Modulating phosphatidic acid metabolism decreases oxidative injury in rat lungs

David M. Guidot1,2,3, Stuart L. Bursten4, Glenn C. Rice4, Robert B. Chaney4, Jack W. Singer4, Alexander J. Repine1, Brooks M. Hybertson1, and John E. Repine1

1 Webb-Waring Institute for Biomedical Research, University of Colorado Health Sciences Center, Denver, Colorado 80262; 2 Atlanta Department of Veterans Affairs Medical Center, Decatur 30033; 3 Department of Medicine, Emory University, Atlanta, Georgia 30322; and 4 Cell Therapeutics, Seattle, Washington 98119

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

We determined that lisofylline, a potent inhibitor of oleate- and linoleate-containing phosphatidic acid formation (half-maximal inhibitory concentration = 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 O<SUP>−</SUP><SUB>2</SUB>⋅ 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 + oleate]/[palmitate]). We conclude that lisofylline prevented both neutrophil-dependent and neutrophil-independent 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.

acute respiratory distress syndrome; adherence; chemotaxis; cytokine

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

NEUTROPHIL RETENTION 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 neutrophil-derived oxygen radicals because capillary leak is not seen when neutrophils that cannot produce superoxide are added to the perfusate (16). Oxidative metabolism 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 end-organ 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)= 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-8-induced, 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-independent, oxidant-mediated capillary leak in isolated rat lungs perfused with purine and xanthine oxidase.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 × 107/ml.

Isolation and Perfusion of Lungs

After adult male Sprague-Dawley rats (350 ± 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 wt-1 · min-1.

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 × 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.

Assessment of Lung Ficoll Retention

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 × 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) (10-6 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 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

Superoxide anion was detected by measuring the SOD-inhibitable 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.

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 × 25 cm, 5-µm Spherisorb C8, Alltech Associates, Deerfield, IL) was placed in series with a reverse-phase C18 column (4.6 × 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)], alpha -linoleate [18:3-(n-6)], gamma -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 congruent 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 detectable from standards was 34 ± 8 ng (121 ± 25 pmol), with contamination varying from congruent 30 to 51 ng total background fatty acids/sample in this study (quantitation and background noise were cross-checked against gas chromatographic-mass 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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.


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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 ± SE of 5 or more determinations.

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 at the end of the 360-s incubation. Each value represents the mean ± 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 × 107 neutrophils is equivalent to ~300-600 pmol and increases to ~2-4 nmol (i.e., an increase of about 6- to 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.


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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 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 ± SD of 9 determinations.

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, 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).


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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; 10-6 M) in the presence or absence of lisofylline (1 µM) (D). Cyt c, cytochrome c. For A, B, and D, values are means ± 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).

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 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.


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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 ± SE of 4 or more determinations.

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).

                              
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Table 1.   Direct effect of lisofylline on superoxide anion and hydrogen peroxide levels in vitro

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 + 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 + 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 + linoleate]/[palmitate] compared with perfusates from untreated control lungs (0.85 ± 0.11 vs. 0.67 ± 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 + linoleate]/[palmitate] compared with perfusates from lungs given purine and xanthine oxidase (0.74 ± 0.12 vs. 0.85 ± 0.11, P < 0.05; not shown in Fig. 5).


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Fig. 5.   Membrane fraction ratios, [oleate + linoleate]/[palmitate], in untreated isolated rat lungs, lungs given IL-8 (200 ng) intratracheally and perfused with human neutrophils (4 × 107 PMN/30 ml), and lungs given IL-8 intratracheally and perfused with PMN and lisofylline (1 µM). Values are means ± 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.

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 ± H/z)], it was found that unsaturated phosphatidic acid was increased by an average of 345% (P < 0.01 vs. control lungs for n = 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 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.


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Fig. 6.   PA content in isolated lungs in a characteristic high-performance liquid chromatographic tracing of lipids separated from rat lungs given IL-8 (200 ng) intratracheally and perfused with human neutrophils (4 × 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.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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-8-stimulated 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 1,4,5-trisphosphate, 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 activation 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, lisofylline-mediated 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-characterized 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 + 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 lisofylline. 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 + 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 zymosan-activated 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 molecules 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.

    ACKNOWLEDGEMENTS

All work was supported by grants from the National Institutes of Health (P50-HL-40784 and K11-02690) and the Colorado Advanced Technology Institute.

    FOOTNOTES

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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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