Alveolar macrophages contribute to alveolar barrier dysfunction in ventilator-induced lung injury

Abstract

In patients requiring mechanical ventilation for acute lung injury or acute respiratory distress syndrome (ARDS), tidal volume reduction decreases mortality, but the mechanisms of the protective effect have not been fully explored. To test the hypothesis that alveolar macrophage activation is an early and critical event in the initiation of ventilator-induced lung injury (VILI), rats were ventilated with high tidal volume (HV(T)) for 10 min to 4 h. Alveolar macrophage counts in bronchoalveolar lavage (BAL) fluid decreased 45% by 20 min of HV(T) (P < 0.05) consistent with activation-associated adhesion. Depletion of alveolar macrophages in vivo with liposomal clodronate significantly decreased permeability and pulmonary edema following 4 h of HV(T) (P < 0.05). BAL fluid from rats exposed to 20 min of HV(T) increased nitric oxide synthase activity nearly threefold in naïve primary alveolar macrophages (P < 0.05) indicating that soluble factors present in the air spaces contribute to macrophage activation in VILI. Media from cocultures of alveolar epithelial cell monolayers and alveolar macrophages exposed to 30 min of stretch in vitro also significantly increased nitrite production in naïve macrophages (P < 0.05), but media from stretched alveolar epithelial cells or primary alveolar macrophages alone did not, suggesting alveolar epithelial cell-macrophage interaction was required for the subsequent macrophage activation observed. These data demonstrate that injurious mechanical ventilation rapidly activates alveolar macrophages and that alveolar macrophages play an important role in the initial pathogenesis of VILI.

Full text

doi:10.1152/ajplung.00055.2006 291:1191-1198, 2006. First published Jul 28, 2006;Am J Physiol Lung Cell Mol Physiol
Michael A. Matthay
James A. Frank, Charlie M. Wray, Danny F. McAuley, Reto Schwendener and
dysfunction in ventilator-induced lung injury
Alveolar macrophages contribute to alveolar barrier
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Alveolar macrophages contribute to alveolar barrier dysfunction in
ventilator-induced lung injury
James A. Frank,
1,3,4,5
Charlie M. Wray,
4
Danny F. McAuley,
3
Reto Schwendener,
6
and Michael A. Matthay
1,2,3
University of California, San Francisco, Departments of
1
Medicine and
2
Anesthesia;
3
Cardiovascular Research
Institute;
4
Northern California Institute for Research and Education;
5
San Francisco Veterans Affairs Medical
Center, San Francisco, California; and
6
The Paul Scherrer Institute, Villigen, Switzerland
Submitted 13 February 2006; accepted in final form 18 July 2006
Frank JA, Wray CM, McAuley DF, Schwendener R, Matthay
MA. Alveolar macrophages contribute to alveolar barrier dysfunc-
tion in ventilator-induced lung injury. Am J Physiol Lung Cell Mol
Physiol 291: L1191–L1198, 2006. First published July 28, 2006;
doi:10.1152/ajplung.00055.2006.—In patients requiring mechanical
ventilation for acute lung injury or acute respiratory distress syndrome
(ARDS), tidal volume reduction decreases mortality, but the mecha-
nisms of the protective effect have not been fully explored. To test the
hypothesis that alveolar macrophage activation is an early and critical
event in the initiation of ventilator-induced lung injury (VILI), rats
were ventilated with high tidal volume (HV
T
) for 10 min to 4 h.
Alveolar macrophage counts in bronchoalveolar lavage (BAL) fluid
decreased 45% by 20 min of HV
T
(P⬍0.05) consistent with
activation-associated adhesion. Depletion of alveolar macrophages in
vivo with liposomal clodronate significantly decreased permeability
and pulmonary edema following4hofHV
T
(P⬍0.05). BAL fluid
from rats exposed to 20 min of HV
T
increased nitric oxide synthase
activity nearly threefold in naı¨ve primary alveolar macrophages (P⬍
0.05) indicating that soluble factors present in the air spaces contribute
to macrophage activation in VILI. Media from cocultures of alveolar
epithelial cell monolayers and alveolar macrophages exposed to 30
min of stretch in vitro also significantly increased nitrite production in
naı¨ve macrophages (P⬍0.05), but media from stretched alveolar
epithelial cells or primary alveolar macrophages alone did not, sug-
gesting alveolar epithelial cell-macrophage interaction was required
for the subsequent macrophage activation observed. These data dem-
onstrate that injurious mechanical ventilation rapidly activates alveo-
lar macrophages and that alveolar macrophages play an important role
in the initial pathogenesis of VILI.
alveolar epithelial barrier function; ventilator-associated lung injury;
acute lung injury; acute respiratory distress syndrome
MECHANICAL VENTILATION with excessive end-inspiratory volume
contributes to mortality in patients with acute lung injury and
the acute respiratory distress syndrome (ARDS). The patho-
genesis of ventilator-associated lung injury is incompletely
understood; however, both clinical and experimental ventila-
tor-attributable lung injury are characterized by activation of
the inflammatory response (12). For example, a variety of pro-
and anti-inflammatory mediators have been correlated with
mechanical ventilation strategy in both experimental and clin-
ical studies, including IL-1␤, IL-6, IL-8, and IL-10 (5, 28, 33,
34). Previous reports have indicated that in vitro mechanical
strain induces IL-8 release from alveolar epithelial-like (A549)
cells and alveolar macrophages (32, 38). Because neutrophils
contribute to ventilator-induced lung injury (VILI) (3), the
release of neutrophil chemokines such as IL-8 likely consti-
tutes an early step in the pathogenesis of VILI. Although
animal studies using immunohistochemistry and in situ hybrid-
ization techniques have shown that a variety of lung cells
produce inflammatory mediators in VILI (7), the relative con-
tributions of resident lung cell types to the pathogenesis of
VILI has not been fully explored. We hypothesized that alve-
olar macrophages have a central role in the initiation of VILI.
The primary objective of the present study was to determine if
alveolar macrophages contribute to the increase in lung vascu-
lar and alveolar epithelial permeability characteristic of exper-
imental VILI using a previously described macrophage deple-
tion technique (19). A second objective was to determine if
injurious mechanical strain activates alveolar macrophages by
inducing the release of soluble mediators from alveolar epithe-
lial cells, or by a direct affect on alveolar macrophages.
METHODS
This protocol conforms to National Institutes of Health animal care
and use guidelines and was approved by the University of California,
San Francisco Institutional Animal Care and Use Committee
(IACUC).
Clodronate liposome preparation and delivery. Liposomes were
prepared as previously described (19). Briefly, liposomes were com-
posed of phosphatidylserine, phosphatidylcholine, and cholesterol at a
molar ratio of 1:6:4 in chloroform. The lipid solution was dried under
low vacuum and dissolved in diethyl ether. A clodronate (Sigma, St.
Louis, MO) stock solution or PBS was added, and the mixture was
placed under nitrogen and sonicated for 3 min. The ether was then
removed by rotary evaporation under reduced pressure at 30°C. Any
gel phase that formed was disrupted by vortexing the sample to
facilitate the removal of ether. The liposome suspension was then
repeatedly extruded through 200-nm filters. The liposome solution
was then delivered to anesthetized (ketamine 90 mg/kg ip) animals by
aerosol (19). Fluorescent liposomes were prepared separately by
adding the dye DiO to the lipid mixture (35). Compared with direct
intratracheal instillation, aerosol delivery is associated with more
macrophage depletion and fewer air space neutrophils (19).
VILI model. A previously described VILI model (17) with slight
modification was used. Briefly, rats were anesthetized with 4% isoflu-
rane and 50 mg/kg ip pentobarbital. A tracheostomy tube (15-gauge
luer adapter) was placed, and mechanical ventilation was started with
a tidal volume of 6 ml/kg, a positive end-expiratory pressure (PEEP)
of 5 cmH
2
O, a respiratory rate of 60 breaths/min, and 21% oxygen. A
Address for reprint requests and other correspondence: J. A. Frank, Cardio-
vascular Research Institute, Univ. of California San Francisco, Dept. of
Medicine, Division of Pulmonary and Critical Care, Medical ICU, San Fran-
cisco VA Medical Center, Box 111D, San Francisco, CA 94121 (e-mail:
james.frank@ucsf.edu).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Am J Physiol Lung Cell Mol Physiol 291: L1191–L1198, 2006.
First published July 28, 2006; doi:10.1152/ajplung.00055.2006.
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right common carotid artery catheter (PE-50 tubing, BD) was placed
for blood pressure monitoring and arterial blood gas measurement.
Respiratory rate was adjusted to maintain normal arterial pH. Follow-
ing a 10-min stable baseline period, tidal volume was increased to 30
ml/kg without PEEP. Tidal volume was decreased if necessary to limit
airway pressure to 30 cmH
2
O throughout the protocol. These settings
were used to approximate ventilation at total lung capacity. Respira-
tory rate was decreased to 30 breaths/min, and additional dead space
was added to the ventilator circuit as needed to maintain normal
arterial pH. Anesthesia was maintained with isoflurane (0.5–2%),
and muscle relaxation was maintained with pancuronium (2
mg䡠kg
⫺1
䡠h
⫺1
). Ventilation was continued from 10 min to 4 h.
Normal saline (1.5 mg䡠kg
⫺1
䡠h
⫺1
) was administered throughout the
protocol. Airway pressures, blood pressure, and heart rate were
monitored continuously using a computer-integrated data collection
system. Arterial blood gases were measured at 1, 2, and 4 h, and
plasma samples were collected at the start and end of the protocol.
Pulmonary edema, permeability, and bronchoalveolar lavage. Pul-
monary edema was measured as the blood-free, excess lung water
determined by gravimetric methods (16). Lung endothelial and alve-
olar epithelial permeability to albumin were measured by determining
the extravasation of intravascular
125
I-labeled albumin into the lung as
previously described (16). Permeability is expressed as the extravas-
cular plasma equivalents in microliters. BAL was performed with
three aliquots of 7 ml of warmed normal saline instilled into the lungs
and gently withdrawn. A differential cell count was determined on an
aliquot of the total BAL fluid using a hemocytometer, and cytological
centrifuge preparation was stained with Wright’s stain and eosin. For
BAL fluid used in the nitrite assay, a single aliquot of 7 ml of warmed
RPMI 1640 was instilled three times, centrifuged, and stored for later use.
Experimental groups for the macrophage depletion studies. A total
of 44 rats were used for these studies. There were four groups: 1)
empty liposomes, unventilated (n⫽8); 2) clodronate liposomes,
unventilated (n⫽8); 3) empty liposomes, ventilated (n⫽14); and 4)
clodronate liposomes, ventilated (n⫽14). BAL was done at the end
of the experimental protocol on 4 subjects from each group. These
four animals were not used for the pulmonary edema or permeability
measurements.
Measurement of plasma CXC ligand 1. Plasma samples were
collected from rats at the beginning of the protocol and at the end of
the high tidal volume ventilation period. Levels of the chemokine
CXC ligand 1 (CXCL1) were measured using a rat-specific ELISA
with a detection threshold of 40 pg/ml (R&D Systems, Minneapolis,
MN). Like human IL-8, CXCL1 is a ligand for CXC receptors 1 and
2. CXCL1 is also known as GRO, KC, MIP-2, and CINC-1 in rodents.
All samples were tested in duplicate. CXCL1 levels were measured on
four matched pairs of empty liposome-treated and clodronate lipo-
some-treated rats (i.e., 4 subjects in each group of 14 subjects).
Macrophage nitrite production assay. Nitrite production was mea-
sured as a surrogate of macrophage nitric oxide production, a recog-
nized functional marker of macrophage activation (25). Primary
alveolar macrophages from 10 rats were plated at 75,000 cells/well in
96-well culture plates. Nitrite production was then measured 18 h after
adding either BAL fluid from rats or after adding supernatants from
cultured alveolar epithelial cells and macrophages exposed to me-
chanical strain in vitro. Nitrite concentration was determined with a
colorimetric nitric oxide assay kit (Caymen, San Diego, CA) as
previously described (17). For the BAL fluid studies, 200 ␮lof
cell-free BAL fluid from unventilated rats (n⫽4) or from rats
exposed to mechanical ventilation for 10 (n⫽3) or 20 min (n⫽4)
were added to the primary macrophages in 96-well plates, and cells
were incubated for 18 h at 37°C in 5% CO
2
. There was a minimum of
six wells of macrophages per experimental condition, and the exper-
iment was repeated five times. To normalize nitrite production to cell
number, cell density was determined as follows. Cells were washed
with PBS and fixed in 70% ethanol for 10 min. Cells were then stained
with 1% crystal violet (15 min) and thoroughly rinsed with tap water.
The stain was extracted from cells by adding 50 ␮l of 0.2% Triton
X-100. Relative quantification was done by reading absorbance at 570
nm on plate reader. Total nitrite is reported as the increase in nitrite
per 5 ⫻10
4
cells over the initial level in the BAL fluid (30). For the
in vitro mechanical strain studies, alveolar epithelial type II cells were
isolated from rat lungs (n⫽10) as previously described (13).
Epithelial cells were plated at 3 ⫻10
6
on 35-mm flexible membranes
coated with fibronectin (Bioflex, Hillsborough, NC) and recoated with
fibronectin (100 ␮g/ml, Calbiochem no. 341631; San Diego, CA). On
day 5, epithelial monolayer confluence was confirmed, and media was
changed to serum-free DME-H21, and primary rat alveolar macro-
phages (3 ⫻10
5
) were added to some epithelial monolayers. Macro-
phages were allowed to settle to the epithelial monolayer for 1 h. In
separate studies, primary alveolar macrophages (3 ⫻10
5
) were plated
on fibronectin-coated membranes and allowed to adhere for 1 h. Cells
were exposed to mechanical strain (30% membrane surface area
change at 0.5 Hz) for 30 min (Flexcell 4000T; Hillsborough, NC).
Media was collected, centrifuged, and then added to naı¨ve primary
alveolar macrophages on 96-well plates for measurement of nitrite
production as described above. There was a minimum of three wells
of cells per experimental condition, and the experiment was repeated
four times.
Statistics. Comparisons between two groups were made using an
unpaired, two-tailed t-test for normally distributed data and the Mann-
Whitney test for nonparametric data. Comparisons among three or
more groups were made using one-way analysis of variance and
Tukey post hoc test for multiple comparisons. Pvalues less than 0.05
were considered significant. Data are expressed as means ⫾SD unless
otherwise noted.
RESULTS
High tidal volume ventilation rapidly decreases BAL mac-
rophage counts. BAL macrophage counts from rats ventilated
with high tidal volume and without PEEP decreased with
increasing duration of mechanical ventilation (Fig. 1). Macro-
Fig. 1. Bronchoalveolar lavage (BAL) fluid macrophage counts after high tidal
volume (HV
T
) ventilation. Alveolar macrophage recovery in BAL fluid
sharply decreased within minutes of starting HV
T
ventilation. BAL alveolar
macrophage counts (cells/ml) were significantly decreased (45%) by 20 min of
ventilation and remained low for up to3h(*P⬍0.05 compared with
unventilated rats, n⫽3– 6 at each time point).
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phage recovery in BAL fluid was significantly decreased by 20
min and continued to decrease at 180 min. At baseline the total
BAL cell count was 313 ⫾50 ⫻10
3
cells/ml, of which 291 ⫾
44 ⫻10
3
were macrophages, 7 ⫾9⫻10
3
were neutrophils,
and 15 ⫾4⫻10
3
were lymphocytes. After 30 min of
ventilation, the total BAL cell count decreased to 190 ⫾32 ⫻
10
3
cells/ml with 169 ⫾25 ⫻10
3
macrophages, 9 ⫾5⫻10
3
neutrophils, and 12 ⫾4⫻10
3
lymphocytes. Only the change
in macrophages was statistically significant at the 30-min time
point (P⬍0.05).
Macrophage depletion. Rats were given increasing doses
(0 –20 mg/ml) of clodronate liposomes by aerosol. Maximal
macrophage depletion was observed with a clodronate concen-
tration of 20 mg/ml (Fig. 2A). The nadir of BAL macrophage
counts was 3– 4 days after administration (Fig. 2B). Macro-
phage uptake of liposomes occurred early and was confirmed
by fluorescently labeled liposomes. Two hours after liposomes
were given, ⬃70% of alveolar macrophages recovered in
lavage fluid contained liposomes (not shown).
Macrophage depletion preserves respiratory system elas-
tance and oxygenation. During baseline ventilation with low
tidal volume and a PEEP level of 5 cmH
2
O, elastance was
comparable between animals treated with empty liposomes and
clodronate containing liposomes (Fig. 3A). Upon increasing
Fig. 2. Clodronate liposomes depleted alveolar macrophages. A: dose response
of alveolar macrophage depletion 4 days after clodronate was given. B: time
course of macrophage depletion following clodronate treatment. An alveolar
macrophage nadir representing 82% depletion was found at 4 days with a
10-ml aerosolized dose of 20 mg/ml clodronate liposome solution.
Fig. 3. Effect of macrophage depletion on respiratory system elastance and
arterial oxygenation in ventilator-induced lung injury. A: compared with empty
liposomes (vehicle), clodronate liposome administration resulted in signifi-
cantly lower respiratory system elastance (cmH
2
O/ml; *P⬍0.05 compared
with vehicle-treated group). B: arterial oxygenation (mmHg) was significantly
higher in alveolar macrophage-depleted rats compared with empty liposome-
treated rats after4hofHV
T
ventilation (*P⬍0.05).
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tidal volume and decreasing PEEP, elastance decreased to a
similar level in both groups. One hour later, elastance was
significantly lower in the clodronate-treated group (P⬍0.05).
This effect persisted for at least 4 h. Arterial oxygenation was
comparable between the two groups for the first2hofthe
protocol, but by 4 h, Pa
O
2
was significantly higher in the
clodronate-treated group (Fig. 3B). There were no significant
differences in arterial PCO
2
, tidal volume, mean arterial blood
pressure, or heart rate at any time in the protocol.
Effect of macrophage depletion on pulmonary edema and
protein permeability. Macrophage depletion with liposomal
clodronate significantly decreased pulmonary edema in this
model of VILI. Excess lung water in the vehicle-treated ani-
mals was significantly higher than in clodronate-treated ani-
mals (Fig. 4A). Although excess lung water in the ventilated
group that received clodronate was significantly higher than in
unventilated controls, the increase was less than 50% of that in
the empty liposome-treated, ventilated group. There was no
difference in excess lung water in unventilated rats given either
empty liposomes or clodronate liposomes (not shown).
Alveolar epithelial and lung endothelial permeability to
labeled albumin measured as the
125
I-albumin activity in the
extravascular space of the lung was significantly increased in
both groups by high tidal volume ventilation. However, per-
meability was significantly lower in rats given clodronate
liposomes compared with controls (P⬍0.05) (Fig. 4B). There
was no difference in permeability in unventilated rats given
either empty liposomes or clodronate liposomes (not shown).
Macrophage depletion decreases chemokine levels and air
space neutrophils. Plasma levels of the neutrophil chemokine
CXCL1 were significantly higher in the vehicle-treated rats
than in the clodronate-treated rats after high tidal volume
ventilation (Fig. 5A). BAL neutrophil counts after4hof
ventilation were significantly higher in rats receiving empty
liposomes (Fig. 5B).
Fig. 4. Effect of alveolar macrophages on alveolar barrier dysfunction in
ventilator-induced lung injury (VILI). A: pulmonary edema measured as excess
lung water (␮l) increased with HV
T
ventilation (*P⬍0.05 compared with no
ventilation). Macrophage depletion with clodronate resulted in significantly
less pulmonary edema compared with rats treated with empty liposomes
(vehicle; †P⬍0.05 compared with the ventilated, clodronate group). B: lung
permeability to albumin measured as the extravasation of intravascular
125
I-
albumin into the extravascular space [extravascular plasma equivalents
(EVPE) in microliters] was significantly decreased by macrophage depletion
(*P⬍0.05 compared with no ventilation; †P⬍0.05 compared with the
ventilated, clodronate group). Excess lung water and permeability were not
different in unventilated rats given either empty liposomes or clodronate
liposomes. Data from these two groups are combined here (No Ventilation).
Fig. 5. Effect of macrophage depletion on plasma chemokine levels and air
space neutrophils in VILI. A: macrophage depletion with clodronate resulted in
significantly lower plasma CXC ligand 1 (CXCL1) levels compared with
empty liposome (vehicle)-treated rats following4hofhigh volume mechanical
ventilation (*P⬍0.05 by Mann Whitney test, data are medians ⫾25% and
75% confidence intervals). B: BAL fluid neutrophil counts (cells/ml) with
clodronate treatment and after4hofHV
T
ventilation. There was no significant
difference in BAL neutrophil counts in unventilated rats treated with either
clodronate liposomes or empty liposomes. Following4hofhigh volume
ventilation, BAL neutrophil counts increased to a greater extent in empty
liposome-treated (vehicle) rats than clodronate-treated rats (*P⬍0.05 com-
pared with all other groups).
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Macrophage nitrite production in vitro. Nitrite production in
macrophages incubated with BAL fluid from ventilated rats
was significantly higher than in macrophages incubated with
BAL from unventilated animals (Fig. 6A). Nitrite levels from
macrophages incubated with BAL from unventilated rats were
not significantly different from levels in macrophages incu-
bated with media alone (not shown). Cell-free media from
cultured alveolar epithelial cells exposed to cyclic mechanical
strain for 30 min did not significantly increase nitrite produc-
tion in naı¨ve alveolar macrophages compared with media from
unstrained alveolar epithelial cells (Fig. 6B). Similarly, media
from cultured primary alveolar macrophages exposed to me-
chanical strain did not increase nitrite production in naı¨ve
macrophages. However, media from alveolar epithelial cell and
macrophage cocultures exposed to mechanical strain for 30
min significantly increased nitrite production in naı¨ve mac-
rophages compared with media from unstrained cocultures
(Fig. 6B).
DISCUSSION
High tidal volume ventilation induces alveolar epithelial and
lung endothelial injury, increases barrier permeability, and
decreases alveolar epithelial fluid clearance from the air spaces
(14, 17, 18, 23, 39). Previous animal studies have shown that
tidal volume reduction preserves epithelial and endothelial
permeability and decreases pulmonary edema (14). In patients
with acute lung injury and ARDS, tidal volume reduction
reduces mortality (2). The precise mechanisms by which me-
chanical forces are translated into more severe lung injury are
not fully understood. In many experimental and clinical stud-
ies, low tidal volume ventilation with PEEP results in signifi-
cantly lower plasma and air space levels of inflammatory
mediators (9, 10, 15, 28, 33, 34). Accordingly, it has been
postulated that pathological mechanical forces are converted
into proinflammatory signals early in the development of VILI
(12). Although many resident lung cells can produce inflam-
matory mediators, alveolar macrophages have a large capacity
for cytokine and chemokine production, as well as nitric oxide
and reactive nitrogen species elaboration. Alveolar macro-
phages have been shown previously to be important in the
pathogenesis of alveolar barrier dysfunction in Pseudomonas
pneumonia (19, 22, 37), endotoxin (6, 8), and ische-
mia-reperfusion lung injury (11, 26, 27) models. The role of
alveolar macrophages in the initial pathogenesis of VILI has
not been fully explored; however, previous reports have shown
that injurious mechanical ventilation results in decreased alve-
olar macrophage counts in bronchoalveolar lavage (BAL) fluid
(40). We reasoned that alveolar macrophage activation in
response to high tidal volume ventilation was an early event in
the initiation of ventilator-attributable lung injury.
Initial studies confirmed that BAL macrophage counts pre-
cipitously dropped with the initiation of high tidal volume, zero
PEEP ventilation. Ventilation for 20 min decreased macro-
phage counts 45% (Fig. 1). To determine the contribution of
alveolar macrophages to the increase in alveolar epithelial and
lung endothelial permeability characteristic of VILI, alveolar
macrophages were depleted using a previously reported lipo-
somal clodronate technique (19). Although macrophage deple-
tion was not complete (82%; Fig. 2), depletion was sufficient to
significantly decrease lung injury severity in this model. Mac-
rophage depletion resulted in significantly lower respiratory
system elastance (Fig. 3A) and preserved arterial oxygenation
(Fig. 3B). It is notable that when ventilator settings were
changed from a tidal volume of 6 ml/kg and a PEEP level of 5
Fig. 6. Macrophage activation by mechanical ventilation and in vitro mechan-
ical strain. A: BAL fluid from rats ventilated with high volume ventilation
activates naı¨ve alveolar macrophages. Macrophage activation was measured in
primary alveolar macrophages as nitrite production, a marker of nitric oxide
synthase activity. Naı¨ve macrophages were incubated with BAL fluid from
unventilated rats or from rats ventilated with HV
T
for 10 or 20 min. BAL fluid
from rats ventilated for 20 min significantly increased nitrite production in
naı¨ve primary macrophages (*P⬍0.05). Data are expressed as means ⫾SE.
B: mechanical strain of alveolar macrophages (M⌽) alone and primary alve-
olar epithelial cell monolayers (AEC) with or without alveolar macrophages
(3 ⫻10
5
) in vitro. Media from alveolar epithelial cells cocultured with alveolar
macrophages and exposed to strain in vitro for 30 min significantly increased
nitrite production in naı¨ve macrophages compared with media from stretched
cultures of either cell type alone (black bars; *P⬍0.05). Nitrite production
was not different in naı¨ve primary macrophages cultured with media from
unstrained alveolar macrophages alone or alveolar epithelial cells with or
without alveolar macrophages (white bars). Data are expressed as means ⫾SE.
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cmH
2
O to a tidal volume of 30 ml/kg without PEEP, elastance
decreased to a similar level in both groups. However, in the
clodronate-treated rats, elastance continued to decrease for up
to 1 h while elastance remained higher in the vehicle-treated
controls. The initial decrease in elastance in both groups was
likely the result of the change in ventilator settings. The
subsequent decrease in elastance in the clodronate-treated
group may have been due to changes in lung recruitment and
surfactant secretion that were counterbalanced in the vehicle-
treated rats by increased pulmonary edema. It is likely that the
final differences in elastance and oxygenation are partly ex-
plained by differences in pulmonary edema severity between
the groups (Fig. 4A). Furthermore, alveolar barrier permeabil-
ity was significantly lower in macrophage-depleted rats follow-
ing high tidal volume ventilation. These data support the
hypothesis that alveolar barrier dysfunction occurs rapidly in
VILI, and alveolar macrophages are important in the initial
pathogenesis of VILI. The data also show that alveolar mac-
rophages are not solely responsible for the increase in perme-
ability in the model as macrophage depletion decreased, but
did not completely prevent lung injury. For example, others
have demonstrated that high volume ventilation results in
endothelial cell activation, P-selectin expression, and lung
neutrophil recruitment (41).
Previous studies have shown that VILI is in part dependent
on the recruitment of neutrophils into the lung (3). One study
found that blocking CXCL1 and CXCL2/3 signaling signifi-
cantly decreased air space neutrophil counts and lung injury
severity in experimental VILI (3). Therefore, one mechanism
by which macrophages could initiate VILI is via the release of
neutrophil chemokines and subsequent neutrophil recruitment
into the lung. In the present study, circulating levels of the
neutrophil chemokine CXCL1 and BAL neutrophil counts
were significantly lower in macrophage-depleted rats (Fig. 5, A
and B). Part of the protective effect of macrophage depletion
could be attributable to decreased lung neutrophil recruitment.
Due to the rapidity of the change in macrophage counts with
high volume, zero PEEP ventilation, we suspected that in-
creased macrophage activation and adhesion, rather than cell
death or emigration, was most likely. Although macrophage
adhesion was not directly measured, increased adhesion is
indicative of activation. In addition, previous studies have
reported that high volume ventilation induced increased intra-
cellular expression of GADD45 (1) and membrane expression
of CD14 (24) in alveolar macrophages (markers of activation)
and that LPS-induced TNF-␣production was significantly
higher in macrophages isolated from rabbits ventilated with
high volumes for 6 h (24). Macrophage activation during high
tidal volume ventilation could be the result of direct mechan-
ical sensing by alveolar macrophages. For example, Pugin and
colleagues (32) have previously shown that alveolar macro-
phages respond to mechanical stress in vitro by increasing IL-8
and IL-6 production. Alternatively, macrophage activation
could be initiated by mediators released from alveolar epithe-
lial cells or other resident lung cells in response to mechanical
stress; however, this hypothesis has not been thoroughly in-
vestigated. We postulated that BAL fluid from rats ventilated
with high tidal volume would induce nitric oxide synthase
activity and nitrite production in primary alveolar macrophages
in vitro. When macrophages were incubated with BAL fluid
from rats exposed to 20 min of high volume ventilation, nitrite
production was significantly increased compared with BAL
fluid from unventilated rats (Fig. 6A). These data indicated that
soluble mediators in the BAL fluid accounted for at least part
of the observed increase in macrophage activation. To deter-
mine if alveolar epithelial cells were a source of soluble
mediators responsible for macrophage activation, we cultured
alveolar epithelial cells and alveolar macrophages separately
and in coculture and exposed the cells to mechanical strain.
Media from stretched or unstretched alveolar epithelial cells or
alveolar macrophages alone did not increase nitrite production
in naı¨ve primary alveolar macrophages (Fig. 6B). However,
media from epithelial monolayers cultured with alveolar mac-
rophages during a 30-min period of stretch increased macro-
phage nitrite production by 39% (P⬍0.05) in the subsequent
macrophage nitrite assay (Fig. 6B). Taken together, these data
provide evidence that alveolar macrophage activation is up-
regulated early in VILI and that soluble mediators contribute to
additional macrophage activation in VILI. Although either
epithelial cells or macrophages could be potential sources of
these mediators, the data suggest that an interaction between
the cell types is required for mediator release. These data are
not inconsistent with the hypothesis that alveolar macrophages
detect and respond to mechanical strain; however, in the
culture conditions studied, mechanical strain alone was not
sufficient to induce the release of mediators capable of induc-
ing nitrite production in naı¨ve macrophages. Of course, me-
chanical forces may affect alveolar epithelial cell membranes
directly and influence cytoskeletal organization (4, 18, 39);
however, data from the present study support an important role
for macrophage-epithelial cell interaction in the loss of epithe-
lial barrier function in this model.
Although clodronate treatment is a widely used tool for
alveolar macrophage depletion, it is possible that clodronate
had other effects on resident lung cells as well. These data must
be interpreted in this context. For example, in one study, up to
30% of alveolar epithelial cells contained liposomes following
intratracheal instillation (31). The precise effects of clodronate
liposomes on epithelial function are not clear, but, consistent
with previous reports (19, 22), we did not observe a significant
difference in permeability in unventilated rats treated with
clodronate liposomes or empty liposomes. It has also been
reported that clodronate liposomes inhibit cytokine production
in a macrophage-like cell line (29); therefore, decreased cyto-
kine production by macrophages remaining in the lung is an
additional potential mechanism for the observed protective
effect in the present study. It is noteworthy that the absence of
alveolar macrophage function has been shown to result in more
severe lung injury in some models, as macrophage phagocy-
tosis of neutrophils and other apoptotic cells is an important
step in the regulation of the inflammatory response (20 –22,
36). Therefore, it is uncertain if sustained macrophage inacti-
vation would have a net beneficial effect in clinical ventilator-
associated lung injury.
In summary, we found that depletion of alveolar macro-
phages in vivo decreased lung endothelial and alveolar epithe-
lial permeability, resulting in lower plasma levels of CXCL1
and lower neutrophil counts in the BAL fluid. These data
suggest that alveolar macrophages are a key contributor to the
early proinflammatory milieu and increased permeability pul-
monary edema characteristic of VILI. In addition, soluble
mediators released into the air spaces during high volume
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ventilation activated naı¨ve macrophages, as did media from
stretched alveolar epithelial cell and macrophage cocultures.
These data support the hypothesis that soluble factors released
into the air spaces as a result of the interaction between
alveolar epithelial cells and alveolar macrophages trigger ad-
ditional macrophage activation in VILI. However, the potential
role of the alveolar epithelium or other resident lung cells in
initiating macrophage activation has not been entirely eluci-
dated. This study provides important insights into the patho-
genesis of ventilator-attributable lung injury. First, VILI is
initiated in part by alveolar macrophages. These data also
suggest that one mechanism by which alveolar macrophages
contribute to VILI is through recruitment of neutrophils into
the air spaces. Second, the onset of ventilator-attributable lung
injury is rapid and begins within minutes of starting mechan-
ical ventilation. Accordingly, in the clinical setting, protective
ventilation strategies may need to be initiated as early as
possible in the course of mechanical ventilation.
ACKNOWLEDGMENTS
We acknowledge Teiji Sawa for invaluable advice on the liposome delivery
protocol and Jeanine Wiener-Kronish for the use of liposome delivery equip-
ment.
Present address of D. F. McAuley: Respiratory Research Group, Queen’s
University of Belfast, Belfast, UK.
GRANTS
This work was supported by grants from the Northern California Institute
for Research and Education (J. A. Frank), the Veterans Affairs Administration
(J. A. Frank), the University of California, San Francisco Department of
Medicine (J. A. Frank), National Heart, Lung, and Blood Institute Grants
HL-69900 (J. A. Frank) and HL-51854 (M. A. Matthay), and a research
fellowship grant from the Peel Medical Research Trust (D. F. McAuley).
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