Pulmonary Inflammation and Emphysema Role in Protection

Transcription Factor Nrf2 Plays a Pivotal Role in Protection against Elastase-Induced Pulmonary Inflammation and Emphysema

This information is current as Yukio Ishii, Ken Itoh, Yuko Morishima, Toru Kimura, of September 24, 2021. Takumi Kiwamoto, Takashi IIzuka, Ahmed E. Hegab, Tomonori Hosoya, Akihiro Nomura, Tohru Sakamoto, Masayuki Yamamoto and Kiyohisa Sekizawa J Immunol 2005; 175:6968-6975; ;

doi: 10.4049/jimmunol.175.10.6968 Downloaded from http://www.jimmunol.org/content/175/10/6968

References This article cites 37 articles, 8 of which you can access for free at: http://www.jimmunol.org/content/175/10/6968.full#ref-list-1 http://www.jimmunol.org/

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists by guest on September 24, 2021 • Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

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 © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Transcription Factor Nrf2 Plays a Pivotal Role in Protection against Elastase-Induced Pulmonary Inﬂammation and Emphysema1

Yukio Ishii,2* Ken Itoh,†‡ Yuko Morishima,* Toru Kimura,* Takumi Kiwamoto,* Takashi IIzuka,* Ahmed E. Hegab,* Tomonori Hosoya,†‡ Akihiro Nomura,* Tohru Sakamoto,* Masayuki Yamamoto,†‡ and Kiyohisa Sekizawa*

Emphysema is one of the major pathological abnormalities associated with chronic obstructive pulmonary disease. The protease/ antiprotease imbalance and inflammation resulting from oxidative stress have been attributed to the pathogenesis of emphysema. Nrf2 is believed to protect against oxidative tissue damage through the transcriptional activation of a battery of antioxidant enzymes. In this study, we investigated the protective role of Nrf2 in the development of emphysema using elastase-induced Downloaded from emphysema as our model system. We found that elastase-provoked emphysema was markedly exacerbated in Nrf2-knockout (KO) mice compared with wild-type mice. The severity of emphysema in Nrf2-KO mice correlated intimately with the degree of lung inflammation in the initial stage of elastase treatment. The highly inducible expression of antioxidant and antiprotease genes observed in wild-type alveolar macrophages was significantly attenuated in the lungs of Nrf2-KO mice. Interestingly, transplantation of wild-type bone marrow cells into Nrf2-KO mice retarded the development of initial lung inflammation and subsequent emphysema, and this improvement correlated well with the appearance of macrophages expressing Nrf2-regulated antiprotease http://www.jimmunol.org/ and antioxidant genes. Thus, Nrf2 appears to exert its protective effects through the transcriptional activation of antiprotease and antioxidant genes in alveolar macrophages. The Journal of Immunology, 2005, 175: 6968–6975.

hronic obstructive pulmonary disease, including pulmo- emphysema in both smokers and nonsmokers. However, other can- nary emphysema, chronic airway obstruction, and didate host factors influencing the risk of emphysema require iden- C chronic bronchitis, is a major public health problem pre- tification because the majority of patients with emphysema have dicted to emerge as one of the top five causes of death and dis- normal serum levels of ␣1-antitrypsin.

ability worldwide by the year 2020 (1). Emphysema is patholog- Nrf2 (NF-E2-related factor 2), a member of the cap’n’collar by guest on September 24, 2021 ically characterized as a breakdown in alveolar architecture, with family of basic leucine zipper type transcription factors, has been enlargement of alveolar airspaces due to the destruction of alveolar shown to bind to the antioxidant response element (ARE)3 as an walls. Lung parenchymal inflammation, protease/antiprotease im- obligatory heterodimer with small Maf protein in the induction of balance, and oxidative stress are thought to be important processes phase 2 enzymes and antioxidant genes (8, 9). Nrf2 binds to the in the development of emphysema (2–4). These processes may Kelch-like ECH-associated protein (Keap1), a cytosolic actin- interact or synergize with each other in provoking the character- binding protein, which retains it in the cytoplasm under normal istic pathological changes associated with emphysema. physiological conditions. When cells encounter oxidative or xeno- Cigarette smoking is a major cause of emphysema. Importantly, biotic stress, Nrf2 is released from Keap1, allowing its rapid however, not all smokers develop clinically significant emphy- translocation to the nucleus (10–13). Nrf2-knockout (Nrf2-KO) sema, and this observation suggests that some additional factors mice grow normally and are fertile, but are susceptible to oxi- may be involved in determining individual susceptibility to em- dative stress and reactive electrophiles (14–18). Nrf2 plays es- physema (5). A well-documented host factor is a hereditary defi- sential roles in protection against pulmonary inflammation ciency of ␣1-antitrypsin, a major circulating inhibitor of serine caused by environmental toxins such as butylated hydroxytolu- protease that is synthesized in the liver (6, 7). This hereditary dis- ene (19) and hyperoxia (20). Moreover, it is reported that Nrf2 ease shows premature and accelerated development of panlobular regulates inflammation in carrageenan-induced pleurisy and acute lung injury (21, 22). To understand the pathogenesis of emphysema, rodent models † *Department of Respiratory Medicine, Exploratory Research for Advanced Tech- of elastase-inducible emphysema have been established (23, 24). nology (ERATO) Environmental Response Project, and ‡Institute of Basic Medical Sciences and Center for Tsukuba Advanced Research Alliance, University of Intratracheal instillation of porcine pancreatic elastase (PPE) first Tsukuba, Tsukuba, Japan induces lung inflammation and subsequently results in alveolar Received for publication September 29, 2004. Accepted for publication September wall destruction. These pathological changes closely mimic those 1, 2005. in human emphysema. In the present study, we therefore investi- The costs of publication of this article were defrayed in part by the payment of page gated the susceptibility of Nrf2-KO mice to PPE-induced emphy- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. sema to explore the role of Nrf2 in protection against emphysema. 1 This work was supported in part by grants from the Japan Science and Technology Agency-ERATO. 2 Address correspondence and reprint requests to Dr. Yukio Ishii, Department of 3 Abbreviations used in this paper: ARE, antioxidant response element; PPE, porcine Respiratory Medicine, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki pancreatic elastase; BAL, bronchoalveolar lavage; KO, knockout; SLPI, secretory 305-8577, Japan. E-mail address: [email protected] leukoprotease inhibitor; BMT, bone marrow transplantation.

Materials and Methods presence of 2-ME and electrotransferred onto Immobilon membrane (Mil- Animals and treatment lipore). The membrane was subsequently probed by monoclonal anti-Nrf2 Ab. For making mAb against mouse Nrf2, rats were immunized by GST- Wild-type BALB/c mice were purchased from Charles River Breeding Nrf2 fusion protein encoding aa 140–318 of mouse Nrf2. As a loading Laboratories. Nrf2-KO mice with a BALB/c background were established control, the membrane was also probed with anti-lamin B Ab. To detect by specific deletion of the Nrf2 gene segment (8). All mice used in this immunoreactive proteins, we used HRP-conjugated anti-rabbit IgG and study were 6- to 8-wk-old and maintained in our animal facilities under ECL blotting reagents (Amersham). specific pathogen-free conditions. All animal studies were approved by the Institutional Review Board. The mice were anesthetized with fluothane Luciferase reporter gene assay (Takeda Pharmaceuticals) and given an intratracheal instillation of 25 ␮g The 1.25 kb of the mouse SLPI 5Ј regulatory region was amplified by PCR of PPE (Elastin Products) in 0.1 ml of sterile saline solution or 0.1 ml of Ј saline alone. using mouse genomic DNA as template and the primers 5 -TGA GCA GCA CTA ACC TGA CT-3Ј and 5Ј-AAG GGG AGC TCT GAT GAC Lung histology and quantitation of emphysema CA-3Ј, then cloned into pGL3-basic vector (SLPI-luc). Deletion of the SLPI 5Ј regulatory region was done by removing the SpeI fragment from On days 1, 4, and 21 after PPE administration, mice were anesthetized by SLPI-luc (⌬SpeI-luc). The murine macrophage cell line RAW 264.7 i.p. injection of pentobarbital (50 mg/kg body weight). The trachea and (American Type Culture Collection) was grown in DMEM (Invitrogen Life lung were removed together and inflated with 4% paraformaldehyde in Technologies) containing 10% FBS and seeded in 6-well dishes 24 h be- PBS to a water pressure of 25 cm. The tissue was then embedded in paraffin fore transfection. Cells were transfected with reporter and Nrf2 expression and 2-␮m thick sections were stained with H&E. Air space enlargement plasmids (9) using Lipofectamine Plus reagent (Invitrogen Life Technol- was quantified by the mean linear intercept (Lm) in 20 randomly selected ogies) according to the manufacturer’s instructions. The luciferase assay fields of tissue sections (25). was performed with the Dual-Luciferase Reporter Assay kit (Promega)

following the supplier’s protocol and measured by a Biolumat Luminom- Downloaded from Bronchoalveolar lavage (BAL) eter (Berthold). Transfection efficiencies were routinely normalized to the On days 1 and 4 after PPE administration, the lungs of terminally anes- activity of a cotransfected Renilla luciferase. The means of four indepen- thetized mice were lavaged with six sequential 1-ml aliquots of PBS. The dent experiments are shown with SE. supernatant of the first BAL was used for analysis of albumin concentration (Sigma-Aldrich). The supernatant was also used for analysis of neutrophil Immunocytochemistry elastase by spectrophotometrical measurement as previously described Paraffin sections (4-␮m thick) were cut and mounted on poly-L-lysine-

(26). Hemoglobin content in a pooled aliquot was represented as the ab- coated glass slides. After removing the paraffin, endogenous peroxidases http://www.jimmunol.org/ sorbance at 412 nm. The remaining pool of BAL was centrifuged and were quenched with 0.3% H2O2 in methanol. After washing, sections were resuspended in PBS. Cells were counted using a hemocytometer and a incubated with anti-F4/80 Ab (Serotec), anti-HO-1 Ab (16), anti-PrxI Ab differential cell count was performed by standard light microscopic tech- (17), or anti-SLPI (Santa Cruz Biotechnology) Ab and incubated for an- niques based on staining with Diff-Quik (American Scientific Products). other hour with Histofine Simple Stain MAX-PO (Nichirei). Diaminoben- Lung mechanics zidine was used as a chromogen. Twenty-one days after PPE administration, terminally anesthetized mice Bone marrow transplantation (BMT) were tracheostomized, and the trachea was cannulated. After opening the Femurs and tibiae were obtained from 6- to 8-wk-old wild-type or Nrf2-KO chest wall, the cannula was connected to a computer-controlled small an- mice. Bone marrow cells were harvested in RPMI 1640 medium and re- imal ventilator (flexiVent; Scireq). The compliance was determined by suspended in sterile PBS. Nrf2-KO recipient mice were irradiated at a dose by guest on September 24, 2021 recording the relaxation pressures during inflation and deflation in 0.1-ml of 3.0 Gy, followed by caudal vein injection of 20 ϫ 106 cells. At the steps between 0 and 25 cm H O. Due to variation in the weight of each 2 specified time points, the lungs of the recipient mice were lavaged and cells animal, the lung volumes were normalized by body weight. Pressures from positive for F4/80 were sorted using FACSVantage (BD Biosciences). the normalized compliance curves were then extrapolated at 0.1-ml incre- DNA was extracted from the sorted cells and Nrf2 gene expression was ments and used to establish the mean static lung compliance in each group. determined by PCR using the primers 5Ј-TGG ACG GGA CTA TTG AAG RNA blot analysis GCTG-3Ј and 5Ј-GCC GCC TTT TCA GTA GAT GGA GG-3Ј.Tode- termine the efficiency of donor cell influx to the recipient’s alveolar space, Total RNA was extracted from the lungs of anesthetized mice using an genome DNA was extracted from alveolar macrophages and the contribu- RNeasy Mini kit (Qiagen). Electrophoresis of RNA was conducted on tion of male cells to alveolar macrophages of female recipients was ex- formaldehyde-agarose gels and RNA was transferred to nylon membranes amined by real-time PCR as previously described (28). The male genomic (Hybond-Nϩ; Amersham Biosciences). Hybridization was performed us- DNA level was assessed through determining the SRY gene content in the ing hybridization solution (ExpressHyb; BD Clontech) containing 1 ϫ 106 sample by PCR. The results were normalized by ␤-actin gene content. cpm/ml [␣-32P]-labeled mouse NAD(P)H:quinine oxidoreductase (NQO1), Primer sequences are as follows; SRY, 5Ј-GGG TGC TTA TGT CTC TTT GST-Yc, heme oxygenase-1 (HO-1), peroxyredoxin I (PrxI), or GAPDH CTC TGG-3Ј and 5Ј-GGT CCT TGT CCT GTA TCT ATG GCT C-3Ј; and cDNA fragments following the manufacturer’s protocol. After washing, ␤-actin, 5Ј-CCA TAG GCT TCA CAC CTT CCT G-3Ј and 5Ј-GCA CTA autoradiograms were made using a bioimaging analyzer (BAS5000; Fuji ACA CTA CCT TCC TCA ACC G-3Ј. Known mixtures of male and fe- Photo Film). male DNA were used to establish standard curves. Individual samples were normalized to the 100% male reference standard according to the following Expression of neutrophil elastase and antiproteinase 100%Ϫ ␤ 100%Ϫ formula: 1.9 exp(CT SRY CT SRY)/1.9 exp(CT -actin CT ␤-actin), where the threshold cycle C represents the crossing point for the Neutrophils were isolated from blood and BAL by standard Percoll dis- T samples of SRY or ␤-actin. The level of male DNA (SRY) was estimated continuous gradient centrifugation (Pharmacia Biotech). Total RNA was by linear regression using all control mixture standards. extracted and the expression of neutrophil elastase was analyzed by RT-PCR using the primer set 5Ј-CAT TGC CAG GAA CTT CGT CA-3Ј and 5Ј-GAG TGC ATA CGT TCA CAC GA-3Ј. Total RNA was also Statistics extracted from the lungs and livers of anesthetized mice. The expressions Data were evaluated by ANOVA and Scheffe’s test. Values for p Ͻ 0.05 of ␣1-antitrypsin and secretory leukoprotease inhibitor (SLPI) were ana- were considered to be statistically significant. lyzed by RT-PCR using the following primers: ␣1-antitrypsin, 5Ј-CAA CCT CAC ACA AAC ATC GG-3Ј and 5Ј-AAC TGT GTC TTC GTC AGG GT; and SLPI, 5Ј-TGC CAC CTA CTT CTG TAG-3Ј and 5Ј-CCA Results CAC TGG TTT GCG AAT-3Ј. The serum level of ␣1-antitrypsin was Development of emphysema is enhanced in Nrf2-KO mice determined by enzymatic assay as previously described (27). To elucidate the role of Nrf2 in protection against emphysema, we Immunoblot analysis first evaluated the development of emphysema 21 days following Alveolar macrophages obtained from BAL were untreated or treated with PPE treatment. No pathological differences were observed be- diethylmaleate for 3 h. For the detection of Nrf2 and HO-1, 20 or 2 ␮g, tween wild-type and Nrf2-KO mice before PPE treatment upon respectively, of the total cell extracts were separated by SDS-PAGE in the microscopic examination (Fig. 1, A and B). After PPE treatment, 6970 Nrf2 PROTECTION AGAINST THE DEVELOPMENT OF EMPHYSEMA

Magnitude and duration of elastase-induced pulmonary inflammation are enhanced in Nrf2-KO mice To delineate the contribution of preceding lung inflammation to the development of emphysema, the severity of the initial hemorrhagic inflammation was compared between wild-type and Nrf2-KO mice 1 and 4 days after PPE treatment. In wild-type mice, hemorrhagic lung inflammation characterized by alveolar hemorrhage, alveolar edema, and the infiltration of neutrophils into airspaces was observed 1 day after PPE treatment (Fig. 2A). How- ever, the hemorrhagic inflammation had resolved by day 4 following PPE treatment and emphysema had not developed by that time (Fig. 2B). In contrast, the pathological changes were much more severe in Nrf2-KO mice (Fig. 2C) than in wild-type mice by day 1 (Fig. 2A) and hemorrhagic lung inflammation persisted by day 4 (Fig. 2D). It should be noted that enlargement of the airspaces had already been observed in the lungs of Nrf2-KO mice by day 4 (Fig. 2D). To quantify the enhancement of initial lung inflammation in Nrf2-KO mice, we analyzed several inflammatory parameters us- Downloaded from ing BAL fluid. By day 1, the hemoglobin concentration in BAL fluid was significantly higher in Nrf2-KO mice than in wild-type FIGURE 1. PPE-induced emphysema was exacerbated in Nrf2-KO mice (Fig. 3A). Although the hemoglobin concentration had de- mice. H&E staining of lung tissue sections from wild-type mice (A)21 creased to control values in wild-type mice by day 4, the level of days after treatment with saline, Nrf2-KO mice (B) 21 days after treatment hemoglobin was still high in Nrf2-KO mice. Similarly, the albumin with saline, wild-type mice (C) 21 days after treatment with PPE, and concentration, an indicator of lung vascular permeability, in BAL http://www.jimmunol.org/ Nrf2-KO mice (D) 21 days after treatment with PPE. Bar ϭ 100 ␮m. E, fluid was significantly higher in Nrf2-KO mice than in wild-type ϩ/ϩ Static lung compliance of wild-type mice (Nrf2 )orNrf2-KO mice mice both 1 and 4 days after PPE treatment (Fig. 3B). (Nrf2Ϫ/Ϫ) 21 days after treatment with PPE or saline. Data are expressed The number of neutrophils present in BAL fluid was also sig- as the mean Ϯ SEM of four mice from each group. Significantly different p Ͻ 0.01). nificantly greater in Nrf2-KO mice than in wild-type mice 1 day ,ء) from the other groups after PPE treatment (Fig. 3C). By day 4, the number of neutrophils remained high in Nrf2-KO mice, but recovered to a basal value in enlargement of airspaces and alveolar wall destruction were ob- wild-type mice (Fig. 3C). Alveolar macrophages were induced by PPE treatment in both wild-type and Nrf2-KO mice (Fig. 3D). served in both wild-type (Fig. 1C) and Nrf2-KO mice (Fig. 1D). by guest on September 24, 2021 However, these pathological changes were much more severe in However, the induction was transient in wild-type mice and Nrf2-KO mice than in wild-type mice. As can be seen in Fig. 1D, peaked at day 1, yet the induction persisted in Nrf2-KO mice. The the alveolar walls are more severely damaged in the lungs of neutrophil elastase activity in BAL fluid was significantly elevated Nrf2-KO mice treated with PPE compared with wild-type mice. To in Nrf2-KO mice compared with that in wild-type mice both 1 and further characterize the development of emphysema in Nrf2-KO 4 days after PPE treatment (Fig. 3E). These results demonstrate animals, we determined the airspace enlargement by measuring the that Nrf2-KO animals develop severe pulmonary inflammation and mean linear intercept in 20 randomly selected fields from H&E exhibit a higher protease activity in the airspaces in the early pe- stained tissue sections. The mean linear intercept values in riod following PPE treatment. Consequently, emphysema was ap- Nrf2-KO mice were approximately three times higher than values parent by day 4 in Nrf2-KO animals. found in wild-type mice after PPE treatment (Table I). A pulmo- Neutrophil elastase activity in BAL fluids reflects the balance in nary function test revealed that static lung compliance was signif- elastase and anti-elastase activity in the airspaces. To clarify icantly increased in PPE-treated Nrf2-KO mice compared with whether a high protease activity in Nrf2-KO mice is due to the compliance in saline-treated Nrf2-KO animals 21 days after enhanced expression of neutrophil elastase, its expression was treatment, showing very good agreement with histological ob- evaluated in neutrophils obtained from blood or BAL fluids 1 day servations (Fig. 1E). This increase in static lung compliance after PPE treatment in both wild-type and Nrf2-KO mice. In both was not observed in wild-type mice following PPE treatment genotypes of mice, only a few mRNA transcripts were expressed (Fig. 1E). Taken together, these histological and physiological in blood neutrophils. Although neutrophils infiltrated into the air- evaluations demonstrate that Nrf2-KO animals are more sus- spaces by stimulation with PPE exhibited significantly higher ex- ceptible to PPE-induced emphysema. pression of neutrophil elastase, expression was not different between wild-type and Nrf2-KO mice (Fig. 3F). Thus, a high protease activity in Nrf2-KO mice may not be due to increased Table I. Mean linear intercept elastase balance but due to decreased anti-elastase balance.

Mean Linear Intercept The inducible expression of host defense genes is attenuated in Group (␮m) Nrf2-KO mice Wild-type mice, saline-treated 58.2 Ϯ 4.2 Nrf2-KO mice, saline-treated 56.7 Ϯ 2.5 To clarify whether a high protease activity in Nrf2-KO mice is due Wild-type mice, elastase-treated 86.7 Ϯ 14.2 to the inactivation of antiprotease, we examined the expression of Nrf2-KO mice, elastase-treated 258.8 Ϯ 15.9b two antiprotease genes, ␣1-antitrypsin and SLPI. The constitutive ␣ a Results are represented as mean Ϯ SEM in each of ﬁve animals. expression level of 1-antitrypsin observed in both lung and liver b Signiﬁcantly different from other groups ( p Ͻ 0.01). was unaltered by PPE treatment and equivalent to that in Nrf2-KO The Journal of Immunology 6971

FIGURE 2. Inflammation was exacerbated in Nrf2-KO lung in the initial stage of PPE-induced emphysema. H&E staining of lung tissue sections from wild-type (A and B) and Nrf2-KO (C and D) mice 1 day (A and C) and 4 days (B and D) after treatment with PPE. Bar ϭ 100 ␮m. Downloaded from http://www.jimmunol.org/ mice (Fig. 4A). The activity of serum ␣1-antitrypsin was consistent (Fig. 6, D and H). F4/80-positive macrophages were similarly de- with this result (Fig. 4B). Interestingly, the SLPI gene was signif- tected in alveoli of both wild-type and Nrf2-KO mice (Fig. 6, A icantly induced in the lungs of wild-type mice 1 and 4 days after and E). In wild-type mice, macrophages were strongly stained with PPE treatment (Fig. 4C). In Nrf2-KO mice, however, SLPI mRNA PrxI, HO-1, and SLPI 1 day after PPE treatment (Fig. 6, B–D). In was not induced by the stimulation of PPE (Fig. 4C). The expres- contrast, no positive staining by these Abs was observed at any sion of SLPI in the liver was unaltered by PPE treatment in both time point in the lungs of Nrf2-KO mice (Fig. 6, F–H and data not genotypes of mice (Fig. 4C). A database search revealed that sev- shown). To assess further whether Nrf2 is actually expressed in eral ARE consensus-like sequences exist in the 1.2-kb promoter alveolar macrophages, we performed immunoblot analysis using region of the mouse SLPI gene (EMBL Accession no. AF205374). anti-Nrf2 Ab and alveolar macrophage obtained from BAL. The by guest on September 24, 2021 Cotransfection of the 1.2-kb SLPI promoter linked to a luciferase result demonstrated that the expression of Nrf2 and its target gene reporter gene and Nrf2 expression plasmid into RAW 264.7 mac- HO-1 were induced in response to the Nrf2 activator diethylmal- rophages revealed that the overexpression of Nrf2 activates re- eate in wild-type alveolar macrophages, but not in Nrf2-KO alve- porter gene expression in a dose-dependent manner (Fig. 4D). De- olar macrophages (Fig. 6I). letion of the region 950 bp upstream from the SLPI promoter that contains several potential AREs resulted in a marked decrease in Transplantation of Nrf2-positive bone marrow cells attenuated Nrf2-mediated luciferase gene expression. These results indicated elastase-induced lung inflammation and emphysema in Nrf2-KO that the expression of SLPI is under Nrf2 regulation. mice In addition to protease/antiprotease imbalance, oxidative stress The results thus far imply that alveolar macrophages are the main is thought to be an important contributor to the pathogenesis of site of Nrf2-mediated gene expression. To further clarify the con- emphysema. Several studies have demonstrated that Nrf2 regulates tribution of macrophages to Nrf2-mediated protection against ela- the gene expression of a wide range of antioxidant enzymes. We stase-induced lung damage, bone marrow cells from wild-type therefore examined the induction of GST, NQO1, HO-1, and PrxI mice were transplanted into Nrf2-KO mice. We first examined the in the lungs of both wild-type and Nrf2-KO mice before and after appearance of donor cells in the alveolar spaces of the recipient PPE treatment. Small amounts of these mRNA transcripts were mice. BAL was performed on days 5, 14, and 35 after BMT and constitutively expressed in the lungs of both genotypes before PPE macrophages were isolated using a macrophage marker. The Nrf2 treatment. In wild-type mice, the expressions of PrxI and HO-1 wild-type allele derived from BMT was detectable in these isolated were significantly elevated 1 and 4 days after PPE treatment (Fig. cells by PCR 5 days after BMT and reached a saturated level by 14 5, A and B). In Nrf2-KO mice, however, these antioxidant enzymes days (Fig. 7A). were not observed to be up-regulated upon PPE treatment (Fig. 5, We then examined the effects of Nrf2 expression on the survival A and B). Comparably, the pulmonary expressions of GST-Yc and of macrophages. To this end, we used female wild-type mice as NQO1 were significantly increased in the lungs of wild-type mice, recipients and male Nrf2-KO or wild-type mice as donors, and but not in Nrf2-KO mice, 1 and 4 days after PPE treatment (Fig. 5, examined the contribution of male cells to alveolar macrophages C and D). of female recipients using SRY gene as a marker (28). We found that significant numbers of donor cells actually flowed in the al- Nrf2-regulated antioxidant and antiprotease proteins are veolar space of the lung of recipient mice without stimuli at day 14 expressed in alveolar macrophages after BMT. The contribution of male cells to alveolar macrophages To clarify which cell types express Nrf2-regulated genes, lungs of female recipients was 8.1% with wild-type mouse-derived bone were immunostained with anti-F4/80 (Fig. 6, A and E), anti-PrxI marrow cells and 12.8% with Nrf2-KO mouse-derived bone mar- (Fig. 6, B and F), anti-HO-1 (Fig. 6, C and G), and anti-SLPI Abs row cells, showing that there was no significant difference in the 6972 Nrf2 PROTECTION AGAINST THE DEVELOPMENT OF EMPHYSEMA Downloaded from

FIGURE 4. Expression of SLPI is severely affected in Nrf2-KO mice. mRNA expression of ␣1-antitrypsin (A) and SLPI (C) in the lung (top) and the liver (bottom) of wild-type mice (Nrf2ϩ/ϩ)orNrf2-KO mice (Nrf2Ϫ/Ϫ) before (Before) and 1 and 4 days after treatment with PPE. The band http://www.jimmunol.org/ density was normalized by GAPDH. B, Serum ␣1-antitrypsin activity in wild-type mice (Nrf2ϩ/ϩ)orNrf2-KO mice (Nrf2Ϫ/Ϫ) before (Before) and 1 and 4 days after treatment with PPE. Signiﬁcantly different in comparison -p Ͻ 0.05). D, A schematic repre ,ء) with corresponding Nrf2-KO mice sentation (top) of the luciferase reporter constructs is shown. SLPI-luc or the SpeI fragment from SLPI-luc (⌬SpeI-luc) reporter constructs were transfected into RAW 264.7 cells along with an increasing amount of mouse Nrf2 expression plasmid. The luciferase activity of reporter alone

was set to 100% and the means of four independent experiments are shown by guest on September 24, 2021 with the SEM.

FIGURE 3. Quantitative analysis of lung inflammation in the initial stage of PPE-induced emphysema. Hemoglobin content (A), albumin con- ingly, Nrf2-regulated host defense proteins such as HO-1, PrxI, centration (B), number of neutrophils (C), number of macrophages (D), and and SLPI were expressed in macrophages 1 day after PPE treat- neutrophil elastase activity (E) in BAL fluids from wild-type mice ment in mice transplanted with wild-type mouse bone marrow (Nrf2ϩ/ϩ)orNrf2-KO mice (Nrf2Ϫ/Ϫ) before (Before) and 1, 4, 8, and 21 (Fig. 7E). These results demonstrate that the Nrf2-mediated de- days after treatment with PPE. Data are expressed as the mean Ϯ SEM of fense system works mainly in macrophages in the protection eight mice from each group. Significantly different in comparison with against PPE-induced emphysema. p Ͻ 0.05). F, Expression of neutrophil ,ء) corresponding wild-type mice elastase in the neutrophils obtained from blood and BAL fluids 1 day after Discussion treatment with PPE. RT-PCR analyses (top panels) and quantification of This study unveiled for the first time that Nrf2-KO mice are highly the bands by densitometric analysis (bottom panels) are represented. Sig- nificantly different from blood neutrophils (‡, p Ͻ 0.05). susceptible to PPE-induced emphysema. We found that lung inflammation in both the initial and subsequent stages of PPE-provoked emphysema was markedly exacerbated in Nrf2-KO mice. influx of Nrf2-null donor cells from that of wild-type donor cells The expressions of several antioxidant enzyme genes, such as (Fig. 7B). These results demonstrate that Nrf2 does not influence NQO1, GST-Yc, HO-1, and PrxI and the antiprotease gene SLPI the influx of macrophages from bone marrow cells without stimuli. were markedly induced by PPE treatment in wild-type lung. How- Therefore, using this model system, we administered PPE 14 days ever, the inducible expression of these genes was largely abolished after BMT. in the lungs of Nrf2-KO mice. Immunohistochemical analysis re- Destruction of the alveolar architecture 21 days after PPE treat- vealed that the inducible expression of HO-1, PrxI, and SLPI was ment was markedly less severe in mice transplanted with wild-type observed in alveolar macrophages in wild-type lung. In Nrf2-KO mouse bone marrow (Fig. 7C, left) than in mice transplanted with mice transplanted with wild-type mouse bone marrow, both initial Nrf2-KO bone marrow (Fig. 7C, right). The degree of initial lung lung inflammation and subsequent emphysema were significantly inflammation was also attenuated in mice transplanted with wild- ameliorated with the appearance of Nrf2-positive macrophages. type bone marrow, compared with mice transplanted with Thus, Nrf2 is a key regulator in the macrophage-mediated defense Nrf2-KO bone marrow. The BAL hemoglobin level (Fig. 7D, left), system against lung injury. albumin concentration (Fig. 7D, center), and neutrophil number Elastase, especially PPE, has been used to provoke emphysema (Fig. 7D, right) were significantly decreased 1 day after PPE treat- in animal models. Kinetic studies and analysis of mutant mice ment in mice transplanted with wild-type bone marrow, compared lacking inflammatory mediators have demonstrated that emphy- with mice transplanted with Nrf2-KO bone marrow. Correspond- sema induced by a low dose of PPE, as is the case for our study, The Journal of Immunology 6973 Downloaded from

FIGURE 5. Expression of antioxidant and antiprotease genes is severely http://www.jimmunol.org/ affected in Nrf2-KO mice. RNA blot analysis showing the expression of ϩ/ϩ antioxidant defense enzymes in the lungs of wild-type mice (Nrf2 )or FIGURE 6. Expression of antioxidant and antiprotease genes was in- Nrf2-KO mice (Nrf2Ϫ/Ϫ ) before (Before) and 1 and 4 days after treatment duced in alveolar macrophages in the lung. Immunocytochemical localiza- with PPE. A blot representing each expression profile is shown atop each tion of macrophage (A and E), PrxI (B and F), HO-1 (C and G), and SLPI graph. The blots (bottom) were then quantified by densitometric analysis. (D and H) in the lungs of wild-type (A–D) and Nrf2-KO (E–H) mice 1 day ,ء) Significantly different in comparison with corresponding Nrf2-KO mice after treatment with PPE. Arrows indicate positive cells. Bar ϭ 100 ␮m. I, p Ͻ 0.05). Alveolar macrophages obtained from BAL were either untreated or treated with diethylmaleate for 3 h, and total cell extracts were analyzed by anti-

Nrf2 (left) or anti-HO-1 (right) Ab. As a loading control, membranes were by guest on September 24, 2021 is not due to the immediate proteolytic activity of PPE, but is also probed with anti-lamin B Ab. mostly due to the subsequent inflammation caused by PPE (29– 32). Thus, the susceptibility of Nrf2-KO mice to PPE-induced emphysema might be due to a decreased antiprotease activity against trophil elastase damage (33). Unlike ␣1-antitrypsin, SLPI is pro- PPE itself or a decreased anti-inflammatory activity against PPE- duced at the site of inflammation by several inflammatory stimuli induced inflammation, or both. The latter possibility is in close including LPS (34), neutrophil elastase (35), and proinflammatory agreement with the observation that Nrf2-KO mice are highly sen- cytokines (36). SLPI, therefore, may participate in the first line of sitive to pulmonary inflammation caused by environmental toxins defense against protease damage at the inflamed site. In addition to such as butylated hydroxytoluene (8) and hyperoxia (9). the antiprotease activity, murine SLPI has been shown to attenuate In addition to parenchymal inflammation and oxidative stress, inflammatory cytokine production in macrophages (37, 38). More- an imbalance between proteases and antiproteases in the lung is over, human SLPI has been demonstrated to prevent activation of important in the pathogenesis of emphysema. Neutrophil elastase the inflammatory transcription factor NF-␬B by inhibition of I␬B has been implicated as the critical protease responsible for lung degradation (39). Thus, reduced production of SLPI, which acts as destruction. In the present study, neutrophil elastase activity in both antiprotease and anti-inflammatory factor at the site of lung BAL fluids was significantly higher in Nrf2-KO mice than in wild- inflammation, may be an additional important factor that explains type mice after PPE treatment, suggesting that an imbalance be- the susceptibility of Nrf2-KO mice to emphysema. To our knowl- tween protease and antiprotease levels is conspicuous in Nrf2-KO edge, this is the first report demonstrating that the antiprotease mice. This elevated neutrophil elastase activity in Nrf2-KO mice gene is under Nrf2 regulation. might be due to increased neutrophil infiltration or due to a de- In the present study, induction of Nrf2 target genes, such as crease in antiprotease gene expression. HO-1, PrxI, and SLPI was observed in alveolar macrophages in ␣1-Antitrypsin is the major antiprotease that inhibits a number wild-type mice after PPE treatment. This observation is consistent of serine proteases such as neutrophil elastase. Individuals with a with a series of studies in which Nrf2 target genes, including HO-1 hereditary deficiency in ␣1-antitrypsin are known to develop pro- and PrxI, were induced abundantly in macrophages (16, 17). It has gressive emphysema at a relatively early age (6, 7). However, both also been demonstrated that macrophages, as well as epithelial the expression of ␣1-antitrypsin in the liver and its activity in cells, are the major sources of SLPI (34, 38). Moreover, we dem- serum did not differ between wild-type and Nrf2-KO mice. Inter- onstrated that the promoter activity of SLPI up-regulated by Nrf2 estingly, whereas the SLPI gene was highly inducible in the lungs in the macrophage cell line RAW 264.7 (Fig. 4D). Because mac- of wild-type mice after PPE treatment, induction of the SLPI gene rophages are the major source of reactive oxygen species and pro- did not occur in the lungs of Nrf2-KO mice. teases, as well as inflammatory cytokines, it is reasonable to hy- SLPI is an 11.7-kDa mucosal secretory protein identified as a pothesize that the aberrant gene expression in Nrf2-KO serine protease inhibitor and involved in protection against neu- macrophages may cause an overwhelming inflammatory response 6974 Nrf2 PROTECTION AGAINST THE DEVELOPMENT OF EMPHYSEMA

Acknowledgments We thank Dr. Tania O’Connor for critical reading of the manuscript and Mark J. Kiel for help and advice.

Disclosures The authors have no ﬁnancial conﬂict of interest.

References 1. Murray, C. J., and A. D. Lopez. 1997. Alternative projections of mortality and disability by cause 1990–2020: Global Burden of Disease Study. Lancet 349: 1498–1504. 2. Barnes, P. J. 2000. Chronic obstructive pulmonary disease. N. Engl. J. Med. 343: 269–280. 3. Repine, J. E., A. Bast, and I. Lankhorst. 1997. Oxidative stress in chronic obstructive pulmonary disease. Am. J. Respir. Crit. Care Med. 156: 341–357. 4. Barnes, P. J. 2003. New concepts in chronic obstructive pulmonary disease. Annu. Rev. Med. 54: 113–129. 5. Siafakas, N. M., and E. G. Tzortzaki. 2002. Few smokers develop COPD: why? Respir. Med. 96: 615–624. 6. Hubbard, R. C., and R. G. Crystal. 1991. Antiproteases. In The Lung: Scientiﬁc Foundations. R. G. Crystal, J. B. West, and P. J. Barnes, eds. Raven Press. New

York. pp. 1775–1787. Downloaded from 7. McElvaney, N. G., and R. G. Crystal. 1997. Inherited susceptibility of the lung to proteolytic injury. In The Lung: Scientific Foundations. R. G. Crystal, J. B. West, E. R. Weibel, and P. J. Barnes, eds. Lippincott-Raven. Philadelphia, pp. 2537–2553. 8. Itoh, K., T. Chiba, S. Takahashi, T. Ishii, K. Igarashi, Y. Katoh, T. Oyake, N. Hayashi, K. Satoh, I. Hatayama, et al. 1997. An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements. Biochem. Biophys. Res. Commun. 236: 313–322. 9. Ishii, T., K. Itoh, S. Takahashi, H. Sato, T. Yanagawa, Y. Katoh, S. Bannai, and http://www.jimmunol.org/ M. Yamamoto. 2000. Transcription factor Nrf2 coordinately regulates a group of oxidative stress-inducible genes in macrophages. J. Biol. Chem. 275: 16023–16029. 10. Itoh, K., K. I. Tong, and M. Yamamoto. 2004. Molecular mechanism activating Nrf2-Keap1 pathway in regulation of adaptive response to electrophiles. Free FIGURE 7. Susceptibility of Nrf2-KO mice to PPE-induced emphy- Radical Biol. Med. 36: 1208–1213. sema was rescued by transplantation of wild-type bone marrow cells. A, 11. Kobayashi, A., T. Ohta, and M. Yamamoto. 2004. Unique function of the Nrf2- Expression of Nrf2 wild-type allele in sorted macrophages from Nrf2-KO Keap1 pathway in the inducible expression of antioxidant and detoxifying en- mice 5, 14, and 35 days after transplantation of wild-type bone marrow zymes. Methods Enzymol. 378: 273–286. ϩ/ϩ Ϫ/Ϫ 12. Itoh, K., N. Wakabayashi, Y. Katoh, T. Ishii, T. O’Connor, and M. Yamamoto. cells (Nrf2 BMT) or Nrf2-KO bone marrow cells (Nrf2 BMT). B, 2003. Keap1 regulates both cytoplasmic-nuclear shuttling and degradation of ϩ/ϩ Ϫ/Ϫ by guest on September 24, 2021 Male wild-type (Nrf2 )orNrf2-KO (Nrf2 ) bone marrow cells were Nrf2 in response to electrophiles. Genes Cells 8: 379–391. transplanted to female recipient mice. Fourteen days after BMT, the ratio 13. McMahon, M., K. Itoh, M. Yamamoto, and J. D. Hayes. 2003. Keap1-dependent of male to female DNA amount in alveolar macrophages was examined by proteasomal degradation of transcription factor Nrf2 contributes to the negative regulation of antioxidant response element-driven gene expression. J. Biol. Chem. quantitative PCR analysis of the SRY gene. The mean of the ratios obtained 278: 21592–21600. from three mice in each group is presented with SEM. C, Lung tissue from 14. Yoh, K., K. Itoh, A. Enomoto, A. Hirayama, N. Yamaguchi, M. Kobayashi, Nrf2ϩ/ϩ BMT mice (left) or Nrf2Ϫ/Ϫ BMT mice (right) 21 days after N. Morito, A. Koyama, M. Yamamoto, and S. Takahashi. 2001. Nrf2-deficient treatment with PPE. H&E stain with bar ϭ 100 ␮m. D, Hemoglobin con- female mice develop lupus-like autoimmune nephritis. Kidney Int. 60: 1343–1353. left center tent ( ), albumin concentration ( ), and the number of neutrophils 15. Chan, K., X. D. Han, and Y. W. Kan. 2001. An important function of Nrf2 in ϩ/ϩ Ϫ/Ϫ (right) in the BAL fluids of wild-type (Nrf2 ), Nrf2-KO (Nrf2 ), and combating oxidative stress: detoxification of acetaminophen. Proc. Natl. Acad. Nrf2-KO mice transplanted with wild-type bone marrow (Nrf2ϩ/ϩ BMT), Sci. USA 98: 4611–4616. and Nrf2-KO mice transplanted with Nrf2-KO bone marrow (Nrf2Ϫ/Ϫ 16. Enomoto, A., K. Itoh, E. Nagayoshi, J. Haruta, T. Kimura, T. O’Connor, BMT) mice 1 day after treatment with PPE. Data are expressed as the mean T. Harada, and M. Yamamoto. 2001. High sensitivity of Nrf2 knockout mice to acetaminophen hepatotoxicity associated with decreased expression of ARE-reg- Ϯ SEM of four mice from each group. Significantly different from wild- ulated drug metabolizing enzymes and antioxidant genes. Toxicol. Sci. 59: ϩ ϩ .p Ͻ 0.05) or from Nrf2 / BMT mice (#, p Ͻ 0.05). E, 169–177 ,ء) type mice Immunocytochemical localization of HO-1 (left), PrxI (center), and SLPI 17. Aoki, Y., H. Sato, N. Nishimura, S. Takahashi, K. Itoh, and M. Yamamoto. 2001. (right) in the lungs of Nrf2-KO mice transplanted with wild-type bone Accelerated DNA adduct formation in the lung of the Nrf2 knockout mouse exposed to diesel exhaust. Toxicol. Appl. Pharmacol. 173: 154–160. marrow. Arrows indicate positive cells. 18. Ramos-Gomez, M., M. K. Kwak, P. M. Dolan, K. Itoh, M. Yamamoto, P. Talalay, and T. W. Kensler. 2001. Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor-deficient mice. Proc. Natl. Acad. Sci. USA 98: 3410–3415. 19. Chan, K., and Y. W. Kan. 1999. Nrf2 is essential for protection against acute and thus evoke lung injury. In Nrf2-KO mice transplanted with pulmonary injury in mice. Proc. Natl. Acad. Sci. USA 96: 12731–12736. wild-type mouse bone marrow, both initial lung inflammation and 20. Cho, H. Y., A. E. Jedlicka, S.P. Reddy, T. W. Kensler, M. Yamamoto, subsequent emphysema were significantly attenuated with the ap- L. Y. Zhang, and S. R. Kleeberger. 2002. Role of NRF2 in protection against hyperoxic lung injury in mice Am. J. Respir. Cell Mol. Biol. 26: 175–182. pearance of Nrf2-positive macrophages. A defense system based 21. Itoh, K., M. Mochizuki, Y. Ishii, T. Shibata, Y. Kawamoto, T. Ishii, V. Kelly, on macrophages, therefore, is thought to be important in protection K. Sekizawa, K. Uchida, and M. Yamamoto. 2004. Transcription factor Nrf2 12,14 against lung injury. regulates inflammation by mediating the effect of 15-deoxy-⌬ -prostaglandin- J2. Mol. Cell. Biol. 24: 36–45. Chronic obstructive pulmonary disease is a major cause of 22. Mochizuki, M., Y. Ishii, K. Itoh, T. Iizuka, Y. Morishima, T. Kimura, chronic morbidity and mortality worldwide, so an effective treat- T. Kiwamoto, Y. Matsuno, A. E. Hegab, A. Nomura, et al. 2005. Role of 15- ⌬12,14 deoxy- -prostaglandin-J2 and Nrf2 pathways in protection against acute lung ment is in great want. Recently, the anti-inflammatory activities of injury. Am. J. Respir. Crit. Care Med. 171: 1260–1266. the Nrf2/ARE system have been widely recognized (40). Thus, 23. Hayes, J. A., A. Korthy, and G. L. Snider. 1975. The pathology of elastase- activation of Nrf2 in alveolar macrophages might be a useful ther- induced panacinar emphysema in hamsters. J. Pathol. 117: 1–14. 24. Kaplan, P. D., C. Kuhn, and J. A. Pierce. 1973. The induction of emphysema with apeutic approach for protection against the development of lung elastase. I. The evolution of the lesion and the influence of serum. J. Lab. Clin. inflammation and emphysema. Med. 82: 349–356. The Journal of Immunology 6975

25. Saetta, M., R. J. Shiner, G. E. Angus, W. D. Kim, N. S. Wang, M. King, J. B. West, E. R. Weibel, and P. J. Barnes, eds. Lippincott-Raven. Philadelphia. H. Ghezzo, and M. G. Cosio. 1985. Destructive index: a measurement of lung pp. 2219–2235. parenchymal destruction in smokers. Am. Rev. Respir. Dis. 131: 764–769. 34. Jin, F. Y., C. Nathan, D. Radzioch, and A. Ding. 1997. Secretory leukocyte 26. Sakamaki, F., A. Ishizaka, T. Urano, K. Sayama, H. Nakamura, T. Terashima, protease inhibitor: a macrophage product induced by and antagonistic to bacterial Y. Waki, S. Tasaka, N. Hasegawa, K. Sato, et al. 1996. Effect of a specific lipopolysaccharide. Cell 88: 417–426. neutrophil elastase inhibitor, ONO-5046, on endotoxin-induced acute lung injury. 35. Abbinante-Nissen, J. M., L. G. Simpson, and G. D. Leikauf. 1993. Neutrophil Am. J. Respir. Crit. Care Med. 153: 391–397. elastase increases secretory leukocyte protease inhibitor transcript levels in air- ␣ 27. Dietz, A. A., H. M. Rubinstein, and L. Hodges. 1976. Use of -N-benzoyl-L- way epithelial cells. Am. J. Physiol. 265: L286–L292. ␣ arginine-p-nitroanilide as trypsin substrate in estimation of 1-antitrypsin. Clin. 36. Sallenave, J. M., J. Shulmann, J. Crossley, M. Jordana, J. Gauldie. 1994. Regu- Chem. 22: 1754–1755. ¨ lation of secretory leukocyte proteinase inhibitor (SLPI) and elastase-specific 28. Kiel, M. J., O. H. Yilmaz, T. Iwashita, O. H. Yilmaz, C. Terhorst, and inhibitor (ESI/elafin) in human airway epithelial cells by cytokines and neutro- S. J. Morrison. 2005. SLAM family receptors distinguish hematopoietic stem and philic enzymes. Am. J. Respir. Cell Mol. Biol. 11: 733–741. progenitor cells and reveal endothelial niches for stem cells. Cell 121: 1109–1121. 37. Betsuyasu, T., K. Takeyabu, M. Tanino, and M. Nishimura. 2002. Role of se- 29. Lucey, E. C., P. J. Stone, T. G. Christensen, R. Breuer, and G. L. Snider. 1988. cretory leukocyte protease inhibitor in the development of subclinical emphy- Eur. Respir. J. An 18-month study of the effects on hamster lungs of intratracheally administered sema. 19: 1051–1057. human neutrophil elastase. Exp. Lung Res. 14: 671–686. 38. Odaka, C., T. Mizuochi, J. Yang, and A. Ding. 2003. Murine macrophages pro- 30. Stone, P. J., E. C. Lucey, J. D. Calore, M. P. McMahon, G. L. Snider, and duce secretory leukocyte protease inhibitor during clearance of apoptotic cells: C. Franzblau. 1988. Defenses of the hamster lung against human neutrophil and implications for resolution of the inflammatory response. J. Immunol. 171: porcine pancreatic elastase. Respiration 54: 1–15. 1507–1514. 31. Lucey, E. C., J. Keane, P. P. Kuang, G. L. Snider, and R. H. Goldstein. 2002. 39. Lentsch, A. B., J. A. Jordan, B. J. Czermak, K. M. Diehl, E. M. Younkin, Severity of elastase-induced emphysema is decreased in tumor necrosis factor-␣ V. Sarma, and P. A. Ward. 1999. Inhibition of NF-␬B activation and augmen- and interleukin-1␤ receptor-deficient mice. Lab. Invest. 82: 79–85. tation of I␬B␤ by secretory leukocyte protease inhibitor during lung inflamma- 32. Snider, G. L., and C. B. Sherter. 1977. A one-year study of the evolution of tion. Am. J. Pathol. 154: 239–247. elastase-induced emphysema in hamsters. J. Appl. Physiol. 43: 721–729. 40. Chen, X. L., and C. Kunsch. 2004. Induction of cytoprotective genes through 33. McElvaney, N. G., and R. G. Crystal. 1997. Inherited susceptibility of the lung nrf2/antioxidant response element pathway: a new therapeutic approach for the Downloaded from to proteolytic injury. In The Lung: Scientific Foundations. R. G. Crystal, treatment of inflammatory diseases. Curr. Pharm. Des. 10: 879–891. http://www.jimmunol.org/ by guest on September 24, 2021