|
|
Prepublished online as a Blood First Edition Paper on August 8, 2002; DOI 10.1182/blood-2002-04-1102.
 Previous Article | Table of Contents | Next Article 
Blood, 1 December 2002, Vol. 100, No. 12, pp. 4193-4200
PHAGOCYTES
GM-CSF, via PU.1, regulates alveolar macrophage Fc R-mediated
phagocytosis and the IL-18/IFN--mediated molecular connection
between innate and adaptive immunity in the lung
Pierre-Yves Berclaz,
Yoko Shibata,
Jeffrey A. Whitsett, and
Bruce C. Trapnell
From Children's Hospital Medical Center, Cincinnati,
OH.
 |
Abstract |
Severely impaired pulmonary microbial clearance was observed in
granulocyte-macrophage colony-stimulating factor (GM-CSF)-deficient mice. To determine mechanisms by which GM-CSF mediates lung host defense, Fc R-mediated phagocytosis (opsonophagocytosis) by alveolar macrophages (AMs) was assessed in GM-CSF-sufficient
(GM+/+) and -deficient (GM /) mice and in
GM/ mice expressing GM-CSF only in the lungs from a
surfactant protein C (SPC) promoter
(SPC-GM+/+/GM/). Opsonophagocytosis
by GM/ AMs was severely impaired and was restored by
pulmonary GM-CSF expression in vivo or by PU.1 expression in vitro.
Defective opsonophagocytosis by GM/ AMs was associated
with decreased FcR expression. Because interferon- (IFN-)
augments macrophage FcR levels, the role of GM-CSF/PU.1 in the
regulation of AM FcR expression by IFN- was assessed during
adenoviral lung infection. Adenoviral infection stimulated IFN-
production and augmented FcR levels on AMs in GM-CSF-expressing but
not GM/ mice. However, IFN- exposure ex vivo
stimulated FcR expression on GM/ AMs. Because
interleukin-18 (IL-18) and IL-12 stimulate IFN- production during
adenoviral infection, their role in GM-CSF/PU.1 regulation of
IFN--augmented FcR expression on AMs was assessed. Adenoviral
infection stimulated IL-18 and IL-12 production in GM-CSF-expressing
mice, but both were markedly reduced or absent in GM/
mice. IL-18 expression by GM/ AMs was severely impaired
and was restored by pulmonary GM-CSF expression in vivo or by PU.1
expression in vitro. Pulmonary administration of IL-18 in
GM/ mice stimulated IFN- production and restored
FcR expression on AMs. These results show that GM-CSF, via PU.1,
regulates constitutive AM FcR expression and opsonophagocytosis and
is required for the IFN--dependent regulation of AM FcR
expression, enabling AMs to release IL-18/IL-12 during lung infection.
(Blood. 2002;100:4193-4200)
© 2002 by The American Society of Hematology.
| |
Introduction |
The alveolar macrophage (AM) plays a central role
in lung host defense through both effector and regulatory functions. As the resident professional phagocyte, AMs provide a first line of host
defense by internalizing and degrading microbial pathogens encountered
on the respiratory surface.1 Upon pathogen exposure, AMs
express cytokines that influence recruitment and activation of
inflammatory cells and modify adaptive immune responses in a
pathogen-selective fashion.2 In this way, cytokines
released from pathogen-exposed AMs provide important molecular
"links" between innate and adaptive immunity in the lung. AMs also
present processed antigens to lymphocytes, resulting in production of opsonizing, pathogen-specific immunoglobulins (Igs).
Opsonins such as IgG further enhance phagocytic pathogen clearance and
modulate inflammatory responses through interaction with cell-surface
receptors that recognize the Fc region of pathogen-bound IgG
(FcRs).3 Two general classes of FcRs are currently
recognized activation receptors (eg, FcRIA, FcRIIIA) and
inhibitory receptors (eg, FcRIIB), both of which are present and
functional in human and murine macrophages and other cells (reviewed by
Ravetch and Bolland4). In macrophages, activating FcRs
initiate a complex intracellular signaling cascade culminating in
enhanced phagocytosis and secretion of inflammatory
cytokines.5-8 Inhibitory FcRs, which are also present
on macrophages and activated concomitantly, regulate the threshold of
activation responses and ultimately terminate IgG-mediated effector
stimulation.9 Phagocytosis mediated by FcRs is
morphologically and functionally distinct from uptake of unopsonized
particles or that mediated by other receptors (eg, complement or
mannose receptors).10 Phagocytic pathways are under
differential regulatory control in macrophages as demonstrated by the
observation that phagocytosis mediated by FcRs (ie, IgG-coated
beads), in contrast to nonspecific phagocytosis (unopsonized latex
beads), can be blocked by inhibiting Src family kinases.11
Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates
growth and differentiation of cultured AMs12-14
and plays a critical role in surfactant homeostasis and in lung host
defense (reviewed by Trapnell and Whitsett15). Defects in
AM functions in GM-CSF gene-targeted
(GM/)16,17 mice result in impaired
pulmonary clearance of and increased susceptibility to bacterial and
fungal pathogens.18-20 For example, nonopsonic
phagocytosis and bacterial killing are reduced in GM/
AMs.20 GM-CSF stimulates FcR levels on macrophages and
enhances macrophage FcR-mediated phagocytosis 21;
however, the mechanism by which this occurs has not been defined. The
ets family transcription factor, PU.1, is required for macrophage
production,22,23 promotes the differentiation of myeloid
progenitors,24 and stimulates the terminal maturation of
AMs in the lung.20 PU.1 is deficient in AMs of
GM/ mice but is restored by local expression of GM-CSF
in the lungs using the human 3.7-kb surfactant protein C (SPC) promoter
(eg, SPC-GM+/+/GM/ mice) rescuing defects
in lung host defense.20 Because PU.1 stimulates FcRI
and FcRIII transcription in monocyte/macrophage cell lines, we
hypothesized that GM-CSF might regulate FcR-mediated phagocytosis by
mature AMs via PU.1.
Interferon- (IFN-) plays an important role in lung host
defense by integrating pulmonary responses to microbial infection through pleiotropic effects on both innate and adaptive immunity. For
example, IFN- stimulates macrophage activation25 and
enhances FcR-mediated phagocytosis by augmenting FcR
levels.26 IFN- also influences
TH1/TH2 cell proliferation and function,
enhancing humoral antibody production.27,28 Infection by
microbial pathogens including adenovirus29 stimulates
IFN- production by various cells, eg, natural killer (NK) and T
helper-1 (TH1) cells.30 IFN-
secretion by these cells is stimulated by either interleukin-18 (IL-18) or IL-12, both of which are released by AMs after exposure to
pathogens such as adenovirus.30 Thus,
IL-18/IL-12-stimulated IFN- secretion forms a feedback loop that
enhances AM FcR-mediated opsonophagocytosis during microbial lung
infection. GM/ mice have a blunted IFN- response to
in vivo lipopolysaccharide exposure although GM/ T
cells have a normal IFN- response in vitro.31 These and other findings suggest that GM-CSF has an indirect effect on IFN- release from T cells in vivo, possibly through regulation of secretion of an "IFN--releasing factor."32 IL-18 and/or
IL-12 are candidates for such a factor because (1) both are released by
AMs after exposure to adenovirus, (2) both strongly stimulate IFN-
production by T cells, and (3) blocking IL-18 and IL-12 signaling
impairs IFN- production following pulmonary adenovirus
infection.30
Together, these observations suggest that GM-CSF might regulate both
FcR-mediated phagocytosis by AMs and the IL-18/IFN- pathway that
is required for augmentation of AM FcR expression following
pulmonary infection. To address this hypothesis, the role of GM-CSF and
PU.1 in FcR-mediated phagocytosis was assessed in primary AMs from
GM+/+, GM/, or
SPC-GM+/+/GM/ mice and in cultured AM cell
lines from GM+/+ and GM/ mice.
| |
Materials and methods |
Mice
GM-CSF gene-targeted mice were previously
created,16 bred into the C57BL/6 background, and
maintained for several years (referred to as GM/ mice
hereafter).33 GM/ mice in which GM-CSF was
selectively expressed in the lung by the human 3.7-kb surfactant
protein C promoter (SPC) were previously described34 and
maintained in the C57BL/6 background (referred to as
SPC-GM+/+/GM/ mice hereafter). C57BL/6 mice
(Charles River, Wilmington, MA) were used for comparison
(referred to as GM+/+ hereafter). Mice were housed in a
barrier facility and studied under procedures approved by the
Institutional Animal Care and Use Committee of the Cincinnati
Children's Hospital Research Foundation. Sentinel mice were tested
periodically and were free of known viral and bacterial pathogens.
Alveolar macrophages and alveolar macrophage cell lines
Primary AMs were obtained from GM/,
GM+/+, or SPC-GM+/+/GM/ mice by
bronchoalveolar lavage (BAL) as described.35 MH-S
(American Type Culture Collection, CRL-2019, Mannasas, VA) is
an AM cell line with morphologic features and functions of normal
mature AMs, previously derived from GM+/+ BALB/cJ
mice36; mAM is an AM cell line with an incompletely differentiated phenotype due to absence of expression of the
transcription factor PU.1 previously derived from GM/
mice without viral or other transformation.20 The
mAM cells constitutively expressing murine PU.1 (referred to as
mAMPU.1+ hereafter) were previously created by retroviral
transduction.20 These cells also expressed green
fluorescent protein (GFP), which is included in the vector as a
selectable marker.37 As a transduction control, mAM cells
expressing only the GFP marker were used (referred to as
mAMGFP+ hereafter).20 Retrovirally transduced alveolar macrophage cell lines (mAMGFP+,
mAMPU.1+) were maintained as unselected populations
following transduction rather than as isolated clonal lines. Cultured
AMs (MH-S, mAMGFP+, or mAMPU.1+) were maintained
as previously described.20 For clarity, AMs used in
experiments after recovery from mice by BAL are referred to as primary
AMs, while the various cultured AM cell lines are referred to as
cultured AMs.
Adenovirus
The adenovirus used in this study is a replication-deficient
derivative of human type 5 adenovirus whose structure has been previously reported.38 Methods for growth and purification
of viruses in endotoxin-free media, media supplements, and solutions (supplied routinely or by special arrangement from BioWhittaker, Walkersville, MD) have been previously
described.39,40 Virus concentration was determined
from the optical density of the purified virions at 260 nm
(OD260) using the formula 1 OD260 = 1 × 1012 particles per milliliter
and expressed as optical particle units (opu) as previously
described.41 Adenovirus was administered to the lungs by
transoral intubation and tracheal instillation as previously
described.29 In vitro infection of cultured AMs was done
as previously described.40
FcR-mediated phagocytosis assay
Albumin-coated fluorescent latex beads (referred to as
unopsonized beads) were prepared using 2-µm-diameter fluorescein
isothiocyanate (FITC)-labeled latex microspheres (Spherotech,
Libertyville, IL) by incubation with bovine serum albumin (BSA)
(faction V; Sigma, St Louis, MO; 10 mg/mL, 37°C, 60 minutes) followed
by washing (3 ×) and resuspension in phosphate-buffered
saline (PBS) at a concentration of 1.5 × 109
beads per milliliter. IgG-opsonized beads were prepared by incubating albumin-coated beads with antialbumin antibody (Pharmingen; 1500 µg/mL, 37°C, 30 minutes) followed by washing and resuspension as
above. FcR-mediated phagocytosis was quantified in primary AMs ex
vivo immediately after recovery by BAL20 and adherence to
plastic42 and in cultured AM cell lines plated the day
prior to analysis. Cells (105 per well, plated in 35-mm
dishes) were exposed to unopsonized beads or IgG-opsonized beads at a
concentration of 0.5 × 107/mL for 1 hour. In some
experiments, cells were preincubated with rat antimouse CD16/32
(Pharmingen) for 30 minutes prior to and during incubation with
IgG-opsonized beads to block FcRII/FcRIII-mediated opsonophagocytosis. Cells were then washed extensively in FACS buffer
to remove noninternalized particles, detached by brief trypsinization,
and evaluated by flow cytometry on a FACScan flow cytometer (Becton
Dickinson, San Jose, CA). Results were analyzed using CellQuest
software (Becton Dickinson) on a Macintosh microcomputer. The
phagocytic index was calculated from the following formula: phagocytic
index = percent of cells containing beads × mean
fluorescence of cells containing beads. All determinations
represent the mean of at least 3 separate measurements, and each
experiment was performed 2 or more times with similar results.
FcR expression on AMs
Primary AMs collected by BAL or cultured AMs collected by
scraping in Versene (Life Technologies, Grand Island, NY) were
resuspended in fluorescence-activated cell sorter (FACS)
buffer (PBS, 0.2% BSA, 0.01% sodium azide), washed in FACS buffer,
counted, and divided into aliquots (105 cells) in 100 µL
FACS buffer. Cells were incubated with phycoerythrin-conjugated antibody directed at mouse FcR (rat antimouse CD16/32; Pharmingen; 30 minutes, 4°C). As controls, cells were also evaluated with primary
isotype- and species-matched antimouse immunoglobulins. After
incubation, immunostained cells were washed twice in FACS buffer and
kept on ice and then analyzed by single-color flow cytometry using a
FACScan flow cytometer as above. Fluorescence data were collected using
logarithmic amplification on 10 000 viable cells as determined by
forward light scattering. To determine if AMs in GM/
mice were capable of responding to IFN- by increasing FcR
expression, primary AMs were obtained by BAL, collected by
centrifugation, and freed of nonadherent cells by adherence in plastic
dishes for 45 minutes as previously described.42 AMs were
then exposed to IFN- (200 U/mL, 24 hours) and detached with Versene,
and cell-surface FcR levels were assessed by FACS as above. In other
experiments, AM FcR levels were also similarly assessed by FACS 48 hours after intrapulmonary administration of IL-18 (100 ng per mouse in
60 µL 0.9% NaCl) to stimulate increased levels of IFN- levels in the lung. All determinations were done on primary AMs from 4 mice per
group or 4 plates of cultured AMs analyzed separately. Results were
similar in corresponding samples, and representative examples are
shown. Each experiment was done twice.
Reverse transcriptase-polymerase chain reaction amplification
Messenger RNA transcript levels were quantified in primary
or cultured AMs using reverse transcriptase-polymerase chain reaction (RT-PCR) as previously described.20,29 The following
primer sets were used: glyceraldehyde-3-phosphate dehydrogenase
[GAPDH]: 5'-ATTCTACCCACGGCAAGTTCAATGG-'3 and
5'-AGGGGCGGAGATGATGACCC-3'; IL-18: 5'-AGACCTGGAATCAGACAACTTTGG-'3 and
5'-AAACTCCATCTTGTTGTGTCCTGG-3'; FcRIA:
5'-GAGCAGGGAAAGAAAGCAAATTCC-3' and 5'-TTAAGAGTTGCATGCCATGGTCC-3'; FcRIIB: 5'-CCCAAGTCCAGCAGGTCTTTACC-3' and
5'-TTCTGGCTTGCTTTTCCCAATGCC-3'; and FcRIIIA:
5'-GATCCAGCAACTACATCCTCCATC-3' and 5'-GCCTTGAACTGGTGATCCTAAGTC-3'. All
determinations were done in triplicate using either primary AMs from 3 mice analyzed separately or plates of cultured AMs. Each
experiment was done twice.
Cytokine levels
IFN-, IL-18, and IL-12 concentrations in the lungs were
measured by enzyme-linked immunosorbent assay (ELISA) as previously described.29 Briefly, BAL fluid was obtained from groups
of mice (4-6 per group) and cleared of cells and debris by low-speed centrifugation (450g, 10 minutes, 4°C). Cleared BAL fluid
from each mouse was then assayed individually for various cytokines by
using the appropriate murine Quantikine kit (R&D Systems, Minneapolis, MN) as directed by the manufacturer. Assessments of IL-18 and IL-12
levels were done twice. IFN- levels in the BAL fluid were also
similarly assessed in mice 48 hours after receiving IL-18 via pulmonary
administration as described above. In vitro determination of IL-18
release by cultured AMs (MH-S, mAMGFP+, and
mAMPU.1+) was done essentially as described.40 Briefly, cells (2.5 × 105 per well in 24-well plates)
were incubated in the absence or presence of adenovirus
(1010 optical particle units [opu] per well) for 24 hours. Culture supernate was then aspirated, cleared by low-speed
centrifugation, and evaluated for the presence of IL-18 by
ELISA as above. All determinations represent the mean of 4 determinations done on separate plates of cells per group. Experiments
with mAMPU.1+ and mAMGFP+ cells were done twice.
Statistics
Numeric data are presented as mean ± SEM. Statistical
comparisons were made using the Student t test. Statistical
calculations were performed with Sigma Plot (version 7.0) software on
an IBM-compatible microcomputer.
| |
Results |
GM-CSF is required for FcR-mediated phagocytosis by
primary AMs
The role of GM-CSF in FcR-mediated phagocytosis by AMs was
assessed in primary AMs from GM+/+, GM/,
and SPC-GM+/+/GM/ mice by ex vivo challenge
with fluorescent latex beads coated either with albumin alone
(unopsonized beads) or with albumin and then antialbumin antibody
(IgG-opsonized beads) followed by flow cytometry. In GM+/+
AMs, FcR-mediated phagocytosis was demonstrated by a phagocytic index for IgG-opsonized beads 117% ± 4% greater than that for unopsonized beads (Figure 1A). In
contrast, FcR-mediated phagocytosis was absent in
GM/ AMs (Figure 1B). Phagocytosis of unopsonized beads
was also reduced in GM/ AMs. Pulmonary expression of
GM-CSF restored FcR-mediated phagocytosis as demonstrated in
SPC-GM+/+/GM/ AMs, which had a phagocytic
index for IgG-opsonized beads 150% ± 23% greater than that for
unopsonized beads (Figure 1C). Thus, exposure to GM-CSF in the lung is
required for FcR-mediated phagocytosis by primary AMs.
View larger version (22K):
[in this window]
[in a new window]
| Figure 1.
FcR-mediated phagocytosis by alveolar macrophages
(AMs) is regulated by GM-CSF in the lungs and expression of PU.1 in
AMs.
(A-C) Primary AMs were recovered by BAL from mice in which GM-CSF
expression was normal (GM+/+), absent
(GM/), or present only in the lungs
(SPC-GM+/+/GM/) and challenged with
unopsonized beads (Beads alone) or IgG-opsonized beads (Beads + Ab) as described in "Materials and methods." Phagocytic indices are
shown. Data represent means ± SEM; n = 4 (GM/
and SPC-GM+/+/GM/) or n = 3
(GM/) mice per group; AMs from each mouse were analyzed
individually. Differences in phagocytic indices for FcR-mediated and
non-FcR-mediated phagocytosis (Beads + Ab, Beads alone,
respectively) by AMs from GM+/+ and
SPC-GM+/+/GM/ were significant
(P < .01). Corresponding phagocytic indices for AMs from
GM/ mice were not significantly different
(P = .86). (D-F) Cultured AM cell lines were challenged as
above except that plates of cells were also challenged in the presence
of FcR-blocking antibody (Beads + Ab +Fc block). Phagocytic
indices are shown. Data represent means ± SEM; n = 4
determinations per group. Differences in phagocytic indices for
FcR-mediated and non-FcR-mediated phagocytosis (Beads + Ab,
Beads alone, respectively) in MH-S and mAMPU.1+ cells were
significant (P < .0001). FcR-mediated phagocytosis was
completely blocked by addition of anti-FcR antibody. Phagocytosis
was not detected in mAMGFP+ cells (*).
|
|
PU.1 expression rescues FcR-mediated phagocytosis by cultured
GM/ AMs
The role of PU.1 in the regulation of FcR-mediated phagocytosis
by AMs, downstream of GM-CSF, was assessed as above in cultured GM/ AM cell lines wherein PU.1 expression was absent
(mAMGFP+) or restored by retroviral gene transfer
(mAMPU.1+) and a cultured GM+/+ AM cell line
expressing PU.1 normally (MH-S). In MH-S cells, FcR-mediated
phagocytosis was demonstrated by a phagocytic index for IgG-opsonized
beads 574% ± 40% greater than that for unopsonized beads (Figure
1D). Rat antimurine FcRII/III antibody completely blocked
FcR-dependent uptake. In GM/ mAM cells,
FcR-mediated phagocytosis did not occur in the absence of PU.1
(mAMGFP+; Figure 1E) but was restored by PU.1 expression
(mAMPU.1+; Figure 1F). FcR-mediated phagocytosis in
mAMPU.1+ cells was completely blocked by antimurine
FcRII/III antibody. Because pulmonary GM-CSF is required for
expression of PU.1 in AMs,20 these data indicate that
GM-CSF regulates FcR-mediated phagocytosis by AMs via PU.1.
GM-CSF, via PU.1, regulates constitutive FcR levels on
AMs
To determine if impaired FcR-mediated phagocytosis by
GM/ AMs could be explained by failure to stimulate
expression of FcRs, levels of cell-surface FcRs on primary and
cultured AMs were assessed by flow cytometry. FcRs were present on
all primary GM+/+ AMs as demonstrated using a rat antimouse
FcRII/III antibody (Figure 2A). In
contrast, FcRs were undetectable on GM/ AMs but were
restored by pulmonary GM-CSF expression as demonstrated with AMs from
SPC-GM+/+/GM/ mice. Because PU.1 stimulates
transcription of the human FcRIB43 and murine
FcRIIIA44 genes in myeloid cell lines, the role of PU.1
in regulation of AM FcR levels was assessed using MH-S, mAMGFP+, and mAMPU.1+ cells. FcRs were readily
and uniformly detected on cultured MH-S cells as demonstrated using the
anti-FcRII/III antibody (Figure 2A). In sharp contrast, FcRs were
absent on mAMGFP+ cells but were restored by PU.1 expression
in mAMPU.1+ cells (Figure 2A). Importantly,
mAMGFP+ cells did not contain detectable mRNA encoding
either FcRIIIA or FcRIIB (Figure 2B), both of which are detected
by the antimurine FcR antibody45,46 used for flow
cytometry above. Nor did these cells contain mRNA for FcRIA. In
contrast, both wild-type (MH-S) and PU.1-expressing GM/
(mAMPU.1+) cultured AMs expressed mRNA for all 3 FcRs
(Figure 2B). Together with the results from primary AMs, these data
indicate that GM-CSF, via PU.1, is required for expression of both
activating FcRs (FcRIA, FcRIIIA) and inhibiting FcRs (FcRIIB)
in AMs.
View larger version (24K):
[in this window]
[in a new window]
| Figure 2.
FcR expression on AMs is regulated by levels of
GM-CSF in the lungs and expression of PU.1 in AMs, and levels
are increased during pulmonary infection by adenovirus.
(A) FcR expression on primary AMs (GM+/+,
GM/, SPC-GM+/+/GM/) or
cultured AMs (MH-S, mAMGFP+, mAMPU.1+) was
quantified by FACS as described in "Materials and methods." Shown
are data for FcRII/III-specific antibodies (shaded histogram) and
isotype controls (open histogram). Differences in autofluorescence
among cultured AM cell lines, due in part to the presence of the GFP
marker in mAMGFP+ and mAMPU.1+, have been
compensated. Results are representative of 4 separate determinations in
AMs from mice analyzed individually. (B) The cultured AM cell line from
GM/ mice (mAMGFP+) failed to express mRNA
for either activating (FcRIA, FcRIIIA) or inhibiting (FcRIIB)
FcRs, but expression was stimulated by PU.1 as shown in
mAMPU.1+ cells. Total RNA was prepared from cultured AM cell
lines (MH-S, mAMGFP+, mAMPU.1+) and subjected to
RT-PCR analysis using gene-specific primers as described in
"Materials and methods." Shown are photographs of ethidium
bromide-stained RT-PCR reaction products separated on 2% agarose
gels. H20 PCR controls were negative (not shown). This
experiment was repeated twice with identical results. (C) Primary AMs
were recovered 36 hours after pulmonary adenoviral infection of
GM+/+ or GM/ mice, and levels of FcR
expression were assessed as above. Levels of FcR on AMs are
represented as the mean fluorescence intensity as determined using the
FcRII/III-specific antibody. Data represent means ± SEM;
n = 4 mice per group; AMs from each mouse were analyzed individually.
Differences in FcR expression on AMs from infected and uninfected
GM+/+ mice were significant (P < .001).
FcR expression was not detected (*) on GM/ AMs in
the absence or presence of adenovirus infection. AMs recovered from
GM/ mice and exposed ex vivo to IFN- for 24 hours
showed a marked up-regulation of cell-surface FcRII/III expression
at levels significantly different from untreated mice
(P < .001).
|
|
FcR expression is deficient on primary AMs in
GM/ mice following adenoviral infection
In GM+/+ mice, pulmonary adenovirus infection
augmented FcR levels on AMs by 253% ± 95% as determined by flow
cytometry using the anti-FcRII/III antibody (Figure 2C). In
contrast, FcRs were absent on GM/ AMs following
pulmonary adenoviral infection. Importantly, ex vivo exposure of
GM/ AMs to IFN- restored FcR expression as
indicated by the marked increase in fluorescence of anti-FcRII/III
antibody-stained cells (Figure 2C). Thus, enhancement of FcR II/III
expression on AMs following adenoviral infection does not occur in
GM/ mice and is not due to an inability of
GM/ AMs to respond to IFN-.
Impairment of IFN-, IL-18, and IL-12 expression in
GM/ mice during pulmonary adenoviral infection
To determine whether GM-CSF is required for regulation of IFN-
expression by IL-18/IL-12, levels of these cytokines were quantified in
the lungs after pulmonary adenoviral infection. IFN- was not
detected in the lungs of uninfected GM+/+,
GM/, or SPC-GM+/+/GM/ mice
(Figure 3A). In GM+/+ or
SPC-GM+/+/GM/ mice, but not
GM/ mice, high levels of IFN- were detected after
infection (392 ± 136, 384 ± 114, and 57 ± 29 pg/mL,
respectively; Figure 3A). Thus, pulmonary GM-CSF expression is required
for the high-level expression of IFN- accompanying pulmonary
adenoviral infection.
View larger version (10K):
[in this window]
[in a new window]
| Figure 3.
The increase in IFN-, IL-18, and IL-12 levels in the
lungs stimulated by pulmonary adenovirus infection is severely impaired
in GM/ mice.
(A) BAL fluid was collected from GM+/+,
GM/, and SPC-GM+/+/GM/ mice
36 hours after pulmonary adenovirus infection and evaluated for the
presence of IFN- by ELISA. Asterisks indicate that no IFN- was
detected in the lungs of any mice in the absence of adenovirus lung
infection. Data represent means ± SEM; n = 4
(GM/ or SPC-GM+/+/GM/) or
n = 3 (GM+/+) mice per group; BAL fluid from each mouse
was analyzed individually. Differences in IFN- levels in infected
(+) and uninfected ( ) GM+/+ and
SPC-GM+/+/GM/ mice were significant
(P < .02) but were not significant in GM/
mice (P = .10). (B) Mice exposed to adenovirus as above
were also evaluated for the presence of IL-18 in BAL. IL-18 levels in
BAL fluid of infected animals are shown. Data represent the means ± SEM; n = 6 (GM/ or
SPC-GM+/+/GM/) or n = 3
(GM+/+) mice per group; BAL fluid from each mouse was
analyzed individually. IL-18 was not detected in BAL fluid from
adenovirus-exposed GM/ mice (*). Differences in IL-18
levels in GM+/+ and GM/ mice were
significant (P < .05). The adenovirus-simulated increase
in IL-18 levels in the lungs was restored in
SPC-GM+/+/GM/ mice. (C) The same
animals evaluated in panel A were also evaluated for the presence of
IL-12 in BAL. IL-12 levels in BAL fluid of infected animals are shown.
Data represent the means ± SEM; n = 7 mice per group.
Differences in IL-12 levels in GM+/+ and
GM/ mice were significant (P < .05). The
adenovirus-simulated increase in IL-18 levels in the lungs was restored
in SPC-GM+/+/GM/ mice.
|
|
Because IL-18 and IL-12 stimulate IFN- production in the lung during
pulmonary adenovirus infection,30 these cytokines were
also quantified after pulmonary adenoviral infection. Neither IL-18 nor
IL-12 was detected in the lungs of uninfected mice (data not shown). In
GM+/+ and SPC-GM+/+/GM/ mice,
but not GM/ mice, pulmonary adenovirus infection
increased IL-18 levels (756 ± 340, 294 ± 93, 0 ± 0 pg/mL BAL,
respectively; Figure 3B). Thus, GM-CSF is required for IL-18 production
during pulmonary adenovirus infection. Further, the absence of
IL-18-stimulated IFN- production in GM/ mice
following adenoviral infection explains the lack of augmentation of
FcR expression on primary AMs. To confirm this hypothesis, IL-18 was
administered to the lungs of GM/ mice and IFN-
levels were measured 48 hours later. IL-18 significantly stimulated
IFN- levels (84 ± 29 pg/mL BAL fluid; n = 7;
P < .04) and restored FcR levels on primary AMs (mean
fluorescence intensity of anti-FcRII/III antibody-stained AMs
92 ± 11; n = 7; P < .001) compared with controls
(n = 5; data not shown). In GM+/+ and
SPC-GM+/+/GM/ mice, pulmonary adenovirus
infection also increased IL-12 levels, but in GM/ mice
levels were elevated to a much lower degree (213 ± 129, 134 ± 60,
26 ± 7.3 pg/mL BAL, respectively; Figure 3C). These data indicate
that GM-CSF is required for stimulation of IL-18 and IL-12 production
in the lung during pulmonary adenovirus infection and provide a
molecular explanation for the decreased IFN- levels seen during
adenovirus infection in GM/ mice.
PU.1 rescues IL-18 production in cultured GM/
AMs
The role of GM-CSF/PU.1 in regulation of IL-18 production by AMs
was assessed in primary and cultured AMs. Consistent with the ELISA
data above, IL-18 mRNA was detected in primary AMs from GM+/+ and SPC-GM+/+/GM/ mice
but not GM/ mice (Figure
4A). IL-18 mRNA was more abundant in
primary AMs from SPC-GM+/+/GM/ than from
GM+/+ mice, likely reflecting the markedly increased
pulmonary GM-CSF levels in SPC-GM+/+/GM/
mice.34 To further assess the role of GM-CSF/PU.1 in
regulation of IL-18 expression in AMs, IL-18 release from
adenovirus-infected and uninfected MH-S, mAMGFP+, and
mAMPU.1+ cells was quantified by ELISA. Adenoviral infection
of MH-S cells, but not mAMGFP+ cells, markedly increased
IL-18 release (25.2 ± 7.9 vs 5.3 ± 1.2 pg/mL medium,
respectively; Figure 4B). PU.1 restored IL-18 release by
mAMPU.1+ cells following adenoviral infection (18.5 ± 4.5
pg/mL medium). Because pulmonary GM-CSF is required for expression of
PU.1 in primary AMs,20 these data indicate that GM-CSF
regulates IL-18 production by AMs via PU.1.
View larger version (17K):
[in this window]
[in a new window]
| Figure 4.
IL-18 expression in AMs from GM/ mice is
severely impaired but was restored by retrovirus-mediated expression of
PU.1.
(A) Primary AMs from GM+/+, GM/, and
SPC-GM+/+/GM/ mice were assessed for the
presence of mRNA transcripts encoding IL-18 or GAPDH, as a control to
demonstrate evaluation of equal amounts of total RNA, using RT-PCR as
described in "Materials and methods." Photographs of ethidium
bromide-stained agarose electrophoresis gels of the PCR products are
shown. Each lane represents AMs from one mouse. The experiment was
repeated twice with the same results. (B) Cultured AMs were exposed to
adenovirus (Adenovirus infected) or not exposed (Control) for 24 hours,
and then IL-18 release into the medium was quantified by ELISA as
described in "Materials and methods." The sensitivity of detection
of IL-18 was 5 pg/mL. Data represent means ± SEM; n = 3 to 6 (uninfected) or 4 to 10 (infected). Differences in IL-18 release by
adenovirus-infected and uninfected MH-S and mAMPU.1+ cells
were significant (P < .03). IL-18 release by
adenovirus-infected and uninfected mAMGFP+ cells was not
significantly different (P = .88).
|
|
| |
Discussion |
The present study was designed to determine the role of
GM-CSF in FcR-mediated opsonophagocytosis by AMs and the mechanism by which GM-CSF influences FcR expression during pulmonary
infection. FcR-mediated phagocytosis by AMs from GM/
mice was severely impaired in vivo and in vitro and was restored by
expression of GM-CSF in the lungs or by constitutive expression of PU.1
in primary and cultured AMs, respectively. Impaired opsonophagocytosis was associated with the absence of FcRs on AMs in vivo and in vitro,
and AM FcR expression was dependent on PU.1. Enhancement of AM
FcR expression following pulmonary adenoviral infection did not
occur in GM/ mice due to impaired IFN- production,
which was associated with impaired IL-18/IL-12 production. Pulmonary
expression of GM-CSF restored adenovirus-stimulated production of all 3 cytokines and augmented FcR levels on AMs. GM/ AMs
did not express IL-18 mRNA or protein, but expression was restored by
PU.1 expression in cultured AMs in vitro, and pulmonary administration
of IL-18 in GM/ mice stimulated both IFN- production
in vivo and augmented FcR levels on primary AMs. These data show
that GM-CSF, via PU.1, regulates AM FcR expression and
opsonophagocytosis and also the positive feedback mechanism by which
pathogen-exposed AMs enhance FcR expression during pulmonary infection.
The correlation between FcR expression and opsonophagocytosis by
primary and cultured AMs observed here suggests that the presence or
absence of the FcR may itself be a principal point of control in the
molecular mechanism regulating the constitutive opsonophagocytic
capacity of AMs. Thus, GM-CSF, via PU.1, may program differentiating
myeloid cells for opsonophagocytosis primarily by stimulating
expression of FcRs. Several lines of evidence support this concept.
First, PU.1 expression was required for expression of FcR mRNA and
protein in cultured AMs. Further, FcRs were present on primary AMs
in vivo only when GM-CSF was present in the lung, consistent with the
prior observation that the pulmonary GM-CSF regulates PU.1 expression
in primary AMs in vivo.20 Thus, opsonophagocytosis and
expression of GM-CSF, PU.1, and FcR were precisely correlated in the
present study. These results are consistent with reports showing that
GM-CSF stimulates FcR expression and opsonophagocytosis in murine
macrophages21 and increased FcR mRNA expression in
monocytes during in vitro differentiation47 and also with
data showing that PU.1 stimulates transcription of FcRIA in a human
monocytic cell line43 and FcRIIIA in a murine
peritoneal macrophage cell line.44 Second, transfection-mediated FcRI expression in COS cells confers
opsonophagocytic capacity, demonstrating that cells that are not
professional phagocytes and do not normally express FcRs have the
molecular machinery for phagocytosis.5 Finally, PU.1 is a
principal regulator of AM terminal differentiation and stimulates
expression of other receptors on AMs, including the mannose receptor
and several Toll-like receptors.20 Although PU.1 was
previously shown to stimulate FcRIA and FcRIIIA expression in
myeloid cells, our data demonstrate that PU.1 is required for
expression of these activating receptors, and also the inhibiting
receptor FcRIIB. Further, their parallel regulation by
GM-CSF/PU.1 suggests that coordinate expression of the balanced
activation and inhibitory Fc signaling pathways during myeloid
differentiation may be important to macrophage opsonophagocytosis
and/or FcR signaling. Our findings do not rule out the possibility
that other critical molecular events may also be simultaneously
regulated by GM-CSF and/or PU.1 in AMs and thus may also be rate
limiting in GM/ AMs. For example, several distinct
protein tyrosine kinase families are important in the regulation of
opsonophagocytosis, including Syk,48 Src,49
and phosphatidylinositol 3-kinase.50,51 Consistent with
this latter possibility is the finding that, although FcR |
Top
Abstract
Introduction