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Published ahead of print on September 25, 2003, doi:10.1165/rcmb.2003-0309OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 479-490, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0309OC

Mononuclear Phagocyte Xanthine Oxidoreductase Contributes to Cytokine-Induced Acute Lung Injury

Richard M. Wright, Lisa A. Ginger, Noi Kosila, Nancy D. Elkins, Brendan Essary, James L. McManaman and John E. Repine

Webb-Waring Institute for Cancer, Aging and Antioxidant Research; School of Medicine, Department of Pulmonary Sciences; and Department of Physiology and Biophysics, University of Colorado Health Sciences Center, Denver, Colorado

Address correspondence to: Dr. Richard M. Wright, Webb-Waring Institute, Campus Box C-322, 4200 East 9th Ave. Denver, CO 80262. E-mail: richard.m.wright{at}uchsc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Acute lung injury (ALI) is characterized by increased alveolar cytokines, inflammatory cell infiltration, oxidative stress, and alveolar cell apoptosis. Previous work suggested that xanthine oxidoreductase (XOR) may contribute to oxidative stress in ALI as a product of the vascular endothelial cell. We present evidence that cytokine induced lung inflammation and injury involves activation of XOR in the newly recruited mononuclear phagocytes (MNP). We found that XOR was increased predominantly in the MNP that increase rapidly in the lungs of rats that develop ALI following intratracheal cytokine insufflation. XOR was recovered from the MNP largely converted to its oxygen radical generating, reversible O-form, and alveolar MNP exhibited increased oxidative stress as evidenced by increased nitrotyrosine staining. Cytokine insufflation also increased alveolar cell apoptosis. A functional role for XOR in cytokine-induced inflammation was demonstrated when feeding rats two different XOR inhibitors, tungsten and allopurinol, decreased MNP XOR induction, nitrotyrosine staining, inflammatory cell infiltration, and alveolar cell apoptosis. Transfer of control or allopurinol treated MNP into rat lungs confirmed a specific role for MNP XOR in promoting lung inflammation. These data indicate that XOR can contribute to lung inflammation by its expression and conversion in a highly mobile inflammatory cell population.

Abbreviations: acute lung injury, ALI • bronchoalveolar lavage fluid, BALF • fluorescence-activated cell sorter, FACS • interferon-{gamma}, IFN-{gamma} • interleukin, IL • monocyte chemotactic protein, MCP • mononuclear phagocytes, MNP • superoxide anion, O2-. • nitric oxide, NO • phosphate-buffered saline, PBS • reactive oxygen species, ROS • xanthine oxidoreductase, XOR


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Acute lung injury (ALI) is a highly fatal inflammatory disorder characterized by increased alveolar cytokine expression, neutrophil and monocyte recruitment into the lung, macrophage activation, oxidative stress, alveolar cell apoptosis, and lung edema (1, 2). Bronchoalveolar lavage fluid (BALF) from patients with ALI contains increased concentrations of numerous cytokines and chemokines including interleukin (IL)-1, interferon (IFN)-{gamma}, IL-6, IL-8, tumor necrosis factor-{alpha}, IL-10, transforming growth factor-ß, and monocyte chemotactic protein (MCP)-1 (3–5). Although the contribution of individual cytokines to the pathophysiology of ALI is not understood, intratracheal insufflation of IL-1 in rats produces lung inflammation with many characteristics typical of ALI. For example, in rats, IL-1 insufflation increased lung edema, BALF protein levels, lung neutrophil recruitment, and oxidative stress (6, 7). In cultured lung epithelial cells, IL-1 stimulated expression of inflammatory chemokines (IL-8, MCP-1) and adhesion molecules (ICAM-1) (8), suggesting its potential role in inflammatory cell recruitment. Likewise, insufflation of IFN-{gamma} (9), or its induced expression as a Clara cell transgene (10), also promoted inflammation and macrophage activation in the lung. Therefore, IFN-{gamma} may also contribute to many of the events that arise during lung inflammation, including the stimulation of inflammatory cell recruitment (11, 12) and the induction of alveolar epithelial cell apoptosis (13). Although neutrophils are widely recognized as possible mediators of ALI pathophysiology (14, 15), alveolar macrophages also appear to contribute to ALI as sources of proinflammatory cytokines (IL-1), chemokines (IL-8, MCP-1), and reactive oxygen species (ROS) (1, 2). The recruitment, infiltration, and differentiation of monocytes into macrophages are important steps in the life cycle of the mononuclear phagocytes (MNP) (16–18) that may also impact ALI.

Oxidative stress is a common feature of ALI whose contribution to pathogenesis is not well understood (19–22), and although ROS can be derived from many sources during inflammation, xanthine oxidoreductase (XOR) emerged as a possible source because it and its substrate, hypoxanthine, are elevated in the blood and lung lavage of patients with ALI (23–25). Furthermore, feeding animals tungsten or allopurinol, XOR inhibiting diets, reduced ALI induced by hemorrhage (26–28) and vascular permeability induced by ischemia/reperfusion (29). XOR generates ROS with high efficiency following proteolytic or oxidative conversion of D-form XOR (xanthine dehydrogenase, XDH) to O-form XOR (xanthine oxidase, XO). Although proteolytic conversion of D-form to O-form has been well studied in vitro (30, 31), XOR conversion by thiol oxidation may be a key biological mediator because it is both reversible and potentially subject to regulation. XOR thiol oxidation can be reversed by incubation with reducing agents, like dithiothreitol, to produce D-form XOR (32), and rat liver O-form XOR content has been directly linked to reduced glutathione status (33). O-form XOR is an efficient source of the superoxide anion (O2-.) (34, 35) that can promote protein tyrosine nitration by reacting with nitric oxide (NO) to form peroxynitrite, the nitrating species (36, 37). Protein nitrotyrosine is a relatively stable modification that provides in vivo evidence of the concomitant presence of NO and O2-. (38, 39).

Alveolar epithelial cell apoptosis has been recognized in the lungs of patients with ALI, and in animal models that develop ALI (1, 2), it can be induced by oxidative stress, and may be responsible for loss of alveolar epithelial function (2). Unlike necrosis, apoptotic cell death involves the ordered activation of effector caspases, such as caspase-3, and the activation of endonucleases that generate DNA nicks that are routinely assayed by fluorescence TUNEL stain (40).

The potential relationship between inflammation and XOR activation was reinforced by the observation that proinflammatory cytokines IL-1 and IFN-{gamma} induced XOR in cultured epithelial cells (41–44), whereas intraperitoneal injection of IFN-{gamma} induced XOR in the lung (45). In the present work, we hypothesized that insufflation of IL-1 and IFN-{gamma} would induce lung XOR activity and contribute to lung inflammation. Our data not only supported this premise, but revealed XOR induction in the differentiating MNP that increased rapidly in the alveoli following cytokine insufflation. Importantly, MNP XOR induction, MNP oxidative stress, lung inflammation, and alveolar cell apoptosis were all attenuated in rats fed tungsten or allopurinol diets. Cell transfer experiments provided additional evidence for the potential contribution of MNP XOR in the inflammatory process.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Most reagents, sodium tungstate, buffers, substrates, and inhibitors were purchased from Sigma Chemical Co. (St. Louis, MO). Recombinant rat IL-1{alpha} (IL-1; 500-RL-005) and IFN-{gamma} (285-IF-100) were purchased from R&D Systems (Minneapolis, MN). TUNEL staining kits were obtained from Trevigen, Inc. (Gaithersburg, MD). Nitrotyrosine and immunoaffinity purified anti-nitrotyrosine antibody (IgG) were purchased from Upstate Biotechnology (Lake Placid, NY). Alexa Flour-488 and Alexa Flour-594 fluorescent antibodies were purchased from Molecular Probes (Eugene, OR). Normal goat serum was obtained from ICN Biomedicals (Aurora, OH).

Intratracheal Cytokine Insufflation
Healthy male Sprague-Dawley rats (300–400 g body weight; Sasco, Omaha, NE) were fed control, tungsten-enriched, or allopurinol-supplemented diets as previously described (46, 47). IL-1 and/or IFN-{gamma} were delivered intratracheally into anesthetized rats as described previously (7). Dose–response optimization of IL-1 revealed no further inflammatory response to IL-1 at doses beyond 100 ng, and because the difference between 100 ng and 50 ng was small, 50 ng per rat was selected for use in the present experiments. Similar experiments and results were obtained with IFN-{gamma}, which was subsequently tested in high and low doses in combination with IL-1. Routinely, 50 ng of recombinant rat IL-1{alpha} and/or 50 ng of IFN-{gamma} in 0.5 ml of normal saline were pumped into the airway. Sham treated control rats were insufflated with normal saline alone. Differential and total inflammatory cell counts were determined on BALF obtained 24 h after cytokine insufflation (6, 7). TUNEL, nitrotyrosine staining, histology, and immunofluorescence were performed on lungs harvested 24 h after cytokine insufflation. Lungs were perfused until blood-free, removed surgically, and divided. One fraction was immediately fixed in paraformaldehyde for histology, TUNEL, or nitrotyrosine staining. One fraction was frozen immediately in liquid N2 for subsequent biochemical analyses. Livers were obtained from each rat, perfused blood-free, and immediately frozen in liquid N2. BALF cells were collected from separate rats by pumping 5.0 ml of normal saline into the trachea. Lavage fluid was pumped in and out of the lung three times before being collected. The use of rats in this study was approved by the University of Colorado Institutional Review Board under the protocol number 4980199(04)1E.

XOR Assay
Total XOR activity in lung and liver tissue was determined using whole tissue protein extracts. Briefly, tissues that had been perfused blood-free were placed into liquid N2 and stored at –80°C before generating enzyme extracts. Enzyme extracts were prepared by placing frozen specimens in extract buffer (100 mM K-Phosphate, pH 7.8, 1 mM EDTA, 1 mM PMSF). Tissues were thawed in extract buffer on ice and minced. Minced tissues were broken in a rotating dounce using exactly three strokes of the pestle. Tissue homogenates were then centrifuged at 15,000 x g at 4°C for 30 min. Clarified extracts were desalted on 2 cm x 20 cm Sephadex G25 columns to remove low molecular weight substances that potentially interfere with enzyme activity (48), and 3 ml of the flow through front were collected. Allopurinol inhibitable XOR activity was determined spectroscopically by measuring uric acid formation from xanthine at 290 nm. The stability of uric acid added to the flow through fraction was determined in the presence and absence of the uricase inhibitor, oxonic acid (OA). Because these analyses revealed high levels of uricase in rat lung and liver extracts, XOR assays were performed in the presence of 0.8 mM OA. Evaluation of D-form XOR and O-form XOR was determined by measuring enzymatic activity in the presence and absence of NAD+ (49).

Tissue Fixation, Immunostaining, and Microscopy
Lung tissue was fixed in 10% neutral-buffered formalin overnight and embedded in paraffin. Four-micron sections were prepared and hydrated by exposure to xylene for 2- to 5-min periods followed by sequential 1-min exposures to 100, 90, 70, and 30% ethanol and finally to phosphate-buffered saline (PBS) for 5 min. Hydrated sections were either stained with H&E or prepared for antibody staining using the antigen retrieval procedure (Vector Labs, Burlingame, CA) according to the manufacturer's instructions. Sections were blocked and permeablized by incubation in PBS containing 0.2% glycine for 30 min followed by PBS containing 5% goat serum and 0.1 mg/ml saponin for 1 h and then incubated with anti-mouse XOR antibodies (1/100) at room temperature for 1 h. Immunoreactivity was detected using a Cy3-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA) secondary antibody (1/150) and visualized with a Nikon Diaphot inverted fluorescent microscope. Anti-XOR antibodies used for immunocytochemistry were generated against affinity purified XOR (49) and purified on Protein-A sepharose. Specificity of the XOR antibody was demonstrated by Western immunoblot analysis of crude tissue extracts (49).

Flow Cytometric Analyses
Cells were recovered from the BALF by centrifugation, washed in PBS, distributed at 2.5 x 105 cells in PBS/5.0% FCS into 96-well plates, and then brought up to 100 µl with saline. All cells were preincubated for 5 min on ice with mouse anti-rat CD32 monoclonal clone D34–485 (BD Pharmingen, San Jose, CA) to minimize nonspecific binding. Phycoerythrin-labeled anti-rat CD11b clone MRC OX-42 (Biosource International, Camarillo, CA) and biotin-conjugated anti-rat MNP monoclonal clone 1C7 (BD Pharmingen) were directly added to the wells 10 min in the dark on ice. The cells were washed in PBS/5.0% FCS followed by sedimentation for 5 min at 1,200 rpm in a bench top Sorvall centrifuge and then incubated with either streptavidin-phycoerythrin or streptavidin–fluorescein isothiocyanate (both from BD Pharmingen) in the appropriate wells. Subsequently, the cells were washed, fixed with 2% paraformaldehyde in PBS, and then washed again. Reacted and washed cells were then resuspended in PBS and analyzed on a FACSCalibur analyzer (Beckton-Dickinson, San Jose, CA). The gates were set by a blank and the appropriate controls were used to indicate nonspecific binding. A total of 10,000 cells were acquired for each sample and analyzed with Cell Quest (Beckton-Dickinson) version 3.1 software.

Lung Nitrotyrosine Staining
Paraffin-embedded lung tissue sections were deparaffinized in ethanol and rehydrated in H2O and PBS. Slides were blocked with a solution of 7.5% normal goat serum, 2.5% ß-casein, 0.1% triton X-100 for 3 h at room temperature. The primary antinitrotyrosine antibody was applied at a dilution of 1:1,000 for 15 min at room temperature in the blocking solution. Slides were then washed in PBS and the Alexa Flour goat anti-rabbit IgG was applied for 15 min at a dilution of 1:100 in the blocking solution at room temperature. Subsequently, the slides were washed extensively in PBS and stained with Hoescht dye for 5 min in PBS, after which they were washed in PBS, covered in Anti-Fade, and sealed under glass coverslips. Positive immunofluorescent controls were exposed to 24–77 mM peroxynitrite for 20 min at room temperature, washed with PBS, and then processed as above. Prebinding negative controls were performed by mixing the antinitrotyrosine antibody with 10 mM nitrotyrosine in PBS for 1 h at room temperature before its addition to blocked slides. All subsequent steps were performed as above. Prebinding blocked the reactivity of antinitrotyrosine antibody to nitrotyrosine but had no effect on heme-dependent autofluorescence of red blood cells. Slides were visualized under red (nitrotyrosine), green (tissue architecture), and blue (nuclei) fluorescence using a Nikon Diaphot inverted confocal fluorescence microscope at x100 magnification. Digitally derived blue and green photographs were combined with the MetaMorph software and printed from 24-bit digital files. A black background was set consistently for each figure using a section of the open air-space in which no cells were present in the field.

TUNEL Staining and Morphometric Analysis
The TUNEL assay was used to detect apoptotic cells in fixed lung sections. After fixation with 4% buffered formalin, lung tissue sections were embedded in paraffin, sectioned at 5 uM, and mounted on glass slides. Slides were deparaffinized, rehydrated for 10 min in PBS, and treated with 0.002% proteinase K (Sigma) in distilled water for 5–15 min at room temperature. Terminal deoxynucleotidyl transferase (TdT) was used for labeling of DNA nicks using the TACS kit (Trevigen, Inc., Gaithersburg, MD). Counterstaining with DAPI was done to stain nuclear DNA and with Rhodamine coupled wheat germ agglutinin (WGA; Molecular Probes, Eugene, OR) to stain the structural architecture. TUNEL-positive cell staining appeared as green fluorescence in the nuclei of clearly defined alveolar cells. Background nuclear staining appeared as blue, whereas the tissue architecture was red.

Activated Caspase-3 Detection
Detection of the cleaved, mature, actived caspase-3 was performed on paraffin-embedded sections using the Cell Signaling Technology, Inc. (Beverly, MA) protocol. First, paraffin-embedded slides were deparaffinized and rehydrated. Incubation with 1% H2O2 for 10 min blocked endogenous peroxidase activity. For antigen unmasking, the tissue sections were microwaved in 10 mM sodium citrate buffer (pH 6.0) for 10 min. Next, the sections were blocked in 5% goat serum for 1 h at room temperature. Slides were then incubated at 4°C overnight with caspase-3 antibody specific for the cleaved, mature, form of caspase-3 diluted 1:200 in PBS (Asp-175; Cell Signaling Technology, Inc). This was followed by a 30-min incubation with a 1:200 dilution of goat anti-rabbit antibody (PK-6101; Vector Labs). A subsequent 30-min incubation with VectaStain Elite ABC reagent (PK-6101; Vector Labs) ensued. This reaction was followed by the addition of the DAB substrate kit for peroxidase (SK-4100; Vector Labs). The slides were finally counterstained with hematoxylin (H-3401; Vector Labs), dehydrated, and mounted. Caspase-3–positive cells were quantitated by counting the percent positive alveolar cells per field under x100 magnification.

Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis and Immunoblot Analysis
Protein was electrophoresed on sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Osmonics, Inc, Minnetonka, MN). Membranes were sliced for staining with Coomassie brilliant blue or processed for immunoblot analysis. For reaction with antisera, membrane strips were blocked with 0.05% dried milk overnight before reaction with preimmune sera or anti-XOR antisera. Antigen–antibody complexes were detected by reaction with an enhanced chemiluminescence Western blotting detection kit according to manufacturer's instruction (Amersham Life Sciences, Piscataway, NJ).

Statistical Analyses
Data are expressed as the mean and standard error of the mean and were assessed for significance using the Student's t test. A P value of < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
XOR Was Induced in the Lungs of Rats following IL-1 and IFN-{gamma} Insufflation
XOR was quantitated in desalted whole-lung protein extracts obtained from lungs and livers of rats subjected to intratracheal insufflation with either IL-1 and IFN-{gamma}, IL-1, IFN-{gamma}, or saline (the cytokine vehicle). The combined insufflation of IL-1 and IFN-{gamma} produced a 3-fold induction of total XOR activity 24 h following cytokine insufflation compared with saline alone (Figure 1A). By comparison, there was an approximate doubling of XOR after IL-1 insufflation and a negligible response following IFN-{gamma} insufflation alone. In contrast, liver XOR did not increase following cytokine insufflation (Figure 1B). Western immunoblot analysis of lung protein extracts showed that lung XOR protein also increased several fold following insufflation with IL-1 and IFN-{gamma} (Figure 1C). XOR recovered from cytokine insufflated lung revealed no evidence of elevated proteolysis.





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  		Figure 1. Lung XOR induction following cytokine insufflation. Total lung and liver XOR activity was quantitated in the presence of NAD+ in soluble desalted protein extracts from rats that were insufflated 24 h before with saline, IL-1, IFN-{gamma}, or IL-1 and IFN-{gamma}. Data show the mean and SE for six rats in each group. IL-1– and IFN-{gamma}–insufflated rats had increased (**P < 0.02) lung (A), but not (P > 0.05) liver (B), XOR activity compared with rats insufflated with saline, IL-1 (*P < 0.05), or IFN- {gamma} (P > 0.05). (C) Western immunoblot analysis of lung XOR from cytokine-insufflated rats. Antibody to rat ß-actin was used to control for gel loading. Gels were scanned with a Perkin-Elmer phosphoimager for semi-quantitation. Data are shown for two rats in each group. These data demonstrate that lungs from rats insufflated with IL-1 and IFN-{gamma} had increased XOR immunoreactive protein compared with rats insufflated with saline, IL-1, or IFN-{gamma}. (D) The percentage of O-form XOR was quantitated in soluble protein extracts of cytokine-insufflated rat lungs. Data show the mean and SE for six rats in each group and are expressed as the percentage of the total XOR activity. Rats insufflated with IL-1 and IFN-{gamma} had increased (*P < 0.05) lung O-form XOR activity compared with rats insufflated with saline, IL-1, or IFN- {gamma}. (E) Soluble protein extracts were reduced in the presence of 5 mM dithiolthreitol for 1 h at 37°C, after which they were rechromatographed on Sephadex G25 and reassayed as above. O-form XOR activity in the soluble protein extracts from all insufflated rats was comparably reduced by DTT. Data show the mean and SE for six rats in each group.

 
XOR Was Recovered in Predominantly O-Form from the Lungs of Rats Insufflated with IL-1 and IFN-{gamma}
We quantitated the levels of O-form XOR in untreated or cytokine insufflated rat lungs. Untreated, normal rat lungs exhibited 23% O-form XOR, an amount that is typical of most rat tissues, whereas O-form XOR increased to 78% of the total XOR activity in lungs insufflated with IL-1 and IFN-{gamma}. In contrast, lung O-form content increased to 42% following saline insufflation, 57% following IL-1 insufflation, and 52% following IFN-{gamma} insufflation (Figure 1D). O-form XOR recovered from cytokine-insufflated lungs was in a reversible state. Soluble lung protein extracts from insufflated rats were exposed to 5 mM DTT at 37°C for 14 h and were rechromatographed on sephadex G25 after which D-form and O-form content were remeasured. Regardless of insufflation, O-form XOR in all lung samples was reduced to ~ 23% of the total XOR by DTT (Figure 1E). Furthermore, native O-form XOR from untreated rat lungs or livers was not further reduced by DTT treatment and remained at ~ 23% of the total XOR following DTT reduction.

Lung Inflammation Increased following IL-1 and IFN-{gamma} Insufflation
The total number of inflammatory cells recovered from the BALF 24 h following IL-1 and IFN-{gamma} insufflation was increased compared with saline-, IL-1–, or IFN-{gamma}–insufflated lungs (Figure 2A). Differential analysis revealed ~ 60% neutrophils and 40% macrophages in BALF from IL-1– and IFN-{gamma}–insufflated lungs compared with ~ 80% neutrophils and 20% macrophages from IL-1– or IFN-{gamma}–insufflated lungs and essentially 100% macrophages from noninsufflated lungs of control rats. Lymphocytes were a small, but comparable, percentage of the cells recovered in all instances. Histologic examination of cytokine insufflated lungs confirmed the increase in inflammatory cells recovered in BALF from the lungs of IL-1– and IFN-{gamma}–insufflated rats and revealed increased numbers of inflammatory cells in the airway and in the perivascular regions of the lung (Figure 2B). The cellularity observed histologically with IFN-{gamma} alone appears greater than the cells recovered in the BALF, suggesting a possible interstitial localization of MNP in the IFN-{gamma}–insufflated lungs.







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  		Figure 2. Induction of XOR in lung inflammatory cells. (A) BALF from rats insufflated 24 h before with IL-1 and IFN-{gamma} had increased (**P < 0.02) numbers of inflammatory cells compared with rats insufflated with saline, IL-1, or IFN-{gamma} alone. Data show the mean and SE for eight rats in each group. (B) Lung tissue slices were mounted on glass slides, H&E-stained, examined under brightfield microscopy, and digitized using the Roper Scientific Digital Imaging program (Roper Scientific, Trenton, NJ). Lungs from rats insufflated with IL-1 and IFN- {gamma} 24 h before had increased numbers of airway and perivascular inflammatory cells compared with rats insufflated with saline, IL-1, or IFN- {gamma}. (C) Immunofluorescent detection of XOR immunoreactive protein in paraffin-embedded lung tissue sections using rhodamine-conjugated anti-XOR antisera (XOR; A, C, E, G, and I). Lung architecture was delineated by staining with fluorescein isothiocyanate–conjugated wheat germ agglutinin (WGA; B, D, F, H, and J). Higher magnifications (I and J) depict increased XOR immunofluorescence primarily associated with the inflammatory cells in the alveolar space of rats insufflated with IL-1 and IFN-{gamma}. (D) Western immunoblot analysis of XOR protein in cells recovered from the BALF of cytokine-insufflated rats. Antibody to rat ß-actin was used to control for gel loading. Data are shown for two rats in each group. XOR protein was elevated in the BALF cells following IL-1 and IFN-{gamma} insufflation compared with the response observed following saline, IL-1, or IFN-{gamma} insufflation. (E and F) Quantitation of D-form and O-form XOR in BALF cells recovered from cytokine insufflated rat lungs. D-form XOR activity (E) was increased in the BALF cells from rats insufflated with IL-1 and IFN-{gamma} compared with rats insufflated with saline (P < 0.02), IL-1, or IFN-{gamma} alone. XOR was recovered in predominantly O-form in BALF cells from rats insufflated with IL-1 and IFN-{gamma} (F). Data show the mean and SE for six rats in each group.

 
XOR Was Induced in Lung Inflammatory Cells following IL-1 and IFN-{gamma} Insufflation
The predominance of O-form XOR in IL-1– and IFN-{gamma}–insufflated rat lungs was surprising because XOR exists in native lung tissue in predominantly D-form. Accordingly, we sought to identify lung cells that expressed XOR following cytokine insufflation. Immunofluorescent staining of rat lung tissue preparations demonstrated that XOR was induced primarily in inflammatory cells following IL-1 and IFN-{gamma} insufflation (Figure 2C). By comparison, XOR immunoreactivity was virtually undetectable in lungs following saline, IL-1, or IFN-{gamma} insufflation. Western immunoblot analysis indicated that XOR was induced dramatically in the BALF cells by the combined action of IL-1 and IFN-{gamma} compared with the response produced by saline, IL-1, or IFN-{gamma} insufflation (Figure 2D). D-form (Figure 2E) and O-form (Figure 2F) XOR activity were also elevated in BALF cells recovered from rats insufflated with IL-1 and IFN-{gamma} compared with the response to saline, IL-1, or IFN-{gamma} insufflation. Furthermore, predominantly O-form XOR was again recovered in the inflammatory cells recovered from IL-1– and IFN-{gamma}–insufflated rat lungs.

XOR Was Induced in the Infiltrating and Differentiating MNP following IL-1 and IFN-{gamma} Insufflation
Differential analysis of cells obtained in the BALF following IL-1 and IFN-{gamma} insufflation revealed nearly equivalent numbers of MNP and neutrophils by 24 h that declined slowly over the next 18 d (Figure 3A). BALF cells from IL-1– and IFN-{gamma}–insufflated rats were stained for the alveolar macrophage marker, ED1, or with the neutrophil/monocyte marker, CD11b, and were then subjected to analysis by fluorescence-activated cell sorter (FACS) (Figure 3B). The CD11b antigen increased dramatically 4 h following cytokine exposure, reflecting the appearance of newly infiltrating CD11b positive monocytes and neutrophils. CD11b staining then gradually declined over the next 18 d to a pattern identical to that found in the 0 time resident macrophages. ED1 was well expressed on the 0 time resident macrophages, was absent on the newly infiltrating monocytes and neutrophils at 4 h, and was gradually restored to high level expression throughout the 18-d time course. These data reflect the rapid migration of neutrophils and monocytes into the lung following IL-1 and IFN-{gamma} insufflation, followed by the gradual maturation of the newly infiltrating monocytes into mature, ED1-expressing macrophages and by the relatively rapid decrease of the neutrophil population. Scatter diagrams revealed the broad forward and side scatter produced by resident macrophages at time 0, and subsequently revealed the low scattering, compact nature of infiltrating monocytes and neutrophils, a pattern that was restored to the original pattern typical of the mature macrophages over the course of 18 d.






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  		Figure 3. Induction of XOR in the infiltrating and differentiating MNP. (A) Differential analysis of inflammatory cells recovered sequentially over an 18-d time course from the lungs of rats insufflated with IL-1 and IFN-{gamma}. (B) FACS analysis of cells recovered from the BALF of IL-1– and IFN-{gamma}–insufflated rats. As described in detail in the text, BALF cells were stained with the monocyte and neutrophil marker, CD11b, or the alveolar macrophage marker, ED1, and were then analyzed by FACS. Ten thousand cells were analyzed for each staining reaction, and representative scatter diagrams are depicted for each time point. For each fluorescence curve, cell numbers are plotted on the ordinate and fluorescence intensity on the abscissa. Filled curves represent net fluorescence, whereas the black line indicates fluorescence due to the nonspecific antisera. In the scatter diagrams, forward scatter is plotted along the abscissa and side scatter along the ordinate. These data demonstrate the rapid influx of neutrophils and monocytes and the maturation of monocytes in the lung following cytokine insufflation. (C) Western immunoblot analysis of Percoll-gradient purified neutrophils and MNP recovered from the BALF of rats 24 h following insufflation with IL-1 and IFN-{gamma} shows the expression of XOR in MNP but not neutrophils. (D and E) Quantitation of D-form (D) and O-form (E) XOR in adherent MNP recovered over a 24-h time course from rats insufflated with IL-1 and IFN-{gamma}. Data show the mean and SE for three rats at each time point. The inset depicts a Western immunoblot showing increased XOR activity for cells recovered at each time point. Cells recovered from the BALF of rats insufflated with saline alone 8 or 24 h previously (sham-treated controls) showed only negligible XOR activity and immunoreactivity (not shown) similar to that observed at the 0 time control.

 
Neutrophils and MNP were recovered from the BALF 24 h following IL-1 and IFN-{gamma} insufflation and purified on Percoll gradients. Western immunoblot analysis of protein extracted from these cells showed that XOR was induced in MNP, but not neutrophils (Figure 3C). BALF cells were also recovered over a 24-h time course from rats following insufflation of IL-1 and IFN-{gamma}, collected by centrifugation and washed. MNP were then allowed to adhere to plastic dishes by cultivation at 37°C for 1 h in rich medium. Nonadherent cells were then washed off the plates and the adherent MNP were assayed for D-form and O-form XOR activity and for XOR immunoreactive protein. These data demonstrated that D-form XOR (Figure 3D) and O-form XOR (Figure 3E) activity were induced in the adherent MNP, and that most of this XOR was recovered in O-form. Although XOR was induced over a 24-h period in the infiltrating MNP recovered from the lungs of IL-1– and IFN-{gamma}–insufflated rats, neither XOR activity nor antigen was detected in circulating rat monocytes (not shown). These observations indicate that XOR induction is associated with monocyte infiltration and/or differentiation following cytokine insufflation.

Tungsten or Allopurinol Feeding Decreased Lung and MNP XOR Activity, Lung Inflammation, MNP Nitrotyrosine Staining, and Alveolar Cell Apoptosis in Rats Insufflated with IL-1 and IFN-{gamma}
The induction of XOR in MNP recovered from lungs following IL-1 and IFN-{gamma} insufflation and its conversion into largely O-form XOR suggested that XOR may participate in the inflammatory process as a source of ROS. We used two systemic inhibitors to assess the involvement of XOR in cytokine-induced inflammation. Rats were fed diets deficient in molybdenum and supplemented with tungsten or diets supplemented with allopurinol. Subsequently, rats were insufflated with IL-1 and IFN-{gamma} or the saline vehicle and prepared for analysis 24 h later. Tungsten feeding decreased XOR activity in the lungs (Figure 4A) and in the BALF cells (Figure 4B) from cytokine-insufflated rats. Furthermore, tungsten or allopurinol feeding also reduced the accumulation of inflammatory cells (Figure 4C) and attenuated histologic evidence of airway and perivascular inflammation (Figure 4D) in the lungs of IL-1– and IFN-{gamma}–insufflated rats. Lung tissue specimens from rats insufflated with IL-1 and IFN-{gamma} exhibited increased nitrotyrosine staining of the alveolar MNPs (88% positive staining) compared with saline-insufflated control lungs (14% positive staining), and MNP nitrotyrosine staining was decreased to background levels (14% positive staining and autofluorescent cells) in IL-1– and IFN-{gamma}–insufflated rats previously fed allopurinol or tungsten diets (Figure 4E). Finally, because a common feature of ALI and a consequence of inflammation can be the induction of alveolar cell apoptosis, we examined cytokine insufflated rat lungs for apoptosis. By quantitative morphometric analysis, lungs from rats insufflated 24 h before with IL-1 and IFN-{gamma} had a 13-fold increase in TUNEL-positive alveolar nuclei staining compared with saline-insufflated rat lungs, and prior feeding with tungsten or allopurinol diets attenuated the development of alveolar apoptosis (Figures 4F and 4G). We quantitated activated caspase-3 in a similar fashion to corroborate the results from TUNEL assay. Lungs from rats insufflated 24 h before with IL-1 and IFN-{gamma} had a 12-fold increase in activated caspase-3 compared with saline-insufflated control lungs. Prior feeding with tungsten or allopurinol diets blocked the activation of caspase-3 in cytokine insufflated rat lungs (Figure 4H).







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  		Figure 4. Effect of tungsten or allopurinol feeding on lung and MNP XOR, BALF cell numbers, MNP nitrotyrosine staining, and alveolar cell apoptosis. Lung XOR activity (A), MNP XOR activity (B), BALF cell number (C), lung histologic abnormalities (D), MNP nitrotyrosine staining (E), and alveolar cell apoptosis (F–H) were all increased (**P < 0.02) following insufflation of IL-1 and IFN-{gamma} compared with saline insufflation. In contrast, rats fed tungsten or allopurinol diets before IL-1 and IFN-{gamma} insufflation had decreased (*P < 0.05 or **P < 0.02) lung XOR activity (A), MNP XOR activity (B), BALF cell numbers (C), lung histologic abnormalities (D), MNP nitrotyrosine staining (E), alveolar cell TUNEL stain (F and G), and alveolar cell caspase-3 activation (H) compared with rats fed a control diet and then insufflated with IL-1 and IFN-{gamma}. Nitrotyrosine immunofluorescence staining (E) was conducted on lung tissue sections 24 h following IL-1 and IFN-{gamma} insufflation. Nitrotyrosine immunofluorescence is shown in red and tissue architecture in green and blue. Allopurinol and tungsten inhibition of MNP nitrotyrosine immunofluorescence and the effect of nitrotyrosine prebinding (NT prebinding) are also shown (E). These data demonstrate increased oxidative stress in the lungs of rats insufflated with IL-1 and IFN-{gamma} and that immunofluorescence in the alveolar located MNP was attenuated by prior inhibition of XOR. In G, panels B1 and B2 show higher magnification fields of fluorescence TUNEL staining associated with alveolar cells. Each data point in the quantitative TUNEL assay represents the counting of at least 8,000 nuclei derived from tandem serial sections of clearly identifiable alveolar cells. Data were acquired for each tissue slice by counting 200 nuclei per field and 20 fields per slide in parallel transects across each slide. These data demonstrate the quantitative increase in TUNEL stain following cytokine insufflation and the subsequent decrease in TUNEL stain in rats previously fed allopurinol or tungsten diets. Active caspase-3 (H) was quantitated in a similar fashion by counting the percentage of activated caspase-3–positive cells per high field view in 20 fields per tissue for two rats in each group. Activated caspase-3 was increased in the lungs of rats insufflated with IL-1 and IFN-{gamma} compared with rats insufflated with saline (**P < the lungs of rats insufflated with IL-1 and IFN-{gamma} compared with rats insufflated with saline (**P < 0.02) or tungsten (*P < 0.05) or allopurinol (**P < 0.02) fed rats insufflated with IL-1 and IFN-{gamma}. Because allopurinol dissociates from XOR during preparation of the protein extracts, allopurinol inhibition of XOR was not assayed (n.d.) in A and B.

 
MNP XOR Contributed to the Inflammatory Response In Vivo
To determine whether XOR induction in MNP could contribute to lung inflammation, we performed a cell transfer experiment. We induced a pulmonary inflammatory response in rats with IL-1 and IFN-{gamma} insufflation, obtained these cells by bronchoalveolar lavage 8 h later, and then treated these cells with either allopurinol or the vehicle for allopurinol in vitro. After a 15-min exposure to allopurinol or the vehicle, these cells were washed, resuspended in PBS, and then insufflated into control rats. Subsequently, cells were harvested from the BALF of these control rats, stained, and quantitated. BALF recovered from the lungs of rats insufflated 24 h before with untreated cells had increased numbers of neutrophils compared with BALF recovered following insufflation with allopurinol-pretreated cells (Figures 5A and 5B). Insufflation of allopurinol-treated BALF cells attenuated the subsequent recovery of neutrophils in the BALF (Figure 5C).




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  		Figure 5. MNP XOR augments lung inflammation in vivo. A lung inflammatory response was induced in rats with IL-1 and IFN-{gamma} insufflation. After 8 h, cells from BALF were recovered from each rat in PBS, pooled, and quantitated. Cells were then divided into two equivalent fractions and exposed to 1 mM KOH or 1 mM KOH with 150 µM allopurinol in vitro. The neutral pH of the PBS and cell mixture was unchanged by adding 1 mM KOH. After 15 min, the cells were washed and resuspended in PBS. Subsequently, 2 x 106 of control or allopurinol treated cells were insufflated into control rats. Twenty-four hours after cell insufflation, BALF cells were recovered from these rats, Wright's stained, and quantitated. Insufflation of untreated BALF cells increased the recovery of neutrophil 24 h later (A) compared with insufflation of allopurinol-treated BALF cells (B). Quantitation of neutrophils from both groups is shown in C. The numbers of neutrophils recoverable in BALF from rats insufflated with control cells is significantly increased (*P < 0.05) compared with the number of neutrophils recovered following insufflation of allopurinol-treated cells. Data are the mean and SE of six rats in each group.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We observed the rapid induction of XOR in the infiltrating, differentiating MNP that were recruited into the lungs of rats insufflated with IL-1 and IFN-{gamma}. Lungs of rats insufflated with IL-1 and IFN-{gamma} developed a vigorous inflammatory response characterized by rapid neutrophil and MNP infiltration, oxidative stress, and alveolar cell apoptosis. MNP XOR was recovered in predominantly its reversible O-form, and MNP from the lungs of IL-1– and IFN-{gamma}–insufflated rats had increased nitrotyrosine staining compared with MNP in control lungs. Pretreatment of rats with two different XOR inhibitors decreased MNP XOR induction, MNP nitrotyrosine staining, lung inflammation, and alveolar cell apoptosis. Transfer of allopurinol-inhibited MNP into normal rat lungs in vivo decreased the recruitment of neutrophils into the lung. Although vascular endothelial cell XOR may contribute to inflammation (35), our observations point to a dynamic, previously unrecognized process by which XOR induction in the MNP contributes to lung inflammation, oxidative stress, and alveolar cell apoptosis.

BALF from untreated rats contained primarily alveolar macrophages and few, if any, neutrophils or monocytes. These resident macrophages had high levels of the macrophage marker, ED1, but expressed low levels of XOR immunoreactive protein and activity. However, this pattern changed rapidly after cytokine insufflation. Beginning 4 h after cytokine insufflation, CD11b expressing, ED1-deficient cells exhibiting the compact, low scattering pattern characteristic of monocytes and neutrophils dominated the population of cells recoverable by lung lavage. Over the initial 24 h, XOR expression increased in the CD11b-positive ED1-negative MNP. This pattern gradually reverted to the zero time control pattern over the next 18 d when a majority of the recoverable cells were again ED1-positive macrophages. Simultaneous histologic and biochemical analyses of these BALF cells revealed that XOR activity was increased in MNP, but not neutrophils. Because XOR activity was not detected in circulating monocytes, the present observations suggest that increased XOR expression occurred in the infiltrating and differentiating MNP.

Predominantly O-form XOR was recovered in MNP throughout the 24 h following IL-1 and IFN-{gamma} insufflation. As early as 8 h after insufflation, O-form content comprised 70–80% of the total XOR activity. Importantly, O-form XOR could be reversed to D-form XOR by DTT reduction, leaving a constant O-form content of ~ 23% regardless of cytokine exposure, and this is exactly the level of O-form XOR found in the lungs of untreated rats or in livers. Thus, the increased amount of O-form XOR recovered from the lung was dependent upon prior cytokine insufflation. Because we observed no increase in XOR proteolysis following cytokine treatment, the combined action of IL-1 and IFN-{gamma} elevated both total XOR activity and most likely promoted the reversible conversion of D-form XOR to the O-form in vivo. Although inflammatory cytokines increased XOR in cultured epithelial cells, they did not cause conversion of D-form to O-form XOR in vitro (42). Although these contrasting observations undoubtedly reflect the different response of immortalized cells in culture and in the rat lung in vivo, the increase in XOR along with the increase in O-form content could enhance the ROS generating capacity of MNP in the alveoli.

The development of increased alveolar MNP nitrotyrosine staining after IL-1 and IFN-{gamma} insufflation suggests that oxidative stress was increased in MNP, and nitrotyrosine modification can be supported by O-form XOR, which can serve as source of O2-. (34–36). Inhibition of MNP nitrotyrosine staining in rats fed tungsten or allopurinol indicates that XOR served as a source of O2-. in the MNP following cytokine insufflation. Although other sources of O2-. exist in MNP, they are unlikely to be inhibited by both tungsten and allopurinol. We observed that 88% of the alveolar MNP stained positive for nitrotyrosine following cytokine insufflation, with a background of 14% positive MNP. The increase of 74% due to cytokine-induced inflammation is nearly identical to the level of nitrotyrosine staining seen at the onset of ARDS in humans (50), and suggests that oxidative stress in the MNP may be a general, early feature of acute lung inflammation.

In addition to inducing XOR expression in the MNP and generating an inflammatory response, insufflation of IL-1 and IFN-{gamma} also increased alveolar cell apoptosis that was quantitated by TUNEL stain and caspase-3 activation. Alveolar cell apoptosis has been frequently observed in clinical ALI and in experimental lung inflammation and appears to depend on activation of the Fas/FasLigand proteins (51–52), which are highly expressed on both inflammatory cells and lung epithelial cells (53). Although the exact mechanism responsible for alveolar cell apoptosis following cytokine insufflation is unclear, the inhibition of alveolar cell TUNEL stain and caspase-3 activation by tungsten or allopurinol treatment implicates the involvement of XOR.

We established the contribution of XOR to cytokine induced lung inflammation using tungsten and allopurinol inhibition. Both of these inhibitors decreased lung inflammation, MNP nitrotyrosine staining, and alveolar cell apoptosis. Allopurinol is a highly specific inhibitor in vivo that inhibits XOR noncompetitively following its conversion into oxypurinol (alloxanthine), which then inhibits the molybdenum center by tight, but reversible, binding (54). In our experiments, rats were fed allopurinol at a dose of 50 mg/kg for 7 d, a regimen well known to inhibit XOR in vivo (26, 27). Although high concentrations of allopurinol may theoretically have ROS scavenging capability in vivo (55), this results primarily from quenching hydroxyl radical and not O2-. (56). Tungsten feeding is another relatively selective method for inhibiting XOR in vivo (26–29). Tungsten acts by displacing molybdenum from the molybdopterin cofactor necessary for the activity of XOR and other MoCo enzymes (57). The similar effect achieved by treatment with either of these distinct inhibitors indicates that XOR activity contributed to the cytokine induced inflammatory response, MNP nitrotyrosine staining, and alveolar cell apoptosis seen in rats insufflated with IL-1 and IFN-{gamma}.

Because the use of systemic inhibitors like tungsten or allopurinol would not be limited to inhibiting XOR in the MNP, we performed a cell transfer experiment in which XOR was inhibited in MNP in vitro. Insufflation of allopurinol-treated, XOR-inhibited MNP demonstrated that MNP XOR could contribute to cytokine-induced lung inflammation. BALF cells recovered from rats insufflated with allopurinol-treated cells had reduced numbers of neutrophils compared with the numbers of neutrophils in the BALF following transfer of cells treated with the vehicle alone. This experiment indicated that XOR specifically located in MNP participated in the pulmonary inflammatory process. These experiments do not exclude the possible contribution of endothelial or epithelial XOR to the inflammatory process, nor do they address the potential role of interstitial or parenchymal MNP. In fact, our data suggest that substantial levels of interstitial MNP arose following IFN-{gamma} insufflation that were not recovered in the BALF. Nonetheless, our data do point to the unanticipated participation of alveolar cell MNP XOR in cytokine induced lung inflammation and underscore a possible functional interaction between neutrophils and MNP that is dependent on XOR.

Our study demonstrates that XOR can be induced rapidly in infiltrating and differentiating MNP following cytokine insufflation in rats, and that this phenomenon is linked to the inflammatory process. We imagine that MNP XOR generates ROS that contribute to increased pulmonary oxidative stress and inflammatory cell recruitment. These observations may explain the oft-postulated "two hit" process leading to the exuberant inflammation characteristic of ALI. If XOR is increased in MNP for as long as 18 d after an initial cytokine or other insult, then the lung may be more susceptible to a second insult for a considerable time. Whether allopurinol would be beneficial in treating or preventing ALI or other inflammatory diseases is unknown. However, the protective effect of allopurinol in diabetes (58, 59), renal ischemia-perfusion injury (60), and chronic heart failure (61–63) may be related to this mechanism inasmuch as MNP infiltration appears to be a component of each of these diseases.


   Acknowledgments
 
These studies were supported in part by grants from the National Institutes of Health (HL52509 and HL45582), the Department of Defense (BC980149), the Robert and Helen Kleberg Foundation, and Mr. Brian Fitzgerald in honor of his son, David Fitzgerald.

Received in original form August 18, 2003

Received in final form September 17, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Ware, L. B., and M. A. Matthay. 2000. The acute respiratory distress syndrome. N. Engl. J. Med. 342:1334–1349.[Free Full Text]
  2. Matthay, M. A., G. A. Zimmerman, C. Esmon, J. Bhattacharya, B. Coller, C. M. Doerschuk, J. Floros, M. A. Gimbrone Jr., E. Hoffman, R. D. Hubmayr, M. Leppert, S. Matalon, R. Munford, P. Parsons, A. S. Slutsky, K. J. Tracey, P. Ward, D. B. Gail, and A. L. Harabin. 2003. Future research directions in acute lung injury: summary of a NHLBI working group. Am. J. Respir. Crit. Care Med. 167:1027–1035.[Abstract/Free Full Text]
  3. Goodman, R. B., R. M. Strieter, D. P. Martin, K. P. Steinberg, J. A. Milberg, R. J. Maunder, S. L. Kunkel, A. Walz, L. D. Hudson, and T. R. Martin. 1996. Inflammatory cytokines in patients with persistence of the acute respiratory distress syndrome. Am. J. Respir. Crit. Care Med. 154:602–611.[Abstract]
  4. Meduri, G. U., G. Kohler, S. Headley, E. Tolley, F. Stentz, and A. Postlethwaite. 1995. Inflammatory cytokines in the BALF of patients with ALI. Persistent elevation over time predicts poor outcome. Chest 108:1303–1314.[Abstract/Free Full Text]
  5. Suter, P. M., S. Suter, E. Girardin, P. Roux-Lombard, G. E. Grau, and J. M. Dayer. 1992. 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.[Medline]
  6. Leff, J. A., J. W. Baer, M. E. Bodman, J. M. Kirkman, P. F. Shanley, L. M. Patton, C. J. Beehler, J. M. McCord, and J. E. Repine. 1994. Interleukin-1{alpha}-induced lung neutrophil accumulation and oxygen metabolite mediated lung leak in rats. Am. J. Physiol. Lung Cell. Mol. Physiol. 266:2–8.
  7. Guidot, D. M., E E. Stevens, M. J. Repine, A. E. Lucca-Broco, and J. E. Repine. 1994. Intratracheal but not intravascular interleukin-1 causes acute edematous injury in isolated neutrophil-perfused rat lungs through an oxygen radical mediated mechanism. J. Lab. Clin. Med. 123:605–609.[Medline]
  8. Krakauer, T. 2000. Pentoxifylline inhibits ICAM-1 expression and chemokine production induced by proinflammatory cytokines in human pulmonary epithelial cells. Immunopharmacology 46:253–261.[CrossRef][Medline]
  9. Steinmuller, C., G. Franke-Ullman, M. L. Lohmann-Matthes, and A. Emmendorffer. 2000. Local activation of nonspecific defense against a respiratory model infection by application of interferon-{gamma}. Am. J. Respir. Cell Mol. Biol. 22:481–490.[Abstract/Free Full Text]
  10. Wang, Z., T. Zheng, and Z. Zhu. 2000. Interferon–{gamma} induction of pulmonary emphysema in the adult murine lung. J. Exp. Med. 192:1587–1599.[Abstract/Free Full Text]
  11. Zhang, P., L. J. Quinton, G. J. Bagby, W. R. Summer, and S. Nelson. 2003. Interferon-{gamma} enhances the pulmonary CXC chemokine response to intratracheal lipopolysaccharide challenge. J. Infect. Dis. 187:62–69.[CrossRef][Medline]
  12. Ribeiro, R. A., F. Q. Cunha, and S. H. Ferreira. 1990. Recombinant gamma interferon causes neutrophil migration mediated by the release of a macrophage neutrophil chemotactic factor. Int. J. Exp. Pathol. 71:717–725.[Medline]
  13. Wen, L. P., K. Madani, J. A. Fahrni, S. R. Duncan, and G. D. Rosen. 1997. Dexamethasone inhibits lung epithelial cell apoptosis induced by IFN-{gamma} and Fas. Am. J. Physiol. Lung Cell. Mol. Physiol. 273:L921–L929.[Abstract/Free Full Text]
  14. Bratt, J., and J. Palmblad. 1997. Cytokine-induced neutrophil-mediated injury of human endothelial cells. J. Immunol. 159:912–918.[Abstract]
  15. Fujita, M., K. Kuwano, R. Kunitake, N. Hagimoto, H. Miyazaki, Y. Kaneko, M. Kawasaki, T. Maeyama, and N. Hara. 1998. Endothelial cell apoptosis in lipopolysaccharide-induced lung injury in mice. Int. Arch. Allergy Immunol. 117:202–208.[CrossRef][Medline]
  16. Rosseau, S., P. Hammerl, U. Maus, H. D. Walmrath, H. Schutte, F. Grimminger, W. Seeger, and J. Lohmeyer. 2000. Phenotypic characterization of alveolar monocyte recruitment in acute respiratory distress syndrome. Am. J. Physiol. Lung Cell. Mol. Physiol. 279:L25–L35.[Abstract/Free Full Text]
  17. Rosseau, S., J. Selhorst, K. Wiechmann, K. Leissner, U. Maus, U. Mayer, F. Grimminger, W. Seeger, and J. Lohmeyer. 2000. Monocyte migration through the alveolar epithelial barrier: adhesion molecule mechanisms and impact of chemokines. J. Immunol. 164:427–435.[Abstract/Free Full Text]
  18. Maus, U., S. Herold, H. Muth, R. Maus, L. Ermert, M. Ermert, N. Weissmann, S. Rosseau, W. Seeger, F. Grimminger, and J. Lohmeyer. 2001. Monocytes recruited into the alveolar air space of mice show a monocytic phenotype but upregulate CD14. Am. J. Physiol. Lung Cell. Mol. Physiol. 280:L58–L68.[Abstract/Free Full Text]
  19. Brigham, K. L. 1990. Oxidant stress and adult respiratory distress syndrome. Eur. Respir. J. 11:482S–484S.
  20. Lamb, N. J., J. M. Gutteridge, C. Baker, T. W. Evans, and G. J. Quinlan. 1999. Oxidative damage to proteins of bronchoalveolar lavage fluid in patients with acute respiratory distress syndrome: evidence for neutrophil mediated hydroxylation, nitration, and chlorination. Crit. Care Med. 27:1738–1744.[CrossRef][Medline]
  21. Leff, J. A., C. P. Wilke, B. M. Hybertson, P. F. Shanley, C. J. Beehler, and J. E. Repine. 1993. Post-insult treatment with N-acetyl-L-cysteine decreases IL-1-induced neutrophil influx and lung leak in rats. Am. J. Physiol. Lung Cell. Mol. Physiol. 265:501–506.
  22. Bernard, G. R., A. P. Wheeler, M. M. Arons, P. E. Morris, H. L. Paz, J. A. Russell, and P. E. Wright. 1997. A trial of antioxidants N-acetylcysteine and procysteine in ALI. The antioxidant in ALI study group. Chest 112:164–172.[Abstract/Free Full Text]
  23. Quinlan, G. J., N. J. Lamb, R. Tilley, T. W. Evans, and J. M. Gutteridge. 1997. Plasma hypoxanthine levels in ALI: implications for oxidative stress, morbidity, and mortality. Am. J. Respir. Crit. Care Med. 155:479–484.[Abstract]
  24. Grum, C. M., R. A. Ragsdale, L. H. Ketai, and R. H. Simon. 1987. Plasma xanthine oxidase activity in patients with adult respiratory distress syndrome. J. Crit. Care 2:22–27.
  25. Pinamonti, S., M. Muzzoli, and M. C. Chicca. 1996. Xanthine oxidase activity in bronchoalveolar lavage fluid from patients with chronic obstructive pulmonary disease. Free Radic. Biol. Med. 21:147–155.[CrossRef][Medline]
  26. Anderson, B. O., E. E. Moore, F. A. Moore, J. A. Leff, L. S. Terada, A. H. Harken, and J. E. Repine. 1991. Hypovolemic shock promotes neutrophil sequestration in lungs by a xanthine oxidase related mechanism. J. Appl. Physiol. 71:1862–1865.[Abstract/Free Full Text]
  27. Shenkar, R., and E. Abraham. 1996. Plasma from hemorrhaged mice activates CREB and increases cytokine expression in lung mononuclear cells through a xanthine oxidase-dependent mechanism. Am. J. Respir. Cell Mol. Biol. 14:198–206.[Abstract]
  28. Modelska, K., M. A. Matthay, L. A. Brown, E. Deutch, L. N. Lu, and J. F. Pittet. 1996. Inhibition of B-adrenergic-dependent alveolar epithelial clearance by oxidant mechanisms after hemorrhagic shock. Am. J. Physiol. 276:L844–L857.
  29. Adkins, W. K., and A. E. Taylor. 1990. Role of xanthine oxidase and neutrophils in ischemia reperfusion injury in rabbit lung. J. Appl. Physiol. 69:2012–2018.[Abstract/Free Full Text]
  30. Enroth, C., B. T. Eger, K. Okamoto, T. Nishino, T. Nishino, and E. F. Pai. 2000. Crystal structure of bovine milk xanthine dehydrogenase and xanthine oxidase: structure based mechanism of conversion. Proc. Natl. Acad. Sci. USA 97:10723–10728.[Abstract/Free Full Text]
  31. McManaman, J. L., and D. L. Bain. 2002. Structural and conformational analysis of the oxidase to dehydrogenase conversion of xanthine oxidoreductase. J. Biol. Chem. 277:21261–21268.[Abstract/Free Full Text]
  32. Nishino, T. 1997. NishinoT. The conversion from the dehydrogenase type to the oxidase type of rat liver xanthine dehydrogenase by modification of cysteine residues with fluorodinitrobenzene. J. Biol. Chem. 272:29859–29864.[Abstract/Free Full Text]
  33. Cighetti, G., S. Debiasi, and R. Paroni. 1993. Effect of glutathione depletion on the conversion of xanthine dehydrogenase to oxidase in rat liver. Biochem. Pharmacol. 45:2359–2361.[CrossRef][Medline]
  34. Nishino, T. 1994. The conversion of xanthine dehydrogenase to xanthine oxidase and the role of the enzyme in reperfusion injury. J. Biochem. (Tokyo) 116:1–6.[Abstract/Free Full Text]
  35. Harrison, R. 2002. Structure and function of xanthine oxidoreductase: where are we now? Free Radic. Biol. Med. 33:774–797.[CrossRef][Medline]
  36. Sawa, T., T. Akaike, and H. Maeda. 2000. Tyrosine nitration by peroxynitrite formed from nitric oxide and superoxide generated by xanthine oxidase. J. Biol. Chem. 275:32467–32474.[Abstract/Free Full Text]
  37. Reiter, C. D., R. J. Teng, and J. S. Beckman. 2000. Superoxide reacts with nitric oxide to nitrate tyrosine at physiological pH via peroxynitrite. J. Biol. Chem. 275:32460–32466.[Abstract/Free Full Text]
  38. Haddad, I. Y., G. Pataki, P. Hu, C. Galliani, J. S. Beckman, and S. Matalon. 1994. Quantitation of nitrotyrosine in lung sections of patients and animals with acute lung injury. J. Clin. Invest. 94:2407–2413.
  39. Baldus, S., J. P. Eiserich, M. L. Brennan, R. M. Jackson, C. B. Alexander, and B. A. Freeman. 2002. Spatial mapping of pulmonary and vascular nitrotyrosine reveals the pivotal role of myeloperoxidase as a catalyst for tyrosine nitration in inflammatory diseases. Free Radic. Biol. Med. 33:1010–1019.[CrossRef][Medline]
  40. Rudin, C. M., and C. B. Thompson. 1997. Apoptosis and disease: regulation and clinical relevance of programmed cell death. Annu. Rev. Med. 48:267–281.[CrossRef][Medline]
  41. Pfeffer, K. D., T. P. Heucksteadt, and J. R. Hoidal. 1994. Xanthine dehydrogenase and xanthine oxidase activity and gene expression in renal epithelial cells. J. Immunol. 153:1789–1797.[Abstract]
  42. Page, S., M. Benboubetra, C. R. Stevens, D. R. Blake, F. Selase, A. J. Wolstenholme, and R. Harrison. 1998. Xanthine oxidoreductase in human mammary epithelial cells: activation in response to inflammatory cytokines. Biochim. Biophys. Acta 1381:191–202.[Medline]
  43. Hassoun, P. M., F. S. Yu, C. G. Cote, J. J. Zulueta, R. Sawhney, K. A. Skinner, H. B. Skinner, D. A. Parks, and J. J. Lanzillo. 1998. Upregulation of xanthine oxidase by lippopolysaccharide, interleukin-1, and hypoxia. Am. J. Respir. Crit. Care Med. 158:299–305.
  44. Falciani, F., P. Ghezzi, M. Terao, G. Cazzaniga, and E. Garattini. 1992. Interferons induce xanthine dehydrogenase gene expression in L929 cells. Biochem. J. 285:1001–1008.
  45. Terao, M., G. Cazzaniga, P. Ghezzi, M. Bianchi, F. Falciani, P. Perani, and E. Garattini. 1992. Molecular cloning of a cDNA coding for mouse liver xanthine dehydrogenase: regulation of its transcript by interferons in vivo. Biochem. J. 283:863–870.
  46. Brown, J. M., L. S. Terada, M. A. Grosso, G. J. Whitmann, S. E. Velasco, A. Patt, A. H. Harken, and J. E. Repine. 1988. Xanthine oxidase produces hydrogen peroxide which contributes to reperfusion injury of ischemic, isolated, perfused rat hearts. J. Clin. Invest. 81:1297–1301.
  47. Rodell, T. C., J. C. Cheronis, C. L. Ohnemus, D. J. Piermattei, and J. E. Repine. 1987. Xanthine oxidase mediates elastase induced injury to isolated lungs and endothelium. J. Appl. Physiol. 63:2159–2163.[Abstract/Free Full Text]
  48. Terada, L. S., D. J. Piermattei, G. N. Shibao, J. L. McManaman, and R. M. Wright. 1997. Hypoxia regulates xanthine dehydrogenase activity at pre- and post-translational levels. Arch. Biochem. Biophys. 348:163–168.[CrossRef][Medline]
  49. McManaman, J. L., M. C. Neville, and R. M. Wright. 1999. Mouse mammary gland xanthine oxidoreductase: purification, characterization, and regulation. Arch. Biochem. Biophys. 371:308–316.[CrossRef][Medline]
  50. Sittipunt, C., K. P. Steinberg, J. T. Ruzinski, C. Myles, S. Zhu, R. B. Goodman, L. D. Hudson, S. Matalon, and T. R. Martin. 2001. Nitric oxide and nitrotyrosine in the lungs of patients with acute respiratory distress syndrome. Am. J. Respir. Crit. Care Med. 163:503–510.[Abstract/Free Full Text]
  51. Serrao, K. L., J. D. Fortenberry, M. J. Owens, F. L. Harris, and L. A. Brown. 2001. Neutrophils induce apoptosis of lung epithelial cells via release of soluble Fas ligand. Am. J. Physiol. Lung Cell. Mol. Physiol. 280:L298–L305.[Abstract/Free Full Text]
  52. Matute-Bello, G., R. K. Winn, M. Jonas, E. Y. Chi, T. R. Martin, and W. C. Liles. 2001. Fas (CD95) induces alveolar epithelial cell apoptosis in vivo. Am. J. Pathol. 158:153–161.[Abstract/Free Full Text]
  53. Albertine, K. H., M. F. Soulier, Z. Wang, A. Ishizaka, S. Hashimoto, G. A. Zimmerman, M. A. Matthay, and L. B. Ware. 2002. Fas and Fas ligand are up regulated in pulmonary edema fluid and lung tissue of patients with acute lung injury and the adult respiratory distress syndrome. Am. J. Pathol. 161:1783–1796.[Abstract/Free Full Text]
  54. Hille, R., and V. Massey. 1985. Molybdenum containing hydroxylases: xanthine oxidase, aldehyde oxidase, and sulfite oxidase. In Molybdenum Enzymes. T. G. Spiro, editor. John Wiley & Sons, New York. 443–519.
  55. Faure, M., E. A. Lissi, and L. A. Videla. 1990. Antioxidant capacity of allopurinol in biological systems. Biochem. Int. 21:356–366.
  56. Zimmerman, B. J., D. A. Parks, M. B. Grisham, and D. N. Granger. 1988. Allopurinol does not enhance antioxidant properties of extracellular fluid. Am. J. Physiol. 255:H202–H206.
  57. Hille, R. 2002. Molybdenum and tungsten in biology. Trends Biochem. Sci. 27:360–367.[CrossRef][Medline]
  58. Desco, M. C., M. Asensi, R. Marquez, J. Martinez-Valls, M. Vento, F. V. Pallardo, J. Sastre, and J. Vina. 2002. Xanthine oxidase is involved in free radical production in type 1 diabetes: protection by allopurinol. Diabetes 51:1118–1124.[Abstract/Free Full Text]
  59. Butler, R., A. D. Morris, J. J. Belch, A. Hill, and A. D. Struthers. 2000. Allopurinol normalizes endothelial dysfunction in type 2 diabetes with mild hypertension. Hypertension 35:746–751.[Abstract/Free Full Text]
  60. Rhoden, E., C. Teloken, M. Lucas, C. Rhoden, M. Mauri, C. Zettler, A. Bello-Klein, and E. Barros. 2000. Protective effect of allopurinol in the renal ischemia-reperfusion in uninephrectomized rats. Gen. Pharmacol. 35:189–193.[Medline]
  61. Cappola, T. P., D. A. Kass, G. S. Nelson, R. D. Berger, G. O. Rosas, Z. A. Kobeissi, A. Marban, and J. M. Hare. 2001. Allopurinol improves myocardial efficiency in patients with idopathic dilated cardiomyopathy. Circulation 104:2407–2411.[Abstract/Free Full Text]
  62. Doehner, W., N. Schoene, M. Rauchhaus, F. Leyva-Leon, D. V. Pavitt, D. A. Reaveley, G. Schuler, A. J. Coats, S. D. Anker, and R. Hambrecht. 2002. Effects of xanthine oxidase inhibition with allopurinol on endothelial function and peripheral blood flow in hyperuricemic patients with chronic heart failure. Circulation 105:2619–2624.[Abstract/Free Full Text]
  63. Farquharson, C. A., R. Butler, A. Hill, J. J. Belch, and A. D. Struthers. 2002. Allopurinol improves endothelial dysfunction in chronic heart failure. Circulation 106:221–226.[Abstract/Free Full Text]



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