Exposure to ozone, which is a major component of air pollution, induces a form of asthma that occurs in the absence of adaptive immunity. Although ozone-induced asthma is characterized by airway neutrophilia, and not eosinophilia, it is nevertheless associated with airway hyperreactivity (AHR), which is a cardinal feature of asthma. Because AHR induced by allergens requires the presence of natural killer T (NKT) cells, we asked whether ozone-induced AHR had similar requirements. We found that repeated exposure of wild-type (WT) mice to ozone induced severe AHR associated with an increase in airway NKT cells, neutrophils, and macrophages. Surprisingly, NKT cell–deficient (CD1d−/− and Jα18−/−) mice failed to develop ozone-induced AHR. Further, treatment of WT mice with an anti-CD1d mAb blocked NKT cell activation and prevented ozone-induced AHR. Moreover, ozone-induced, but not allergen-induced, AHR was associated with NKT cells producing interleukin (IL)-17, and failed to occur in IL-17−/− mice nor in WT mice treated with anti–IL-17 mAb. Thus, ozone exposure induces AHR that requires the presence of NKT cells and IL-17 production. Because NKT cells are required for the development of two very disparate forms of AHR (ozone- and allergen-induced), our results strongly suggest that NKT cells mediate a unifying pathogenic mechanism for several distinct forms of asthma, and represent a unique target for effective asthma therapy.

Bronchial asthma is a heterogeneous syndrome associated with diverse factors, including allergen sensitization, infection, obesity, as well as exposure to air pollution (1). Regardless of the trigger, asthma is associated with reversible airway obstruction and airway hyperreactivity (AHR) and an increased sensitivity of the airways to nonspecific stimuli such as cold air or respiratory irritants, and quantitated by responsiveness to methacholine or histamine (2). Diverse mechanisms have been proposed to explain the pathogenesis of various asthma phenotypes, including allergic inflammation, Th2 cells, eosinophils, basophils (3), neutrophils (4), and oxidative stress (5). Thus, different forms of asthma appear to depend on distinct cell types and pathways, and a shared disease mechanism for all forms of asthma has not been established.

Invariant TCR natural killer T (iNKT) cells comprise a newly described, unique subset of lymphocytes that express features of both classical T cells and NK cells. iNKT cells express a conserved/invariant TCR repertoire consisting of Vα14-Jα18 (in mice) or Vα24-Jα18 (in humans) (6). iNKT cells are activated by recognition of glycolipid antigens presented by the nonpolymorphic MHC class I–like protein CD1d, which is widely expressed by airway and intestinal epithelial cells, B cells, macrophages, and dendritic cells. Recognition of CD1d-presented glycolipids by iNKT cells is highly conserved across phylogeny, suggesting that iNKT cells play a pivotal role in immunity (7, 8). Activation of iNKT cells results in an innate-like immune response, with rapid production of large quantities of cytokines, including IL-4 and IFN-γ (6, 9), and this rapid response endows iNKT cells with the capacity to critically amplify adaptive immunity and regulate the development of polarized T cells. The rapid production of cytokines by iNKT cells has been shown to regulate the development of several diseases, including diabetes mellitus, colitis, autoimmune neurological disease, rejection of malignant tumors, infectious diseases, and asthma (1014).

In animal models of allergic asthma, iNKT cell–deficient mice (CD1d−/− or Jα18−/− mice) fail to develop AHR when sensitized and challenged with allergens (13, 14). This requirement for iNKT cells in the development of allergen-induced AHR is independent of Th2 responses, which develop normally in these mice (15). The requirement for iNKT cells is also specific because adoptive transfer of WT iNKT cells reconstituted the capacity of Jα18−/− mice to develop AHR. Furthermore, direct activation of iNKT cells by the respiratory administration of glycolipids such as α-Galactosylceramide (α-GalCer) or glycolipids isolated from Sphingomonas bacteria caused AHR and airway inflammation, even in the complete absence of class II MHC–restricted T cells and adaptive immunity (16). Moreover, iNKT cells are present in the lungs of patients with asthma (1719), although the specific factors that regulate their frequency and function in the lungs has not been fully established. Together, these studies suggest that iNKT cells play a very important role in the development of asthma and AHR.

To determine whether iNKT cells form the basis for a shared pathogenic mechanism for different forms of asthma, we examined the role of iNKT cells in a form of AHR not thought to involve immune mechanisms, i.e., that induced by exposure to ozone, a major component of air pollution. In children, ambient exposure to ozone increases asthmatic symptoms even at concentrations below the U.S. Environmental Protection Agency standard (20), and hospital admissions for asthma are higher on days of high ambient ozone concentrations (21, 22). Even in healthy individuals, exposure to ozone induces the development of AHR that is associated with airway epithelial cell damage and an increase in neutrophils and inflammatory mediators (TNF-α, CXCL-8/IL-8, IL-6, and GM-CSF) in proximal airways (23, 24), and asthmatics appear to be more susceptible to the adverse effects of this pollutant (25). In our current studies, we demonstrated that repeated exposure of mice to ozone induced AHR that required the presence of iNKT cells and the production of IL-17, which is a potent neutrophil chemotactic factor. These results suggest that innate immunity involving iNKT cells may play a critical role as a unifying pathogenic mechanism for several very distinct forms of asthma.

Results

NKT cells are required for AHR induced by repeatedozone exposure

We exposed mice to ozone (1 part per million [ppm] for 3 h) every other day over a 5-d period, using a semiacute protocol that maximized airway inflammatory cell recruitment over a brief period of time. Exposure of WT BALB/c mice to ozone in this manner resulted in higher baseline airway resistance, and in the development of severe AHR (Fig. 1 A) and significant airway inflammation, consisting of increased macrophages, lymphocytes, and neutrophils, but not eosinophils, in the bronchoalveolar lavage (BAL) fluid (Fig. 1 C). Repeated ozone exposure also increased the number of iNKT cells in the BAL fluid by >10-fold (Fig. 1 D, and Fig. S1). To determine the role of these iNKT cells in the development of AHR, we compared WT mice and CD1d−/− mice, which lack the restriction element of NKT cells, and therefore lack NKT cells (6). Surprisingly, CD1d−/− mice exposed to ozone failed to develop AHR (Fig. 1 A). The CD1d−/− mice exposed to repeated ozone exposure also had fewer airway macrophages, and significantly less airway lymphocytes and neutrophils in BAL fluid compared with that of WT mice (Fig. 1 C). The requirement for iNKT cells was not dependent on the genetic background of our mice (all backcrossed to BALB/c), as we observed the same effects in C57BL/6 mice (unpublished data). Pretreatment of WT mice with an anti-CD1d–blocking mAb to prevent iNKT cell activation also reduced ozone-induced AHR (Fig. 1 B), and decreased the number of lymphocytes, neutrophils, and iNKT cell numbers in the BAL fluid (Fig. 1, C and D). Thus, repeated exposure of mice to ozone induced AHR that depended on the presence of CD1d restricted T cells.

Jα18−/− mice fail to develop ozone-induced AHR

To confirm that iNKT cells were specifically required for the development of ozone-induced AHR, we examined another iNKT cell–deficient strain, Jα18−/− mice, which lack the invariant TCR-α chain, and therefore lack iNKT cells (6). Like CD1d−/− mice, Jα18−/− mice repeatedly exposed to ozone over 5 d failed to develop AHR, whereas WT mice exposed to ozone developed severe AHR (Fig. 2 A). Compared with WT mice, Jα18−/− mice exposed to ozone also had significantly reduced numbers of airway macrophages, lymphocytes, and neutrophils (Fig. 2 B) and no detectable iNKT cells (Fig. 2 C) in BAL fluid. The specific requirement for iNKT cells in the development of ozone-induced AHR was demonstrated by adoptive transfer of purified WT iNKT cells into Jα18−/− mice, which partially restored the capacity of Jα18−/− mice to develop ozone-induced AHR (Fig. 2 D). Full restoration of the AHR response was not achieved by adoptive transfer of WT iNKT cells, presumably because insufficient numbers of iNKT cells migrated into the lungs of recipients (the number of iNKT cells in the BAL fluid of recipients was only 30% of that found in WT mice; unpublished data).

IL-17 is required for ozone-induced neutrophilia and AHR

Because ozone-induced AHR was associated with airway neutrophilia, we evaluated the role of IL-17, which has been shown to enhance neutrophil migration to sites of inflammation (26, 27). We found that repeated ozone exposure was associated with the induction of IL-17 synthesis in iNKT cells and small numbers of T cells in the lungs (Fig. 3 A), but not in the spleen (not depicted). Note that the total number of cells in the lungs was greatly increased after ozone treatment (three- to sixfold), and therefore the percentages of cells indicated by flow cytometry underestimated the increases in IL-17–producing cells induced with ozone exposure. In addition, we found that the pulmonary iNKT cells produced IL-4, but not IFN-γ, unlike resting or activated splenic iNKT cells, which produce both IL-4 and IFN-γ, suggesting that IL-4, but not IFN-γ, production by iNKT cells also played a role in the induction of ozone AHR. Ozone also induced IL-10 expression in iNKT cells, but not T cells, in the lungs (Fig. 3 A). To assess expression of NK1.1 by the iNKT cells (not present in BALB/c mice), we exposed C57BL/6 mice to ozone and found that the IL-17–producing T cells were NK1.1 (Fig. 3 C). This suggests that the IL-17–producing iNKT cells induced with ozone were distinct from the IL-4/IL-13–producing iNKT cells, which are generally NK1.1+, at least in C57BL/6 mice.

To determine whether the development of ozone-induced AHR required IL-17 production by conventional CD4+ T cells (Th17 cells) or by iNKT cells, we exposed MHC class II−/− mice, which lack conventional CD4+ T cells but have iNKT cells, to ozone. Fig. 3 B shows that the MHC class II−/− mice developed severe ozone-induced AHR and severe airway inflammation (not depicted), as did the WT mice exposed to ozone. These results clearly indicated that IL-17–producing iNKT cells and not Th17 cells are required for the development of ozone-induced AHR, although it is possible that Th17 cells may contribute to the development of AHR.

Ozone-induced, but not allergen-induced, AHRrequires IL-17

Importantly, IL-17 production in the lungs was absolutely required for ozone-induced AHR because treatment of mice with an anti–IL-17 blocking mAb greatly reduced ozone-induced AHR (Fig. 4 A), airway lymphocytes, and neutrophils (but not macrophages; Fig. 4 B). These results were confirmed in IL-17−/− mice, which failed to develop ozone-induced AHR (Fig. 4 E) and inflammation (Fig. S2). IL-17 production by iNKT cells or T cells was required specifically for ozone-induced, but not allergen-induced, AHR because OVA-induced AHR and inflammation occurred normally in WT mice treated with anti–IL-17 blocking mAb (Figs. 4, C and D) and in IL-17−/− mice (Fig. 4 F). In addition, IL-17–producing cells were not detected in the lungs of mice sensitized and challenged with OVA to induce AHR (unpublished data). Therefore, these results demonstrate that IL-17 plays a critical role in the development of AHR and airway inflammation induced by repeated ozone exposure, but not by allergen. Thus, although both allergen- and ozone-induced AHR require the presence of iNKT cells, the cytokines produced by these cells are distinct, with IL-17 playing a pathogenic role in ozone-induced, but not allergen-induced AHR.

IL-4 and -13 are also involved in ozone-induced AHRand airway inflammation

We previously showed that iNKT cells producing IL-4 and -13 are essential in the development of allergen-induced AHR (13). Because IL-4 was present in most of the iNKT cells in the lungs of ozone-exposed mice (Fig. 3), we sought to determine whether IL-4 and -13 were also required for AHR induced by repeated ozone exposure. To that end, we exposed IL-4−/−/IL-13−/− double knockout, IL-13−/−, and IL-4−/− mice to ozone over a 5-d period. After exposure to ozone, the IL-4−/−/IL-13−/− mice failed to develop AHR and had reduced airway inflammation, including significantly fewer macrophages and lymphocytes, compared with WT mice (Figs. 5, A and B). The requirement of IL-4 and -13 production by iNKT cells for ozone-induced AHR was confirmed by additional studies demonstrating that adoptive transfer of iNKT cells purified from IL-4−/−/IL-13−/− double knockout mice into Jα18−/− mice failed to reconstitute ozone-induced AHR (Fig. 5 C), whereas transfer of WT iNKT cells partially restored AHR (Fig. 2 D). Furthermore, IL-13−/− and -4−/− mice exposed to ozone both failed to develop AHR (Figs. 5, D and E, and Fig. S2). These results indicate that although allergen- and ozone-induced AHR differ in their requirement for IL-17 and in the types of inflammatory cells present in the airways (eosinophils versus neutrophils), both forms of AHR require iNKT cells producing IL-4 and -13.

Discussion

In this study, we demonstrated that repeated exposure to ozone induces AHR mediated by a novel mechanism involving innate-like immunity, iNKT cells, and IL-17. Thus, iNKT cell–deficient mice, including CD1d−/− and Jα18−/− strains, generated by disruption of two completely independent genes failed to develop ozone-induced AHR. Blockade of iNKT cell activation with an anti-CD1d mAb also prevented ozone-induced AHR and the recruitment of iNKT cells into the lungs. Furthermore, ozone-induced AHR was associated with IL-17 production by pulmonary iNKT, and neutralization of IL-17 with anti–IL-17 mAb or by disruption of the IL-17 gene prevented the development of ozone-induced, but not allergen-induced, AHR. Together, these results demonstrate for the first time that repeated exposure to ozone induces the development of AHR in a process that requires iNKT cells (presumably responding to oxidized self-lipids), as well as IL-17, but not adaptive immunity, conventional Th2 cells, or eosinophils.

Although respiratory exposure to ozone is known to trigger clinical symptoms of asthma and to induce toxic responses that have been studied extensively in laboratory animals and humans, the precise mechanisms by which ozone causes asthma have not been fully defined. We and others have shown previously that a single acute exposure to ozone (2 ppm for 3 h) results in the pulmonary expression of proinflammatory cytokines, including TNF-α, IL-6, and IL-1, which play important roles in the airway inflammation and/or AHR induced by such ozone exposures (2832). Although in our current studies we used repeated ozone exposure to maximize airway inflammation and iNKT cell recruitment over a brief period of time and have not yet compared our current model with the single acute exposure model, it is conceivable that these acute phase cytokines lead to the production of chemokines that recruit iNKT cells, which then induce AHR upon subsequent ozone exposure. It is also known that exposure to ozone induces the generation of reactive oxygen species, hydrogen peroxide, and aldehydes (from the ozonation of unsaturated fatty acids), leading to oxidative stress responses (33). As such, polymorphisms in the genes that regulate intracellular levels of the antioxidant glutathione (GSTM1 and GSTP1) are associated with an increased susceptibility to respiratory symptoms related to ozone exposure in asthmatic children (34). Disruption of the nuclear erythroid 2 p45-related factor 2 (Nrf 2) gene, which transcriptionally regulates many antioxidant genes, also leads to severe allergen-driven airway inflammation and AHR (35), which is consistent with the observation that exposure to ozone and to ultrafine particulates in air pollution increases airway inflammation and potentiates immune responses to inhaled allergens (5). Thus, oxidative stress appears to play a role in the airway responses to both allergy and ozone. We now suggest that asthma associated with air pollution/oxidative stress shares another very important pathogenic disease mechanism with allergen-induced asthma, i.e., AHR that requires the presence of iNKT cells.

Although our data indicate that iNKT cells are required for both allergen-induced (13) and ozone-induced AHR (Fig. 1 A and Fig. 2 A), the pulmonary inflammation induced by ozone exposure is very distinct from that induced by allergen. In our mouse model, ozone-induced AHR was characterized by the presence of neutrophils, IL-17, and a small number of cells expressing IL-4, whereas the allergen-induced form of AHR was characterized by the presence of eosinophils and with high levels of IL-4 and -13 (13). These differences may reflect the fact that the cytokine profile of the iNKT cells involved in these two different forms of AHR is distinct, with iNKT cells in allergen-induced AHR producing IL-4, but not IL-17, and iNKT cells in ozone-induced AHR producing IL-17 and -4 (Fig. 3) and not expressing NK1.1 (when performed in C57BL/6 mice). Indeed, IL-17 production by iNKT cells appears to be critical for ozone-induced, but not allergen-induced, AHR because IL-17−/− mice and WT mice treated with anti–IL-17 mAb failed to develop ozone-induced, but not allergen-induced AHR. Moreover, MHC class II−/− mice developed ozone-induced AHR, indicating that IL-17–producing iNKT cells and not class II MHC–restricted Th17 cells, which have been shown to critically regulate the development of autoimmune disease (36), are required for the development of ozone-induced AHR. Recently, respiratory exposure to α-GalCer has been shown to induce IL-17 production by pulmonary NK1.1iNKT cells, associated with airway neutrophils, suggesting that IL-17 production by iNKT cells can occur in some circumstances, although the specific function of these IL-17–producing NK1.1iNKT cells in terms of AHR was not clear (37).

Previous studies have provided conflicting results regarding the role of IL-17 in asthma. IL-17 is known to play an important role in activating T cells (38), enhancing neutrophil accumulation, and inducing IL-6, TNF-α, and TGF-β production, which increase airway inflammation (39). Although IL-17 (protein and mRNA) is present in the lungs of patients with severe asthma, along with eosinophils and neutrophils (4042), or in the lungs of mice chronically exposed to OVA (43), other studies have indicated that IL-17 has a potent inhibitory role (44), or that it has minimal effects (38), on allergen-induced AHR. Our results help to resolve these discrepancies, and suggest that IL-17 production by iNKT cells (and possibly by Th17 cells) mediates and is required for a specific form of AHR (i.e., ozone-induced AHR associated with airway neutrophilia), but may have a minor role in other forms of AHR (i.e., allergen-induced AHR, associated primarily with Th2-driven eosinophilic inflammation) (44). However, it is possible that under some circumstances, when adaptive allergen-specific Th2 responses develop in the context of oxidative stress and/or exposure to ozone in a single individual, both IL-4– and IL-17–producing iNKT cells may be present to synergistically drive the development of lung inflammation and AHR. This is consistent with the observation that exposure to air pollution and allergens together results in severe airway inflammation and asthma (45, 46).

The idea that ozone exposure leads to the activation of iNKT cells and to the development of AHR may be related to the observation that iNKT cells can exacerbate atherosclerotic heart disease (47). These observations together suggest that ozone exposure alters/oxidizes lipids that are sensed by iNKT cells, which then initiate the inflammation leading to the development of asthma or atherosclerotic heart disease. Moreover, the role of iNKT cells in sensing oxidized lipids may explain the observation that obesity increases the risk of developing asthma in humans (48) and in mice (49). Thus, oxidized lipids, which may develop readily in obese individuals or in obese mice, might serve as an important “antigen” that activates iNKT cells, leading to the development of AHR and atherosclerotic heart disease.

In summary, we identified novel cellular and molecular mechanisms (iNKT cells and IL-17) required for the development of AHR induced by repeated ozone exposure. Importantly, because both ozone-induced and allergen-induced AHR require the presence of iNKT cells, these results strongly suggest that iNKT cells mediate a common unifying pathogenic mechanism for diverse forms of asthma. The specific cytokines produced by iNKT cells in ozone- versus allergen-induced AHR are distinct, which may account for the different inflammatory cell types that accompany ozone- and allergen-induced AHR. Thus, ozone-induced AHR is associated with neutrophils and IL-17, whereas allergen-induced AHR is associated with eosinophils, but primarily with IL-4 and not IL-17. These results suggest that targeting iNKT may provide an important and effective therapy for diverse and mixed forms of asthma.

Materials And Methods

Mice.

Wild-type BALB/c ByJ and C57BL/6N mice were purchased from The Jackson Laboratory. CD1d−/− and Jα18−/− mice (both backcrossed to BALB/c) were gifts from M. Grusby (Harvard School of Public Health, Boston, MA) (50) and M. Taniguchi and T. Nakayama (Chiba University, Chiba, Japan) (51), respectively. IL-17−/− mice on the C57BL/6 background were generated as previously described (38), and were bred in our mouse colony. IL-13−/− and IL-4−/−/IL-13−/− double-knockout mice (BALB/c background) were obtained from A. McKenzie (Medical Research Council Laboratory of Molecular Biology, Cambridge, UK). IL-4−/− and MHC class II−/− mice were obtained from Jackson ImmunoResearch Laboratories. Female mice were studied at approximately eight weeks of age. The Animal Care and Use Committee at Children's Hospital Boston and Harvard Medical School approved all animal protocols.

Antibodies and reagents.

α-GalCer was synthesized by P.B. Savage (Brigham Young University, Provo, Utah), and it was used as positive control to induce AHR (unpublished data). Neutralizing rat anti–mouse CD1.1 (CD1d) mAb (hybridoma HB323; American Type Culture Collection) was used as previously described (16). Anti–mouse IL-17A blocking antibody (clone eBioTC11-18H10.1) and the control rat IgG1k isotype control antibody (eBioscience) were used in the blocking experiments.

ELISA.

Supernatants of lung cell suspensions were collected 48 h after α-GalCer restimulation (100 ng/ml). Mouse IL-4 and IFN-γ levels were measured by ELISA, as previously described (13).

Flow cytometry.

Cells were preincubated with anti-Fcγ blocking mAb (2.4G2) and washed before staining. Cells were stained with anti–mouse FITC NK1.1, PE-Texas red–conjugated CD45, PerCPCy5.5-conjugated CD3, and Alexa Fluor 750–conjugated CD4 mAb (clone RM4-5). iNKT cells were identified using APC-conjugated TCRβ (clone H57-597; eBioscience) and PE-conjugated, PBS57-loaded, CD1d-tetramers (with empty CD1d tetramers always used as control; National Institute of Allergy and Infectious Disease Major Histocompatibility Complex Tetramer Core Facility, Atlanta, GA). For intracellular staining, after permeabilization (Cytofix/Cytoperm kit; BD Biosciences), cells were incubated with FITC-conjugated IL-4, FITC-conjugated IFN-γ, APC-conjugated IL-10, Alexa Fluor 647–conjugated IL-17, or the respective isotype control antibodies, as follows: FITC-conjugated rat IgG1k, APC-conjugated rat IgG2b, or Alexa Fluor 647–conjugated rat IgG2a (eBioscience). Cells were collected on a BD Canto flow cytometer (BD Biosciences) and analyzed with FlowJo 8.3.3 software (Tree Star, Inc.).

Adoptive transfer of iNKT cells.

iNKT cells were purified from splenocytes of wild-type BALB/c or IL-4−/−/IL-13−/− double-knockout mice using magnetic cell sorting (MACS), as previously described (13). Splenic iNKT cells were labeled with PE-conjugated CD1d-tetramer, followed by anti-PE microbeads (Miltenyi Biotec), and then sorted with AutoMACS according to the manufacturer's instruction. Purity of iNKT cells was >80%. Purified iNKT cells (106) were adoptively transferred into recipient Jα18−/− mice by intravenous injection 1 d before the first exposure to ozone or air.

Ozone-exposure and measurement of airway responsiveness in the ozone model.

Mice were placed awake in individual wire mesh cages inside a stainless steel and Plexiglas exposure chamber and exposed to ozone (1 ppm) for 3 h. For room air exposure, a separate and identical exposure chamber was used. Details of the ozone exposure and monitoring have been previously described (52). Exposure was repeated every other day (days 0, 2, and 4) to optimize airway inflammation and iNKT cell recruitment into the BAL fluid over the shortest period of time, and did not induce any weight changes in the mice (unpublished data). As a positive control for lung mechanics assessment, control mice received one intranasal injection of α-GalCer (0.5 μg) the day before the measurement of AHR (unpublished data). 24 h after the last ozone (day 6) or α-GalCer exposure, mice were anesthetized with 50 mg/kg pentobarbital and instrumented for the measurement of pulmonary mechanics (BUXCO Electronics). Mice were tracheostomized, intubated, and mechanically ventilated at a tidal volume of 0.2 ml and a frequency of 150 breath/min, as previously described (52). Baseline lung resistance (RL) and responses to aerosolized saline (0.9% NaCl) were measured first, followed by responses to increasing doses (0.32 to 40 mg/ml) of aerosolized acetyl-β-methylcholine chloride (methacholine; Sigma-Aldrich). The three highest values of RL obtained after each dose of methacholine were averaged to obtain the final values for each dose.

Induction of AHR and measurement of airway responsiveness in the OVA model.

To induce AHR, we sensitized BALB/c mice with 100 μg of OVA (Sigma-Aldrich) in alum administered i.p. After 8 d, mice were exposed to intranasal antigen (50 μg OVA/d) or normal saline on 3 consecutive days, as previously described (13). AHR was assessed on day 12. C57BL/6 mice were sensitized with 100 μg of OVA adsorbed to 2 mg of an aqueous solution of alum i.p. on days 0 and 14, followed by 50 μg of OVA in 50 μl of PBS administered intranasally on days 14 and 25–27. Control mice received i.p. injection of PBS and intranasal administrations of normal saline. AHR was assessed on day 28.

BAL fluid.

After measurement of AHR and euthanasia, the lungs were lavaged twice with 1 ml of PBS 2% FCS, and the fluid was pooled as previously described (16). For some experiments, total BAL for each mouse or pooled BAL was stained and analyzed by flow cytometry. BAL iNKT cell numbers were quantified by multiplying hemacytometer cell counts, excluding red blood cells, by the percentage of iNKT within a cellular gate that included lymphocytes, monocytes, large epithelial cells, and granulocytes (by forward scatter and side scatter).

Lung tissue.

After measurement of AHR, killing of the mice, and collection of BAL fluids, lungs were perfused with PBS 2% FCS, and portions of lung tissues were treated with collagenase type 4 (Worthington Biochemical Corp.). The lymphocyte-enriched fraction was collected at the 50–70% interface of Percoll gradients (GE Healthcare), and plated for 2 h with BD GolgiStop (BD Biosciences) before intracellular staining.

Statistical tests.

Differences between groups with parametric distributions were analyzed by Student's t test; otherwise, the Mann-Whitney U test was used. Significance for all statistical tests was shown in figures for P ≤ 0.05 (*) and P ≤ 0.01 (**).

Online supplemental material.

Airway inflammation was evaluated in BAL fluid after ozone exposure (Figs. S1 and S2).

Acknowledgments

We thank Drs. E. Betteli and V.K. Kuchro (Center for Neurological Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts) for their comments and discussion; L.A. Albacker for his help with the invasive measurement; Y.L. Chim for her work in the animal facility; M. Grusby for the CD1d−/− mice; M. Taniguchi and T. Nakayama for the Jα18−/− mice; and A. McKenzie for the IL-4−/−/ IL-13−/− double-knockout mice.

This work has been supported by grants HL062348 and AI054456 from the National Institutes of Health, and by funds from the Bunning Food Allergy Project.

Dale Umetsu and Paul Savage are consultants for Innate Immune, Inc., but the other authors have no conflicting financial interests.

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Abbreviations used: α-GalCer, α-galactosylceramide; AHR, airway hyperreactivity; ppm, parts per million.