in a new window]
| Fig 7.
The ability of mouse bone marrow cells from WT and
c-null mice to take up the glucose analogue 3H-2-DOG
after incubation in GM-CSF (concentration range, 500 to 50,000 U/mL) or
carrier. Cells were incubated in 2-DOG and then 3H-2-DOG, 1 µCi per 1 mL culture, was added for 10 minutes' incubation at
37°C. The fold change in glucose uptake after
incubation in GM-CSF compared with carrier was calculated for each cell
preparation. Results are means of 14 WT mice and 22 c-null mice.
Error bars represent SD.
|
|
| |
DISCUSSION |
GM-CSF enhances the activity of a range of mature hematopoietic cell
functions, including mature cell survival,24
phagocytosis,42,43 leukocyte adhesion,44 and
proliferation and activation of alveolar macrophages.45-47
To date, the function of mature blood cells in hematopoietic cytokine
and cytokine receptor null mice has largely been examined by assessing
the response to infection with parasitic and bacterial
organisms.48-51 In this study we used a range of in vivo
and in vitro assays to examine the function of neutrophils and
macrophages from c-null mice.
We administered G-CSF to c-null and WT mice to obtain a
neutrophil-rich cell population for analysis.22 The in
vivo exposure to G-CSF did not impair the ability of cells to
bind GM-CSF. In both WT and c-null cell preparations neutrophil
responses at baseline or in response to IL-3 were normal for survival,
phagocytosis, and hydrogen ion secretion. In c-null cells a
GM-CSF-mediated increase in these functions was not seen.
A role for the GM-CSF R in mediating glucose uptake has been
described in two studies. In one, Xenopus laevis oocytes were injected with RNA encoding human GM-CSF R, and in another melanoma cell lines that endogenously expressed only low-affinity receptors for
GM-CSF were used.11,12 In contrast with these studies, an
increase in glucose uptake in response to GM-CSF was not seen in
c-null neutrophils expressing only the low-affinity GM-CSF R
chain. Moreover, the lack of an extracellular acidification response to
GM-CSF in neutrophils that express GM-CSF R alone suggests that
occupancy of GM-CSF R leads to little change in the metabolic state
of the cells.
The in vitro survival of c-null neutrophils cultured in the presence
of saline or IL-3 did not differ from WT. Iversen et al52,53 have shown that the GM-CSF analog E21R, which binds normally to the GM-CSF R but abnormally to c, causes apoptosis of
hematopoietic cells in the presence of the high-affinity GM-CSF receptor. These studies, together with our observations, demonstrate that the GM-CSF R alone is insufficient to mediate a survival signal
in hematopoietic cells.
In keeping with the pathological findings, examination of the
bronchoalveolar lavage fluid from c-null mice showed increased total
cellularity and the presence of large foamy cells. In this study we
used immunohistochemistry to demonstrate that these large cells were of
the macrophage lineage. In in vitro assays of macrophage function, we
found that adhesion and phagocytosis of colloidal carbon by c-null
alveolar macrophages were reduced. Immunohistochemistry showed the
c-null macrophages did express CR3 and mMSR, which have been
implicated in these macrophage functions.35 The defects may
be secondary to the lung disease, because a similar adhesion defect has
been described in patients with human pulmonary alveolar proteinosis.54 Overall, we were unable to demonstrate
defects in macrophage function that could account for the alveolar
proteinosis-like disease seen in these mice. To address this further,
we are now undertaking studies examining the surfactant catabolism by
c-null alveolar macrophages.
Production of NO in response to IFN-/LPS was similar in both WT and
c-null peritoneal macrophages. Similarly, the production of NO by
c-null peritoneal macrophages in response to heat-killed L
monocytogenes was normal, consistent with reports that the response of c-null mice to infection with this organism is similar to that of
WT.48 NO production by c-null alveolar macrophages was
unable to be assessed because of the adhesion defect of these cells.
These results are similar to those in a recent study in which the NO
response of peritoneal macrophages from GM-CSF null mice, when
stimulated with IFN-/LPS, was similar to that of WT.55 The investigators did observe a decrease in NO production when peritoneal macrophages from GM-CSF null mice were stimulated with LPS
alone.
In summary, this study showed that neutrophils and peritoneal
macrophages from c-null mice were capable of normal survival, phagocytosis, and NO production. In contrast, alveolar macrophages, implicated in the lung disease alveolar proteinosis, showed impaired adhesion and reduced ability to phagocytose colloidal carbon. No
GM-CSF-elicited responses were seen in cells from c-null mice. In
particular, the GM-CSF R alone was unable to mediate glucose transport in hematopoietic cells lacking the c chain.
| |
ACKNOWLEDGMENT |
We thank Drs Yifan Zhan and Christina Cheers for performing the
Listeria experiments, and Bette Papaevangeliou for technical assistance. We are grateful to Prof Donald Metcalf for comments on the
manuscript.
| |
FOOTNOTES |
Submitted April 20, 1998;
accepted July 28, 1998.
Supported by the National Health and Medical Research Council,
Canberra, the Anti-Cancer Council of Victoria, National Institutes of
Health Grant No. CA22556, and the Australian Government Cooperative Research Centres Scheme.
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 C.L. Scott, MD, The Walter and
Eliza Hall Institute of Medical Research, PO Royal Melbourne Hospital,
Victoria 3050 Australia.
| |
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Top
Abstract
Introduction