Modulating phosphatidic acid metabolism decreases
oxidative injury in rat lungs
DAVID M. GUIDOT,1–3 STUART L. BURSTEN,4 GLENN C. RICE,4
ROBERT B. CHANEY,4 JACK W. SINGER,4 ALEXANDER J. REPINE,1
BROOKS M. HYBERTSON,1 AND JOHN E. REPINE1
1Webb-Waring Institute for Biomedical Research, University of Colorado Health Sciences Center,
Denver, Colorado 80262; 2Atlanta Department of Veterans Affairs Medical Center, Decatur 30033;
3Department of Medicine, Emory University, Atlanta, Georgia 30322;
and 4Cell Therapeutics, Seattle, Washington 98119
acute respiratory distress syndrome; adherence; chemotaxis;
cytokine
and activation in the lung, with
subsequent capillary endothelial damage, is an early
feature of acute respiratory distress syndrome (ARDS).
The proinflammatory cytokines interleukin-8 (IL-8)
and interleukin-1 (IL-1) are elevated in lung lavages of
ARDS patients (9, 26), and administering IL-8 or IL-1
intratracheally causes acute capillary leak in isolated
rat lungs perfused with human neutrophils (15, 16).
Moreover, lung injury appears to depend on neutrophilderived oxygen radicals because capillary leak is not
seen when neutrophils that cannot produce superoxide
are added to the perfusate (16). Oxidative metabolism
NEUTROPHIL RETENTION
of linoleate has also been implicated in inflammatory
damage (3, 6, 7, 11, 14, 20). IL-1 stimulates formation of
lipoxygenase-derived linoleate oxidation products, including 9-hydroxyoctadecadienoic acid (9-HODE) and
13-hydroxyoctadecadienoic acid (13-HODE) (6); these
substances are mitogenic and proinflammatory (3, 7,
20). In addition, they have been found to be present in
esterified form in lipids (11). Lipid oxidation products
such as these recently have been implicated in endorgan damage in human sepsis, including lung injury
(14). Although these findings suggest important interactions between cytokines, neutrophils, lipids (especially
oxidized forms), and oxygen radicals in the development of acute inflammatory lung injury, the responsible mechanisms remain unclear.
Intracellular phospholipid signaling pathways are
critical to cytokine-mediated inflammatory responses.
For example, in addition to promoting peroxidation and
oxidation of linoleate, IL-1 activates lysophosphatidic
acyl transferase (LPAAT), which produces specific phosphatidic acids containing both sn-1 and sn-2 oleate and
linoleate, as well as the free 18:1(n-9) and 18:2(n-6) acyl
chains, and diacylglycerol species that initiate cellular
responses to IL-1 (25). Lisofylline [(R)-1-(5-hydrohexyl)3,7-dimethylxanthine], a specific inhibitor of this pathway [half-maximal inhibitory concentration (IC50 )5 40
nM], prevented intrapulmonary cytokine signaling and
inflammatory injury in mice after hemorrhage and
resuscitation (1) and inhibited formation of oleate- and
linoleate-containing phosphatidic acid species by neutrophils exposed to hypoxia-reoxygenation in vitro (25).
Phosphatidic acids also appear to act as intracellular
second messengers in neutrophils (10), but their precise role in neutrophil responsiveness to cytokines is
unknown. Importantly, lisofylline does not inhibit IL-8
production by lipopolysaccharide-activated human
whole blood ex vivo (24) or superoxide anion production
by activated neutrophils in vivo (1). Therefore, its
efficacy in ARDS, in which IL-8-dependent neutrophil
sequestration and subsequent oxidant production appear to be important mechanisms in the early phase of
the syndrome, is unknown. In the present investigation, we determined the effects of lisofylline on IL-8induced, neutrophil-dependent acute capillary leak in
isolated rat lungs. We then extended these studies and
determined the effects of lisofylline on IL-8-stimulated
neutrophil responsiveness in vitro, as well as neutrophil-
1040-0605/97 $5.00 Copyright r 1997 the American Physiological Society
L957
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Guidot, David M., Stuart L. Bursten, Glenn C. Rice,
Robert B. Chaney, Jack W. Singer, Alexander J. Repine,
Brooks M. Hybertson, and John E. Repine. Modulating
phosphatidic acid metabolism decreases oxidative injury in
rat lungs. Am. J. Physiol. 273 (Lung Cell. Mol. Physiol. 17):
L957–L966, 1997.—We determined that lisofylline, a potent
inhibitor of oleate- and linoleate-containing phosphatidic acid
formation (half-maximal inhibitory concentration 5 40 nM),
prevented oxidant-mediated capillary leak in isolated rat
lungs given interleukin-8 (IL-8) intratracheally and perfused
with human neutrophils. Lung leak was prevented by lung,
but not neutrophil, lisofylline pretreatment. Furthermore,
although lisofylline inhibited IL-8-stimulated neutrophil production of phosphatidic acid in vitro, it did not prevent
IL-8-stimulated neutrophil adherence, chemotaxis, or intracellular calcium mobilization or N-formyl-Met-Leu-Phe
(fMLP)-stimulated oxidant production in vitro. Lisofylline
also prevented acute capillary leak in isolated rat lungs
perfused only with the oxidant generator purine-xanthine
oxidase but did not scavenge O2
2 z or H2O2 in vitro. Finally,
lisofylline-mediated protection against lung leak in both
models was associated with alterations in lung membrane
free fatty acid acyl composition (as reflected by the decreased
ratio [linoleate 1 oleate]/[palmitate]). We conclude that lisofylline prevented both neutrophil-dependent and neutrophilindependent oxidant-induced capillary leak in isolated rat
lungs and that protection appears to be mediated by blocking
intrinsic lung linoleoyl phosphatidic acid metabolism. We
speculate that lisofylline, in addition to our previously reported effects on cytokine signaling by intrapulmonary mononuclear cells, alters intrinsic pulmonary capillary membrane
composition and renders this barrier less vulnerable to
oxidative damage.
L958
PHOSPHATIDIC ACID METABOLISM AND ACUTE LUNG INJURY
independent, oxidant-mediated capillary leak in isolated rat lungs perfused with purine and xanthine
oxidase.
MATERIALS AND METHODS
Purification of Human Neutrophils
Neutrophils were isolated using a Percoll gradient and
differential centrifugation from heparinized blood obtained
from healthy volunteers. Each preparation contained highly
purified (.99%) neutrophils that were suspended in Hanks’
balanced salt solution (Sigma, St. Louis, MO) at a concentration of 2 3 107/ml.
Isolation and Perfusion of Lungs
Isolated Lung Experimental Protocol
In all experiments, a 20-min equilibration period was
followed by a 60-min experimental period. Untreated lungs
served as controls. In some experiments, IL-8 (250 ng) was
diluted in 0.5 ml of saline and then injected intratracheally
immediately on lung isolation. In some experiments, freshly
purified human neutrophils (a total of 4 3 107, comparable to
an initial circulating concentration of ,1,300 neutrophils/µl)
were added to the perfusate chamber following a 20-min
equilibration period. In some experiments, lungs were given
IL-8 and perfused with neutrophils. In some of the lisofylline
treatment experiments, lisofylline (1 µM final concentration)
was added to the perfusate at the beginning of the equilibration period. In the experiments in which the isolated lungs
were pretreated with lisofylline before neutrophil perfusion,
lisofylline (0.5 ml of a 10 µM solution) was administered
intratracheally with IL-8 (total volume 0.5 ml in saline), and
lisofylline (1 µM) was added to the perfusate; after the
equilibration period, the perfusate was replaced and neutrophils were added to the fresh perfusate without lisofylline. In
the experiments in which neutrophils were pretreated with
lisofylline, neutrophils were incubated with 10 µM lisofylline
for 20 min and then washed before their addition to the
perfusate. Finally, in some experiments, isolated lungs were
treated with xanthine oxidase (0.02 U/ml, grade 1, purified
from cow buttermilk and kindly provided by Dr. Joe McCord)
with or without allopurinol (50 µM, Sigma) and purine (2
mM, Sigma) added to the perfusate after the equilibration
period. In all experiments, lung weights were monitored
continuously throughout the 60-min experimental protocol
with a force transducer. After each experiment, lungs were
freeze-clamped in liquid N2 for assessment of lipid composition, or they were subsequently homogenized and centrifuged
at 15,000 g for 10 min, and their supernatants were recovered
for Ficoll determinations.
Samples of lung homogenate supernatants were added to a
solution of 0.05% anthrone (Sigma) in sulfuric acid, mixed
well, and allowed to equilibrate for 20 min. Total Ficoll
concentrations retained per lung were determined by measuring absorbance spectrophotometrically at 627 nm (22).
Assessment of Neutrophil Linoleoyl Phosphatidic Acid
Synthesis in Response to IL-8 Stimulation In Vitro
Neutrophils were stimulated with IL-8 (20 ng/ml) with and
without lisofylline (1 or 10 µM) and then fixed in ice-cold
methanol at serial time points from 0 to 360 s after IL-8
stimulation. High-performance liquid chromatography
(HPLC) analysis of phosphatidic acid was then performed as
previously described (4, 5). Briefly, lipids were extracted and
separated by HPLC with a Waters µ-Porasil silica column
with a mobile phase consisting of a gradient of 1–9% water in
hexane-isopropanol (3:4, vol/vol) run at a flow rate of 1 ml/min
(4, 5, 27) using an anisocratic gradient, with column effluents
monitored at 206–224 nm. HPLC fractions were also analyzed for hydrolyzed acyl content (as below) and mass spectrometry to confirm peak identities (1). Fast-atom bombardment (FAB) mass spectrometry spectra were acquired using a
VG 70 SEQ tandem hybrid instrument of EBqQ geometry
(VG Analytical, Altrincham, UK) (10). Unsaturated phosphatidic acid was expressed as relative mass (1).
Assessment of Neutrophil Responsiveness In Vitro
Chemotaxis. Neutrophil chemotactic activity was determined using a 96-well chamber assay (Neuro Probe, Cabin
John, MD). IL-8 (20 ng/ml) was placed in the bottom well with
or without lisofylline (1 µM); neutrophils were added to the
top well with or without lisofylline. After the chambers were
incubated for 1 h at 37°C, neutrophil migration into the
bottom wells was quantitated spectrophotometrically at 450
nm (reflecting myeloperoxidase activity as an index of neutrophils in the bottom well) (19).
Adherence. Neutrophil adherence was assessed by quantitating the percentage of neutrophils adhering to nylon fibers
(21) after addition of IL-8 (20 ng/ml) with or without lisofylline (1 µM).
Calcium flux. After isolation, human neutrophils were
divided into 5-ml tubes at 1 3 106/ml in cold RPMI 1640
(containing 100 mg/l of calcium nitrate, 5 mM glucose, and
0.04% gelatin). After incubation with lisofylline (10–200 µM)
for 45 min at room temperature, neutrophils were loaded
with an intracellular fluorescent probe, indo 1-acetoxymethyl
ester (AM) (10 µM, Molecular Probes). After a 30-min incubation, neutrophils were centrifuged at 1,100 revolutions/min
for 5 min, resuspended in cold phosphate-buffered saline
(PBS), centrifuged again at 1,100 revolutions/min for 5 min,
and resuspended in cold RPMI 1640. Neutrophils were then
assayed by a fluorescence-activated cell sorter (FACS; Coulter
Epics Elite model). Once a baseline (no stimulation) was
established (8- to 12-min equilibration period), human recombinant IL-8 (100 nM) was added, and the tubes were mixed
and rapidly assayed for intracellular calcium flux (17). In this
method, the relative change in the ratio of fluorescence
intensity of indo 1-loaded cells at 405 and 525 nm reflects
relative intracellular calcium levels in the cells (17).
Superoxide production. Neutrophil superoxide production
was determined by quantitating superoxide dismutase (SOD)inhibitable reduction of cytochrome c (8) in response to
N-formyl-Met-Leu-Phe (fMLP) (1026 M) in the presence or
absence of lisofylline (1 µM). Briefly, neutrophils were incubated at 37°C for 30 min in the presence of cytochrome c with
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After adult male Sprague-Dawley rats (350 6 50 g) were
anesthetized with pentobarbital sodium (60 mg/kg ip), the
lungs were excised, buffer perfused, and ventilated continuously with a tidal volume of 3 ml at a rate of 60 breaths/min
with 5% CO2-21% O2-74% N2. Immediately, lungs were perfused essentially blood free and then perfused continuously
with Earle’s balanced salt solution (Sigma) containing (in g/l)
40 Ficoll-70 (Sigma), 0.265 calcium chloride, 0.09767 magnesium sulfate, 0.4 potassium chloride, 6.8 sodium chloride,
0.122 monobasic sodium phosphate, 1 D-glucose, and 2.2
sodium bicarbonate, with the final pH adjusted to 7.40.
Perfusate (30 ml) was passed through the lungs to remove
residual blood. The system was then closed, and 30 ml of
perfusate were recirculated continuously at a rate of 40 ml · kg
body wt21 · min21.
Assessment of Lung Ficoll Retention
PHOSPHATIDIC ACID METABOLISM AND ACUTE LUNG INJURY
or without fMLP or lisofylline. The reduction of cytochrome c
was determined by spectrophotometric analysis at 550 nm at
the end of each experiment.
Measurement of Superoxide Anion and Hydrogen Peroxide
Scavenging In Vitro
Assessment of Lung and Perfusate Free Fatty Acid
Acyl Distribution
Free fatty acids were analyzed by HPLC. Briefly, after
lipids were extracted from isolated lungs or perfusate (4, 5),
free fatty acids were separated from phospholipids and
repurified using normal-phase HPLC performed on a Gilson
system 45 using a Waters µ-Porasil silica column with a
mobile phase consisting of a gradient of 1–9% water in
hexane-isopropanol (3:4, vol/vol) run at a flow rate of 1 ml/min
(4, 5, 27). Collected free fatty acids [retention factor (Rf ) 3–4.5
min, previously characterized as a pure fraction (4)] were
diluted with methanol and then derivatized with 9-anthroyldiazomethane (9-ADAM) (23, 30). This solution (20 µl) was
then analyzed by a reverse-phase HPLC method that separates and quantitates derivatized anthroyl free fatty acids. A
reverse-phase C8 column (4.6 3 25 cm, 5-µm Spherisorb C8,
Alltech Associates, Deerfield, IL) was placed in series with a
reverse-phase C18 column (4.6 3 15 cm, 3-µm Spherisorb
ODS2, Alltech Associates) to separate saturated and unsaturated fatty acids, which could not be separated in toto by
either column alone. Anthroyl free fatty acids were separated
using an anisocratic gradient, with acetonitrile-water (70:30,
vol/vol) used in tandem with 100% acetonitrile (2). Separated
anthroyl free fatty acids were detected both by ultraviolet
absorption (4) and by fluorescence with excitation between
305 and 395 nm and emission at 430–470 nm (23). Separation
of free fatty acids including laurate (12:0), myristate (14:0),
myristoleate (14:1), palmitate (16:0), palmitoleate [16:1(n-9)], heptadecanoate (17:0), stearate (18:0), oleate [18:1(n-9)], petroselinate (18:1), linoleate [18:2-(n-6)], a-linoleate
[18:3-(n-6)], g-linolenate [18:3-(n-3)], eicosatrienoate (20:3),
and arachidonate (20:4) occurred with an interassay coefficient of variation for Rf of ,12%. Anthroyl free fatty acids
were quantitated against fatty acid standards (Avanti Polar
Lipids) derivatized as above, with extraction efficiency quantitated against the added internal standard heptadecanoate
(17:0) converted to 17:0-anthroyldiazomethane. The amount
of each fatty acid recovered was a linear function of the
amount added in a range from >30–40 ng (lower limits of
linearity and detection) to 10 µg, with r values in the
0.96–0.995 range. Intra-assay coefficients of variation ranged
from 12–15% for saturated fatty acids, oleate, and linoleate to
17–22% for polyunsaturated fatty acids including arachidonate. The lowest reliable level of individual fatty acids detect-
able from standards was 34 6 8 ng (121 6 25 pmol), with
contamination varying from >30 to 51 ng total background
fatty acids/sample in this study (quantitation and background noise were cross-checked against gas chromatographicmass spectrometric detection of free fatty acids).
Statistical Analyses
Values were compared by analysis of variance and corrected by
the Student-Newman-Keuls test for differences between groups.
A P value of ,0.05 was considered significant.
RESULTS
Effect of Lisofylline on Leak in Isolated Rat Lungs
Given IL-8 Intratracheally and Perfused
With Human Neutrophils
Isolated rat lungs given IL-8 intratracheally and
perfused with human neutrophils developed increased
(P , 0.05) weights (Fig. 1A) and Ficoll retention (Fig.
1B) compared with untreated control lungs, lungs
given only IL-8 intratracheally, and lungs only perfused with human neutrophils. In contrast, isolated rat
lungs given IL-8 intratracheally and perfused with
human neutrophils and lisofylline (1 µM) had decreased
(P , 0.05) weights (Fig. 1A) and Ficoll retention (Fig. 1B)
compared with lungs given IL-8 intratracheally and perfused with human neutrophils. Furthermore, isolated rat
lungs that were pretreated with lisofylline intratracheally
(0.5 ml of a 10 µM solution) and via perfusion (1 µM during
equilibration period) and then given IL-8 intratracheally
and perfused with human neutrophils also had decreased
(P , 0.05) weights (Fig. 1A) and Ficoll retention (Fig.
1B) compared with lungs given IL-8 intratracheally
and perfused with human neutrophils. In contradistinction, nonpretreated lungs given IL-8 intratracheally
and then perfused with human neutrophils that had
been pretreated with lisofylline (10 µM for 20 min and
then washed) had the same (P . 0.05) weights (Fig. 1A)
and Ficoll retention (Fig. 1B) as lungs given IL-8
intratracheally and perfused with human neutrophils.
Effect of Lisofylline on IL-8-Stimulated Neutrophil
Phosphatidic Acid Metabolism In Vitro
Human neutrophils stimulated with IL-8 (20 ng/ml)
had a significant (P , 0.05) increase compared with
baseline in unsaturated phosphatidic acid relative mass
(Fig. 2). In contrast, human neutrophils stimulated
with IL-8 in the presence of lisofylline (1 or 10 µM) had
no significant (P . 0.05) increase in unsaturated phosphatidic acid relative mass (Fig. 2). Human neutrophils
that were not stimulated with IL-8 had an unsaturated
phosphatidic acid relative mass of 27.4 6 6 at the end of
the 360-s incubation. Each value represents the mean 6
SD of nine determinations. The HPLC methods used
and described detect unsaturated acyl chains (primarily oleate and linoleate). Saturated acyl chains such as
stearate are not detected by this method. We calculated
that the resting mass of phosphatidic acid in 2.5 3 107
neutrophils is equivalent to ,300–600 pmol and increases to ,2–4 nmol (i.e., an increase of about 6- to
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Superoxide anion was detected by measuring the SODinhibitable reduction of cytochrome c during a 30-min assay
using hypoxanthine (50 µM) and 0.2 ml of a stock solution
containing bovine xanthine oxidase (1.6 U/ml) in the presence
or absence of lisofylline (20 or 200 µM). Each determination
was done in quadruplicate. Hydrogen peroxide was detected
with a peroxidase assay using o-dianisidine dihydrochloride
as a chromogenic donor. Briefly, hydrogen peroxide (200 µM)
was added to cuvettes containing 1 ml of reagent [10 mg
o-dianisidine dihydrochloride, 18.6 mg EDTA, 5.0 ml 0.1%
horseradish peroxidase, 1.0 ml 10% Triton X-100, all diluted
in 0.05 M sodium acetate, pH 5.0] with or without lisofylline
(20 or 200 µM) and allowed to incubate for 15 min at room
temperature. Absorbance at 460 nm was measured and
recorded. Assays were done in quadruplicate.
L959
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PHOSPHATIDIC ACID METABOLISM AND ACUTE LUNG INJURY
12-fold) after stimulation with IL-8. Analysis of this
increased mass reveals that the majority of it is accounted for by increases in oleate-, linoleate-, and
palmitate-containing phosphatidic acids.
Effect of Lisofylline on Neutrophil
Responsiveness In Vitro
Adherence. Human neutrophils treated with IL-8
had increased (P , 0.05) adherence activity compared
with untreated neutrophils (Fig. 3A). In comparison,
Fig. 2. Unsaturated phosphatidic acid (PA) relative mass in neutrophils stimulated with IL-8 (20 ng/ml) and neutrophils stimulated
with IL-8 in the presence of lisofylline (1 or 10 µM). Human
neutrophils that were not stimulated with IL-8 had an unsaturated
PA relative mass of 27.4 6 6 at the end of the 360-s incubation.
Identity of unsaturated PA was verified by mass spectrometry and
acyl chain analysis of hydrolyzed PA fractions, which revealed that
the majority of this increased mass is accounted for by increases in
oleate-, linoleate-, and palmitate-containing PA. Values are means 6
SD of 9 determinations.
neutrophils treated with IL-8 in the presence of lisofylline had the same (P . 0.05) adherence activity as
neutrophils stimulated with IL-8 in the absence of
lisofylline (Fig. 3A).
Chemotaxis. Human neutrophils treated with IL-8
had increased (P , 0.05) chemotaxis activity compared
with unstimulated neutrophils (Fig. 3B). In comparison, neutrophils treated with IL-8 in the presence of
lisofylline had the same (P . 0.05) chemotaxis activity
as neutrophils stimulated with IL-8 in the absence of
lisofylline (Fig. 3B).
Calcium flux. Indo 1-loaded human neutrophils analyzed at 16–64 s after treatment with IL-8 had qualitatively similar profiles of calcium flux by FACS analysis
in the presence or absence of lisofylline. Shown in Fig.
3C is a representative pair of histograms for the 150
µM dose of lisofylline; each experiment was performed
three times at 10, 150, and 200 µM lisofylline. The peaks
represent relative change in the ratio of fluorescence;
lisofylline had no effect at 10 or 200 µM (not shown).
Superoxide production. Human neutrophils treated
with fMLP made more (P , 0.05) superoxide than
unstimulated neutrophils (Fig. 3D). In comparison,
neutrophils treated with lisofylline and then fMLP
made the same (P . 0.05) amount of superoxide as
neutrophils treated only with fMLP (Fig. 2D).
Effect of Lisofylline on Isolated Rat Lungs Perfused
With Xanthine Oxidase and Purine
Isolated rat lungs perfused with xanthine oxidase
and purine had increased (P , 0.05) weights (Fig. 4A)
and Ficoll retention (Fig. 4B) compared with untreated
control lungs. In contrast, isolated rat lungs perfused
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Fig. 1. Lung weight gain (A) and Ficoll retention (B) in
(left to right) untreated isolated rat lungs, lungs given
only interleukin-8 (IL-8) intratracheally, lungs only
perfused with human neutrophils [polymorphonuclear
leukocytes (PMN)], lungs given IL-8 intratracheally and
perfused with human neutrophils, lungs given IL-8
intratracheally and perfused with neutrophils and lisofylline, lungs pretreated with lisofylline and then given
IL-8 intratracheally and perfused with neutrophils, and
lungs given IL-8 intratracheally and then perfused with
neutrophils that had been pretreated with lisofylline.
Values are means 6 SE of 5 or more determinations.
PHOSPHATIDIC ACID METABOLISM AND ACUTE LUNG INJURY
L961
with lisofylline, xanthine oxidase, and purine had no
significant (P . 0.05) weight gain (Fig. 4A) or Ficoll
retention (Fig. 4B) compared with untreated control
lungs. In parallel, isolated rat lungs perfused with
allopurinol (an inhibitor of xanthine oxidase enzymatic
activity), xanthine oxidase, and purine had no significant (P . 0.05) weight gain (Fig. 4A) or Ficoll retention
(Fig. 4B) compared with untreated control lungs.
Effect of Lisofylline on Superoxide Anion
and Hydrogen Peroxide Levels Generated
by Xanthine Oxidase In Vitro
Lisofylline at concentrations of 20 or 200 µM did not
reduce (P . 0.05) superoxide anion levels or hydrogen
peroxide levels in vitro (Table 1).
Effect of Lisofylline on Lung and Perfusate Free Fatty
Acid Acyl Chain Composition
Isolated rat lungs given IL-8 intratracheally and
then perfused with human neutrophils had increased
membrane ratios, [oleate 1 linoleate]/[palmitate], compared with untreated control lungs (P , 0.05) (Fig. 5).
In contrast, lisofylline-treated isolated rat lungs given
IL-8 intratracheally and then perfused with human
neutrophils had decreased [oleate 1 linoleate]/[palmitate] (P , 0.05) compared with untreated lungs given
IL-8 and then perfused with neutrophils (Fig. 5). Membrane ratios from lungs given only IL-8 and from lungs
perfused only with neutrophils were not determined. In
addition, perfusates from lungs given purine and xanthine oxidase had significantly increased [oleate 1
linoleate]/[palmitate] compared with perfusates from
untreated control lungs (0.85 6 0.11 vs. 0.67 6 0.08,
P , 0.05; not shown in Fig. 5). In contrast, perfusates
from lungs given purine and xanthine oxidase and
treated with lisofylline had significantly reduced [oleate 1 linoleate]/[palmitate] compared with perfusates
from lungs given purine and xanthine oxidase (0.74 6
0.12 vs. 0.85 6 0.11, P , 0.05; not shown in Fig. 5).
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Fig. 3. Human neutrophil adherence activity stimulated by IL-8 (20 ng/ml) in the presence or absence of lisofylline
(1 µM) (A), chemotaxis activity [absorbance at 450 nm (Abs450)] stimulated by IL-8 (20 ng/ml) in the presence or
absence of lisofylline (1 µM) (B), calcium flux stimulated by IL-8 (100 nM) in the absence (control) or presence of
lisofylline (LSF; 150 µM) (C), and superoxide production stimulated by N-formyl Met-Leu-Phe (fMLP; 1026 M) in
the presence or absence of lisofylline (1 µM) (D). Cyt c, cytochrome c. For A, B, and D, values are means 6 SE of 5 or
more determinations. C: a representative fluorescence-activated cell sorter analysis; peaks represent relative
change in the ratio of fluorescence intensity of indo 1-loaded cells at 405 and 525 nm, reflecting relative intracellular
calcium levels in the cells after stimulation with IL-8. Time scale is in seconds after addition of IL-8 (arrow). Each
experiment was performed 3 times at 10, 150, and 200 µM lisofylline. Lisofylline had no effect at 10 or 200 µM either (not
shown).
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PHOSPHATIDIC ACID METABOLISM AND ACUTE LUNG INJURY
Effect of Lisofylline on Phosphatidic Acid Content
in Stimulated Lungs
When isolated rat lungs given IL-8 intratracheally
and perfused with human neutrophils were fixed in
ice-cold methanol, followed by total lipid extraction and
lipid separation by HPLC [lipid fractions were characterized by FAB mass spectrometry, positive and negative molecular ions (200–2,000 M 6 H/z)], it was found
that unsaturated phosphatidic acid was increased by
an average of 345% (P , 0.01 vs. control lungs for n 5
6). Lysophosphatidic acid was also increased by 110–
130% (P , 0.05 vs. control). In contrast, isolated rat
lungs given IL-8 intratracheally and perfused with
neutrophils and lisofylline demonstrated a reduction of
phosphatidic acid to baseline amounts (P . 0.10 vs.
controls; P , 0.001 vs. IL-8 and neutrophils) but a
further increase in lysophosphatidic acid to 195% over
baseline (P , 0.01 vs. controls). A characteristic experiment is shown in Fig. 6, in which lipids extracted from a
lung exposed to IL-8 and perfused with neutrophils is
contrasted with lipids extracted from a lung given IL-8
and then perfused with neutrophils and lisofylline. It
can easily be observed that highly unsaturated phosphatidic acid species (PAa, Rf 6 min; PAb, Rf 9 min) stimulated by IL-8 and neutrophil perfusion are greatly
inhibited in the presence of lisofylline (the peak of
which can be seen with an Rf of 14–15 min). In contrast,
lysophosphatidic acid (Rf values of 12 and 13 min)
increases moderately, while there is no change in
phosphatidylethanol. PAa represents diunsaturated
phosphatidic acid species (mainly oleoyl and linoleoyl),
PAb represents alkyl unsaturated phosphatidic acid
Table 1. Direct effect of lisofylline on superoxide anion
and hydrogen peroxide levels in vitro
Test Conditions
O2
2 · Levels
H2O2 Levels
XO 1 purine
32.96 6 4.49 (6) 1.11 6 0.01 (4)
XO 1 purine 1 lisofylline (20 µM) 29.08 6 0.20* (4) 1.11 6 0.1* (4)
XO 1 purine 1 lisofylline (200 µM) 31.27 6 1.41* (4) 1.11 6 0.02* (4)
Values are means 6 SE; nos. in parentheses are no. of determinations. O2
2 · levels are in nmol cytochrome c reduced/30 min; H2O2
levels are oxidation of o-dianisidine/15 min based on absorbance at
460 nm (see MATERIALS AND METHODS for details). * Value not significantly different (P . 0.05) from value for xanthine oxidase (XO) 1
purine alone.
Fig. 5. Membrane fraction ratios, [oleate 1 linoleate]/[palmitate], in
untreated isolated rat lungs, lungs given IL-8 (200 ng) intratracheally and perfused with human neutrophils (4 3 107 PMN/30 ml),
and lungs given IL-8 intratracheally and perfused with PMN and
lisofylline (1 µM). Values are means 6 SE of 5 or more determinations. Membrane ratios from lungs perfused with purine and XO with
or without lisofylline are given in text. Membrane ratios from lungs
given only IL-8 and from lungs only perfused with PMN were not
determined.
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Fig. 4. Lung weight gain (A) and Ficoll retention (B) in
untreated control lungs, lungs perfused with purine (2
mM) and xanthine oxidase (XO, 0.02 U/ml), lungs
perfused with purine, XO, and lisofylline (1 µM), and
lungs perfused with allopurinol (an inhibitor of XO
enzymatic activity), XO, and purine. Values are means 6
SE of 4 or more determinations.
PHOSPHATIDIC ACID METABOLISM AND ACUTE LUNG INJURY
species (mainly 1-o-hexadecanyl, 2-oleoyl, and 2-linoleoyl), and PAc represents a mixture of alkenyl and
monounsaturated phosphatidic acid species. The phosphatidic acid content in control lungs, lungs given only
IL-8, and lungs perfused only with neutrophils was not
determined.
DISCUSSION
We found that lisofylline, a specific inhibitor of
oleate- and linoleate-containing phosphatidic acid formation, prevented acute capillary leak in isolated rat
lungs given IL-8 intratracheally and perfused with
human neutrophils. Although lisofylline blocked IL-8stimulated neutrophil production of oleate- and linoleate-containing phosphatidic acid, it did not prevent
IL-8-stimulated neutrophil calcium mobilization or chemotaxis in vitro. This is as expected, given that neutrophil calcium mobilization and chemotaxis are dependent on phospholipase C-mediated hydrolysis of
phosphoinositide to diacylglycerol and inositol 1,4,5trisphosphate, which is not inhibited by lisofylline. In
addition, lisofylline did not inhibit IL-8-induced neutrophil adherence or fMLP-stimulated neutrophil superoxide production in vitro, additional evidence that lisofylline-mediated protection in this model was not
associated with demonstrable effects on neutrophil
responsiveness. Therefore, although lisofylline prevented neutrophil-dependent lung injury in this model,
we did not identify any inhibition of neutrophil activa-
tion by lisofylline in vitro, suggesting there might be an
unanticipated mechanism for lisofylline-mediated protection.
We previously demonstrated that neutrophil oxidant
production is required for acute edematous injury in
isolated rat lungs given either IL-1 or IL-8 intratracheally and perfused with human neutrophils (15, 16).
However, as we have discussed in these previous studies, IL-1 is not a potent direct activator of neutrophils
and the direct effects of IL-8 on neutrophils appear to be
dominated by its chemokinetic effects (i.e., adhesion
and migration). To wit, we could not detect significant
superoxide production by IL-8-stimulated neutrophils
in vitro (data not shown) in our assay conditions. A
recent report demonstrated that IL-8 does stimulate
the neutrophil respiratory burst but that, compared
with other well-studied activators including fMLP and
phorbol 12-myristate 13-acetate (PMA), the effect is
transient (18). However, as neutrophils that are unable
to generate superoxide anion (but can adhere and
migrate in response to stimuli in vitro) do not injure
either IL-1- or IL-8-treated lungs (15, 16), it appears
that neutrophil NADPH activation, either directly or
indirectly, contributes to acute edematous injury caused
by these cytokines in the intact lung. In this study we
found that lisofylline did not inhibit fMLP-stimulated
superoxide production by neutrophils in vitro, and we
previously determined that lisofylline did not inhibit
PMA-stimulated superoxide production by neutrophils
(1). Furthermore, in the present study, neutrophils
pretreated with lisofylline (which irreversibly inhibits
neutrophil LPAAT activity) still caused leak in isolated
lungs given IL-8, whereas lungs pretreated with lisofylline and then given IL-8 did not develop leak with
neutrophil perfusion. In total, these observations suggest that although neutrophil-derived reactive oxygen
species appear to be important mediators of acute lung
injury in this model, lisofylline-mediated protection
cannot be explained by an inhibition of the neutrophil
respiratory burst. In this study we used IL-8 because
we had developed this model previously and had already characterized the synergistic effects of IL-8 and
human neutrophils in producing a neutrophil-dependent, oxidant-mediated acute edematous injury in rat
lungs perfused ex vivo. To date we have not determined
the effects of lisofylline on other inflammatory mediators, including IL-1, in this model, and therefore we
cannot at present generalize these findings to include
other activators of neutrophil function. However, our
present findings suggest that, in contrast to our initial
hypothesis when we initiated this project, lisofyllinemediated protection is not explained by an inhibition of
neutrophil activation in the lung.
Therefore, we extended this project to test the hypothesis that lisofylline could be acting as an antioxidant by
some other mechanism in the lung. We therefore tested
its ability to prevent an oxidant-mediated edematous
injury that was independent of neutrophils. Importantly, lisofylline prevented acute capillary leak in
isolated rat lungs perfused with the cell-free oxidant
generator purine-xanthine oxidase, a well-character-
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Fig. 6. PA content in isolated lungs in a characteristic highperformance liquid chromatographic tracing of lipids separated from
rat lungs given IL-8 (200 ng) intratracheally and perfused with
human neutrophils (4 3 107/30 ml) (dashed line) or given IL-8 and
perfused with neutrophils and LSF (1 µM; solid line). PA content in
control lungs, lungs given only IL-8, and lungs only perfused with
neutrophils was not determined. PAa, diunsaturated PA species
(mainly oleoyl and linoleoyl); PAb, alkyl unsaturated PA species
(mainly 1-o-hexadecanyl, 2-oleoyl, and 2-linoleoyl); PAc, a mixture of
alkenyl and monounsaturated PA species; lyso-PA, lysophosphatidic
acid; PG, phosphatidylglycerol; CL, cardiolipin, PE, phosphatidylethanol; A206, absorbance at 206 nm.
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L964
PHOSPHATIDIC ACID METABOLISM AND ACUTE LUNG INJURY
line. This pool in serum and in other tissues is profoundly affected by inflammatory stimuli and is consistently reduced or ablated by lisofylline. Interestingly,
patients who are at risk for ARDS or who have ARDS
have increased serum [oleate 1 linoleate]/[palmitate]
compared with control subjects, and these ratios are
decreased by treating the patients with lisofylline
(Bursten, Singer, and Repine, unpublished observation). It is also possible that the inflammatory effects on
cell membranes are mediated by reesterified oxidized
linoleate species such as 9-HODE and 13-HODE or an
epoxified derivative such as leucotoxin (3, 7, 11, 14), all
of which would be reduced by lisofylline.
The present finding that lisofylline confers intrinsic
lung protection but does not reduce neutrophil responsiveness during IL-8-mediated inflammation provides
a striking contradistinction to our previous finding, i.e.,
that intrinsic neutrophil 5-lipoxygenase is a critical
intracellular lipid second messenger in IL-8 signaling,
with its action on neutrophils rather than on the lung
per se. Indeed, in that situation, inhibiting neutrophil
5-lipoxygenase activity prevented IL-8-stimulated chemotaxis in vitro and IL-8-mediated lung injury, whereas
inhibiting lung 5-lipoxygenase activity did not prevent
lung leak (15). Intracellular signaling via lipid second
messengers, such as leukotrienes, 9- and 13-HODE,
and phosphatidic acid species, is critical in the inflammatory cascade. The precise roles for each pathway
remain to be delineated and obviously may differ
depending on the inflammatory signal and the responding cell. For example, we found that although IL-8
rapidly stimulates neutrophil production of oleate- and
linoleate-containing phosphatidic acids, these moieties
are not required for neutrophil responsiveness to IL-8,
at least as measured by calcium mobilization, adherence, and chemotaxis in vitro, as well as acute capillary
leak in the isolated rat lung. In contrast, lisofylline did
inhibit neutrophil chemotaxis stimulated by zymosanactivated serum in vitro (1), observations that reveal
divergent signaling in response to complement stimulation. Another candidate role for phosphatidic acid metabolism in neutrophils is priming by IL-8, by which
IL-8 is known to potentiate neutrophil oxidant production to a subsequent stimulus, such as fMLP (29).
Activation of this pathway might enhance neutrophil
responsiveness at sites of inflammation. However, neutrophils incubated with lisofylline, which irreversibly
inhibits oleate- and linoleate-containing phosphatidic
acid formation for at least 2 h (25), still caused lung
damage. Thus the precise role(s) for this pathway in
neutrophil responsiveness to IL-8 and other agents is
unknown.
Lisofylline may also protect targets such as the
pulmonary capillary endothelium by blocking multiple
cytokine signal responses in these cells. At present, the
role(s) of phosphatidic acid signaling in nonimmune
cells is even less well understood. However, changes in
membrane composition could have profound consequences for surface expression of cell adhesion mol-
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ized method of inducing oxidative stress in biological
systems. We used bovine xanthine oxidase that had
been purified from cow buttermilk rather than commercially available xanthine oxidase, which may be contaminated with immunoglobulins and/or proteases. Furthermore, lungs treated with xanthine oxidase and its
inhibitor allopurinol did not develop lung edema, indicating that the observed injury in these experiments
was related to its enzymatic function (i.e., generation of
superoxide anion and hydrogen peroxide) and was not
caused by either contaminants or nonenzymatic effects
of xanthine oxidase. Thus lisofylline protected against
both neutrophil-dependent and neutrophil-independent oxidant-mediated acute lung injury. However,
lisofylline did not scavenge superoxide anion or hydrogen peroxide or inhibit xanthine oxidase activity in
vitro. This last observation argued that lisofylline was
not acting as a classic antioxidant, such as superoxide
dismutase, catalase, glutathione, or ascorbate.
Because lisofylline did not appear to have any significant effects on oxidant generation by neutrophils and
did not scavenge oxidants in vitro, we questioned
whether it may be acting to protect the targets of
oxidative injury. Consistent with this speculation, we
found that lisofylline treatment lowered lung and perfusate free fatty acid [oleate 1 linoleate]/[palmitate] as
well as phosphatidic acids containing linoleate. Although the mechanism is unclear, inflammatory stimuli
could rapidly produce oleate- and linoleate-containing
phosphatidic acid and diacylglycerol species that could
increase the relative concentration of these acyl groups
in the cell membrane via specific partitioning between
cytosolic and membrane pools. Membranes with relatively higher proportions of unsaturated lipids are
more vulnerable to lipid peroxidation and subsequent
membrane damage (13), and membrane peroxidation
increases exponentially with the number of bis-allylic
(unsaturated) positions in the constituent lipids (28). In
addition, an increase in phosphatidic acids containing
oleate and linoleate will be reflected in free fatty acids
released by phospholipase A2 that has been activated
by inflammatory cytokines and oxidants. Lisofylline
appears to prevent membrane lipid compositional
changes induced by inflammatory stimuli, and this
relative preservation of saturated lipid content may
provide intrinsic protection to parenchymal cells against
oxidative injury. The decrease in phosphatidic acids
containing oleate and linoleate and other unsaturated
fatty acids induced by lisofylline treatment may decrease availability of these unsaturated fatty acids to
phospholipase A2 or decrease shedding of phosphatidic
acid-containing microvesicles (12). Therefore, although
increased membrane fluidity may serve important functions within the inflammatory response, such as cytokine-mediated signal transduction by immune cells, it
may also increase tissue susceptibility to oxidative
injury. We analyzed the total free (nonhydrolyzed) fatty
acid pool from the lung. We have found that this reflects
the exchangeable acyl pool that is affected by lisofyl-
PHOSPHATIDIC ACID METABOLISM AND ACUTE LUNG INJURY
All work was supported by grants from the National Institutes of
Health (P50-HL-40784 and K11–02690) and the Colorado Advanced
Technology Institute.
Address for reprint requests: D. M. Guidot, Atlanta Department of
Veterans Affairs Medical Center (151P), 1670 Clairmont Rd., Decatur, GA 30033.
Received 15 August 1996; accepted in final form 23 July 1997.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
REFERENCES
1. Abraham, E., S. Bursten, R. Shenkar, J. Allbee, R. Tuder, P.
Woodson, D. M. Guidot, G. Rice, J. W. Singer, and J. E.
Repine. Phosphatidic acid signalling mediates lung cytokine
expression and lung inflammatory injury following hemorrhage
in mice. J. Exp. Med. 181: 569–575, 1995.
2. Aveldano, M. I., M. van Rollins, and L. A. Horrocks. Separation and quantitation of free fatty acids and fatty acid methyl
esters by reverse phase high pressure liquid chromatography. J.
Lipid Res. 24: 83–93, 1983.
3. Buchanan, M. R., R. W. Butt, Z. Magas, J. Van Ryn, J. Hirsh,
and D. J. Nazir. Endothelial cells produce a lipoxygenase
derived chemorepellent which influences platelet-endothelial
cell interactions—effect of aspirin and salicylate. Thromb. Haemost. 53: 306–311, 1985.
4. Bursten, S. L., and W. E. Harris. Interleukin-1 stimulates
phosphatidic acid-mediated phospholipase D activity in human
mesangial cells. Am. J. Physiol. 266 (Cell Physiol. 35): C1093–
C1104, 1994.
5. Bursten, S. L., W. E. Harris, K. Bomsztyk, and D. Lovett.
Interleukin-1 rapidly stimulates lysophosphatidate acyltransferase and phosphatidate phosphohydrolase activities in human
mesangial cells. J. Biol. Chem. 266: 20732–20743, 1991.
6. Camacho, M., N. Godessart, R. Anton, M. Garcia, and L.
Vila. Interleukin-1 enhances the ability of cultured human
19.
20.
21.
22.
23.
24.
25.
umbilical vein endothelial cells to oxidize linoleic acid. J. Biol.
Chem. 270: 17279–17286, 1995.
Cowlen, M. S., and T. E. Eling. Effects of prostaglandins and
hydroxyoctadecadienoic acid on epidermal growth factor-dependent DNA synthesis and c-myc proto-oncogene expression in
Syrian hamster embryo cells. Biochim. Biophys. Acta 1174:
234–240, 1993.
Crapo, J., J. McCord, and I. Fridovich. Preparation and
assay of superoxide dismutases. Methods Enzymol. 53: 382–393,
1978.
Donnelly, S., R. Strieter, S. Kunkel, A. Walz, C. Robertson,
D. Carter, I. Grant, A. Pollok, and C. Haslett. Interleukin-8
and development of adult respiratory distress syndrome in
at-risk patient groups. Lancet 341: 643–647, 1993.
English, D. Involvement of phosphatidic acid, phosphatidate
phosphohydrolase, and inositide-specific phospholipase D in
neutrophil stimulus-response pathways. J. Lab. Clin. Med. 120:
520–526, 1992.
Folcik, V. A., and M. K. Cathcart. Predominance of esterified
hydroperoxylinoleic acid in human monocyte-oxidized LDL. J.
Lipid Res. 35: 1570–1582, 1994.
Fourcade, O., M. Simon, C. Viode, N. Rugani, F. Leballe, A.
Ragab, B. Fournie, L. Sarda, and H. Chap. Secretory phospholipase A2 generates the novel lipid mediator lysophosphatidic
acid in membrane microvesicles shed from activated cells. Cell
80: 919–927, 1995.
Garrido, A., F. Garrido, R. Guerra, and A. Valenzuela.
Ingestion of high doses of fish oil increases the susceptibility of
cellular membranes to the induction of oxidative stress. Lipids
24: 833–835, 1989.
Goode, H. F., H. C. Cowley, B. E. Walker, P. D. Howdle, and
N. R. Webster. Decreased antioxidant status and increased lipid
peroxidation in patients with septic shock and secondary organ
dysfunction. Crit. Care Med. 23: 646–651, 1995.
Guidot, D. M., M. J. Repine, J. Y. Westcott, and J. E. Repine.
Intrinsic 5-lipoxygenase activity is required for neutrophil responsivity. Proc. Natl. Acad. Sci. USA 91: 8156–8159, 1994.
Guidot, D. M., E. E. Stevens, M. J. Repine, A. E. LuccaBroco, and J. E. Repine. Intratracheal but not intravascular
interleukin-1 causes acute edematous injury in isolated neutrophil-perfused rat lungs through an oxygen radical dependent
mechanism. J. Lab. Clin. Med. 123: 605–609, 1993.
Holmes, W. E., J. Lee, W. Kuang, G. C. Rice, and W. I. Wood.
Structure and functional expression of a human interleukin-8
receptor. Science 253: 1278–1280, 1991.
Jones, S. A., M. Wolf, S. Qin, C. R. Mackay, and M. Baggiolini. Different functions for the interleukin 8 receptors (IL-*R) of
human neutrophil leukocytes: NADPH oxidase and phospholipase D are activated through IL-8R1 but not IL-8R2. Proc. Natl.
Acad. Sci. USA 6682–6686, 1996.
Junger, W. G., T. A. Cardoza, F. C. Liu, D. B. Hoyt, and R.
Goodwin. Improved rapid photometric assay for quantitative
measurement of PMN migration. J. Immunol. Methods 160:
73–79, 1993.
Kaduce, T. L., P. H. Figard, R. Leifur, and A. A. Spector.
Formation of 9-hydroxyoctadecadienoic acid from linoleic acid in
endothelial cells. J. Biol. Chem. 264: 6823–6830, 1989.
MacGregor, R., P. Spagnulo, and A. Lentnek. Inhibition of
granulocyte adherence by ethanol, prednisone, and aspirin,
measured with an assay system. N. Engl. J. Med. 291: 642–646,
1974.
Morse, E. Anthrone in estimating low concentrations of sucrose.
Anal. Chem. 19: 1012–1013, 1947.
Nakaya, T., T. Tomomoto, and M. Imoto. The syntheses and
the reactions of 9-anthroyldiazomethane and a-naphthyldiazomethane. Bull. Chem. Soc. Jpn. 40: 691–692, 1967.
Rice, G. C., J. Rosen, R. Weeks, J. Michnick, S. Bursten,
J. A. Bianco, and J. W. Singer. CT-1501R selectively inhibits
induced inflammatory monokines in human whole blood ex vivo.
Shock 1: 254–266, 1994.
Singer, J. W., S. L. Bursten, G. C. Rice, W. Perry Gordon,
and J. A. Bianco. Inhibitors of intracellular phosphatidic acid
Downloaded from http://ajplung.physiology.org/ by 10.220.32.247 on June 17, 2017
ecules that mediate interactions between endothelial
cells and neutrophils.
This study shows that inhibiting lung phosphatidic
acid metabolism prevented both neutrophil-dependent
and neutrophil-independent oxidant-mediated vascular leak. Although lisofylline belongs to the class of
methylxanthine drugs, such as theophylline, it does not
share any of their other functions such as phosphodiesterase inhibition (IC50 . 150 µM) or adenosine receptor
blockade (IC50 . 150 µM) at the concentrations used in
this study. Furthermore, although we previously determined that lisofylline inhibits cytokine-dependent signal transduction in intrapulmonary mononuclear cells
during acute inflammation (1), our current study suggests additional effects of lisofylline that cannot be
explained by either delimiting neutrophil activation or
scavenging reactive oxygen species. Clearly, further
study of this relatively recently identified inhibitor of
LPAAT activity may reveal other effects on immune
and nonimmune cells that are at present unknown.
Finally, unlike other agents such as vitamin E that
are also proposed to work by altering membrane lipid
composition and limiting peroxidative damage, lisofylline appears to act quickly, can be delivered easily to
both the pulmonary capillary and alveolar spaces, and
may attenuate inflammatory responses by resident and
recruited immune cells (1). In light of the multiple
integral inflammatory responses affected, inhibition of
the oleate- and linoleate-containing phosphatidic acid
pathway may provide clues as to the mechanisms
responsible for acute lung injury and offer new strategies for its prevention.
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L966
PHOSPHATIDIC ACID METABOLISM AND ACUTE LUNG INJURY
production: novel therapeutics with broad clinical applications.
Exp. Opin. Invest. Drugs 3: 631–643, 1994.
26. Suter, P., S. Suter, E. Girardin, P. Roux-Lombard, G. Grau,
and J. Dayer. High bronchoalveolar levels of tumor necrosis
factor and its inhibitors, interleukin-1, interferon, and elastase,
in patients with adult respiratory distress syndrome after trauma,
shock, or sepsis. Am. Rev. Respir. Dis. 145: 1016–1022, 1992.
27. Van Kessel, W. S. M. G., W. M. A. Hax, R. A. Demel, and J.
deGier. High performance liquid chromatographic separation
and direct ultraviolet detection of phospholipids. Biochim. Biophys. Acta 486: 524–530, 1977.
28. Wagner, B. A., G. R. Buettner, and C. P. Burns. Free
radical-mediated lipid peroxidation in cells: oxidizability is a
function of cell lipid bis-allylic hydrogen content. Biochemistry
33: 4449–4453, 1994.
29. Wozniak, A., W. Betts, G. Murphy, and M. Rokicinski.
Interleukin-8 primes human neutrophils for enhanced superoxide anion production. Immunology 79: 608–615, 1993.
30. Yoshida, T., A. Uetake, H. Yamaguchi, N. Nimura, and T.
Kinoshita. New preparation method for 9-anthroyldiazomethane (ADAM) as a fluorescent labeling reagent for fatty acids and
derivatives. Anal. Biochem. 173: 70–74, 1988.
Downloaded from http://ajplung.physiology.org/ by 10.220.32.247 on June 17, 2017