CXCR2 Is Critical to Hyperoxia-Induced Injury Richard D. Sue, John A. Belperio, Marie D. Burdick, Lynne A. Murray, Ying Ying Xue, Maria C. Dy, Jeffery J. Kwon, This information is as Michael P. Keane and Robert M. Strieter of September 24, 2021. J Immunol 2004; 172:3860-3868; ; doi: 10.4049/jimmunol.172.6.3860 http://www.jimmunol.org/content/172/6/3860 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

CXCR2 Is Critical to Hyperoxia-Induced Lung Injury1

Richard D. Sue,* John A. Belperio,* Marie D. Burdick,* Lynne A. Murray,* Ying Ying Xue,* Maria C. Dy,* Jeffery J. Kwon,* Michael P. Keane,* and Robert M. Strieter2*†

Hyperoxia-induced lung injury is characterized by infiltration of activated neutrophils in conjunction with endothelial and epi- thelial cell injury, followed by fibrogenesis. Specific mechanisms recruiting neutrophils to the lung during hyperoxia-induced lung injury have not been fully elucidated. Because CXCL1 and CXCL2/3, acting through CXCR2, are potent neutrophil chemoat- tractants, we investigated their role in mediating hyperoxia-induced lung injury. Under variable of , murine survival during hyperoxia-induced lung injury was dose dependent. Eighty percent oxygen was associated with 50% mortality at 6 days, while greater oxygen concentrations were more lethal. Using 80% oxygen, we found that harvested at day 6 demonstrated markedly increased neutrophil sequestration and lung injury. Expression of CXCR2 ligands paralleled neutrophil recruitment to the lung and CXCR2 mRNA expression. Inhibition of CXC chemokine ligands/CXCR2 interaction using CXCR2؊/؊ mice exposed to hyperoxia significantly reduced neutrophil sequestration and lung injury, and led to a significant Downloaded from survival advantage as compared with CXCR2؉/؉ mice. These findings demonstrate that CXC chemokine ligand/CXCR2 biological axis is critical during the pathogenesis of hyperoxia-induced lung injury. The Journal of Immunology, 2004, 172: 3860–3868.

anagement of acute lung injury (ALI)3 and acute re- the expression of CXCR2 ligands through their interaction with spiratory distress syndrome (ARDS) consists of low their shared receptor, CXCR2. M tidal volume protective ventilation, treatment of the Our study demonstrates that high concentrations of inspired ox- http://www.jimmunol.org/ inciting cause, prevention of nosocomial infections, and the ad- ygen lead to lung neutrophil sequestration and injury that parallel ministration of high concentrations of inspired oxygen with vari- the expression of CXCL1 and CXCL2/3, and the recruitment of able amounts of positive end-expiratory (1, 2). The high cells expressing CXCR2. Moreover, inhibition of CXCR2/CXCR2 concentrations of inspired oxygen used during the management of ligand interaction markedly attenuates hyperoxia-induced lung in- ALI/ARDS may result in increased reactive oxygen metabolites in jury and prolongs survival under hyperoxic conditions. the lung that may perpetuate the inflammatory response (3, 4). Chemokines are 8- to 10-kDa proteins with 20Ð70% homology Materials and Methods in amino acid sequences that are subdivided into families based on Murine model of hyperoxia-induced lung injury by guest on September 24, 2021 the relative position of the cysteine residues in the mature protein. Male C57BL/6 mice (6Ð8 wk) were purchased from The Jackson Labo- Murine CXCL1 and CXCL2/3 are glutamic acid-leucine-arginine- ratory (Bar Harbor, ME). Mice were maintained in specific pathogen-free positive (ELR-positive) CXC chemokines; are structural homo- conditions and provided with food and water ad libitum. To induce hyper- logues of human growth-related oncogene-␣/CXCL1 and growth- oxia-induced lung injury, mice were allowed to roam free under normo- ␤ ␥ baric in 20-gallon chambers under varying concentrations of hy- related oncogene- / /CXCL2/3; and act as functional homologues peroxia or room air to determine whether different concentrations of to other human ELR-positive CXC chemokines in the mouse (5Ð oxygen led to differences in acute lung injury and survival. Oxygen mix- 9). Both murine chemokines share the ability to signal through a G tures or room air were delivered through the chamber at 3 liters/min and protein-coupled receptor, CXCR2, and are potent neutrophil che- allowed to vent through a distal ventilation port to maintain normobaric moattractants and promote angiogenesis (5Ð7). Their human struc- pressures. In subsequent studies, mice were placed in 80% oxygen, and after 6 days of exposure, anesthetized with ketamine i.p.; then, under sterile tural and functional homologues have been associated with ALI/ conditions, the thorax was exposed. A 22-gauge needle was used to punc- ARDS (10Ð17). We hypothesized that the neutrophil recruitment ture the left atrium. A 22-gauge needle was introduced into the right ven- to the lung during hyperoxia-induced lung injury is due, in part, to tricle, and 3 ml of PBS were infused at 20 cm H2O. The heart and thymus were removed and the lungs were dissected from the hilum. The tissue was frozen in liquid nitrogen and stored at Ϫ80¡C for future processing and analysis. We also performed the same studies using CXCR2Ϫ/Ϫ mice on a ϩ/ϩ *Department of , Division of Pulmonary and Critical Care Medicine, and C57BL/6 background, as compared with CXCR2 (wild-type) †Department of Pathology and Laboratory Medicine, David Geffen School of Med- C57BL/6 mice. icine, University of California, Los Angeles, CA 90095 Reagents Received for publication November 14, 2003. Accepted for publication January 14, 2004. Biotinylated and nonbiotinylated anti-murine CXCL1 was purchased from The costs of publication of this article were defrayed in part by the payment of page R&D Systems (Minneapolis, MN). Polyclonal rabbit anti-murine charges. This article must therefore be hereby marked advertisement in accordance CXCL2/3 used for ELISA was produced by the immunization of a rabbit with 18 U.S.C. Section 1734 solely to indicate this fact. with carrier-free murine rCXCL2/3 (R&D Systems) in multiple intrader- 1 This work was supported, in part, by National Institutes of Health Grants HL04493, mal sites with CFA, followed by at least three boosts of CXCL2/3 in IFA, HL03906, P50HL67665, CA87879, P50CA90388, HL66027, and RG-019-B. as previously described (18, 19). 2 Address correspondence and reprint requests to Dr. Robert M. Strieter, Department Myeloperoxidase (MPO) assay of Medicine, Division of Pulmonary and Critical Care Medicine, and Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of Pulmonary neutrophil sequestration was quantitated using a MPO assay, as California, Room 14-154 Warren Hall, Box 711922, 900 Veteran Avenue, Los An- previously described (20). Briefly, at the time of sacrifice, lungs were per- geles, CA 90024-1922. E-mail address: [email protected] fused free of blood with 3 ml of 0.9% saline via the spontaneously beating 3 Abbreviations used in this paper: ALI, acute lung injury; ARDS, acute respiratory right ventricle. The lungs were excised from 10 mice under conditions of distress syndrome; MPO, myeloperoxidase. hyperoxia and from 6 mice under conditions of room air exposure and

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 The Journal of Immunology 3861 placed in a 50 mM potassium phosphate buffer (pH 6.0) with 5% previously described (20). Briefly, flat-bottom 96-well microtiter plates hexadecyltrimethylammonium bromide (Sigma-Aldrich, St. Louis, MO). (Nunc-Immuno-Plate I 96-F; Nalge Nunc International, Rochester, NY) The lung tissue was homogenized, sonicated, and centrifuged at 12,000 ϫ were coated with anti-murine CXCL1 or CXCL2/3 (1 ng/␮l in 0.6 M NaCl, g for 15 min at 4¡C. The supernatant was then assayed for MPO activity 0.26 M H3BO4, and 0.08 N NaOH, pH 9.6) for 24 h at 4¡C and then washed using a spectrophotometric reaction with o-dianisidine hydrochloride (Sig- with PBS (pH 7.5) and 0.05% Tween 20 (wash buffer). Microtiter plate ma-Aldrich) at 460 nm, as previously described (20). nonspecific binding sites were blocked with 2% BSA in PBS and incubated for 60 min at 37¡C. Plates were washed three times with wash buffer, and Histopathological grading of hyperoxia-induced lung injury samples or standard were added, followed by incubation for1hat37¡C. Plates were washed three times; 50 ␮l/well biotinylated anti-murine Three random 5-␮m paraffin-embedded tissue sections from five different CXCL1 and CXCL2/3 Abs were added; and plates were incubated for 45 lungs taken from the 80% oxygen-exposed and normal room air-exposed min at 37¡C. Plates were washed three times; streptavidin-peroxidase con- mice were stained with H&E at day 6. The histopathology was reviewed in jugate (Jackson ImmunoResearch Laboratories, West Grove, PA) was add- a blinded manner with respect to which group or mouse was being re- ed; and the plates were incubated for 30 min at 37¡C. Plates were washed viewed, using a modified histologic scoring system, as previously de- three times, and 3,3Ј,5,5Ј-tetramethylbenzidine chromogen substrate scribed (7). Four easily identifiable pathological processes were scored on (Kirkegaard & Perry Laboratories, Gaithersburg, MD) was added. The a scale of 0Ð4: 1) alveolar congestion, 2) hemorrhage, 3) leukocyte infil- plates were incubated at room to the desired extinction, and the tration or aggregation of neutrophils in air space or the vessel wall, and 4) reaction was terminated with3MHSO solution. Plates were read at 450 thickness of the alveolar wall. A score of 0 represented normal lungs; 1, 2 4 nm in an automated microplate reader (Bio-Tek Instruments, Winooski, mild, Ͻ25% lung involvement; 2, moderate, 25Ð50% lung involvement; 3, VT). Standards were half-log dilutions of either CXCL1 or CXCL2/3 from severe, 50Ð75% lung involvement; and 4, very severe, Ͼ75% lung in- 100 ng/ml to 1 pg/ml (50 ␮ volvement. An overall score of hyperoxia-induced lung injury was ob- l/well). This ELISA method consistently de- tained based on the summation of all the scores, and then a mean Ϯ SEM tected specific chemokine concentrations greater than 50 pg/ml in a linear was generated from the cohort of normal room air- or hyperoxia-exposed fashion. CXCL1 and CXCL2/3 were specific in our sandwich ELISA with- Downloaded from lungs (three sections from each lung, six lungs per group) at each time out cross-reactivity to a panel of cytokines, including human and murine ␣, IFN-␥, and mem- point to generate a cumulative histological hyperoxia-induced lung injury IL-1 receptor antagonist, IL-1, IL-2, IL-6, IL-4, TNF- bers of the CXC and CC chemokine families. score. ␬ ␣ Evans blue microvascular permeability and wet:dry analysis of ELISA for phosphorylated I- B lung To assess activated NF-␬B in tissue, phosphorylated I-␬B␣ from whole lung homogenates of six mice per group was measured using a modifica-

Microvascular permeability related to lung injury was measured using a tion of a commercially available ELISA kit, I-␬B␣ ActivELISA (BioCarta, http://www.jimmunol.org/ modification of the Evans blue dye extravasation technique, as previously San Diego, CA), as previously described (7). Briefly, a flat-bottom 96-well described (20, 21). Extravasation of Evans blue (Sigma-Aldrich) into the microtiter plate was coated with capture Ab for 24 h at 4¡C and then extravascular compartment was used as a quantitative measure of lung washed. Nonspecific binding sites were blocked with blocking buffer (Bio- injury and changes in pulmonary microvasculature permeability. Briefly, Carta) and incubated for 60 min at room temperature. The plate was each animal received 20 mg/kg Evans blue (pH 7.34) by tail vein injection washed three times with wash buffer (BioCarta), and samples or standard 3 h before sacrifice. At the time of sacrifice, a heparinized sample of blood were added, followed by incubation for4hat4¡C. The plate was washed was harvested, and plasma was removed by centrifugation. Ten lungs from three times with wash buffer (BioCarta), and detection Ab was added, each group were perfused free of blood with 1 ml of 0.9% normal saline via followed by incubation for1hatroom temperature. The plate was further the spontaneously beating right ventricle and removed from the thoracic washed three times, and streptavidin-HRP was added. The plate was then cavity. The , mainstem bronchi, and surrounding mediastinal struc- incubated for additional1hatroom temperature. The plate was washed tures were removed. Evans blue was extracted from pulmonary tissues after three times with wash buffer (BioCarta), and 3,3Ј,5,5Ј-tetramethylbenzi- by guest on September 24, 2021 homogenization in 1 ml of 0.9% normal saline. This volume was added to dine chromogen substrate (Kirkegaard & Perry Laboratories) was added. 2 vol of deionized formamide and incubated at 60¡C for 12 h. The super- The plate was incubated at room temperature to the desired extinction, and natant was separated by centrifugation at 2000 ϫ g for 30 min. Evans blue the reaction was terminated with3MH2SO4 solution. The plate was read in the plasma and lung tissue was quantitated by dual-wavelength spectro- at 450 nm in an automated microplate reader (Bio-Tek Instruments). The photometric analysis at 620 and 740 nm (22). This method corrects the standard curve was generated using half dilutions of phosphorylated I-␬B␣ specimen’s absorbance at 620 nm for the absorbance of contaminating from 1000 ␮g/ml to 0, provided by the manufacturer (BioCarta). heme pigments, using the following formula: corrected absorbance at 620 nm ϭ actual absorbance at 620 nm Ð (1.426 (absorbance at 740) ϩ 0.03). Total RNA isolation and RT-PCR amplification We calculated a permeability index by dividing the corrected pulmonary tissue Evans blue absorbance at 620 nm/g lung tissue by the corrected Total cellular RNA from lung tissue of six mice per group was isolated, as plasma Evans blue absorbance at 620 nm; this index reflects the degree of previously described (20, 23). Total RNA was determined, and 1.0 ␮gof extravasation of Evans blue into the extravascular pulmonary tissue total RNA was reverse transcribed into cDNA using Taqman Gene Ex- compartment. pression Quantification assays (Applied Biosystems (Foster City, CA) kit To quantitate lung edema during hyperoxia-induced lung injury, we ob- 4304134). cDNA was amplified and quantified using the TaqMan 7700 tained wet:dry ratios by ligating 10 lungs per group away from the Sequence Detection System and specific primers for murine CXCL1, mu- hilum. The lungs were blotted dry and weighed. They were then desiccated rine CXCL2/3, murine CXCR2, procollagen I, procollagen III, and a by incubation at 130¡C overnight in a vacuum oven and reweighed to housekeeping gene18S. The primers used were 5Ј-TGA-GCT-GCG-CTG- determine their dry weight. The wet:dry ratio was then calculated. TCA-GTG-CCT-3Ј (sense) and 5Ј-AGA-AGC-CAG-CGT-TCA-CCA- GA-3Ј (antisense) for CXCL1 (259 bp); 5Ј-GCT-GGC-CAC-CAA-CCA- Collagen protein measurement CCA-GG-3Ј (sense) and 5Ј-AGC-GAG-GCA-CAT-CAG-GTA-CG-3Ј (antisense) for murine CXCL2/3 (359 bp); 5Ј-TCA-CCT-ACA-GCG- Total soluble collagen was measured in whole lung homogenates by the TCA-CTG-TCG-3Ј (sense) and 5Ј-CAC-TGT-TGC-CCC-AGC-3Ј (anti- Sircol assay (Biocolor, Belfast, N. Ireland). Ten lungs per group were sense) for procollagen 1 (259 bp); and 5Ј-GCA-GGC-ATG-GTG-GCA-3Ј ground in 1 ml of PBS, sonicated, centrifuged, and filtered through a Ј Ј ␮ ␮ ␮ (sense) and 5 -CCC-CGG-GTT-CTA-GTA-GTG-ATT-CTC-3 (antisense) 1.2- m syringe filter. A total of 50 l of sample was mixed with 50 lof for procollagen III (359 bp). Predeveloped assay reagents (Applied Bio- 0.5 M acetic acid. Samples were mixed by gentle inversion, and 1 ml of ␮ systems kit 4304134) were used for murine CXCR2 and the housekeeping Sirius Red reagent was added to 100 l of test sample and mixed for 30 gene, 18S. Quantitative analysis of gene expression was done using the min at room temperature. The collagen-dye complex was precipitated by ⌬ ϫ comparative CT ( CT) methods, in which CT is the threshold cycle number centrifugation at 5000 g for 10 min and mixed with 1.0 ml of Sircol (the minimum number of cycles needed before the product can be detected) alkali reagent; the absorbance was measured at 540 nm using a microtiter ⌬ (7). The arithmetic formula for the CT method is the difference in thresh- plate reader. The calibration curve was set up on the basis of collagen old cycles for a target (i.e., CXCR2) and an endogenous reference (i.e., standard provided by the manufacturer. The assay was performed in du- housekeeping gene 18S). The amount of target normalized to an endoge- plicate, and the mean of two data was determined for individual sample. nous reference (i.e., CXCR2) and relative to a calibration normalized to an CXC chemokine ELISAs endogenous reference (i.e., CXCR2 in room air controls) is given by 2Ϫ⌬⌬CT (7). The following is an example for comparing CXCR2 expres- CXCL1 or CXCL2/3 protein in whole lung homogenates from six mice per sion from hyperoxia-treated animals and room air controls. Both CXCR2 group was quantitated using a modification of a double-ligand method, as from the hyperoxia-treated and room air controls are normalized to 18S: 3862 CXCR2 IS CRITICAL TO HYPEROXIA-INDUCED LUNG INJURY

ondary Ab used for these experiments was Alexa 488 (Molecular Probes, Eugene, OR). Dual-color-stained cell suspensions were analyzed on a FACScan flow cytometer (BD Immunocytometry Systems, San Jose, CA) using CellQuest 3.2.1f1 software (BD Immunocytometry Systems). Statistical analysis Data were analyzed using the StatView 4.5 statistical package (Abacus Concepts, Berkeley, CA). Two group comparisons were evaluated using the unpaired t test. Three group comparisons were evaluated by the ANOVA test with the post hoc analysis (i.e., Bonferroni/Dunn). Survival was evaluated between groups using the Kaplan-Meier survival analysis and log rank test. Data were expressed as mean Ϯ SEM. Results FIGURE 1. Hyperoxia induces mortality in a -dependent Oxygen induces mortality in a concentration-dependent manner manner (n ϭ 10 mice per each group; p Ͻ 0.0001). Because human studies have suggested that hyperoxia is associ- ated with increased lung injury, we performed translational studies ⌬⌬ ⌬ CT ϭ CT (CXCR2 expression from hyperoxia-treated animals normal- using a murine model system of hyperoxia-induced lung injury to Ϫ⌬ ized to endogenous 18S) CT (CXCR2 expression from room air con- dissect the mechanisms related to this process. Mice exposed to trols normalized to endogenous 18S). The calculation of 2Ϫ⌬⌬CT then gives 100 and 90% oxygen became rapidly ill and demonstrated 100% a relative value when comparing the target with the calibrator, which we Downloaded from designate in this context as fold increase of hyperoxia-treated animals to mortality within 4Ð5 days (Fig. 1). Mice exposed to 80% oxygen room air controls of the target mRNA relative quantification. showed 50% mortality by day 6 and 100% mortality within 12 days (Fig. 1). Finally, mice exposed to 70% oxygen remained mor- FACS analysis of lung neutrophils tality free during the study period of 14 days (Fig. 1). Whole lung single cell suspension preparations were made from harvested lungs from six mice per group using a method, as previously described (7). Hyperoxia is associated with increased lung injury 6 Single cell suspensions (5 ϫ 10 cells/ml) were stained with primary Abs: http://www.jimmunol.org/ Tricolor-conjugated (BD Biosciences, Franklin Lakes, NJ) anti-murine Quantitative analysis of lung histopathology demonstrated more CD45 (Caltag Laboratories, South San Francisco, CA), anti-murine Ly-6G edema, leukocyte infiltration, alveolar hemorrhage, and alveolar (neutrophil surface marker; BD Biosciences), or isotype controls. The sec- wall thickness in lungs harvested at day 6 from the mice exposed by guest on September 24, 2021

FIGURE 2. Hyperoxia (80%) induces lung injury. IA, Lung injury scores by analysis of H&E-stained histopathologic sections from lungs at day 6 (n ϭ 15; ,ء ;random sections per lung and 5 lungs per group 3 p Ͻ 0.05). IB, Evans blue permeability index at day 6 p Ͻ 0.05). IC, Wet:Dry ,ء ;n ϭ 10 lungs per group) .(p Ͻ 0.05 ,ء ;ratio at day 6 (n ϭ 10 lungs per group ID, Extracellular matrix deposition as determined by measurement of collagen at day 6 (n ϭ 10 lungs per -p Ͻ 0.05). II, Representative photomicro ,ء ;group graphs of H&E staining (ϫ200) of the lungs of mice exposed to either room air (A) or 80% oxygen (B) for 6 days. The Journal of Immunology 3863 to hyperoxia (80%) than mice exposed to room air (Fig. 2, IA and CXCL1 and CXCL2/3 mRNA expression and protein levels in II). We found that lungs from the high oxygen concentration-ex- the lung are elevated during hyperoxia-induced lung injury posed group had a significantly higher vascular permeability index We found significantly higher levels of mRNA expression of than those mice exposed to room air (Fig. 2IB). These findings CXCL1 and CXCL2/3 in the lungs from the 80% oxygen-exposed were confirmed by demonstrating a greater wet:dry ratio of lungs animals, as compared with the lung homogenates of room air- of animals exposed to 80% oxygen (Fig. 2IC). Furthermore, be- exposed mice (Fig. 4, A and B, respectively). The expression of cause lung injury during ARDS ultimately leads to extracellular these chemokines paralleled lung neutrophil infiltration and hyper- matrix deposition, we evaluated both groups for collagen deposi- oxia-induced lung injury (Figs. 2 and 3). This was further con- tion in the lung. The lungs of mice exposed to 80% oxygen had firmed by measuring protein levels of CXCL1 and CXCL2/3 in more collagen deposition than the lungs of mice exposed to room whole lung homogenates from mice exposed to 80% oxygen and air at day 6 (Fig. 2ID). Finally, histopathological analysis of lungs room air. There were significantly higher protein levels of CXCL1 from the hyperoxia-exposed mice on day 6 demonstrated markedly and CXCL2/3 in the lung homogenates from mice exposed to 80% increased alveolar congestion, alveolar hemorrhage, leukocyte in- oxygen as compared with lungs from mice exposed to room air filtration, and alveolar wall thickness as compared with room air- (Fig. 4, C and D, respectively). To determine that the elevated exposed mice (Fig. 2II). expression of CXCL1 and CXCL2/3 that resulted in neutrophil infiltration and hyperoxia-induced lung injury was compartmen- talized to the lung and not a result of systemic increases in plasma Neutrophils are recruited into the lung during hyperoxia- CXCL1 and CXCL2/3, we next measured protein levels of CXCL1 induced lung injury and CXCL2/3 in plasma from mice exposed to 80% oxygen and Downloaded from Next, we measured neutrophil sequestration in the lung during room air. There were similar protein levels of CXCL1 and 80% oxygen in comparison with normal room air-exposed control CXCL2/3 in the plasma from mice exposed to 80% oxygen as mice. C57BL/6 mice placed on either 80% oxygen or room air compared with lungs from mice exposed to room air (Fig. 4, E and were sacrificed at 6 days, and their lungs were harvested for MPO F, respectively).

analysis as an indirect measurement of neutrophil infiltration into http://www.jimmunol.org/ the lung. Lung homogenates from the 80% oxygen-exposed group Expression of CXCL1 and CXCL2/3 is associated with increased had significantly greater levels of MPO than lung homogenates of phosphorylated I-␬B␣ in the lung under hyperoxic conditions room air-exposed mice (Fig. 3A). FACS analysis of Ly-6G for neu- To begin to determine a potential mechanism for the increased trophils was used to confirm this finding by demonstrating a greater gene expression of CXCL1 and CXCL2/3 in the lung under hy- number of neutrophils infiltrating the lungs of hyperoxia-exposed peroxic conditions, we assessed the presence of phosphorylated mice when compared with the lungs of control mice (Fig. 3B). I-␬B␣. The analysis of the phosphorylation of I-␬B␣ correlates with nuclear localization and activation of NF-␬B (24Ð26). Lung homogenates of mice were analyzed at 6 days of continuous ex- by guest on September 24, 2021 posure to 80% oxygen as compared with room air-exposed mice. We found significantly higher levels of phosphorylated I-␬B␣ in the lungs from the 80% oxygen-exposed animals than in the room air-exposed mice (Fig. 5).

CXCR2 expression is increased during hyperoxia-induced lung injury The finding of increased levels of CXCL1 and CXCL2/3 associ- ated with neutrophil sequestration and hyperoxia-induced lung in- jury led us to evaluate the expression of CXCR2 mRNA in the lungs of these animals. Lung homogenates from the 80% oxygen- exposed group had significantly greater expression of CXCR2 mRNA than lung homogenates of room air-exposed mice (Fig. 6). The expression of CXCR2 mRNA paralleled its ligand expression, neutrophil sequestration, and hyperoxia-induced lung injury (Figs. 2Ð4).

CXCR2Ϫ/Ϫ mice display reduced neutrophil sequestration in the lung under conditions of hyperoxia With CXCL1 and CXCL2/3 correlating with the recruitment of neutrophils and expression of CXCR2, we next used a genetic strategy using C57BL/6 CXCR2Ϫ/Ϫ vs C57BL/6 CXCR2ϩ/ϩ mice, and compared their responses under conditions of 80% ox- ygen exposure at day 6. We found that lungs from CXCR2Ϫ/Ϫ mice had lower levels of MPO under conditions of 80% oxygen FIGURE 3. Hyperoxia-induced neutrophil sequestration in the lung. A, ϩ/ϩ Lung MPO levels from mice exposed to room air or 80% oxygen for 6 days exposure at day 6, as compared with CXCR2 mice exposed to p Ͻ 0.05). B, FACS analysis of neutrophils the same concentration of oxygen (Fig. 7). Furthermore, the levels ,ء ;n ϭ 10 mice per group) Ϫ/Ϫ from the lungs of mice exposed to either room air or hyperoxia for 6 days of MPO from CXCR2 mice were not significantly different ϩ ϩ .(p Ͻ 0.05). from the CXCR2 / mice exposed to room air (Fig. 7 ,ء ;n ϭ 6 mice per group) 3864 CXCR2 IS CRITICAL TO HYPEROXIA-INDUCED LUNG INJURY

FIGURE 4. Hyperoxia induces the expression of CXCL1 and CXCL2/3 mRNA and protein in the lung. A and B, Quantitative levels of CXCL1 and CXCL2/3 mRNA, respectively, in the lungs from mice exposed to either room air or hyperoxia for 6 days (n ϭ 6 mice p Ͻ 0.05). C and D, Quantitative levels ,ء ;per group of CXCL1 and CXCL2/3 protein in the lungs, respec- tively, from mice exposed to either room air or hyper- Downloaded from .(p Ͻ 0.05 ,ء ;oxia for 6 days (n ϭ 6 mice per group E and F, Quantitative levels of CXCL1 and CXCL2/3 protein, respectively, in plasma from mice exposed to either room air or hyperoxia for 6 days (n ϭ 6 mice per group; no statistical difference found). http://www.jimmunol.org/ by guest on September 24, 2021

CXCR2Ϫ/Ϫ mice were protected from hyperoxia-induced lung ygen at day 6 when compared with CXCR2ϩ/ϩ mice exposed to injury the same conditions (Fig. 8I, B and C). In addition, CXCR2Ϫ/Ϫ Quantitative analysis of lung histopathology confirmed that mice exposed to 80% oxygen for 6 days demonstrated reduced ϩ ϩ CXCR2Ϫ/Ϫ mice were protected from hyperoxia-induced lung in- collagen deposition in the lungs, as compared with CXCR2 / jury and indistinguishable from CXCR2ϩ/ϩ mice exposed to room mice exposed to similar conditions, and the levels were similar to air (Fig. 8IA). Both markers of lung microvascular permeability CXCR2ϩ/ϩ exposed to room air (Fig. 8ID). Histopathological and edema confirmed that the lungs from CXCR2Ϫ/Ϫ mice re- analysis of lungs from the CXCR2Ϫ/Ϫ mice, as compared with tained their microvascular integrity under conditions of 80% ox- CXCR2ϩ/ϩ mice on day 6, demonstrated minimal alveolar con-

FIGURE 5. Hyperoxia induces increased levels of phosphorylated I-␬B␣. Lung injury is associated with increased levels of NF-␬B. Phos- FIGURE 6. Hyperoxia induces increased expression of CXCR2 mRNA phorylated I-␬B␣ was measured from whole lung homogenates in mice in the lung. Quantitative RT-PCR was determined by Taqman analysis for exposed to either room air or hyperoxia for 6 days (n ϭ 6 mice per group; CXCR2 mRNA from the lungs of mice exposed to either room air or 80% .(p Ͻ 0.05 ,ء ;p Ͻ 0.05). hyperoxia for 6 days (n ϭ 6 mice per group ,ء The Journal of Immunology 3865

CXCR2Ϫ/Ϫ mice have a survival advantage over CXCR2ϩ/ϩ mice when exposed to hyperoxia To determine whether the protective effects of CXCR2 depletion on lung injury were relevant to clinical survival, we compared the survival of CXCR2Ϫ/Ϫ mice with CXCR2ϩ/ϩ mice under condi- tions of 80% oxygen exposure. CXCR2ϩ/ϩ mice exposed to 80% oxygen became ill and reached 100% mortality within 8 days. In contrast, CXCR2Ϫ/Ϫ mice exposed to 80% oxygen remained healthy and mortality free out to day 21 (Fig. 9; p Ͻ 0.0001).

Discussion Acute lung injury is characterized by an initial microvascular leak with a neutrophil-predominant inflammatory response that pro- FIGURE 7. Hyperoxia-induced neutrophil infiltration is markedly attenu- motes diffuse alveolar damage (1, 27). High concentrations of ox- ated in CXCR2Ϫ/Ϫ mice as compared with CXCR2ϩ/ϩ mice. Neutrophil se- ygen may perpetuate lung injury and result in persistent inflam- questration was determined by MPO levels of mice exposed to either room air .(p Ͻ 0.016). mation indistinguishable from that seen in ARDS (28Ð30 ,ء ;or 80% hyperoxia for 6 days (n ϭ 10 mice per group Hyperoxia-induced lung injury is characterized by noncardiogenic Downloaded from pulmonary edema, alveolar hyaline membrane formation, type I alveolar epithelial cell injury, type II alveolar epithelial cell hy- gestion, alveolar hemorrhage, leukocyte infiltration, and alveolar perplasia, neutrophil infiltration, alveolar hemorrhage, and in- wall thickness, as compared with the lungs of CXCR2ϩ/ϩ mice creased alveolar wall thickness (31, 32). As the lung injury exposed to 80% oxygen (Fig. 8II). progresses, the interstitium becomes infiltrated with leukocytes, http://www.jimmunol.org/

FIGURE 8. Hyperoxia-induced lung injury, vascular permeability, pulmonary edema, and total by guest on September 24, 2021 collagen deposition were markedly reduced in CXCR2Ϫ/Ϫ mice as compared with CXCR2ϩ/ϩ mice. IA, Lung injury scores from CXCR2Ϫ/Ϫ and CXCR2ϩ/ϩ mice exposed to either room air or hy- peroxia for 6 days. A cumulative score was based on scoring of leukocyte infiltration, exudative edema, hemorrhage, and alveolar wall thickness (n ϭ 15; 3 random sections per lung and 5 lungs -p Ͻ 0.016). IB, Lung Evans blue vas ,ء ;per group cular permeability index from CXCR2Ϫ/Ϫ and CXCR2ϩ/ϩ mice exposed to either room air or hy- p Ͻ ,ء ;peroxia for 6 days (n ϭ 10 mice per group 0.016). IC, Lung wet:dry ratio from CXCR2Ϫ/Ϫ and CXCR2ϩ/ϩ mice exposed to either room air or ,ء ;hyperoxia for 6 days (n ϭ 10 mice per group p Ͻ 0.016). ID, Total lung collagen deposition in whole lung homogenates from CXCR2Ϫ/Ϫ and CXCR2ϩ/ϩ mice exposed to either room air or hy- p Ͻ ,ء ;peroxia for 6 days (n ϭ 10 mice per group 0.016). II, Hyperoxia-induced lung injury is atten- uated in CXCR2Ϫ/Ϫ mice. Representative pho- tomicrographs of H&E staining (ϫ200) of the lung. IIA, Lung histology from CXCR2ϩ/ϩ mice exposed to room air for 6 days. IIB, Lung histology from CXCR2ϩ/ϩ mice exposed to hyperoxia (80%) for 6 days. IIC, Lung histology from CXCR2Ϫ/Ϫ mice exposed to hyperoxia (80%) for 6 days. 3866 CXCR2 IS CRITICAL TO HYPEROXIA-INDUCED LUNG INJURY

pulmonary edema, and fibrosis. In our study, we did not find any significant elevations in the plasma levels of CXC chemokines between groups, suggesting that CXCL1 and CXCL2/3 are up- regulated in a local compartmentalized manner in the lung. Having demonstrated that hyperoxia is associated with in- creased neutrophil sequestration, we determined that CXCL1 and CXCL2/3 expression was significantly greater in the lungs of the 80% oxygen-exposed mice than in the lungs of room air controls. These results are similar to, and extend the findings of Deng et al. (50), who found elevated levels of CXCL1 and CXCL2/3 in 95% oxygen-exposed lungs in neonatal rats after 4 days of exposure. Ϫ Ϫ FIGURE 9. CXCR2 / mice have a survival advantage under condi- The levels of CXCL1 and CXCL2/3 in both our study and that of Ͻ ء ϭ tions of hyperoxia (80%) (n 10 mice in each group; , p 0.0001). Deng et al. (50) demonstrate that these CXC chemokines are ex- pressed under conditions of hyperoxia and are associated with the presence of neutrophils in the lung. In contrast to our study, Deng followed by pulmonary vascular remodeling and eventual devel- et al. (50) used a strategy of neutralizing either CXCL1 or opment of fibrosis (33). Hyperoxia-induced lung injury is mediated CXCL2/3. Although this strategy resulted in a marked reduction of through the generation of that may initiate neutrophils in the bronchoalveolar lavage fluid, it did not suppress and control the activation of NF-␬B and its subsequent gene prod- Downloaded from the levels of lung tissue MPO to the same magnitude that we see ucts such as CXC chemokines (34). In this study, we hypothesized Ϫ/Ϫ that the interaction between CXCR2 and ELR-positive CXC che- in the CXCR2 mice under similar conditions. The magnitude Ϫ/Ϫ mokines expressed under conditions of hyperoxia is critical in me- of reduction of MPO in our model system using CXCR2 mice diating neutrophil recruitment, a pivotal process required for hy- under hyperoxic conditions may reflect that we attenuated the ef- peroxia-induced lung injury. fect of not only CXCL1 and CXCL2/3, but all ELR-positive CXC

Previous studies have demonstrated that mice exposed to 100% chemokines that may have been expressed under these conditions http://www.jimmunol.org/ oxygen rapidly become moribund and reach uniform mortality and use the same shared receptor. within 3 days (35, 36). This present study extends these findings by For example, in a study by Auten et al. (51), they only used determining the concentration-dependent effect of hyperoxia-in- neutralizing Abs to CXCL1 in the same rat newborn model of duced lung injury on mortality. Because of our interest in the hyperoxia as Deng et al. (50). In this study, they found a marked pathophysiological mechanisms leading to hyperoxia-induced lung reduction of neutrophils in the bronchoalveolar lavage fluid in re- injury, the time point and oxygen concentration in which the mice sponse to hyperoxia and neutralization of CXCL1 (51). However, had significant lung injury, yet no overwhelming mortality, were the use of the neutralizing anti-CXCL1 Abs did not fully attenuate focused upon. Histopathological analysis demonstrated increased levels of CXCL1 in the bronchoalveolar lavage fluid, nor did this lung injury, consistent with other animal studies of hyperoxia-in- approach fully protect the lung in hyperoxia-exposed animals (51), by guest on September 24, 2021 duced lung injury (37, 38). In addition, we further characterized suggesting that the effect seen was related to the inability to fully the alveolar-capillary membrane injury for pulmonary edema and deplete CXCL1 in their system. In our study, the more profound measurements of collagen deposition. Interestingly, our findings protective effect on parameters of lung injury under conditions of support the work of previous investigators who demonstrated that hyperoxia in the CXCR2Ϫ/Ϫ mice, as compared with CXCR2ϩ/ϩ increased levels of procollagen III in bronchoalveolar lavage fluid mice, is due to the total loss of CXCR2/CXCR2 ligand biology. were a marker of acute lung injury and early mortality (39, 40). NF-␬B is a transcription factor that can modulate the expression Having characterized the histopathological damage caused by of cytokines and chemokines during cellular stress, and has been hyperoxia, we then focused on the underlying mechanisms respon- implicated in multiple inflammatory processes in the lung (52, 53). sible for promoting the inflammation and subsequent lung injury. Phosphorylation of I-␬B␣ and degradation of I-␬B␣ protein have Our findings of a significant increase in neutrophil infiltration in been shown to correlate with NF-␬B activation and transcription of hyperoxia-exposed mice complement and extend the conclusions CXC chemokines (24, 25). Having found that hyperoxia-induced of other studies that found that high concentrations of oxygen led lung injury is mediated through CXCL1- and CXCL2/3-induced to the release of reactive oxygen species that promoted neutrophil neutrophil infiltration, we demonstrated an increase in phosphor- infiltration into the lung (41, 42). Lungs taken from multiple spe- ylation of I-␬B␣ in the mice exposed to 80% oxygen at day 6, as cies after 24 h of hyperoxia, when studied by electron microscopy, compared with mice exposed to room air. Others have found that demonstrate a diffuse infiltration of activated neutrophils (43). In NF-␬B is up-regulated in response to hyperoxia in both in vitro addition, increases in P-selectin and up-regulation in ICAM-1, a ␤ and ex vivo lung preparations (54Ð57). Our study extends these ligand for neutrophil 2 integrins, have been demonstrated early in the course of hyperoxia exposure, lending further evidence for a findings by suggesting that hyperoxia promotes phosphorylation of ␬ ␣ ␬ neutrophil-mediated injury (44Ð47). This exemplifies that hyper- I- B that may be associated with activation of NF- B, resulting oxia by itself can lead to increased neutrophil infiltration. How- in the subsequent expression of CXCL1 and CXCL2/3, which ever, the molecular and cellular mechanisms involved in recruiting leads to increased neutrophil infiltration and subsequent lung these neutrophils remain to be fully elucidated. injury. Elegant in vitro studies have demonstrated that either hyperoxia The expression of CXCR2 mRNA paralleled the production of or oxidant stress can induce IL-8 expression from human alveolar both CXCL1 and CXCL2/3 ligands and neutrophil sequestration macrophages, endothelial cells, and epithelial cells (48, 49). These during hyperoxia-induced lung injury. Other studies of inflamma- findings suggest that high concentrations of oxygen can induce the tory diseases, such as ventilator-induced lung injury and pneumo- expression of CXCR2 ligands by multiple cell types in the lung, nia, have demonstrated the importance of CXCR2 expression and resulting in increased neutrophil infiltration and hyperoxia-induced its role in neutrophil recruitment during the pathogenesis of these lung injury as characterized by increased vascular permeability, diseases (7, 18, 58). Collectively, these studies demonstrate that The Journal of Immunology 3867 augmented levels of CXCR2 ligands are important in the recruit- 10. Kurdowska, A., J. M. Noble, I. S. Grant, C. R. Robertson, C. Haslett, and ment of neutrophils during the pathogenesis of inflammatory dis- S. C. Donnelly. 2002. Anti-interleukin-8 autoantibodies in patients at risk for acute respiratory distress syndrome. Crit. Care Med. 30:2335. eases and suggest that the interaction between CXCR2 ligands and 11. Goodman, E. R., E. Kleinstein, A. M. Fusco, D. P. Quinlan, R. Lavery, CXCR2 may be pivotal in the recruitment of neutrophils to the D. H. Livingston, E. A. Deitch, and C. J. Hauser. 1998. Role of interleukin 8 in the genesis of acute respiratory distress syndrome through an effect on neutrophil lung during hyperoxia-induced lung injury. apoptosis. Arch. Surg. 133:1234. Based on the above findings, we performed proof of principle 12. Kanazawa, M. 1996. 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Jourdain, C. Gibert, C. Elbim, J. Chastre, and M. A. Gougerot-Pocidalo. 1996. Interactions between neutrophils and cytokines mulation was prevented through the use of a nonpeptide CXCR2 in blood and alveolar spaces during ARDS. Am. J. Respir. Crit. Care Med. antagonist in newborn rats under conditions of 95% oxygen. How- 154:594. 16. Park, W. Y., R. B. Goodman, K. P. Steinberg, J. T. Ruzinski, F. Radella II, ever, this study did not demonstrate the impact of this treatment on D. R. Park, J. Pugin, S. J. Skerrett, L. D. Hudson, and T. R. Martin. 2001. parameters of lung injury. Cytokine balance in the lungs of patients with acute respiratory distress syn- Although previous studies have shown that using nonpeptide drome. Am. J. Respir. Crit. Care Med. 164:1896. 17. Keane, M. P., S. C. Donnelly, J. A. Belperio, R. B. Goodman, M. Dy, CXCR2 antagonist or neutralizing CXCL1 or CXCL2/3 Abs can M. D. Burdick, M. C. Fishbein, and R. M. Strieter. 2002. Imbalance in the ex- Downloaded from reduced neutrophil infiltration into the lung under hyperoxic con- pression of CXC chemokines correlates with bronchoalveolar lavage fluid angio- ditions (50, 51, 58), our study is the first to demonstrate that knock- genic activity and procollagen levels in acute respiratory distress syndrome. J. Immunol. 169:6515. out of a single gene, CXCR2, can result in a marked survival 18. Mehrad, B., R. M. Strieter, T. A. Moore, W. C. Tsai, S. A. Lira, and advantage of adult mice in hyperoxia. This finding was supported T. J. Standiford. 1999. CXC chemokine receptor-2 ligands are necessary com- ponents of neutrophil-mediated host defense in invasive pulmonary aspergillosis. by Kaplan-Meier survival analysis and log rank test, and was in J. Immunol. 163:6086. contrast to studies using only a strategy of neutralization of 19. Moore, T. A., M. W. Newstead, R. M. Strieter, B. Mehrad, B. L. 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