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Chronic Ethanol Ingestion in Rats Decreases
Granulocyte-Macrophage
Colony-Stimulating Factor Receptor
Expression and Downstream Signaling in the
Alveolar Macrophage
Pratibha C. Joshi, Lisa Applewhite, Jeffrey D. Ritzenthaler,
Jesse Roman, Alberto L. Fernandez, Douglas C. Eaton, Lou
Ann S. Brown and David M. Guidot
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2005 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2005; 175:6837-6845; ;
doi: 10.4049/jimmunol.175.10.6837
http://www.jimmunol.org/content/175/10/6837
The Journal of Immunology
Chronic Ethanol Ingestion in Rats Decreases
Granulocyte-Macrophage Colony-Stimulating Factor Receptor
Expression and Downstream Signaling in the
Alveolar Macrophage1
Pratibha C. Joshi,*† Lisa Applewhite,*† Jeffrey D. Ritzenthaler,† Jesse Roman,*†
Alberto L. Fernandez,*† Douglas C. Eaton,‡ Lou Ann S. Brown,§ and David M. Guidot2*†
F
or over a century, alcohol abuse has been well recognized
as a significant risk factor for serious pulmonary infections. For example, alcoholic patients are at increased risk
for infection with necrotizing Gram-negative pathogens such as
Klebsiella pneumoniae (1) or to develop bacteremia and shock
from typical pathogens, most notably Streptococcus pneumoniae
(2). The mechanisms by which alcohol abuse increases the risk of
pneumonia are likely multiple and include increased risk of aspiration of oropharyngeal flora, decreased mucociliary clearance of
bacterial pathogens from the upper airway, and impaired pulmonary host defenses. Perhaps the most prominent effects on host
defense involve the alveolar macrophage, the first cellular line of
defense against pathogens within the lower airways. In experimental models, chronic ethanol ingestion suppresses chemokine responses and pathogen clearance from the airways (3–9) and impairs alveolar macrophage innate immunity, including phagocytic
function and IL-12 secretion in response to endotoxin (10). Such
studies support the evolving recognition that alcohol abuse has
specific effects on innate immune function within the lower airways and that the increased risk of pneumonia in these patients
cannot be ascribed solely to factors such as malnutrition, aspira-
*Atlanta Veterans Affairs Medical Center, †Department of Medicine, ‡Department of
Physiology, and §Department of Pediatrics, Emory University School of Medicine,
Atlanta, GA 30322
Received for publication May 11, 2005. Accepted for publication September 1, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work is supported by National Institutes of Health, National Institute on Alcohol
Abuse and Alcoholism P50 AA013757 and a Veterans Affairs Merit Review (to D.M.G.).
2
Address correspondence and reprint requests to Dr. David M. Guidot, Atlanta Veterans Affairs Medical Center (151-P), 1670 Clairmont Road, Decatur, GA 30033.
E-mail address: [email protected]
Copyright © 2005 by The American Association of Immunologists, Inc.
tion, or poor oral hygiene. However, the precise mechanisms by
which chronic ethanol ingestion impairs alveolar macrophage
function are poorly understood.
Within the alveolar space, relatively undifferentiated circulating
monocytes are recruited and undergo terminal maturation and differentiation into alveolar macrophages in response to stimulation
by GM-CSF. GM-CSF is a 23-kDa protein that was originally
isolated from mouse lung extracts but was named because of its
potent effects on bone marrow development (fully reviewed in Ref.
11). However, when a GM-CSF knockout mouse was constructed
a little more than a decade ago, the phenotype was unexpected
(12). Specifically, the absence of GM-CSF expression had no discernible effect on hematopoiesis. However, the mice developed a
severe pulmonary phenotype that closely resembled pulmonary alveolar proteinosis (PAP)3 in humans. Insights from the mouse
studies ultimately led to the recognition that most patients with
PAP have acquired Abs to GM-CSF that neutralize the protein
within the alveolar space and prevent binding to its receptor on the
alveolar macrophage membrane (13). Although PAP was first described based on the accumulation of surfactant proteins and phospholipids within the alveolar space, we now recognize that it is due
to global defects in GM-CSF-dependent alveolar macrophage
function that include impaired surfactant recycling, as well as depressed innate immune functions (13). Therefore, patients with
PAP have an acquired, functional deficiency in GM-CSF (as opposed to a genetic mutation) that produces alveolar macrophage
dysfunction. With this background, we hypothesized that alcoholmediated suppression of alveolar macrophage function could involve a functional defect in GM-CSF expression and/or signaling
within the alveolar space.
3
Abbreviations used in this paper: PAP, pulmonary alveolar proteinosis; GMCSFR␣, GM-CSF receptor ␣ subunit; GM-CSFR␤, GM-CSF receptor ␤ subunit.
0022-1767/05/$02.00
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Although it is well recognized that alcohol abuse impairs alveolar macrophage immune function and renders patients susceptible
to pneumonia, the mechanisms are incompletely understood. Alveolar macrophage maturation and function requires priming by
GM-CSF, which is produced and secreted into the alveolar space by the alveolar epithelium. In this study, we determined that
although chronic ethanol ingestion (6 wk) in rats had no effect on GM-CSF expression within the alveolar space, it significantly
decreased membrane expression of the GM-CSF receptor in alveolar macrophages. In parallel, ethanol ingestion decreased
cellular expression and nuclear binding of PU.1, the master transcription factor that activates GM-CSF-dependent macrophage
functions. Furthermore, treatment of ethanol-fed rats in vivo with rGM-CSF via the upper airway restored GM-CSF receptor
membrane expression as well as PU.1 protein expression and nuclear binding in alveolar macrophages. Importantly, GM-CSF
treatment also restored alveolar macrophage function in ethanol-fed rats, as reflected by endotoxin-stimulated release of TNF-␣
and bacterial phagocytosis. We conclude that ethanol ingestion dampens alveolar macrophage immune function by decreasing
GM-CSF receptor expression and downstream PU.1 nuclear binding and that these chronic defects can be reversed relatively
quickly with rGM-CSF treatment in vivo. The Journal of Immunology, 2005, 175: 6837– 6845.
6838
Materials and Methods
Ethanol feeding
Adult Male Sprague-Dawley rats (initial weights, 150 –200 g; Charles
River Laboratory) were fed the Lieber-DeCarli liquid diet (Research Diets)
containing either ethanol (36% of total calories) or an isocaloric substitution with maltin-dextrin ad lib for 6 wk as published previously (18). All
work was performed with the approval of the Institutional Care and Use of
Animals Committee at the Atlanta Veterans Affairs Medical Center.
rGM-CSF treatment via upper airway in vivo
In some experiments, control-fed and ethanol-fed rats were treated with
recombinant rat GM-CSF (PeproTech) or PBS vehicle alone via intranasal
instillation for 3 consecutive days as we published previously (18). Briefly,
rats were anesthetized with 2% isofluorane before gently instilling 500 ng
of GM-CSF in 100 ␮l of PBS or 100 ␮l of PBS alone into one nostril with
a pipette, which is then delivered into the airway by reflex sniffing by the
anesthetized rat. Rats were then sacrificed 24 h after the third treatment
with GM-CSF to obtain alveolar macrophages as described below.
Isolation of alveolar macrophages
Following pentobarbital anesthesia (100 mg/kg i.p.), a tracheostomy tube
was placed and rat lungs were lavaged four times with 10 ml of sterile cold
PBS (pH 7.4). The recovered lavage solution was centrifuged at 1500 rpm
for 7 min, and the cell pellet resuspended in sterile medium for functional
studies. This procedure yielded ⬎95% alveolar macrophages.
RNA extractions and RT-PCR for GM-CSF expression
Total RNA was extracted from lung tissue using Qiagen RNA extraction
kit. RNA from each sample was reverse transcribed followed by PCR with
gene-specific primers. The number of cycles (35 for G3PDH and 40 for
GM-CSF) was chosen from our preliminary optimization experiments for
each gene product. PCR conditions were as follows: 5 min of denaturation
at 94°C followed by 35– 40 cycles of 45 s of denaturation at 94°C, 45 s
annealing at 60°C or 53°C, and 90-s extension at 72°C, followed by a final
extension at 72°C for 7 min. PCR products were separated on a 2% agarose
gel containing ethidium bromide. For quantitation, PCR bands were
scanned using an imaging system linked to a computer with analysis software. Relative amounts of G3PDH (983 bp) and GM-CSF (300 bp) were
quantitated and expressed as GM-CSF:G3PDH ratios. Specific primers
were as follows: G3PDH, (sense) 5⬘-GAAGGTCGGTGTCAACGGATT
GGC-3⬘, and (antisense) 5⬘-CATGTAGGCCATGAGGTCCACCAC-3⬘;
and GM-CSF, (sense) 5⬘-TCTGAGCCTCCTAAATGAC-3⬘, and (antisense) 5⬘-CATTTCTGGACCGGCTTC-3⬘.
Rat GM-CSF primers were designed in our lab and were obtained from
Sigma-Genosys. Rat G3PDH primers were purchased from Promega. Molecular mass marker HaeIII digest with fragment sizes 1358 –72 bp was
purchased from Amersham Biosciences.
Determination of GM-CSF protein levels in lung lavage fluid
In selected experiments, rat lungs were lavaged via a tracheostomy tube
with saline (5 cc ⫻ 3). The recovered lavage fluid (12 ⫾ 1 cc in all cases)
was centrifuged at 1500 ⫻ g for 10 min, and GM-CSF levels in the supernatants were determined by a rat-specific ELISA (R&D Systems). The
lower limit of detection was 10 pg/ml. Data are reported as total amount (in
nanograms) of GM-CSF present in the lung lavage fluid.
Flow cytometric detection of membrane and intracellular
receptor expression
Membrane and intracellular expression of GM-CSF receptors on alveolar
macrophages were measured by an established protocol (19). Briefly, cells
were incubated for 30 min at room temperature with rabbit polyclonal Abs
(Santa Cruz Biotechnology) to either the rat GM-CSF receptor ␣ or ␤
subunit or to an isotype-matched control Ab. Cells were washed to remove
unbound Ab followed by 30 min incubation at room temperature with
secondary anti-rabbit Ab conjugated to FITC. For intracellular staining of
the receptors, cells were first permeabilized with 0.1% saponin in PBS,
followed by staining with the Ab. Cells were washed with PBS-saponin
before adding FITC-conjugated secondary Ab (Santa Cruz Biotechnology).
Cells were washed with PBS and were kept in the dark at 4°C until analyzed. The labeled cells were analyzed by FACScan flow cytometer (BD
Biosciences). Data are expressed both as percentage of cells positive for the
␣ subunit or the ␤ subunit, as well as the mean channel fluorescence for
positive cells in each group.
Western blot analyses of PU.1 protein expression
Cell lysates were prepared by adding lysing reagent to isolated alveolar
macrophages. Fifty micrograms of protein from each sample were loaded
onto a 12% acrylamide gel and electrophoresed at 150 V for 75 min as
described previously (17). The separated proteins were transferred to a 0.45
␮M polyvinylidene difluoride membrane at 15 V for 75 min. Membranes
were blocked at room temperature for 1 h in TBS with 0.2% Tween 20
(TBS-T) containing 5% nonfat dry milk in TBS-T. Primary Ab for PU.1
(Santa Cruz Biotechnology) at 1/50 in 5% milk in TBS-T was added to the
membranes and kept at 4°C overnight. After several washing steps to remove unbound primary Ab, membrane was incubated at room temperature
with HRP-labeled anti-rabbit IgG secondary Ab in 5% milk in TBS-T for
2 h. After adding ECL chemiluminescence reagent (Amersham Biosciences) to the membranes, bands were detected using a Bio-Rad Imaging
System. For those experiments involving prior treatment with GM-CSF,
PU.1 expression was normalized to expression of the housekeeping protein
G3PDH to control for any potential proliferative effects of GM-CSF.
PU.1 electromobility shift assay
Cells were washed with cold PBS, and nuclear binding proteins were extracted. Protein concentration was determined by the Bradford method using Bio-Rad protein assay reagent. A double-stranded PU.1 consensus oligonucleotide (5⬘-TGAAAGAGGAACTTGGT-3⬘) was radiolabeled with
[32P]␥-ATP using T4 polynucleotide kinase enzyme. Nuclear protein (10
␮g) was incubated with radiolabeled PU.1 for 30 min at room temperature.
For competition reactions, nonradiolabeled consensus and mutated PU.1
double-stranded oligonucleotides (5⬘-TGAAAGAGCTACTTGGT-3⬘)
were added to the reaction mixture at 50⫻ molar concentration as a control
to confirm the identity of the PU.1-DNA complexes. DNA-protein complexes were separated on 6% native polyacrylamide gel (20:1 acrylamide/
bis ratio) for 2–3 h. Gels were fixed in a 10% acetic acid/10% methanol
solution for 10 min, dried under vacuum, and exposed to phosphoscreen.
Alveolar macrophage bacterial phagocytosis
In some experiments, alveolar macrophages were isolated from control-fed
and ethanol-fed rats that had been treated with either GM-CSF or vehicle
via the upper airway as described above. In those experiments, the macrophages were incubated for 4 h with FITC-labeled Staphylococcus aureus
(Wood strain without protein A; Molecular Probes) in a 1:1 ratio; after
incubation, cells were washed several times with PBS and examined by
confocal microscopy. Phagocytosis images were obtained by laser confocal
microscopy with Fluoview analysis (Olympus). Representative photomicrographs at ⫻60 magnification were obtained at a depth of 3–5 m in the
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GM-CSF is produced by the alveolar epithelium and binds to
specific GM-CSF receptors on the plasma membrane of the alveolar macrophage and thereby activates an intracellular signaling
pathway that ultimately leads to expression and nuclear binding of
the transcription factor PU.1 (13). PU.1 is a member of the ETS
family of transcription factors previously identified as a master
transcription factor in the proliferation and differentiation of myeloid cells (14), and its expression is lost in alveolar macrophages
both in patients with PAP and in GM-CSF knockout mice (11, 15).
Lung-specific transgenic expression of GM-CSF in the type II cells
of these mice restores PU.1 expression and normalizes alveolar
macrophage function (16). In fact, constitutive expression of PU.1
in alveolar macrophages of GM-CSF-deficient mice by transfection with a PU.1-containing vector completely normalizes alveolar
macrophage function (17), confirming the critical role for PU.1 in
GM-CSF signal transduction. Thus, GM-CSF-dependent expression of PU.1 appears to be absolutely required for terminal maturation and function of the alveolar macrophage. However, to our
knowledge, the effects of ethanol ingestion on GM-CSF expression
and/or signaling to the alveolar macrophage within the alveolar
space have not been examined. Therefore, we examined GM-CSF
expression and key elements of its signaling, namely GM-CSF
receptor expression and PU.1 expression, in our rat model of
chronic ethanol ingestion. We then determined the effects of rGMCSF treatment in vivo on restoring GM-CSF signal responsiveness, as well as innate immune function, in the alveolar macrophages of ethanol-fed rats.
ETHANOL AND GM-CSF SIGNALING
The Journal of Immunology
6839
z-plane of the macrophage, and both fluorescent and Nomarski differential
contrast images were obtained. The cell membranes in the differential contrast images were digitally outlined and then these digital outlines were
superimposed on the corresponding fluorescent images. In other experiments, alveolar macrophages from control-fed and ethanol-fed rats were
isolated and then incubated with the FITC-labeled S. aureus in a 1:1 ratio ⫾ rGM-CSF (10 ng/ml) in vitro for 4 h.
TNF-␣ release from rat alveolar macrophages
Freshly isolated alveolar macrophages (106 cells/ml) were incubated overnight ⫾ 100 ng/ml LPS (Escherichia coli 0111:B4). Supernatants were
collected and frozen at ⫺70°C. TNF-␣ in these supernatants was measured
using a rat TNF-␣ ELISA kit from BioSource International.
Statistics
Data are presented as mean ⫾ SEM. Data analysis was done by ANOVA
with Student-Newman-Keuls test for group comparison and were considered statistically significant at a value of p ⬍ 0.05.
Results
The first potential mechanism we examined was whether chronic
ethanol ingestion dampened GM-CSF-dependent macrophage
function by inhibiting expression of GM-CSF within the lung. We
determined that ethanol ingestion in fact had no apparent effect on
GM-CSF expression. As shown in Fig. 1, GM-CSF gene expression, as determined by RT-PCR (Fig. 1A shows a representative
PCR gel and Fig. 1B shows the summary data from all experiments), was the same ( p ⬎ 0.05) in the lungs of control-fed and
ethanol-fed rats. We next examined GM-CSF protein levels in the
alveolar space where GM-CSF priming of alveolar macrophages
occurs. As shown in Fig. 1C, chronic ethanol ingestion had no
effect ( p ⬎ 0.05) on the levels of GM-CSF protein in the lung
lavage fluid when compared with control-fed rats. Taken together,
these initial studies indicate that chronic ethanol ingestion had no
significant effect on GM-CSF expression within the lungs of
ethanol-fed rats.
Chronic ethanol ingestion decreased membrane expression of
the GM-CSF receptor in the alveolar macrophage
As ethanol ingestion did not appear to affect GM-CSF protein
availability within the alveolar space, we next examined whether
ethanol ingestion could be interfering with GM-CSF signaling to
the alveolar macrophage. As a first step in these experiments, we
examined membrane expression of the GM-CSF receptor in alveolar macrophages freshly isolated from control-fed and ethanol-fed
rats. As shown in Fig. 2, chronic ethanol ingestion significantly
( p ⬍ 0.05) decreased membrane expression of both the GM-CSF
receptor ␣ subunit (GM-CSFR␣) and the GM-CSF receptor ␤ subunit (GM-CSFR␤). Fig. 2A shows the relative number of cells that
were positive for the GM-CSFR ␣ and ␤ subunit, with cells from
ethanol-fed rats expressed relative to cells from control-fed rats.
Fig. 2B shows the relative mean channel fluorescence per cell for
those cells that were positive for the ␣ and ␤ subunit and again
with the cells from ethanol-fed rats expressed relative to cells from
control-fed rats. Although ethanol ingestion did not significantly
decrease the percentage of alveolar macrophages that were positive for GM-CSFR␣ membrane expression, the relative expression
(mean channel fluorescence) for GM-CSFR␣ per positive cell was
decreased by ⬃50% ( p ⬍ 0.05). By comparison, ethanol ingestion
not only decreased the percentage of alveolar macrophages that
were positive for GM-CSFR␤ membrane expression by ⬃50%
( p ⬍ 0.05), the relative expression for GM-CSFR␤ per positive
cell was likewise decreased by ⬃50% ( p ⬍ 0.05). Importantly,
decreased membrane expression of the GM-CSF receptor was rel-
FIGURE 1. The effects of chronic ethanol ingestion on lung GM-CSF
expression in rats. Shown in A is a representative RT-PCR gel showing
mRNA levels for GM-CSF as well as the housekeeping gene G3PDH in the
lungs of a control-fed rat and an ethanol-fed rat. B, The summary data for
the ratio of GM-CSF/G3PDH gene expression in control-fed and ethanolfed rats. C, The levels of GM-CSF protein, as determined by a rat-specific
ELISA, in the lung lavage fluids of control-fed and ethanol-fed rats. B and
C, Each value represents the mean ⫾ SEM of four rats in each group.
atively specific, at least as reflected by our determination that
membrane expression for the IL-6R was the same ( p ⬎ 0.05) in
alveolar macrophages from ethanol-fed and control-fed rats (Fig.
3). Therefore, chronic ethanol ingestion significantly decreased
membrane expression for both subunits of the GM-CSF receptor in
alveolar macrophages, and this effect was more pronounced for the
␤ subunit, which is responsible for initiating intracellular signaling
following GM-CSF binding.
In parallel, chronic ethanol ingestion decreased expression of
the transcription factor PU.1 that is required for GM-CSFdependent functions in alveolar macrophages
We next compared the expression of PU.1, the master transcription
factor for GM-CSF-dependent functions, in alveolar macrophages
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Chronic ethanol ingestion had no apparent effect on pulmonary
GM-CSF expression
6840
ETHANOL AND GM-CSF SIGNALING
counts
counts
counts
counts
A
A
FL1-Height
40
35
Membrane
GM-CSFR
(% cells positive)
FL1-Height
FL1-Height
FL1-Height
50
30
Membrane
IL-6R
25
(% cells positive)
20
25
*
15
10
5
0
0
B
B
80
70
60
(Mean Channel
Fluorescence)
50
*
*
40
Membrane
IL-6R
50
(Mean Channel
Fluorescence)
30
25
20
10
0
control
ethanol
Alpha
control
ethanol
Beta
FIGURE 2. The effects of chronic ethanol ingestion on membrane expression of the GM-CSF receptor in alveolar macrophages, as determined
by flow cytometry. A, The relative number of cells that were positive for
the GM-CSFR ␣ and ␤ subunit, with cells from ethanol-fed rats expressed
relative to cells from control-fed rats. Insets, Representative histograms of
cell counts vs fluorescent intensity for the expression of GM-CSFR ␣ and
␤ (gray line) in alveolar macrophages from control-fed animals. The histograms on the left in each inset represent cells stained with an appropriate
isotype-matched control Ab. B, The relative mean channel fluorescence per
cell for those cells that were positive for the ␣ and ␤ subunit and again with
the cells from ethanol-fed rats expressed relative to cells from control-fed
rats. Each value represents the mean ⫾ SEM of six determinations. ⴱ, p ⬍
0.05 compared with control.
from ethanol-fed and control-fed rats. Although ethanol ingestion
significantly decreased GM-CSF receptor expression, these findings did not necessarily mean that GM-CSG signaling was sufficiently impaired to explain the dampened macrophage function.
Therefore, we reasoned that the next target to examine was PU.1
expression because the loss of PU.1 expression in the alveolar
macrophages of patients with alveolar proteinosis and in GM-CSF
knockout mice is causally related to alveolar macrophage dysfunction. As shown in Fig. 4, chronic ethanol ingestion significantly
( p ⬍ 0.05) decreased PU.1 protein expression in alveolar macrophages from ethanol-fed rats compared with control-fed rats. Shown
in Fig. 4A are representative Western blots for PU.1 in macrophages
from two control-fed and two ethanol-fed rats, while shown in Fig. 4B
are the summary data for all of the experimental determinations. Importantly, decreased PU.1 expression by ethanol was associated with
decreased nuclear binding of PU.1 as discussed later.
Treatment with rGM-CSF in vivo restores alveolar macrophage
membrane expression of the GM-CSF receptor in ethanol-fed rats
We had shown previously that treatment with rGM-CSF via the
upper airway restores alveolar epithelial barrier function in chronic
0
control
ethanol
FIGURE 3. Membrane expression of the IL-6R as determined by flow
cytometry in alveolar macrophages from control-fed and ethanol-fed rats.
A, The relative number of cells that were positive for the membrane IL-6R,
with cells from ethanol-fed rats expressed relative to cells from control-fed
rats. Insets, Representative histograms of cell counts vs fluorescent intensity for the expression of IL-6R (gray) in alveolar macrophages from control- and ethanol-fed animals. The histograms on the left in each inset
represent cells stained with an appropriate isotype-matched control Ab. B,
The relative mean channel fluorescence per cell for those cells that were
positive for IL-6R and again with the cells from ethanol-fed rats expressed
relative to cells from control-fed rats. Each value represents the mean ⫾
SEM of six determinations. ⴱ, p ⬍ 0.05 compared with control.
ethanol-fed rats. Therefore, we reasoned that similar treatment
could mitigate the dampening effects of chronic ethanol ingestion
on GM-CSF-dependent functions of the alveolar macrophage. As
a first step in these experiments, we examined the effects of rGMCSF treatment in vivo on membrane expression of the GM-CSF
receptor. As shown in Fig. 5, in these experiments ethanol ingestion again decreased membrane expression (as reflected by mean
channel fluorescence) by ⬃50% for both the ␣ and the ␤ subunits.
However, rGM-CSF treatment significantly ( p ⬍ 0.05) increased
membrane expression for both the ␣ subunit (GM-CSFR␣; Fig.
5A) and the ␤ subunit (GM-CSFR␤; Fig. 5B). In fact, alveolar
macrophage membrane expression for each subunit was increased
⬃3-fold by GM-CSF treatment in ethanol-fed rats. In contrast,
GM-CSF treatment had no significant effect ( p ⬎ 0.05) on membrane expression of either the ␣ or the ␤ subunit in control-fed rats.
This GM-CSF-induced increase in membrane expression of the
GM-CSF receptor in alveolar macrophages from ethanol-fed rats
appeared to be mediated in significant part by increased translocation of the receptor subunits from intracellular pools to the membrane. Specifically, rGM-CSF treatment significantly ( p ⬍ 0.05)
increased the membrane to intracellular ratio by ⬃2-fold for both
the ␣ subunit (Fig. 6A) and the ␤ subunit (Fig. 6B) in alveolar
macrophages from ethanol-fed rats. In contrast, rGM-CSF treatment had no effect ( p ⬎ 0.05) on the relative cellular distribution
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
Membrane
GM-CSFR
The Journal of Immunology
6841
A
-ethanol-
A
counts
-control-
160
40kD a
40
kDa →
→
140
FL1-Height
120
3
B
Cellular
PU.1 protein
(relative
densitometry)
**
Membrane 100
GM-CSFRα
2.5
(Mean Channel
Fluorescence)
2
80
60
1.5
*
40
1
*
20
0.5
0
control
ethanol
FIGURE 4. The effects of chronic ethanol ingestion on alveolar macrophage protein expression of the GM-CSF-dependent transcription factor
PU.1. A, A representative Western blot of total cellular protein from alveolar macrophages from two control-fed and two ethanol-fed rats probed
with an Ab against rat PU.1. B, The summary data of the relative densitometry (in arbitrary units) of PU.1 protein in both experimental groups,
with each value representing the mean ⫾ SEM of six determinations. ⴱ,
p ⬍ 0.05 compared with control group.
B
counts
0
160
**
140
120
FL1-Height
Membrane 100
GM-CSFRβ
(Mean Channel
Fluorescence)
80
60
20
of either subunit in alveolar macrophages from control-fed rats.
Fig. 7 shows representative fluorescent images for GM-CSFR␣ on
the cell membranes of an alveolar macrophage from an ethanol-fed
rat (Fig. 7, left panel) and an alveolar macrophage from an ethanolfed rat treated with rGM-CSF (Fig. 7, right panel). Consistent with
the flow cytometry data in Fig. 6A, there is visual evidence of
increased GM-CSFR␣ expression on the cell membrane following
GM-CSF treatment. Taken together, the results in Figs. 5–7 suggest that rGM-CSF restores membrane expression of the GM-CSF
receptor in alveolar macrophages from ethanol-fed rats, at least in
part, by mobilizing receptor subunits from the intracellular pool to
the plasma membrane.
Treatment with rGM-CSF in vivo restores PU.1 protein
expression as well as PU.1 nuclear binding in alveolar
macrophages in ethanol-fed rats
To determine whether restoration of GM-CSF receptor membrane
expression translated to a restoration of PU.1 expression and therefore signaling capability, we next examined PU.1 expression as
well as nuclear binding in alveolar macrophages from control-fed
and ethanol-fed rats with or without treatment with rGM-CSF in
vivo. For these experiments, PU.1 protein expression was quantitated and expressed relative to the housekeeping protein G3PDH.
This was done to verify that any GM-CSF-mediated increases in
PU.1 protein expression were not due solely to generalized growth
factor effects of GM-CSF on the alveolar macrophages. As shown
in Fig. 8A, rGM-CSF treatment in vivo significantly ( p ⬍ 0.05)
increased cellular PU.1 protein expression in alveolar macrophages from ethanol-fed rats by ⬃44%. By comparison, GM-CSF
treatment induced a much more modest, albeit significant ( p ⬍
0.05), increase in PU.1 protein expression in alveolar macrophages
from control-fed rats. In parallel, rGM-CSF treatment increased
nuclear binding of PU.1 in alveolar macrophages from ethanol-fed
rats. As shown in Fig. 8B, GM-CSF treatment in vivo increased
PU.1 nuclear binding as determined by electromobility shift assay
on nuclear extracts from freshly isolated alveolar macrophages in
each experimental group. Also evident in this representative gel is
that chronic ethanol ingestion decreased PU.1 nuclear binding in
parallel to the decrease in cellular PU.1 protein expression shown
in Fig. 4. In contrast, GM-CSF treatment in vivo increased PU.1
nuclear binding in alveolar macrophages from both control-fed and
ethanol-fed rats, although in general, this effect was more dramatic
in macrophages from ethanol-fed rats. Taken together, the results
0
control
ethanol
control
ethanol
+GM-CSF
FIGURE 5. The effects of rGM-CSF treatment on GM-CSF receptor
expression in alveolar macrophages. Control-fed and ethanol-fed rats were
given either GM-CSF (500 ng in 100 ␮l of PBS) or PBS alone intranasally
daily for 3 consecutive days. Twenty-four hours after the third treatment,
alveolar macrophages were isolated and membrane expression of the GMCSFR␣ (A) and the GM-CSFR␤ (B) determined by quantitating the mean
channel fluorescence (MCF) by flow cytometry and expressed as a percentage of the MCF in macrophages from control-fed rats. Each value
represents the mean ⫾ SEM of six determinations. ⴱ, p ⬍ 0.05 compared
with untreated, control-fed group. ⴱⴱ, p ⬍ 0.05 compared with untreated,
ethanol-fed group. Inset in each panel shows a representative histogram of
cell counts vs fluorescent intensity for membrane GM-CSF receptor expression in alveolar macrophages from ethanol-fed animals after GM-CSF
treatment (gray peak on the right) as compared with no GM-CSF treatment
(peak on the left).
in Fig. 8 suggest that rGM-CSF treatment in vivo restores PU.1
protein expression and nuclear binding in alveolar macrophages
from ethanol-fed rats, and this increased PU.1 expression corresponds to restoration of GM-CSF receptor expression (as shown in
Figs. 5–7).
Treatment with rGM-CSF in vivo restores alveolar macrophage
innate immune function in ethanol-fed rats
Our final step in this study was to determine whether rGM-CSF
treatment in vivo could actually improve the functional status of
the alveolar macrophage in ethanol-fed rats. Clearly, restoration of
GM-CSF receptor and PU.1 protein expression in the alveolar
macrophage of ethanol-fed rats would be of limited significance if
this did not translate into improved immune function. We first
examined endotoxin-induced secretion of TNF-␣ by freshly isolated alveolar macrophages in vitro. As shown in Fig. 9, basal
TNF-␣ secretion was the same ( p ⬎ 0.05) in alveolar macrophages
from control-fed and ethanol-fed rats, and prior GM-CSF treatment had the same ( p ⬎ 0.05) modest effect on increasing basal
TNF-␣ secretion in each group. However, endotoxin-stimulated
TNF-␣ secretion was significantly decreased ( p ⬍ 0.05) in alveolar macrophages from ethanol-fed rats (Fig. 9, third bar in each
group). However, alveolar macrophages from GM-CSF-treated,
ethanol-fed rats had the same ( p ⬎ 0.05) endotoxin-stimulated
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*
40
A
ETHANOL AND GM-CSF SIGNALING
1
counts
6842
*
0.75
GM-CSFRα
Ratio of
Membrane to
Intracellular
Mean Channel
Fluorescence
FL1-Height
0.5
0.25
B
1
counts
0
0.75
Ratio of
Membrane to
Intracellular
Mean Channel
Fluorescence
*
0.5
0.25
0
control
ethanol
control
ethanol
+GM-CSF
FIGURE 6. The effects of rGM-CSF treatment on the relative distribution of the GM-CSF receptor in the membrane vs the intracellular compartment of alveolar macrophages. Control-fed and ethanol-fed rats were
treated with either GM-CSF or PBS alone intranasally as detailed in Materials and Methods and in Fig. 5. Twenty-four hours after the third treatment, alveolar macrophages were isolated, and both membrane expression
and intracellular expression of the GM-CSFR␣ (A) and the GM-CSFR␤
(B) were determined by quantitating the mean channel fluorescence (MCF)
by flow cytometry. In each experimental determination, the ratio of the
membrane MCF and the intracellular MCF was calculated and expressed as
a ratio. Each value represents the mean ⫾ SEM of six determinations. ⴱ,
p ⬍ 0.05 compared with untreated, ethanol-fed group. Inset in each panel
shows representative histogram of cell counts vs fluorescent intensity for
intracellular expression of GM-CSF receptor (gray) in alveolar macrophages from control-fed animals. The histograms on the left in each inset
represent cells stained with an appropriate isotype-matched control Ab.
secretion as alveolar macrophages from control-fed rats (hatched
gray line connects these two groups in Fig. 9). Notably, TNF-␣
secretion was greatest ( p ⬍ 0.05) in endotoxin-stimulated macrophages from control-fed rats, indicating that even under “normal”
conditions, GM-CSF stimulation augments the endotoxin response. We next examined the effects of GM-CSF treatment on the
ability of alveolar macrophages isolated from ethanol-fed rats to
phagocytose bacteria in vitro. We did not include macrophages
from control-fed rats in these studies. The confocal microscopy
images in Fig. 10 illustrate that GM-CSF treatment augmented the
ability of macrophages from ethanol-fed rats to phagocytose the
fluorescent bacteria. Fig. 10, A and B, shows the corresponding
fluorescent and differential contrast images for a macrophage from
an untreated, ethanol-fed rat, in which relatively few bacteria have
been phagocytosed, and the majority of these remain in the periphery of the cell. In contrast, as shown in Fig. 10, C and D,
alveolar macrophages from ethanol-fed rats treated with GM-CSF
were able to ingest and internalize more of the fluorescent bacteria.
FIGURE 7. Immunofluorescence labeling of membrane GM-CSFR␣
chain on rat alveolar macrophages. A, Representative images of intracellular expression of GM-CSFR␣ in alveolar macrophages from control- and
ethanol-fed animals. Cells were made permeable with saponin before staining with anti-GM-CSFR␣ Ab, followed by an appropriate FITC-conjugated secondary Ab. B, Representative images of membrane expression of
GM-CSFR␣ in alveolar macrophages from control- and ethanol-fed rats
that were given either PBS or recombinant rat GM-CSF (500 ng/ml) intranasally for 3 days. Cells were stained with an anti-GM-CSFR␣ Ab,
followed by an appropriate FITC-conjugated secondary Ab. Cells were
fixed in methanol, mounted on slides, and examined by fluorescent microscopy. These qualitative images correlate with the quantitative analyses
of GM-CSFR␣ expression shown in Fig. 5.
Taken together, the results shown in Figs. 9 and 10 indicate that
GM-CSF treatment improved innate immune functions in the alveolar macrophages of ethanol-fed rats.
Treatment with rGM-CSF in vitro restores bacterial phagocytic
capacity in alveolar macrophages from ethanol-fed rats
Although the results shown in Figs. 9 and 10 are striking, it is
possible that GM-CSF treatment in vivo recruited and primed a
new population of alveolar macrophages from peripheral monocyte pools and had no effect on the existing alveolar macrophage pool. To test whether or not GM-CSF treatment could
directly improve immune function in resident alveolar macrophages in the alcoholic lung, we performed additional experiments in which macrophages were isolated and then stimulated
with GM-CSF in vitro. In these conditions, alveolar macrophages adhere tightly to plastic, and therefore, we could not
perform flow cytometry to assess GM-CSF receptor membrane
expression. However, adherent macrophages remain functional,
and therefore, we assessed bacterial phagocytic capacity with
and without GM-CSF treatment in vitro. We reasoned that if
GM-CSF treatment could directly increase a relevant macrophage function in these conditions, this would provide further
evidence that its effects in vivo could not be solely ascribed to
recruiting an entirely new alveolar macrophage population from
an extrapulmonary monocyte pool. In these experiments, isolated macrophages from control-fed and ethanol-fed rats were
incubated with the fluorescent bacteria for 4 h, ⫾ recombinant
rat GM-CSF (10 ng/ml). The cells were examined by fluorescent microscopy and the percentage of macrophages that had
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FL1-Height
GM-CSFRβ
The Journal of Immunology
6843
*
3000
A
TNFα
secretion
(pg/ml)
0.3
**
1000
**
Cellular
PU.1
protein
expression
(relative to
G3PDH)
2000
*
0
0.2
LPS
GM-CSF
-
+
+
-
control
0.1
0
+ GM-CSF
+GM-CSF
control
ethanol
GM-CSF
(10 ng/ml)
0
C
E
C
1
2
3
PU.1
50x
mPU.1
50x
E C
E
C
E
4
6
7
8
5
bound
-
+
+
-
+
+
ethanol
FIGURE 9. The effects of rGM-CSF treatment on endotoxin-induced
secretion of TNF-␣ by alveolar macrophages in vitro. Control-fed and ethanol-fed rats were treated with either GM-CSF or PBS alone intranasally.
Twenty-four hours after the third treatment, alveolar macrophages were
isolated, and 106 cells/ml were incubated overnight ⫾ 100 ng/ml LPS (E.
coli 0111:B4). TNF-␣ concentrations in these supernatants were measured
by ELISA as detailed in Materials and Methods. Each value represents the
mean ⫾ SEM of three determinations. ⴱ, p ⬍ 0.05 increased compared
with LPS-stimulated, untreated (no GM-CSF) control; ⴱⴱ, p ⬍ 0.05 decreased compared with LPS-stimulated, untreated (no GM-CSF) control.
Gray hatched line, p ⬎ 0.05 same compared with LPS-stimulated, untreated (no GM-CSF) control.
vs 92 ⫾ 5%; p ⬍ 0.05) compared with macrophages from control-fed rats, and this was almost completely reversed with exogenous GM-CSF treatment (83 ⫾ 6% vs 56 ⫾ 8%; p ⬍ 0.05).
Discussion
free
FIGURE 8. The effects of rGM-CSF treatment on PU.1 protein expression (A) and nuclear binding (B) in alveolar macrophages. Control-fed and
ethanol-fed rats were treated with either GM-CSF or PBS alone intranasally as detailed in Materials and Methods and in Fig. 5. Twenty-four hours
after the third treatment, alveolar macrophages were isolated. A, The cellular PU.1 protein expression relative to the housekeeping protein G3PDH
in each experimental group, with each value representing the mean ⫾ SEM
of six determinations. The inset shows a representative Western blot of
cellular protein from two ethanol-fed animals treated with vehicle alone
and two ethanol-fed animals treated with GM-CSF that were probed with
a polyclonal Ab for PU.1. The band at 40 kDa is consistent with the known
size of PU.1, and as shown in the right side of the gel, this band is eliminated in the presence of a 20⫻ concentration of the control peptide. ⴱ, p ⬍
0.05 compared with untreated, control-fed group. ⴱⴱ, p ⬍ 0.05 compared
with untreated, ethanol-fed group. B, A representative electromobility shift
assay in which nuclear extracts from alveolar macrophages in each experimental group (C ⫽ control diet and E ⫽ ethanol diet). Lane 0 is free probe
without nuclear extract. Lanes 1– 4 were probed with a 32P-labeled PU.1
consensus oligonucleotide. Lanes 3 and 4, Rats were treated with rGMCSF before macrophage isolation. Lanes 5– 8, The results of probing nuclear extracts of macrophages from control-fed and ethanol-fed rats with
the 32P-labeled PU.1 consensus oligonucleotide and either a 50⫻ concentration of unlabeled PU.1 consensus nucleotide (lanes 5 and 6) or a 50⫻
concentration of a 32P-labeled mutated form of the PU.1 consensus nucleotide (lanes 7 and 8).
ingested one or more bacteria determined. As shown in Fig. 11
(and consistent with the results shown in Fig. 4 above), the
percentage of macrophages from ethanol-fed rats that had any
detectable phagocytic function in vitro was decreased (56 ⫾ 8%
In this study, we determined that although chronic ethanol ingestion in rats did not affect GM-CSF expression within the alveolar
space, it nevertheless dampened GM-CSF signaling capacity by
decreasing expression of GM-CSF receptors on the surface of the
alveolar macrophage. This appeared to be relatively specific, as
membrane expression of the IL-6R was not affected by chronic
ethanol ingestion. In parallel and likely as a consequence of decreased membrane receptors, the expression and nuclear binding of
the GM-CSF-dependent transcription factor PU.1 was also decreased. Remarkably, treatment with rGM-CSF via the upper airway restored membrane expression of the GM-CSF receptor as
well as the downstream expression and nuclear binding of PU.1 in
the alveolar macrophages of ethanol-fed rats. Even more importantly in terms of potential clinical relevance, GM-CSF treatment
restored innate immune functions in alveolar macrophages of ethanol-fed rats, as reflected by endotoxin-induced secretion of
TNF-␣ and bacterial phagocytosis. Taken together, these results
suggest that chronic ethanol ingestion inhibits alveolar macrophage immune function by dampening, but not completely blocking, macrophage responsiveness to the stimulatory effects of ambient GM-CSF within the alveolar microenvironment.
Furthermore, this study raises the possibility that in the appropriate
clinical context impaired pulmonary host defenses in alcoholic patients could be corrected with rGM-CSF treatment.
For more than a century, it has been recognized that chronic
alcohol abuse is a major risk factor for the development of pneumonia. Although factors associated with alcoholism such as malnutrition, poor dentition, aspiration, smoking, and other drug likely
exacerbate the risk, experimental animal models have demonstrated that ethanol ingestion alone (in the absence of these other
factors associated with chronic alcohol abuse in humans) impairs
alveolar macrophage innate immune function (5, 7, 8, 10, 20).
However, the specific mechanisms by which ethanol ingestion
down-regulates macrophage function have not been identified.
This study provides a plausible and specific mechanism by which
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B
+
+
6844
ETHANOL AND GM-CSF SIGNALING
FIGURE 10. Confocal images of bacterial phagocytosis by alveolar
macrophages from ethanol-fed rats ⫾ GM-CSF treatment in vivo. Shown
are representative ⫻60 confocal images of bacterial phagocytosis in vitro
by alveolar macrophages isolated from ethanol-fed rats ⫾ prior treatment
with rGM-CSF in vivo. A and B, The corresponding fluorescent and differential contrast images for a macrophage from an untreated, ethanol-fed
rat, in which relatively few bacteria have been phagocytosed, and the majority of these remains in the periphery of the cell. In contrast, as shown in
C and D, alveolar macrophages from ethanol-fed rats treated with GM-CSF
in vivo before macrophage isolation were able to phagocytose more bacteria, and most of the bacteria have been internalized. The cell membranes
in the differential contrast images in B and C, respectively, were digitally
outlined, and these outlines were superimposed on the corresponding fluorescent images in A and C, respectively, to better illustrate the cellular
localization of the bacteria relative to the plasma membranes.
alveolar macrophage maturation and function is dampened during
chronic ethanol ingestion. Specifically, ethanol ingestion interferes
with GM-CSF priming within the alveolar space that is absolutely
essential for the alveolar macrophage to acquire its full complement of immune functions.
These findings are important because they provide novel insights into the fundamental mechanisms by which chronic alcohol
abuse impairs host defenses and renders patients susceptible to
pulmonary infections. They are also important because they raise
the provocative possibility that the alcoholic macrophage could be
stimulated in vivo by exogenous GM-CSF treatment and thereby
rapidly reacquire the innate immune function that protects the
lower airways from microbial invasion. As GM-CSF has already
been tested in a phase II clinical trial of sepsis and lung injury and
was found to increase alveolar macrophage function (21), it is
reasonable to speculate that treating alcoholic patients with rGMCSF as adjunctive therapy for serious lung infections could augment their pulmonary host defense and improve outcome.
Although we have identified significant defects in GM-CSF receptor expression and parallel decreases in PU.1 expression in the
alcoholic macrophage, we recognize that GM-CSF signaling is
complex and that alcohol abuse likely perturbs other components
of the GM-CSF signal transduction pathway. For example, while
the GM-CSFR␤ initiates intracellular signaling following GMCSF binding, it contains no intrinsic catalytic activity. Rather, it is
constitutively associated with a tyrosine kinase, JAK2, that when
activated initiates the intracellular signaling cascade (22). We did
not examine this kinase or any of the other intracellular components that transduce the GM-CSF signal from receptor binding to
PU.1 expression and nuclear transcription activation, and one
would expect that these components would likewise be dampened.
Another complexity is that the GM-CSFR␤ is actually common to
the IL-3R and IL-5R (22, 23). However, IL-5R expression appears
to be limited to eosinophils (23), whereas IL-3 expression is limited to eosinophils, basophils, and mast cells (23). Therefore, ethanol-mediated changes in ␤ subunit expression in alveolar macrophages, as we observed, are likely specific to GM-CSF receptor
expression in this cell type, particularly as they parallel changes in
the unique ␣ subunit. Whether alcohol abuse has any significant
effects on IL-3 and/or IL-5 function in eosinophils and/or basophils
is an open question.
Our findings provide important new insights but also raise new
questions. In particular, how does ethanol ingestion inhibit mobilization and/or insertion of the GM-CSF receptor into the plasma
membrane? In parallel, how does treatment with supraphysiological levels of GM-CSF correct this defect when physiologic levels,
which are not inhibited by ethanol ingestion, do not? At present we
can only speculate. We know from previous studies that chronic
ethanol ingestion causes oxidative stress and severe glutathione
depletion within the alveolar space in experimental animals (24) as
well as in humans (25), leading to diverse abnormalities in alveolar
epithelial function that are prevented by supplementing the ethanol-containing diet with glutathione precursors (26 –28). It is certainly conceivable that ethanol-induced oxidative stress interferes
with GM-CSF trafficking to the plasma membrane. Acetaldehyde,
the first product of ethanol metabolism, produces endoplasmic reticulum stress in hepatocytes, thereby inhibiting mitochondrial glutathione transport (29). If similar endoplasmic reticulum stresses
occurred within the alveolar macrophage, this could lead to misfolding of the GM-CSF receptor. However, this would have to be
a relatively specific inhibition as we determined that membrane
expression of the IL-6R was not affected. Although we did not find
evidence that ethanol ingestion decreased gene expression of GMCSF and/or its receptors, it is possible that supraphysiological levels of GM-CSF increased expression of one or more components
of the pathway in addition to augmenting membrane expression of
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FIGURE 11. Bacterial phagocytosis in alveolar macrophages from control-fed and ethanol-fed rats ⫾ treatment with rGM-CSF in vitro. Macrophages were incubated in a 1:1 ratio with FITC-labeled, inactivated S.
aureus with or without recombinant rat GM-CSF (10 ng/ml) for 4 h. The
percentage of macrophages that ingested fluorescent bacteria was determined by direct observation under fluorescent microscopy for each experiment. Shown are the means ⫾ SEM for four experiments in each condition. ⴱ, p ⬍ 0.05 decreased compared with untreated control cells; ⴱⴱ, p ⬍
0.05 increased compared with untreated ethanol cells.
The Journal of Immunology
Disclosures
The authors have no financial conflict of interest.
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the receptors. Regardless of the mechanism, it is intriguing that
rGM-CSF treatment restored GM-CSF receptor membrane expression, PU.1 expression, and immune function in the alcoholic macrophage. Clearly, the signaling cascade is dampened, but not completely blocked, by chronic ethanol ingestion. As GM-CSF protein
levels in the alveolar space were not altered, one could speculate
that the significant decrease in GM-CSF receptor expression alone
can explain this dampening. Therefore, when the ambient level of
GM-CSF is markedly increased by treatment with rGM-CSF, a
higher percentage (if not all) of the available GM-CSF receptors
could become activated, thereby amplifying the previously muted
signaling cascade. The subsequent activation of PU.1-responsive
genes could then further activate the macrophage, which in turn
could drive trafficking of the GM-CSF receptor as well as other
membrane components of a “mature” macrophage to the cell surface. Therefore, while the alcoholic macrophage may be relatively
dormant in the face of physiologic GM-CSF stimulation, it appears
that its immune function can be restored by pharmacological treatment with “supraphysiological” concentrations of GM-CSF. In
parallel, we cannot exclude the possibility that GM-CSF treatment
could have increased the recruitment of extrapulmonary monocytes to the alveolar space and their maturation into functional
alveolar macrophages. We did not detect any increase in the alveolar macrophage population in the lung lavage fluid after GM-CSF
treatment (data not shown), but this does not exclude the possibility that recruited cells replaced the dysfunctional pool.
Finally, it is important to note that GM-CSF treatment did not
significantly increase GM-CSF receptor expression in the alveolar
macrophages of control-fed rats. However, this treatment modestly
increased PU.1 protein expression and nuclear binding, and LPSstimulated TNF-␣ secretion, in alveolar macrophages from control-fed rats, albeit less dramatically than in ethanol-fed rats. This
suggests that under normal conditions, GM-CSF receptor surface
expression is at or near maximal density. However, it is likely that
not all of the receptors are occupied at any one time under normal
conditions, as supraphysiological levels of GM-CSF induced a response even in the macrophages of control-fed rats. Whether GMCSF signaling is constitutive or regulated at the receptor or postreceptor level under normal conditions is unknown and is a ripe
area for further investigation.
In summary, we report for the first time that chronic ethanol
ingestion interferes with GM-CSF-dependent alveolar macrophage
immune function by decreasing GM-CSF receptor expression and
subsequent activation, of the master transcription factor PU.1, and
that these defects are reversed by high-dose GM-CSF treatment
delivered via the airway. These findings provide new insights into
the potential mechanisms by which alcohol abuse suppresses pulmonary host immunity and renders patients susceptible to serious
lung infections, including tuberculosis and bacterial pneumonias.
Although the specific cellular mechanisms require further investigation, it is fascinating to consider that pulmonary host defenses
could be rapidly augmented by acute treatment with rGM-CSF,
even in the context of chronic alcohol abuse, and thereby decrease
the morbidity and/or mortality from serious pulmonary infections
in this vulnerable population.
6845
The Journal of Immunology
CORRECTIONS
Joshi, P. C., L. Applewhite, J. D. Ritzenthaler, J. Roman, A. L. Fernandez, D. C. Eaton, L. A. S. Brown, and D. M. Guidot.
2005. Chronic ethanol ingestion in rats decreases granulocyte-macrophage colony-stimulating factor receptor expression
and downstream signaling in the alveolar macrophage. J. Immunol. 175: 6837– 6845.
In Figure 1, panel C was omitted. The corrected figure is shown below. The error has been corrected in the online
version, which now differs from the print version as originally published.
Copyright © 2005 by The American Association of Immunologists, Inc.
0022-1767/05/$02.00
8440
CORRECTIONS
Li, X., K. Malathi, O. Krizanova, K. Ondrias, K. Sperber, V. Ablamunits, and T. Jayaraman. 2005. Cdc2/cyclin B1
interacts with and modulates inositol 1,4,5-trisphosphate receptor (type 1) functions. J. Immunol. 175: 6205– 6210.
In the author line, the sequence of the first two authors is reversed. The corrected author line is shown below.
Krishnamurthy Malathi, Xiaogui Li, Olga Krizanova, Karol Ondrias, Kirk Sperber, Vitaly Ablamunits, and Thottala
Jayaraman
Pasquetto, V., H.-H. Bui, R. Giannino, F. Mirza, J. Sidney, C. Oseroff, D. C. Tscharke, K. Irvine, J. R. Bennink, B. Peters,
S. Southwood, V. Cerundolo, H. Grey, J. W. Yewdell, and A. Sette. 2005. HLA-A*0201, HLA-A*1101, and HLAB*0702 transgenic mice recognize numerous poxvirus determinants from a wide variety of viral gene products. J.
Immunol. 175: 5504 –5515.
The fourth author’s name, Cindy Banh, was omitted. The correct list of authors and affiliations is shown below.
Valerie Pasquetto,* Huynh-Hoa Bui,* Rielle Giannino,* Cindy Banh,* Fareed Mirza,† John Sidney,* Carla Oseroff,*
David C. Tscharke,§¶ Kari Irvine,§ Jack R. Bennink,§ Bjoern Peters,* Scott Southwood,‡ Vincenzo Cerundolo,† Howard
Grey,* Jonathan W. Yewdell,§ and Alessandro Sette2*
*La Jolla Institute for Allergy and Immunology, San Diego, CA 92109; †Tumor Immunology Unit, Weatherall Institute
of Molecular Medicine, Oxford University, Oxford, United Kingdom; ‡Epimmune Incorporated, San Diego, CA 92121;
§
Laboratory of Viral Diseases, National Institutes of Health, Bethesda, MD 20892; and ¶ Division of Immunology and
Infectious Diseases, Queensland Institute of Medical Research, Herston, Queensland, Australia
Zhang, X., P. Shan, S. Qureshi, R. Homer, R. Medzhitov, P. W. Noble, and P. J. Lee. 2005. Cutting edge: TLR4 deficiency
confers susceptibility to lethal oxidant lung injury. J. Immunol. 175: 4834 – 4838.
In Materials and Methods, in the first sentence under the heading Intranasal administration of recombinant adenovirus-containing HO-1 cDNA, the source for adenoviral HO-1 cDNA was incorrectly attributed. The source is stated in
the corrected sentence below.
Mice were anesthetized with methoxyflurane, and then 5 ⫻ 108 PFU of adenoviral HO-1 (Ad-HO-1) (a gift from K.
Kolls, University of Pittsburgh Medical Center, Pittsburgh, PA, and J. Alam, Alton Ochsner Medical Foundation, New
Orleans, LA) (29) or adenoviral ␤-galactosidase (Ad-LacZ) (BD Biosciences) were administered intranasally to each
mouse in a volume of 50 ␮l as described previously (12).
The authors also wish to add the reference shown below.
29. Otterbein, L. E., J. K. Kolls, L. L. Mantell, J. L. Cook, J. Alam, and A. M. K. Choi. 1999. Exogenous administration
of heme oxygenase-1 by gene transfer provides protection against hyperoxia-induced lung injury. J. Clin. Invest. 103:
1047–1054.
The Journal of Immunology
Gays, F., K. Martin, R. Kenefeck, J. G. Aust, and C. G. Brooks. 2005. Multiple cytokines regulate the NK gene complexencoded receptor repertoire of mature NK cells and T cells. J. Immunol. 175: 2938 –2947.
In Figure 1, a sentence regarding the solid and broken lines was omitted from the legend. The corrected legend is shown
below.
FIGURE 1. Specificity of the CM4 mAb. A, YB2 or RNK cells transfected with Ly49 constructs were stained with
medium or first layer Abs followed by AF488 goat anti-mouse Ig. Solid lines: staining by CM4. Left broken line: medium
control. Right broken line: staining by positive control Abs Ly49A ⫽ A1, Ly49B ⫽ 1A1, Ly49C ⫽ 4D12, Ly49D ⫽ 4E5,
Ly49E ⫽ 4D12, Ly49F ⫽ HBF, Ly49G ⫽ 4G11, Ly49H ⫽ 3D10, Ly49I ⫽ YBI. B, Cross-competition between Abs.
YB2 cells transfected with Ly49E (YB2-E) and RNK cells transfected with Ly49F (RNK-F) were incubated with medium
or saturating quantities of the unlabeled Ly49 Abs shown on the y-axis. After 20 min, AF488-labeled CM4, 4D12, or HBF
Ab was added, and incubation was continued for an additional 20 min. Median fluorescence values were determined by
flow cytometry, and the percentage inhbition caused by pretreatment with each unlabeled Ab is plotted on the y-axis. The
likelihood that the inhibition observed was due to chance variation was determined by Student’s t test (*, p ⬍ 0.05,
**, p ⬍ 0.01, ***, p ⬍ 0.001). The experiments shown are representative of three similar experiments of each type that
were performed.
In Figure 9A, the gel image labeled Ly49A is inverted. The corrected figure is shown below.
8441
8442
CORRECTIONS
Rakoff-Nahoum, S., H. Chen, T. Kraus, I. George, E. Oei, M. Tyorkin, E. Salik, P. Beuria, and K. Sperber. 2001.
Regulation of class II expression in monocytic cells after HIV-1 infection. J. Immunol. 167: 2331–2342.
Figure 10, demonstrating intracellular trafficking of HLA-DR after the introduciton of HIV proteins, is incorrect. The
corrected figure is shown below.
Lukacs, N. W., K. K. Tekkanat, A. Berlin, C. M. Hogaboam, A. Miller, H. Evanoff, P. Lincoln, and H. Maassab. 2001.
Respiratory syncytial virus predisposes mice to augmented allergic airway responses via IL-13-mediated mechanisms. J.
Immunol. 167: 1060 –1065.
In Materials and Methods, in the first sentence under the heading RSV infection, the designation of the virus type
should be human RSV A strain, not A2 strain.
Tekkanat, K. K., H. F. Maassab, D. S. Cho, J. J. Lai, A. John, A. Berlin, M. H. Kaplan, and N. W. Lukacs. 2001.
IL-13-induced airway hyperreactivity during respiratory syncytial virus infection is STAT6 dependent. J. Immunol. 166:
3542–3548.
In Materials and Methods, in the first sentence under the heading Virus and infection, the designation of the virus type
should be human RSV A strain, not A2 strain.
The Journal of Immunology
Chen, H., Y. K. Yip, I. George, M. Tyorkin, E. Salik, and K. Sperber. 1998. Chronically HIV-1-infected monocytic cells
induce apoptosis in cocultured T cells. J. Immunol. 161: 4257– 4267.
Figure 3B, demonstrating the apoptotic effect of gp120 on CD4 and CD8 cells; Figure 4B, depicting the apoptotic effect
of Fas-FasL interactions in CD4 and CD8 T cells cocultured with 43HIV cells; and Figure 6B, showing the apoptotic
activity of fractionated supernatant from the 43HIV cell line, are inaccurate. The corrected figures are shown below.
8443
8444
CORRECTIONS
Polyak, S., H. Chen, D. Hirsch, I. George, R. Hershberg, and K. Sperber. 1997. Impaired class II expression and antigen
uptake in monocytic cells after HIV-1 infection. J. Immunol. 159: 2177–2188.
In Figure 5, demonstrating the inability of HIV-1-infected 43 cells to present antigen to HLA-DR2 and DR4 T cells,
panels A and B are the same. The corrected figure is shown below.