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,2
Free Radical Metabolite Section, Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA;
* National Health and Environmental Effects Research Laboratory, Environmental Protection Agency, Research Triangle Park, North Carolina, USA;
Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA; and
Laboratory of Hepatobiology and Toxicology, Department of Pharmacology, and Curriculum in Toxicology, University of North Carolina, Chapel Hill, North Carolina, USA
1Correspondence: Free Radical Metabolite Section, Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, P.O. Box. 12233, 111 TW Alexander Drive, Research Triangle Park, NC 27709, USA. E-mail: sato{at}niehs.nih.gov
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN), we investigated in vivo free radical production by rats treated with intratracheal instillation of LPS. ESR spectroscopy of lipid extract from lungs exposed to LPS for 6 h gave a spectrum consistent with that of a POBN/carbon-centered radical adduct (aN=14.94±0.07 G and aßH=2.42±0.06 G) tentatively assigned as a product of lipid peroxidation. To further investigate the mechanism of LPS-initiated free radical generation, rats were pretreated with the phagocytic toxicant GdCl3, which significantly decreased the production of radical adducts with a corresponding decrease in neutrophil infiltration. NADPH oxidase knockout mice completely blocked phagocyte-mediated, ESR-detectable radical production in this model of acute lung injury. Rats treated intratracheally with LPS generate lipid-derived free radicals via activation of NADPH oxidase.—Sato, K., Kadiiska, M. B., Ghio, A. J., Corbett, J., Fann, Y. C., Holland, S. M., Thurman, R. G., Mason, R. P. In vivo lipid-derived free radical formation by NADPH oxidase in acute lung injury induced by lipopolysaccharide: a model for ARDS.
Key Words: spin trapping • knockout mice • GdCl3
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. LPS activates alveolar macrophages and causes neutrophils to infiltrate and damage the lungs (2
, 3)
. The stimulated leukocytes produce various molecules that mediate lung damage such as platelet-activating factor (4)
, arachidonic acid metabolites (5
, 6)
, cytokines (2)
, proteases (7)
, and free radicals (8
, 9)
.
It has been reported that free radicals such as superoxide, nitric oxide, and peroxynitrite play important roles in the pathogenesis of acute lung injury because SOD (or its chemical mimics), nitric oxide synthase inhibitors, and N-acetylcysteine all inhibit LPS-induced damage (10
11
12)
. Therefore, lung damage must be caused by these reactive species either directly or indirectly. Although no direct evidence has been presented for free radical generation in LPS lung injury in vivo, it is suggested by in vitro observations such as increased lipid peroxidation and xanthine oxidase/dehydrogenase activity (9)
.
Whereas in vitro investigation has demonstrated radical generation after exposure of cells to LPS, in vivo free radical formation by LPS has not been reported in the lung. Therefore, direct evidence for free radical production in the lung was sought by using the electron spin resonance (ESR) spin-trap method, the only method by which reactive free radicals can be detected directly in biological systems. The present work clearly demonstrates that free radicals are produced in vivo in the LPS lung injury model of ARDS.
Moreover, the origin of free radical formation in this system was investigated. Both macrophage activity (13
14
15)
and neutrophil infiltration (16
, 17)
are inhibited by GdCl3; the vital role of macrophages in free radical production by LPS was confirmed by GdCl3 pretreatment. The major oxidant-generating enzyme in neutrophils and macrophages is known to be NADPH oxidase (18)
. Therefore, we have used NADPH oxidase knockout mice to evaluate NADPH oxidase dependency, and we present here the first in vivo evidence that LPS-induced lung injury is mediated by NADPH oxidase-induced production of free radicals.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN), and LPS (Escherichia coli O26: B6; Sigma Chemical, St. Louis, MO), pentobarbital (Abbott Laboratories, North Chicago, IL), and modified Wright’s stain kit (Fisher Chemicals, Pittsburgh, PA) were used as received.
Animals and treatments
Adult male Sprague-Dawley rats weighing 
350 g (8 wk) were used. Rats were anesthetized by pentobarbital (40 mg/kg), and lung injury was caused by intratracheal instillation of LPS at a dose of 500 µg/rat. Five hours after LPS instillation, rats were anesthetized by pentobarbital (30 mg/kg) and injected with POBN intraperitoneally (i.p.) (6 mmol/kg). Six hours after LPS instillation, POBN-treated rats were killed and lipid extracts of the lungs were measured for radical adduct content. Control rats were given 0.25 mL saline. Another group of rats were pretreated with GdCl3 (7 mg/kg, i.v.) and instilled with LPS 24 h later.
The NADPH oxidase-deficient (p47phox-/-) mouse lacks a critical cytosolic component required for the assembly of active NADPH oxidase complex (19)
. This animal is a product of embryonic stem cell 129/Sv-derived sperm and C57BL/6-derived eggs and was housed in a pathogen-free barrier facility accredited as shown in detail in previous reports (19
20
21)
. Six-wk-old males (20 g) were used as models for LPS-induced lung injury.
The experimental procedure of LPS instillation in knockout mice is essentially the same as in rats, but the dose of LPS was 10 µg/mouse.
In vivo ESR studies
Rats were treated with LPS or saline as shown above. Lungs were homogenized in 5 mL of 2:1 chloroform:methanol, 1 mL of 30 mM 2,2'-dipyridyl, and 4 mL of deionized water using a homogenizer (Fisher Scientific PowerGen 125) in an ice bath. The 2,2'-dipyridyl was used to inhibit ex vivo ferrous-dependent reactions. To the homogenate 26 mL of 2:1 chloroform:methanol was added, shaken, then centrifuged at 2000 rpm for 10 min (Beckman TJ-6) as described in refs 22
, 23
. The chloroform layer was isolated and dried by passing through a sodium sulfate column. After evaporating the sample by bubbling with N2, ESR spectra were immediately recorded at room temperature using a quartz flat cell in a Bruker EMX EPR spectrometer equipped with a super high-Q cavity. Spectra were recorded on an IBM-compatible computer interfaced with spectrometer instrument settings of 9.79 GHz, 20.2 mW microwave power, 100 kHz modulation frequency, 1300 ms conversion time, and 655 ms time constant. ESR spectra were simulated with a computer optimization procedure (24)
.
Histopathology
Control or treated lung tissue was removed 6 h after intratracheal instillation of LPS and fixed-inflated to 20 cm H2O pressure with 6% formalin. After fixation, all lobes of the lung were cut sagittally through the center of each lobe. Tissue sections were stained with hematoxylin-eosin.
Bronchoalveolar lavage (BAL) fluid and cell counts
As described previously (25)
, three injections and aspirations with 10 mL of sterile ice-cold saline containing 1 mM EDTA were used to collect the BAL fluid in the rat. Cells from BAL fluid were determined by using a hemocytometer and differential cell counts were performed on 500 cells from BAL fluid with a modified Wright’s stain.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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27
28)
. Because of large individual differences, the LPS dose was critical for reproducibility of radical adduct intensities. A dose of 100 µg/rat was insufficient to produce an ESR signal; 250 µg/rat produced a signal whose intensity varied widely. Six hours after administration of 500 µg/rat LPS, a six-line ESR spectrum could be reproducibly detected in the lung extract of a POBN-injected rat (Fig. 1
A). The instillation of saline instead of LPS resulted in a much weaker signal (Fig. 1B
). Without the spin trap, neither LPS (Fig. 1C
) nor saline (Fig. 1D
) instillation yielded a detectable spectrum. The increase in signal intensity of the POBN radical adduct from LPS-treated vs. saline-treated lungs was statistically significant (Fig. 1E
) (P=0.023).
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Computer simulation of the POBN radical adduct spectrum and confirmation of in vivo generation of free radicals
The ESR spectrum shown in Fig. 2
A was simulated (Fig. 2B
) using a computer program developed in this laboratory (24)
. The hyperfine coupling constants for the POBN radical adducts were aN = 14.94 ± 0.07 G and aßH = 2.42 ± 0.06 G. To evaluate whether the POBN radical adduct detected was derived from lipid, we compared the hyperfine coupling constants with literature values (22
, 23
, 29
30
31)
. As shown in Table 1
, there were only minor variations in hyperfine coupling constants between the LPS-induced radical adducts and other radical adducts identified as probably polyunsaturated fatty acid-derived. As an in vitro model for free radical production in rats treated intratracheally with LPS, we used a system of xanthine/xanthine oxidase + linoleic acid + FeCl3. Linoleic acid-derived free radicals generated in this system produced radical adducts whose coupling constants (aN=14.89±0.01 G and aßH=2.42±0.04 G) were similar to those obtained in vivo (Table 1)
. To evaluate the possibility of ex vivo free radical generation, we performed a series of control experiments. In the lung extract from a rat treated with LPS intratracheal instillation and homogenized with POBN (ex vivo), we detected a much smaller signal than that formed in vivo (Fig. 2C
). In the extract from a rat treated with POBN, then homogenized with LPS (ex vivo), the signal was also quite weak (Fig. 2D
). In the system where POBN and LPS were both added ex vivo to untreated lung and homogenized, there was no detectable ESR spectrum of any radical adduct (Fig. 2F
). The ex vivo POBN concentration of 5 mM was chosen on the basis of concentrations in blood, heart, and liver reported by Liu et al. (32)
. The ex vivo bile concentration of LPS (100 µg/mL) was selected to be high enough to activate neutrophils and macrophages. These experiments indicate that the radical adduct formation detected in lipid extracts of lung was not produced ex vivo.
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Histological analysis of lung instilled with LPS
Six hours after intratracheal instillation of LPS, inflammatory responses were confirmed by histological analysis of neutrophil infiltration and increasing cell counts of BAL fluid. In the BAL fluid of rats treated intratracheally with LPS, neutrophil counts increased significantly (P<0.001) over those of the control group, but alveolar macrophages were not increased (Fig. 3
). These data suggest that in this model LPS caused severe lung inflammation in the form of neutrophil alveolitis.
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Effect of GdCl3 treatment to evaluate the role of phagocytes
As GdCl3 is well known to decrease phagocyte activity, we evaluated its inhibitory effect on production of free radicals in the lungs of rats treated intratracheally with LPS. When GdCl3 was administered to rats 24 h before LPS instillation, the production of free radicals in this system decreased by 67.5% (Fig. 4
A–C, P<0.01) whereas the hyperfine coupling constants (aN=14.94±0.07 G and aßH=2.42±0.06 G) were unchanged. At the same time, levels of neutrophils and macrophages in BAL fluid decreased significantly (Fig. 4D
). Parallel to changes in the neutrophil population, both lung injury parameters—wet weight/dry weight ratio (P<0.01) and the protein concentration of BAL fluid (P<0.01)—were greatly decreased by GdCl3 pretreatment (Table 2
). In the histopathological study, we found a remarkable decrease in lung injury as a result of GdCl3 pretreatment. GdCl3 pretreatment had significant inhibitory effects on diffuse alveolar damage including interstitial edema, infiltration with neutrophils and monocytes, parenchymal hemorrhage, collapse of air space, and fibrin exudation into alveolar space (Fig. 5
). GdCl3 pretreatment had significant inhibitory effects not only on lung injury parameters and histopathological findings but also on direct free radical production by the lung. These results confirm that free radical production in this system depends on phagocytes, either activated macrophages or infiltrating neutrophils.
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Free radical formation in NADPH oxidase knockout mice treated with LPS
Infiltrated neutrophils and macrophages have a high potential to produce oxygen radicals by activating NADPH oxidase. NADPH oxidase knockout mice (p47phox-/-) were used to test the hypothesis that NADPH oxidase is the major trigger of in vivo free radical production in LPS-induced acute lung injury. At least 90% of the production of lipid-derived free radical was mediated by NADPH oxidase (Fig. 6
A–C, P<0.001). The levels of neutrophils and macrophages in BAL fluid were not significantly different between wild-type and knockout mice (Fig. 6D
). In the histopathological study, there was no remarkable change in the lung injury between wild-type and knockout mice on LPS-induced diffuse alveolar damage including interstitial edema, infiltration with neutrophils and monocytes, parenchymal hemorrhage, collapse of air space, and fibrin exudation into alveolar space (Fig. 7
).
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Xanthine oxidase-dependent, lipid-derived free radical production
It has been reported that xanthine oxidase is up-regulated in this lung injury model by LPS, interleukin-1, and hypoxia (9)
. In several reports, LPS increased xanthine oxidase activity and exacerbated its role in bacterial translocation in pulmonary edema (9
, 33
, 34)
. Thus, it is possible that xanthine oxidase has some pathogenic role in this model, especially since GdCl3 was not completely inhibitory; up-regulated xanthine oxidase could generate some of the free radicals detected by ESR in our model. Therefore, we examined the effect of the xanthine oxidase inhibitor allopurinol (3-day pretreatments of 1, 2, 4, and 20 mg/kg). The activity of xanthine oxidase in BAL fluid was completely inhibited by pretreatment of 4 mg/kg of allopurinol (data not shown). However, when mice were pretreated with allopurinol, the intensity of the lipid-derived free radical did not change (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 36)
. In the past, direct detection of free radicals in vivo was limited because the only available method was ESR, which has low sensitivity relative to the reactivity of free radicals and the ensuing low concentrations. To solve this problem, we selected an appropriate spin-trapping agent and an established extraction method for the radical adducts. For extractions, a homogenizing buffer containing the ferrous chelator dipyridyl was used in order to limit any possible artifacts due to iron-catalyzed free radical formation.
The radicals detected with this method have hyperfine couplings that are consistent with radicals derived from lipid peroxidation. Lipid peroxidation is known to cause tissue damage in various inflammations (37
, 38)
. Free radical-induced lipid peroxidation is also of pathogenic relevance during tissue injury, for which antioxidants could be of therapeutic value (12)
.
The free radicals that cause lipid peroxidation damage can be generated in vitro by alveolar macrophages or neutrophils, but in this LPS model free radical production clearly correlated with an increase in neutrophils (Fig. 3)
. Although macrophages are activated by LPS and therefore generate oxygen-derived radicals and cause lipid peroxidation (39)
, many neutrophils are known to infiltrate the lung in response to the intratracheal instillation of LPS (Fig. 3)
. Thus, we propose that the main source of pathogenesis is neutrophils, although a role for activated alveolar macrophages is not excluded.
Neutrophil infiltration can be decreased by blocking enzymes that promote neutrophil migration. In the ozone exposure lung injury model, ß2 integrins from macrophage and ICAM-1 BAL fluid were decreased by pretreatment with the neutrophil infiltration inhibitor GdCl3 (15)
. GdCl3 pretreatment also lessened an increase in lung myeloperoxidase activity, a marker of neutrophils caused by liver ischemia reperfusion (40)
and decreased NO production by LPS and IFN-
(13)
. In the LPS instillation model, GdCl3 decreased the number of alveolar macrophages and consequently their production of factors related to neutrophil migration and adhesion, thus significantly decreasing the number of infiltrated neutrophils by BAL and histopathological studies.
It is well known that phagocytes produce oxygen radicals via NADPH oxidase, and several reports have connected NADPH oxidase to tissue damage. Kubo et al. (21)
were able to prevent complement-induced lung injury in mice with NADPH oxidase deficiency. Kono et al. (20)
reported that NADPH oxidase in phagocytes has a crucial role in alcohol-induced liver disease, producing free radicals detected by ESR. NADPH oxidase was the source of the free radicals detected by ESR in our in vivo experiments. It is unlikely this is an effect of oxidases in general as xanthine oxidase activity had no effect on free radical intensity. In contrast, experiments with NADPH oxidase knockout mice demonstrate that NADPH oxidase is responsible for >90% of the lipid-derived free radical in the LPS instillation model. This is an important result for the resolution of the mechanism of free radical generation in this system. In this histological study, there was no significant change between wild-type and NADPH oxidase knockout mice in an LPS-induced ARDS model. These results indicate that lung injury induced by LPS-IT is caused not only by lipid-derived free radicals (NADPH oxidase-dependent), but also by other inflammatory agents such as proteinase, prostaglandins, cytokines, etc., from phagocytes. In addition, our ARDS model was a very acute phase, because samples for ESR and histology were extracted after 6 h of LPS instillation. Neutrophil infiltration was remarkable during this acute-phase reaction; these pathological findings (including interstitial edema, infiltration with monocytes, parenchymal hemorrhage, collapse of air space, and fibrin exudation into alveolar space) were not severe. Perhaps during longer periods a significant difference between wild and knockout mouse histology will be found.
The reported immunoreactivity of p47phox in nonphagocytic cells such as pulmonary endothelial cells (41)
and smooth muscle cells (42)
opens the possibility of these cells generating reactive oxygen species, but the free radical production potential of these cells is much less than that of phagocytic cells undergoing their respiratory burst. The main target of GdCl3 is the macrophages (13
14
15)
. Anti-neutrophil antisera, which decreased the neutrophil counts in blood and lung, significantly reduced the production of free radical (data not shown). Therefore, the main source of free radical production is the lung phagocytic cells.
The molecular mechanism of the superoxide-mediated formation of these lipid-derived free radicals remains unclear although several possible explanations are consistent with the known chemistry of superoxide. On protonation, superoxide forms the hydroperoxyl radical (HOO•) with a pKa of 4.8, which is a much stronger oxidant than superoxide anion. Aikens and Dix demonstrated that HOO• initiates fatty acid peroxidation by two parallel pathways: a fatty acid hydroperoxide-independent and -dependent pathway (43)
. On the other hand, Thomas et al. showed that iron released from ferritin by superoxide mediates lipid peroxidation (44)
. Superoxide also releases iron by oxidation of the [4Fe-4S] center of dehydrases such as aconitase (45)
. In summary, either the reaction of HOO• or the release of iron by superoxide could lead to superoxide-dependent lipid peroxidation consistent with the dramatic decrease in lipid-derived free radical formation in the NADPH oxidase knockout mice.
In conclusion, this is the first direct evidence of in vivo production of lipid-derived free radicals from intratracheally instilled LPS-induced lung injury. This free radical production originates primarily from infiltrating neutrophils and perhaps, to some extent, from alveolar macrophages. We have further shown, using NADPH oxidase knockout mice, that in vivo free radical production in intratracheally instilled LPS-induced lung injury is mediated by NADPH oxidase.
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FOOTNOTES |
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Received for publication April 9, 2002. Accepted for publication June 26, 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, superoxide dismutases, and related matters. J. Biol. Chem. 272,18515-18517This article has been cited by other articles:
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
s) and neutrophil counts. Bronchoalveolar lavage fluid of rat lung treated with or without GdCl3 was perfused by 3 injections with 10 mL of sterile ice-cold saline containing 1 mM EDTA (n=4). Volumes of BAL recovered were 25.0 ± 0.2 mL for the LPS instillation and 24.9 ± 0.2 mL for the LPS instillation group with GdCl3 pretreatment.
