Endogenous osteopontin promotes ozone-induced neutrophil ...

Am J Physiol Lung Cell Mol Physiol 305: L118–L129, 2013. First published May 10, 2013; doi:10.1152/ajplung.00080.2013.

Endogenous osteopontin promotes ozone-induced neutrophil recruitment to the lungs and airway hyperresponsiveness to methacholine Ramon X. Barreno,1 Jeremy B. Richards,2 Daniel J. Schneider,3 Kevin R. Cromar,4 Arthur J. Nadas,4 Christopher B. Hernandez,5 Lance M. Hallberg,6 Roger E. Price,7 Syed S. Hashmi,8 Michael R. Blackburn,3 Ikram U. Haque,1 and Richard A. Johnston1,5,8 1

Division of Pediatric Critical Care Medicine, Department of Pediatrics, 3Department of Biochemistry and Molecular Biology, and 8Pediatric Research Center, Department of Pediatrics, The University of Texas Medical School at Houston, Houston, Texas; 7 Comparative Pathology Laboratory, Center for Comparative Medicine, Baylor College of Medicine, Houston, Texas; 5Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Internal Medicine, The University of Texas Medical Branch at Galveston School of Medicine, Galveston, Texas; 6Division of Environmental Toxicology, Department of Preventative Medicine and Community Health, The University of Texas Medical Branch at Galveston School of Medicine, Galveston, Texas; 2Division of Pulmonary, Critical Care, and Sleep Medicine, Department of Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts; and 4Department of Environmental Medicine, New York University School of Medicine, Tuxedo, New York Submitted 26 March 2013; accepted in final form 7 May 2013

Barreno RX, Richards JB, Schneider DJ, Cromar KR, Nadas AJ, Hernandez CB, Hallberg LM, Price RE, Hashmi SS, Blackburn MR, Haque IU, Johnston RA. Endogenous osteopontin promotes ozone-induced neutrophil recruitment to the lungs and airway hyperresponsiveness to methacholine. Am J Physiol Lung Cell Mol Physiol 305: L118 –L129, 2013. First published May 10, 2013; doi:10.1152/ajplung.00080.2013.—Inhalation of ozone (O3), a common environmental pollutant, causes pulmonary injury, pulmonary inflammation, and airway hyperresponsiveness (AHR) in healthy individuals and exacerbates many of these same sequelae in individuals with preexisting lung disease. However, the mechanisms underlying these phenomena are poorly understood. Consequently, we sought to determine the contribution of osteopontin (OPN), a hormone and a pleiotropic cytokine, to the development of O3-induced pulmonary injury, pulmonary inflammation, and AHR. To that end, we examined indices of these aforementioned sequelae in mice genetically deficient in OPN and in wild-type, C57BL/6 mice 24 h following the cessation of an acute (3 h) exposure to filtered room air (air) or O3 (2 parts/million). In wild-type mice, O3 exposure increased bronchoalveolar lavage fluid (BALF) OPN, whereas immunohistochemical analysis demonstrated that there were no differences in the number of OPN-positive alveolar macrophages between air- and O3-exposed wild-type mice. O3 exposure also increased BALF epithelial cells, protein, and neutrophils in wild-type and OPN-deficient mice compared with genotype-matched, air-exposed controls. However, following O3 exposure, BALF neutrophils were significantly reduced in OPN-deficient compared with wild-type mice. When airway responsiveness to inhaled acetyl-␤-methylcholine chloride (methacholine) was assessed using the forced oscillation technique, O3 exposure caused hyperresponsiveness to methacholine in the airways and lung parenchyma of wild-type mice, but not OPN-deficient mice. These results demonstrate that OPN is increased in the air spaces following acute exposure to O3 and functionally contributes to the development of O3-induced pulmonary inflammation and airway and lung parenchymal hyperresponsiveness to methacholine. bronchoalveolar lavage fluid; forced oscillation technique; inflammation; interleukin-6; macrophage OZONE (O3) IS A HIGHLY REACTIVE, oxidant gas and the major component of photochemical smog (28, 43). At present, nearly

Address for reprint requests and other correspondence: R. A. Johnston, Rm. 3.125, Medical School Bldg., Division of Pediatric Critical Care Medicine, Dept. of Pediatrics, The Univ. of Texas Medical School at Houston, 6431 Fannin St., Houston, TX 77030-1501 (e-mail: [email protected]). L118

120 million residents of the United States live in areas with ambient O3 concentrations that exceed the U.S. Environmental Protection Agency National Ambient Air Quality Standards (51). Healthy individuals who inhale O3 exhibit pulmonary injury, pulmonary inflammation, cough and substernal soreness, decrements in pulmonary function, and airway hyperresponsiveness (AHR) (3, 5, 15, 23). O3 also exacerbates respiratory symptoms in individuals with preexisting lung disease, including asthma and chronic obstructive pulmonary disease (38a, 68). Furthermore, increases in ambient O3 concentrations are associated with short-term mortality (7). As air pollution is a persistent problem in the U.S., and as a large number of U.S. residents live in areas with unhealthy concentrations of O3 in the ambient air (2, 51), exposure to O3 and its associated detrimental health effects are significant public health concerns. Consequently, it is important to understand the mechanistic basis by which the respiratory system responds to O3. O3 indirectly injures resident airway or lung cells through the action of lipid peroxides, reactive oxygen species (ROS), and oxidized proteins, which are generated when O3 reacts with polyunsaturated fatty acids and proteins in the respiratory tract lining fluid (36, 43, 44). O3-induced pulmonary injury is characterized, in part, by necrosis or apoptosis of cells within the lung (11, 38, 58), increased permeability of the alveolarcapillary membrane (1), and damage to deoxyribonucleic acids within the nucleus or mitochondria of lung cells (9, 14). Injurious ozonation products can also initiate signaling cascades, leading to pulmonary inflammation, which manifests as the increased expression of proinflammatory cytokines [interleukin (IL)-1␣, IL-1␤, IL-6, IL-17, osteopontin (OPN), and tumor necrosis factor (TNF)-␣] and chemokines [eotaxin, IL-8, interferon-␥-inducible protein (IP)-10, keratinocyte chemoattractant (KC), macrophage inflammatory protein (MIP)-2, and monocyte chemoattractant protein (MCP)-1] in the lungs of humans and/or mice (3, 32–34, 37, 54, 56, 75a). The inflammatory response to O3 is also characterized by the migration of neutrophils to the lungs (3, 32, 33, 54). AHR to nonspecific bronchoconstrictors is observed in humans and laboratory animals following exposure to O3 (23, 33, 47, 54, 56). Data from numerous investigators, including ourselves, suggest that O3-induced AHR is mechanistically linked

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OSTEOPONTIN AND AIRWAY AND LUNG PARENCHYMAL RESPONSES TO O3

to several of the proinflammatory cytokines or chemokines induced by O3 exposure. For example, impairment of IL-1, KC, MIP-2, or TNF-␣ signaling attenuates the development of O3-induced AHR (13, 33, 54, 67). However, the precise mechanisms underlying O3-induced AHR are not well understood. Of the cytokines whose levels of expression in the lung are elevated following exposure to O3, there is reason to believe that OPN may be an important contributor to the development of O3-induced pulmonary injury, pulmonary inflammation, and AHR. OPN is a phosphorylated, acidic glycoprotein, which functions as a hormone and a pleiotropic cytokine (17, 18), and is constitutively expressed in a variety of tissues, including the kidney, pancreas, liver, testes, ovaries, skin, and lungs (10). Specifically, OPN is expressed by endothelial cells, smooth muscle cells, T cells, osteoblasts, and macrophages (49, 60, 71). O3, when inhaled into the lungs, can generate ROS (36), which can induce the expression of OPN (62, 80, 81). Indeed, Kim and colleagues (37) previously reported that exposure to O3 can increase the expression of OPN in the lungs of mice. OPN can initiate several proinflammatory events, which include, but are not limited to, the activation of NF-␬B and the recruitment of macrophages, neutrophils, and T cells to sites of inflammation (4, 35, 63, 64, 83). NF-␬B, macrophages, neutrophils, and T cells contribute to the development of O3induced pulmonary injury, pulmonary inflammation, and/or AHR (12, 22, 50, 55, 56). Furthermore, these aforementioned sequelae are reduced in mice genetically deficient in OPN following allergen sensitization and challenge and/or exposure to asbestos or cigarette smoke (61, 65, 69). Finally, mice genetically deficient in CD44, one of several receptors for OPN (17), have reduced numbers of bronchoalveolar lavage (BAL) fluid (BALF) macrophages and decreased airway responsiveness following exposure to O3 (24). Taken together, these data suggest that OPN may contribute to O3-induced pulmonary pathology. Accordingly, the purpose of this study was to test the hypothesis that OPN mediates pulmonary pathology induced by acute exposure to O3. To that end, we assessed pulmonary injury, pulmonary inflammation, and airway responsiveness to inhaled acetyl-␤-methylcholine chloride (methacholine) in mice genetically deficient in OPN and wild-type, C57BL/6 mice 6 and/or 24 h following the cessation of an acute (3 h) O3 [2 parts/million (ppm)] exposure. Pulmonary injury and pulmonary inflammation were assessed via biochemical and histopathological analyses of tissue extracts, whereas airway and lung parenchymal responsiveness to methacholine was assessed using the forced oscillation technique. MATERIALS AND METHODS

Animals. Female mice genetically deficient in the gene encoding OPN [OPN-deficient mice; (40)] were purchased from The Jackson Laboratory (Bar Harbor, ME) at 4 – 8 wk of age or generated in a multi-species, modified barrier animal care facility at The University of Texas Medical School at Houston (Houston, TX) from the descendants of breeding pairs of OPN-deficient mice, which were also purchased from The Jackson Laboratory. Because the OPN-deficient mice were backcrossed onto a C57BL/6J genetic background for at least 10 generations, age-matched, female C57BL/6J mice were purchased from The Jackson Laboratory and used as controls. All mice were housed in individually ventilated microisolator cages (Tecniplast; Buguggiate, Varese, Italy), containing no more than five ani-

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mals per cage, within a multi-species, modified barrier animal care facility, where they were given irradiated food and autoclaved water ad libitum, exposed to a 12-h:12-h light/dark cycle, and acclimated to their new environment for at least 1 wk before entering the experimental protocol when at least 8 wk of age. OPN-deficient mice weighed significantly more than wild-type, C57BL/6 mice (21.4 ⫾ 0.4 and 20.2 ⫾ 0.2 g, respectively). All of the experimental protocols were approved by The University of Texas Medical Branch at Galveston Institutional Animal Care and Use Committee (Galveston, TX) or The University of Texas Health Science Center at Houston Animal Welfare Committee (Houston, TX). Protocol. To perform the subsequently described experiments, three separate cohorts of mice were used. In the first cohort, each animal was euthanized 6 or 24 h following the cessation of exposure to either filtered room air (air) or O3 (2 ppm) for 3 h. Afterward, blood was collected from each animal, and then a BAL was performed. In the second cohort, the mice were euthanized and the lungs were fixed in situ either 6 or 24 h following the cessation of a 3-h exposure to either air or O3 (2 ppm). In the third cohort, airway responsiveness to inhaled methacholine was assessed in the mice 24 h following the cessation of a 3-h exposure to either air or O3 (2 ppm). O3 exposure. The mice from the first and second cohorts were exposed to air or O3 (2 ppm) within the Environmental Exposure Facility at The University of Texas Medical Branch at Galveston School of Medicine. Conscious mice were placed into individual wire mesh cages, which were subsequently placed inside an 850-l stainless steel and glass Hinnars-type exposure chamber (Young and Bertke Air Systems, Cincinnati, OH). The temperature inside the chamber was maintained at 23°C, whereas the humidity within the chamber was 40 –50%. Air within the chamber was renewed at a rate of 15 changes per hour. O3 was generated by passing 100% oxygen (National Gases, South Houston, TX) through a Sander Certizon ozoniser (Erwin Sander Elektroapparatebau, Osterberg, Germany). Air containing O3 was subsequently delivered to the chamber and mixed with air that was run through a high-efficiency particulate air filter. The O3 concentration within the chamber was continuously monitored by an O3 analyzer (Model 49C Ozone Analyzer; Thermo Electron, Waltham, MA), which determined the O3 concentration via ultraviolet photometry. Before each exposure, the baseline of the O3 analyzer (0 ppm O3) was calibrated using air from the exposure chamber with no O3 present. For exposure to air, the experimental protocol was identical to the O3 exposure, except that a separate and identical exposure chamber dedicated to air exposure was used. The third cohort of mice was exposed to air or O3 (2 ppm) at The University of Texas Medical School at Houston. Again, conscious mice were placed into individual wire mesh cages, which were then placed inside a 75.5-l powder-coated aluminum and Plexiglas exposure chamber (Teague Enterprises, Woodland, CA). The average temperature and humidity inside of the chamber was 22.3 ⫾ 0.1°C and 45 ⫾ 1%, respectively. Air within the chamber was renewed at a rate of 9-14 changes per hour. O3 was generated by passing ultra-pure air (Matheson Tri-Gas, Houston, TX) through a Sander Certizon 25 ozoniser that was subsequently mixed with activated charcoal-filtered room air in the chamber. Air containing O3 was delivered to the chamber at a rate of 2.95 l/min via a stainless steel thermal O3 mass flow controller (Model GFC; Aalborg, Orangeburg, NY). The O3 concentration within the chamber was continuously monitored by an O3 analyzer (Model IN-2000-L2-LC; IN USA, Norwood, MA), which determined the O3 concentration via ultraviolet photometry. For exposure to air, the experimental protocol was identical to the O3 exposure, except that the ozoniser was not powered on during the air exposure. Isolation of serum. Six and twenty-four hours following the cessation of air or O3 exposure, mice were euthanized with an intraperitoneal (i.p.) injection of pentobarbital sodium (Vortech Pharmaceuticals, Dearborn, MI) at a dose of no less than 200 mg/kg. Once the animal was unresponsive to any stimuli, the animal’s chest was opened and blood was collected from the heart via cardiac puncture

AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00080.2013 • www.ajplung.org

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with a 25-gauge needle attached to a 1-ml syringe. Next, the blood was allowed to clot at room temperature in Microtainer blood collection tubes (Becton Dickinson, Franklin Lakes, NJ) for at least 30 min, and then it was spun at 15,000 g for 2 min at 4°C to isolate serum, which was subsequently stored at ⫺20°C until needed. BAL. After blood was collected from the heart via cardiac puncture, the animal was prepared for a BAL. First, the trachea was exposed in situ, and a small incision was made in the trachea directly distal to the larynx with micro scissors. Next, a 20-gauge fluorinated ethylene propylene polymer catheter (Becton Dickinson), which was attached to a 1-ml syringe, was inserted into the trachea. Subsequently, the lungs were lavaged twice with 1 ml of ice-cold lavage buffer [phosphate-buffered saline (PBS) containing 0.6 mM of EDTA (Mallinckrodt Baker, Phillipsburg, NJ)]. During each lavage, the lavage buffer was instilled and retrieved twice; afterward, both lavagates were pooled and stored on ice. Once all of the animals were lavaged, the lavagates were centrifuged at 2,000 revolutions/min for 10 min at 4°C, the BALF supernatants were collected, and stored at ⫺80°C until further use, and the BALF cell pellets were resuspended in 1 ml of Hanks’ Balanced Salt Solution (HyClone Laboratories, Logan, UT). Next, the total number of BALF cells was enumerated using a hemacytometer. Finally, to perform a differential count of the BALF cells, ⬃25,000 cells from each mouse were spun at 800 revolutions/ min for 10 min at room temperature onto glass microscope slides using a Shandon Cytospin 4 Cytocentrifuge (Thermo Electron) and then stained with the Hema 3 stain set (Fisher Diagnostics, Middletown, VA). At least 300 cells per mouse were counted under brightfield microscopy for differential cell analysis. Protein, enzyme-linked immunosorbent, and soluble collagen assays. The concentration of protein in the BALF was determined spectrophotometrically according to the Bradford protein assay (Bio-Rad Laboratories, Hercules, CA). The concentrations of IL-6, IL-17, IP-10, KC, MIP-2, and OPN in the BALF and/or serum were determined with enzyme-linked immunosorbent assays (R&D Systems, Minneapolis, MN), whereas the concentration of soluble collagen in the BALF was quantified using a Sircol soluble collagen assay (Biocolor Life Science Assays, Carrickfergus, Northern Ireland). All assays were performed using the manufacturer’s instructions. Lung histology and immunohistochemistry. Six or twenty-four hours following the cessation of air or O3 exposure, mice were euthanized via an i.p. injection of pentobarbital sodium. Next, the chest of each animal was opened, the heart and the circulation were perfused with ice-cold PBS, the trachea was cut and cannulated with a catheter, the lungs were fixed in situ with 10% buffered formalin phosphate (Fisher Scientific, Fair Lawn, NJ), and were removed en bloc. The lungs were fixed for at least 24 h in 10% buffered formalin phosphate at 4°C, dehydrated, cleared, infiltrated, and then embedded in paraffin. Coronal sections, 4 ␮m thick and encompassing the right and left lung lobes, were cut with a microtome from the paraffinembedded lungs. Separate sections were subsequently used for the immunohistochemical detection of OPN or the visualization of collagen in Masson’s trichrome-stained sections. To determine which cell or cells in the airways and lungs of wild-type, C57BL/6 mice express OPN via immunohistochemistry following air or O3 exposure, the sections underwent antigen retrieval (Dako Denmark, Glostrup, Denmark) following deparaffinization and rehydration, respectively. Next, endogenous peroxidase activity in the sections was quenched by submerging the sections in a solution of 1% hydrogen peroxide. The slides were then blocked with goat serum (Vector Laboratories, Burlingame, CA) and then avidin and biotin (Vector Laboratories) to prevent nonspecific binding and high-background staining, respectively. The sections were next incubated overnight at 4°C with an anti-mouse OPN antibody (1 ␮g/ml; R&D Systems). The following day, the sections were washed and incubated with a biotinylated secondary antibody (goat IgG; Vector Laboratories) for 1 h, an avidin DH:biotinylated enzyme complex (Vector Laboratories) for 30 min, and 3,3=-diaminobenzidine tetrahydrochloride (Sigma-

Aldrich, St. Louis, MO) until the sections began to appear brown. After incubation, the sections were washed with either PBS or deionized water. Finally, the sections were counterstained with methyl green (Fisher Scientific) for no more than 30 s, washed in deionized water, dehydrated, mounted, and then examined via bright-field microscopy. Slides were then evaluated and scored on a blinded basis for the number of OPN-positive alveolar macrophages that were present within a 10-mm2 ocular reticle field using a BX50 upright, light microscope (Olympus, Tokyo, Japan) and a ⫻20 objective lens. OPN-positive alveolar macrophages were counted if they were either completely within the boundaries or touched the upper or left boundaries of the 10-mm2 ocular reticle field. Six randomly chosen and properly inflated 10-mm2 areas that did not include primary or secondary bronchi were used for scoring purposes. The number of OPN-positive macrophages counted within each of the six randomly chosen 10-mm2 ocular reticle fields was averaged to calculate a mean value for each mouse. Airway and lung parenchymal responsiveness to methacholine. Twenty-four hours following the cessation of exposure to air or O3, the mice were anesthetized with pentobarbital sodium (50 mg/kg i.p.; Hospira, Lake Forest, IL) and xylazine hydrochloride (7 mg/kg i.p.; Akorn, Decatur, IL). Once the mice were anesthetized, a tracheostomy was performed on each animal, and an 18-gauge tubing adapter (Becton Dickinson) was inserted into the trachea. With intact chest walls, the mice were subsequently ventilated with a tidal volume of 0.3 ml at a frequency of 150 breaths per min (2.5 Hz) using a specialized ventilator (flexiVent; SCIREQ Scientific Respiratory Equipment, Montréal, Québec, Canada) that is equipped with a computer-controlled piston pump to deliver tidal volumes to the animals in a sinusoidal flow pattern. To prevent atelectasis at the end of expiration, a positive end-expiratory pressure of 3 cm H2O was applied to the lungs by placing the expiratory line under water. Total respiratory system impedance (Zrs) was determined by the forced oscillation technique as previously described (25, 29, 57). To measure Zrs, ventilation was perturbed for 3 s, while 13 sinusoidal forcing functions, which ranged in frequency from 1 to 20.5 Hz, were simultaneously delivered to the animal. The flexiVent was used to both ventilate the lungs and deliver the sinusoidal forcing functions. Furthermore, the impedance of the tubing adapter, which was used to intubate the mice, was removed when the system was calibrated (29). Thus our data only represent changes in the mechanical properties of the respiratory system. The constant-phase model, as described by Hantos and colleagues (25), was used to partition Zrs into components representing airway resistance (Raw), the coefficient of lung tissue damping (G), the coefficient of lung tissue elastance (H), and lung tissue hysteresivity (␩ ⫽ G/H), according to the following equation Zrs ⫽ Raw ⫹ (G ⫺ jH)/(2␲f)␣, where j is the imaginary unit (√-1), f is the frequency, and ␣ ⫽ (2/␲)tan⫺1(1/␩). Measurements of the real and imaginary components of Zrs, which were obtained from air-exposed, wild-type C57BL/6 mice that were part of the third cohort, conformed to the constant phase model (data not shown). Although we measured indices of airway (Raw) and lung parenchymal (G and H) oscillation mechanics in animals with the chest wall intact, Sly et al. (70) previously reported that changes in the mechanical properties of the lungs, and not the chest wall, are the major determinant of Raw, G, and H. To measure airway responsiveness to methacholine in our mice, the following protocol was executed. First, ventilation was paused for 6 s, and a pressure of 30 cm H2O was applied to the system to inflate the lungs to capacity to standardize lung volume history. Afterward, ventilation was allowed to resume for at least 6 s, and then a 2.5-Hz sinusoidal forcing function was applied, while ventilation was perturbed for 1.25 s, to measure respiratory system resistance (Rrs), which was derived by multiple linear regression after measurements of pressure and flow within the system were fit to the equation of the single-compartment, linear model of respiratory mechanics (6). We measured Rrs at least five more times using the aforementioned procedures to ensure that a stable baseline value of Rrs was achieved.



Once baseline conditions were established, 100 ␮l of PBS (0 mg/ml of methacholine) were aerosolized with an Aeroneb Lab Micropump Nebulizer (Aerogen Limited, Galway, Ireland) and delivered to the lungs of the animal through the inspiratory line of the flexiVent and the tracheal cannula. The aerosol was delivered for 10 s, and, afterward, a sinusoidal forcing function, as described above, perturbed ventilation for 1.25 s to measure Rrs. Subsequently, Zrs was determined by simultaneously delivering 13 sinusoidal forcing functions, which ranged in frequency from 1 to 20.5 Hz, to the animal. The measurement of Rrs and Zrs was repeated 14 more times for a total of 15 measurements. Once all measurements were complete, the perturbations ceased and ventilation resumed uninterrupted. Next, baseline conditions were reestablished in the lungs such that we could assess airway responsiveness to inhaled methacholine. To reestablish baseline conditions, we standardized lung volume history by inflating the lungs to capacity by applying a pressure of 30 cm H2O to the system. Afterward, we measured Rrs. This process was repeated at least two more times to ensure reproducible, baseline Rrs values. Next, methacholine (Sigma-Aldrich), dissolved in PBS, was delivered to the animal in the same manner as described for PBS and administered in approximate half-logarithm intervals between 0.1 and 100 mg/ml. After the delivery of each dose of methacholine, Rrs and Zrs were measured as previously described. Between the administration of each dose of methacholine, baseline conditions were reestablished. Out of the 15 measurements made for each outcome indicator, the five highest responses were averaged to determine Raw, G, and H for each dose. Baseline Raw, G, and H values refer to those measurements made following the administration of PBS. The flexiVent software calculates a coefficient of determination (COD) to establish the goodness of fit of the constant-phase model to our data. Any measurement with a COD less than 0.9 was excluded from the study. Statistical analysis of data. The effect of genotype and exposure on BALF and serum parameters and baseline airway and lung parenchymal oscillation mechanics were assessed by a two-way ANOVA for normally distributed data or by a Kruskal-Wallis one-way ANOVA for nonnormally distributed data. In all of these analyses, genotype (wild-type and OPN-deficient) and exposure (air and O3) were the main effects. Depending on whether the data were normally or nonnormally distributed, the Fisher’s least significant difference test or the Wilcoxon signed-rank test, respectively, were used as a follow-up to determine the significance of differences between the different groups. Body masses, the mean number of OPN-positive macrophages from each mouse, and BALF collagen levels were

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compared using an unpaired Student’s t-test or a Wilcoxon signedrank test as appropriate. Statistical analysis of the repeated measures comprising the methacholine dose-response curves was completed using the area under the curve (AUC) analysis with respect to increased response compared with the response following PBS administration. AUC analysis was performed using R (Version 2.15.3) (59), whereas Stata 12 was used for all other statistical analyses (StataCorp LP, College Station, TX). The results are expressed as the means ⫾ SE. A P value ⬍0.05 was considered significant. RESULTS

Effect of O3 exposure on the levels of OPN in the BALF and serum. OPN was detectable in the BALF of wild-type mice exposed to air (Fig. 1A). Six hours following the cessation of O3 exposure, the levels of OPN in the BALF of wild-type mice were not different from age- and genotype-matched, wild-type mice exposed to air. Twenty-four hours following the cessation of O3 exposure, the levels of OPN in the BALF of wild-type mice were threefold greater than those of age-matched, wildtype mice exposed to air. OPN was not detectable in the BALF of air- or O3-exposed, OPN-deficient mice at any time point examined (Fig. 1A). To determine whether O3 exposure altered circulating levels of OPN, we determined the concentration of OPN in the serum of wild-type mice exposed to air or O3 (Fig. 1B). OPN was detectable in the serum of wild-type mice exposed to air or O3. However, there was no effect of O3 on the levels of OPN in the serum at either 6 or 24 h following the cessation of exposure. Furthermore, OPN was undetectable in the serum of air- or O3-exposed, OPN-deficient mice. Because our data demonstrate that the levels of OPN in the BALF were significantly increased by O3 24 h following the cessation of exposure, we subsequently examined all of our outcome indicators at this time point. Immunohistochemical localization of OPN in the lungs of wild-type mice exposed to O3. As demonstrated in Fig. 1A, OPN is significantly increased in the BALF of wild-type mice 24 h following the cessation of a 3-h exposure to O3 (2 ppm) compared with wild-type mice exposed to air. To determine the cell or cell types in the lung that express OPN following air and

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Fig. 1. The concentrations of osteopontin (OPN) in the bronchoalveolar lavage fluid (BALF) (A) and serum (B) of wild-type, C57BL/6 mice and mice genetically deficient in OPN 6 or 24 h following the cessation of a 3-h exposure to filtered room air (air) or 2 parts/million (ppm) ozone (O3). Each value is expressed as the mean ⫾ SE. n ⫽ 6 –10 mice for each group. *P ⬍ 0.05 compared with genotype-matched mice exposed to air. #P ⬍ 0.05 compared with OPN-deficient mice with an identical exposure.

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O3 exposure, we used immunohistochemistry to investigate the expression of OPN in lung sections obtained from air- or O3-exposed, wild-type mice. (Fig. 2). Alveolar macrophages from wild-type mice were strongly positive for OPN following air or O3 exposure (Fig. 2, A and B). However, there were no differences in the number of OPN-positive alveolar macrophages between air- or O3-exposed, wild-type mice (Fig. 2C). Effect of OPN deficiency on O3-induced pulmonary injury and pulmonary inflammation. Thus far, our experiments demonstrate that OPN is increased in the BALF and is expressed by alveolar macrophages following O3 exposure (Figs. 1A and 2B). To determine whether O3-induced increases in BALF OPN are functionally linked to pulmonary injury or pulmonary inflammation induced by exposure to O3, we examined indices of these sequelae in wild-type and OPN-deficient mice 24 h after air or O3 exposure ceased. Hyperpermeability of the alveolar-capillary membrane, which is indicated by an increase in BALF protein (1), and epithelial cell sloughing are two features of O3-induced pulmonary injury (8, 11). Following exposure to air, there were no genotype-related differences in the number of BALF epithelial cells (Fig. 3A). O3 exposure significantly increased the number of BALF epithelial cells in both wild-type and OPN-deficient mice. However, there were no genotype-related differences in the number of BALF epithelial cells following the cessation of O3 exposure. Similar observations were made for BALF protein (Fig. 3B).

C Number of OPN-Positive Macrophages per 10 mm2 Ocular Reticule

Fig. 2. Representative sections demonstrating the immunolocalization of OPN in alveolar macrophages (arrows) within the lungs of wildtype, C57BL/6 mice 24 h following the cessation of a 3-h exposure to filtered room air (air) (A) or 2 ppm ozone (O3) (B) via a ⫻20 objective lens. C: number of OPN-positive alveolar macrophages in a 10-mm2 ocular reticle field of lung sections from wild-type, C57BL/6 mice 24 h following the cessation of a 3-h exposure to air or O3 (2 ppm). In A and B, the scale bars represent 100 ␮m. In C, each value is expressed as the mean ⫾ SE. n ⫽ 6 –7 mice for each group.

We assessed O3-induced pulmonary inflammation by enumerating the number of BALF macrophages and neutrophils and quantifying the concentrations of inflammatory cytokines and chemokines in the BALF. Twenty-four hours following the cessation of exposure to air, OPN-deficient mice had significantly fewer BALF macrophages compared with wild-type mice (Fig. 4A), whereas no genotype-related differences existed in the number of BALF neutrophils (Fig. 4B). There was no effect of O3 on the number of BALF macrophages in wild-type or OPN-deficient mice (Fig. 4A). As expected, exposure to O3 increased the number of neutrophils in the BALF of wild-type and OPN-deficient mice (Fig. 4B). However, the number of neutrophils in the BALF of OPN-deficient mice was significantly reduced compared with wild-type mice. Eosinophils and lymphocytes were rarely observed in any genotype or following any exposure (data not shown). To further assess the effect of OPN deficiency on the development of O3-induced pulmonary inflammation, we measured the levels of IL-6, IL-17, IP-10, KC, and MIP-2 in the BALF of wild-type and OPN-deficient mice 24 h following the cessation of air or O3 exposure. We and others have previously reported that IL-6, IL-17, IP-10, MIP-2, and KC are required for maximal neutrophil recruitment to the lungs following O3 exposure (33, 34, 41, 56). No genotype-related differences existed in BALF IL-6 following air exposure (Fig. 4C). O3 exposure significantly increased BALF IL-6 in wild-type but

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not OPN-deficient mice. However, no genotype-related differences existed in BALF IL-6 following O3 exposure. IL-17, IP-10, MIP-2, and KC were below the detection limit of our assay, regardless of genotype or exposure (data not shown). Effect of OPN deficiency and O3 exposure on airway and lung parenchymal responsiveness to methacholine. In Table 1, we report baseline values of airway (Raw) and lung parenchymal (G and H) oscillation mechanics in wild-type and OPNdeficient mice exposed to air or O3, which were determined following the administration of PBS as described in MATERIALS

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AND METHODS. Twenty-four hours following the cessation of air exposure, there were no genotype-related differences in Raw, and, although indices of parenchymal oscillation mechanics (G and H) were lower in OPN-deficient compared with wild-type mice at this time, statistical significance was only achieved for H. In wild-type mice, O3 exposure significantly increased G but did not affect Raw or H, whereas, in OPN-deficient mice, Raw, G, and H were unaffected by O3 exposure. In air-exposed, wild-type mice, methacholine caused significant increases in Raw, G, and H (Fig. 5). However, in OPN-

#

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6

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O3

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Fig. 3. Total number of epithelial cells (A) and the concentration of protein (B) in the BALF of wild-type, C57BL/6 mice and mice genetically deficient in OPN 24 h following the cessation of a 3-h exposure to filtered room air (air) or 2 ppm O3. Each value is expressed as the mean ⫾ SE. n ⫽ 9 –10 mice for each group. *P ⬍ 0.05 compared with genotype-matched mice exposed to air.

Fig. 4. The total number of macrophages (A) and neutrophils (B) as well as the concentration of interleukin (IL)-6 (C) in the BALF of wild-type, C57BL/6 mice, and mice genetically deficient in OPN 24 h following the cessation of a 3-h exposure to filtered room air (air) or 2 ppm O3. Each value is expressed as the mean ⫾ SE. n ⫽ 9 –10 mice for each group. *P ⬍ 0.05 compared with genotypematched mice exposed to air. #P ⬍ 0.05 compared to OPN-deficient mice with an identical exposure.

Wild-type OPN-Deficient

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0

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Table 1. Baseline airway and lung parenchymal oscillation mechanics of wild-type and OPN-deficient mice exposed to filtered room air or ozone Airway and Lung Parenchymal Oscillation Mechanics Genotype (Exposure)

Raw, cm H2O/ml per s

G, cm H2O/ml

H, cm H2O/ml

Wild-type (Air) OPN-Deficient (Air) Wild-type (O3) OPN-Deficient (O3)

0.33 ⫾ 0.01 0.32 ⫾ 0.01 0.33 ⫾ 0.01 0.30 ⫾ 0.01

4.11 ⫾ 0.14 3.73 ⫾ 0.13 4.59 ⫾ 0.17*† 3.79 ⫾ 0.10

25.76 ⫾ 1.05† 21.42 ⫾ 1.12 25.27 ⫾ 0.87† 20.58 ⫾ 0.86

The results are expressed as the means ⫾ SE for 7–8 mice in each group. The measurements were made in each animal 24 h following the cessation of a 3-h exposure to filtered room air (air) or ozone (O3; 2 parts/million). Raw, airway resistance; G, coefficient of lung tissue damping; H, coefficient of lung tissue elastance; OPN, osteopontin. *P ⬍ 0.05 compared with genotype-matched mice exposed to air. †P ⬍ 0.05 compared with OPN-deficient mice with an identical exposure.

deficient mice exposed to air, only Raw was significantly increased by methacholine. In air-exposed mice, responses to methacholine for Raw were significantly less in OPN-deficient compared with wild-type mice at the two highest doses of

methacholine (Fig. 5A). In air-exposed, OPN-deficient mice, responses to methacholine for G and H were significantly less than air-exposed, wild-type mice at several doses of methacholine (Fig. 5, B and C). Methacholine induced significant increases in Raw, G, and H in wild-type and OPN-deficient mice following O3 exposure (Fig. 5). In wild-type mice, O3 exposure significantly increased responses to methacholine for Raw, G, and H compared with genotype-matched, air-exposed controls, whereas O3 exposure only increased responses to methacholine for Raw at the highest dose of methacholine in OPN-deficient mice. Furthermore, responses to methacholine for Raw, G, and H were significantly less in OPN-deficient compared with wild-type mice at numerous doses of methacholine following O3 exposure. Effect of OPN deficiency on collagen matrix organization in the lung. Myers et al. (45) previously reported that, in the absence of any inciting stimulus, arterial compliance was greater in OPNdeficient compared with wild-type mice due to the existence of a disorganized collagen matrix underlying the arteries of OPNdeficient mice. If a similar collagen matrix surrounded the airways

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Fig. 5. Responses to aerosolized acetyl-␤-methylcholine chloride (methacholine) for airway resistance (Raw) (A), the coefficient of lung tissue damping (G) (B), and the coefficient of lung tissue elastance (H) (C) in wild-type, C57BL/6, and mice genetically deficient in OPN 24 h following the cessation of a 3-h exposure to filtered room air (Air) or 2 ppm O3. Each value is expressed as the mean ⫾ SE. n ⫽ 7– 8 mice for each group. *P ⬍ 0.05 compared with genotypematched mice exposed to air. #P ⬍ 0.05 compared with OPN-deficient mice with an identical exposure.

A Raw (cm H2O/ml/s)

Wild-type (Air) Wild-type (O3) OPN-Deficient (Air) OPN-Deficient (O3)

Methacholine (mg/ml) AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00080.2013 • www.ajplung.org

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and blood vessels of OPN-deficient mice, this could explain the decrease in H that we observed in these animals at baseline (Table 1). To that end, we examined the organization of the collagen matrix in Masson’s trichrome-stained sections from wild-type and OPN-deficient mice exposed to air. However, we did not observe any genotype-related differences in the organization of the collagen matrix surrounding either the airways or blood vessels within the lung (data not shown). In addition, we did not find any difference between wild-type and OPN-deficient mice with regard to the amount of BALF collagen (45.7 ⫾ 2.5 and 48.4 ⫾ 3.9 ␮g/ml, respectively). DISCUSSION

In the 24-h period following the cessation of an acute exposure to O3, the results of this study demonstrate that OPN expression increases in the air spaces of wild-type mice. In addition, our results reveal a functional role for OPN in the development of pulmonary inflammation and AHR, which are induced by acute exposure to O3. With regard to the specificity of these sequelae, which result from acute exposure to O3, we demonstrate that OPN is essential for the migration of neutrophils to the air spaces and for the hyperresponsiveness of the airways and lung parenchyma to inhaled methacholine. OPN is a highly acidic glycoprotein, which was initially identified in osteosarcoma cells (52). OPN is presently known to be constitutively expressed in numerous organs, tissues, and cells, including the lung (10). Figure 2A demonstrates that alveolar macrophages from wild-type mice exposed to air constitutively express OPN and thus are a probable source of OPN, which is present in the BALF of wild-type mice exposed to air (Fig. 1A). By reacting with constituents of the respiratory tract lining fluid, O3 can generate ROS in the lung (36). Furthermore, ROS can induce the expression of OPN in a number of different cell types (62, 80, 81). Consistent with these reports, we demonstrate that OPN is significantly increased in the BALF of wild-type mice 24 h following the cessation of exposure to O3 (Fig. 1A). Similar to our observations in wild-type mice exposed to air, alveolar macrophages from wild-type mice exposed to O3 were also strongly positive for OPN (Fig. 2B). Consequently, even after the cessation of exposure to O3, alveolar macrophages remain the probable source of OPN recovered in the BALF. Because OPN is a chemotactic factor for macrophages and neutrophils (4, 35, 64, 83), which contribute to the pathological effects induced by O3 in the lungs (16, 33, 55), we performed a differential analysis of the cells, retrieved from the BALF of wild-type and OPN-deficient mice, to assess the ability of OPN to promote macrophage and neutrophil chemotaxis following acute exposure to O3 (Fig. 4). Interestingly, the number of BALF macrophages was significantly reduced in OPN-deficient compared with wild-type mice following air exposure (Fig. 4A), which suggests that OPN mediates the recruitment of macrophages to the air spaces in the absence of any inciting stimulus. We previously observed the same phenomenon in mice genetically deficient in the type I IL-1 receptor (IL-1RI) (32). IL-1 and OPN have the capacity to induce the expression of each other (26, 82). Consequently, the reduced number of macrophages in the air spaces of OPN-deficient mice could be a result of the inability of IL-1 to induce OPN via IL-1RI in

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OPN-deficient mice or, alternatively, the absence of OPNinduced IL-1 expression in OPN-deficient mice. Because 1) O3 exposure did not affect the number of BALF macrophages in either wild-type or OPN-deficient mice (Fig. 4A), 2) there were no genotype-related differences in the number of BALF macrophages following O3 exposure (Fig. 4A), and 3) there was no difference in the number of OPN-positive macrophages in the air spaces of air- and O3-exposed wild-type mice (Fig. 2C), these data suggest that O3 exposure increases the synthesis and/or release of OPN from macrophages already present in the air spaces. In addition to influencing the chemotaxis of macrophages, our results demonstrate that OPN contributes to the majority of neutrophil recruitment to the air spaces in the 24-h period following the cessation of an acute exposure to O3. In fact, the number of neutrophils in the BALF of O3-exposed, OPNdeficient mice was ⬃75% less than those retrieved in the BALF of O3-exposed, wild-type mice (Fig. 4B). These results are not without precedent, as we and others have demonstrated that OPN contributes to neutrophil chemotaxis under various pathological conditions (4, 64, 65). The decreased migration of neutrophils to the air spaces of OPN-deficient mice following O3 exposure is not due to a diminished number of circulating neutrophils in the blood, as we and others have previously reported no difference in the number of circulating neutrophils between OPN-deficient and wild-type mice (45, 64). Furthermore, diminished neutrophil recruitment to the lungs of OPNdeficient mice following O3 exposure is not due to decreased permeability of the alveolar-capillary membrane in OPN-deficient mice because BALF protein, a sensitive indicator of the integrity of the alveolar-capillary membrane (1), was not different between wild-type and OPN-deficient mice following O3 exposure. Interestingly, we previously reported that the majority of neutrophil recruitment to the air spaces in mice acutely exposed to O3 was dependent on CXCR2, the receptor for KC and MIP-2 (33). In our previous study (33), we reported that the number of neutrophils in the air spaces of O3-exposed mice genetically deficient in CXCR2 was decreased by 82% compared with O3-exposed, wild-type controls. We have recently reported that OPN can induce the expression of KC (64). Thus, during O3 exposure, OPN may induce the expression of KC or its receptor, which could explain the strikingly similar effect of OPN and CXCR2 deficiency on neutrophil recruitment following O3 exposure. We did measure the levels of the CXCR2 ligands, KC and MIP-2, in the BALF of our animals 24 h following the cessation of exposure, but they were below the detection limit of our assay (data not shown). Nevertheless, these results demonstrate that OPN significantly contributes, either directly or indirectly, to neutrophil recruitment to the air spaces following acute exposure to O3. Because neutrophil recruitment to the air spaces was not completely abolished in OPN-deficient mice, this suggests that factors other than OPN influence neutrophil chemotaxis in O3-exposed, OPN-deficient mice. Other potential candidates for OPN-independent neutrophil chemotaxis include IL-6, IL17, IP-10, KC, MIP-2, and MCP-3, which we and others have previously reported contribute to neutrophil migration to the air spaces following acute exposure to O3 (33, 34, 41, 56). Our prior work demonstrates that there are very robust increases in BALF IL-6, IP-10, KC, and MIP-2 at 6 but not 24 h following


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the cessation of acute exposure to O3 (2 ppm for 3 h) (32). Heretofore, we reported that IL-6 contributes to O3-induced neutrophil migration to the lungs (34), and, although IL-6 was barely detectable in the BALF 24 h following the cessation of O3 exposure (Fig. 4C), there were no genotype-related differences in BALF IL-6, suggesting that IL-6 is not likely involved in the recruitment of neutrophils to the lungs via OPN. However, this does not exclude the possibility that robust increases in IL-6, IP-10, KC, and MIP-2, which are observed in lungs during the first several hours following the cessation of an acute exposure to O3 (2 ppm for 3 h) (32), contribute to OPN-independent neutrophil migration observed in OPN-deficient mice (Fig. 4B). In the absence of any inciting stimulus, indices of lung parenchymal mechanics (G and H) were reduced in OPNdeficient mice before and following the administration of methacholine (Table 1 and Fig. 5, B and C). Myers et al. (45) previously reported that arterial compliance was greater or, alternatively, arterial elastance was lower in OPN-deficient compared with wild-type mice. This reduction in arterial elastance was attributed to the presence of a disorganized collagen matrix encircling the arteries of OPN-deficient mice (45). Because collagen is a major determinant of lung elastance (21), the alterations in the oscillatory mechanics of the lung parenchyma, which we observed in OPN-deficient mice, could be explained by the presence of a disorganized collagen matrix and/or a reduction in the amount of collagen in the lungs of these animals. However, we did not observe any genotyperelated differences in the organization of the collagen matrix (data not shown) or in the amount of collagen in the BALF. Thus differences in the distribution of collagen in the lung, at least at the level of the light microscope, and/or the amount of collagen in the lung do appear to account for the differences in lung elastance between OPN-deficient and wild-type mice in the absence of any inciting stimulus. OPN deficiency could lead to changes in the distribution and/or expression of elastin and/or surfactant within the lung, which are critical determinants of the mechanical and viscoelastic properties of the lung parenchyma (66, 72, 73). Nevertheless, regardless of the molecular mechanism, our data reveal that OPN is critically important for the normal, oscillatory behavior of the lung parenchyma, a novel finding that has not yet been reported to date. We and others have previously demonstrated that O3-induced AHR is mechanistically linked to several aspects of O3-induced pulmonary inflammation (13, 33, 67). O3-induced pulmonary inflammation is characterized by the increased expression of proinflammatory cytokines and chemokines in the lungs (3, 32, 33, 37, 54). In addition, neutrophils also migrate to the air spaces following O3 exposure (3, 32, 33, 54). Of the above indices, which we measured, only the number of neutrophils was diminished in the BALF of O3-exposed, OPNdeficient compared with O3-exposed, wild-type mice. Neutrophils can synthesize and release moieties, such as leukotrienes and neutrophil elastase, which can elicit bronchoconstriction (19, 46, 74). Thus it is entirely possible that these moieties act directly on the airway smooth muscle or its neural innervation to enhance the effects of methacholine on Raw in wild-type mice following O3 exposure. Furthermore, these moieties could have the same effect on the contractile elements within the lung parenchyma to simultaneously increase G and H in

O3-exposed, wild-type mice. Increases in both G and H in wild-type mice following exposure to O3 could be due to increased closure of the small airways (76, 77), which could result from a loss of surfactant function (31). Indeed, in addition to O3, neutrophil elastase impairs surfactant function (57a, 58a), which could lead to increased airway closure and increase both G and H. Because the number of neutrophils in the air spaces of OPN-deficient mice are significantly decreased (Fig. 4B), such effects on Raw, G, and H would be absent in OPN-deficient mice. However, the role of neutrophils in O3-induced AHR is very controversial with some studies demonstrating that neutrophils promote O3-induced AHR (16, 30, 50), whereas others suggest no role for neutrophils in O3-induced AHR (20, 39, 53, 84). Similar to our observations in OPN-deficient mice, we reported the same decrease in responsiveness to methacholine in mice genetically deficient in CXCR2 (33), suggesting that a common pathway may be involved in these airway responses to O3. However, from the data presented, we cannot determine whether a diminished number of neutrophils prevent the development of O3-induced AHR in OPN-deficient mice or whether these events are simply coincident, but independent. Alternatively, OPN can induce the expression of IL-1 and TNF-␣ (26, 42), which have been previously shown to mediate O3-induced AHR (13, 54, 67). Thus OPN could elicit AHR through the induction of these cytokines following O3 exposure. OPN binds to a variety of endogenous receptors to initiate its biological effects (17, 18), and any number of these receptors could mediate the observed effects of OPN. We have previously demonstrated that two endogenous receptors for OPN, CD44 and the ␣V␤3 integrin, promote neutrophil chemotaxis in response to OPN (64). However, Garantziotis et al. (24) reported that no differences existed in the number of BALF neutrophils between wild-type and CD44-deficient mice 24 h following the cessation of exposure to O3 (2 ppm). Because Garantziotis et al. reported no differences in the number of BALF neutrophils in wild-type compared with CD44-deficient mice using the same O3 exposure regimen we did, these data suggest that CD44 does not mediate the OPN-induced migration of neutrophils to the lungs using this O3 exposure protocol. Thus ␣V␤3 or one of the other receptors for OPN mediates OPN-induced neutrophil chemotaxis to the lungs in response to O3. Interestingly, in this same study, Garantziotis and colleagues did report that O3-induced AHR was significantly reduced in CD44-deficient mice (24), which suggests that OPN may be mediating its effects on O3-induced AHR via CD44. Taken together, these data suggest that OPN mediates O3induced neutrophil recruitment to the lungs and AHR via two distinct receptors. Consequently, more definitive studies are needed to elucidate the signaling pathways by which OPN contributes to these effects following O3 exposure. Finally, the O3 exposure protocol, which we used in this study, has been previously used by other investigators (13, 24, 54, 67). However, the concentration of O3 (2 ppm) that the mice were exposed to in this study exceeds the current U.S. National Ambient Air Quality Standards for O3 of 0.075 ppm (75). Of note, when human subjects participate in controlled experiments to assess the effect of O3 on pulmonary function, these subjects are exposed to much lower concentrations of O3 (0.12– 0.75 ppm), yet, in all of these experiments, the participants are exercising (3, 5, 15, 23). As the dose of O3 delivered



to the lungs is the product of O3 concentration, exposure time, ˙ E) (78), exercising during exposure and minute ventilation (V ˙ E of these subjects and, subsequently, the total increases the V dose of O3 delivered to their lungs. In contrast, mice decrease ˙ E by 60% during a 3-h exposure to O3 (2 ppm) (67), their V which decreases the dose of O3 delivered to their lungs. Thus, although the O3 concentrations humans and mice are exposed to under experimental conditions are different; the total dose of O3 inhaled into the lungs may be very similar. Indeed, Hatch and colleagues (27) have previously reported that the dose of O3 delivered to the lungs of exercising humans and sedentary rodents is comparable despite the fact these humans and rodents were exposed to very different O3 concentrations (0.4 ppm compared with 2 ppm, respectively). In summary, we demonstrate that the release of OPN into the air spaces of wild-type mice is increased following acute exposure to O3. Furthermore, to our knowledge, this is the first study to demonstrate a functional role for OPN in the development of O3-induced neutrophil recruitment to the air spaces as well as O3-induced AHR. Thus neutralization of OPN may serve as preventative or palliative therapy to alleviate the toxic effects of inhaled O3, especially in susceptible individuals, such as patients with preexisting chronic lung disease. ACKNOWLEDGMENTS The authors thank José G. Molina for maintaining the colony of OPNdeficient mice used in this study. In addition, the authors thank Ms. Ning-Yuan Chen for technical assistance with this study. GRANTS This work was supported, in part, by the National Institute of Environmental Health Sciences of the National Institutes of Health under award number R03ES022378. The content contained within this publication is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors. AUTHOR CONTRIBUTIONS Author contributions: R.X.B., D.J.S., C.B.H., L.M.H., R.E.P., and R.A.J. performed experiments; R.X.B., J.B.R., K.R.C., A.J.N., R.E.P., S.S.H., M.R.B., I.U.H., and R.A.J. analyzed data; R.X.B., J.B.R., K.R.C., A.J.N., R.E.P., S.S.H., M.R.B., I.U.H., and R.A.J. interpreted results of experiments; R.X.B., J.B.R., K.R.C., and R.A.J. drafted manuscript; R.X.B., J.B.R., D.J.S., K.R.C., A.J.N., C.B.H., L.M.H., R.E.P., S.S.H., M.R.B., I.U.H., and R.A.J. edited and revised manuscript; R.X.B., J.B.R., D.J.S., K.R.C., A.J.N., C.B.H., L.M.H., R.E.P., S.S.H., M.R.B., I.U.H., and R.A.J. approved final version of manuscript; D.J.S., M.R.B., I.U.H., and R.A.J. conception and design of research; R.E.P. and R.A.J. prepared figures. REFERENCES 1. Alpert SM, Schwartz BB, Lee SD, Lewis TR. Alveolar protein accumulation. A sensitive indicator of low level oxidant toxicity. Arch Intern Med 128: 69 –73, 1971. 2. American Lung Association. State of the Air 2012. Washington, D.C.: American Lung Association, 2012. 3. Balmes JR, Chen LL, Scannell C, Tager I, Christian D, Hearne PQ, Kelly T, Aris RM. Ozone-induced decrements in FEV1 and FVC do not correlate with measures of inflammation. Am J Respir Crit Care Med 153: 904 –909, 1996. 4. Banerjee A, Apte UM, Smith R, Ramaiah SK. Higher neutrophil infiltration mediated by osteopontin is a likely contributing factor to the increased susceptibility of females to alcoholic liver disease. J Pathol 208: 473–485, 2006.

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