Abstract
Pulmonary macrophages are one of the sources of various antioxidant and detoxification enzymes for which NF-E2–related factor 2 (Nrf2) is a key transcriptional factor. Although Nrf2 deficiency reportedly induces severe emphysema in mice exposed to cigarette smoke (CS), no reports have studied Nrf2 regulation in chronic obstructive pulmonary disease (COPD). In this study, Nrf2 activation in response to CS was evaluated in human alveolar macrophages, and age-related differences in CS-induced Nrf2 regulation in mouse alveolar macrophages were determined. Furthermore, Nrf2 mRNA levels in human macrophages harvested by bronchoalveolar lavage or laser capture microdissection were measured. CS induced nuclear Nrf2 accumulation and up-regulation of Nrf2 target genes without substantial changes in Nrf2 mRNA levels in human alveolar macrophages. In humans, the Nrf2 mRNA level in lavaged macrophages of young subjects (n = 14) was independent of smoking status; however, the Nrf2 mRNA level was down-regulated in the lavaged macrophages of older current smokers (n = 14) compared with older nonsmokers (n = 9) (P < 0.001). Among older subjects, the macrophage Nrf2 mRNA level was inversely correlated with oxidized glutathione and carbonylated albumin levels in bronchoalveolar lavage fluid. In mice, aging suppressed the CS-induced up-regulation of Nrf2 target genes, as well as Nrf2, in alveolar macrophages. Furthermore, the Nrf2 mRNA level was decreased in laser capture microdissection–retrieved macrophages obtained from subjects with COPD (n = 10) compared with control subjects (n = 10) (P = 0.001). In conclusion, CS induces Nrf2 activation in macrophages, and Nrf2 expression is decreased in the macrophages of older current smokers and patients with COPD.
There have been no studies on the relationship between Nrf2 and chronic obstructive pulmonary disease (COPD). Acute cigarette smoke exposure leads to Nrf2 activation in human macrophages, and Nrf2 expression is decreased in pulmonary macrophages in current smokers and patients with COPD.
NF-E2–related factor 2 (Nrf2), a member of the cap'n'collar family of the basic leucine zipper transcription factors, is a key transcription factor that regulates the expression of several antioxidant and detoxification genes (3, 4). Nrf2 heterodimerizes with another basic leucine zipper protein, such as small Maf (MafF, MafG, and MafK) or Jun (c-Jun, Jun-D, and Jun-B) and then binds to the antioxidant response element (ARE) in target gene promoters (5). In normal mouse lungs, immunohistochemical studies have shown that Nrf2 protein is localized in airway epithelium, in type II alveolar epithelial cells, and in resident macrophages (6). Nrf2-deficient mice are highly susceptible to oxidative stress and reactive electrophiles; they develop severe emphysema when exposed to cigarette smoke or elastase (7–9).
There is a marked increase in the number of macrophages in the lungs of smokers and in patients with COPD. Macrophages play an important role in the pathogenesis of COPD because they release many compounds, such as reactive oxygen species, chemotactic factors, inflammatory cytokines, smooth muscle constrictors, mucus gland activators, extracellular matrix proteins, and matrix metalloproteinases (10). On the other hand, pulmonary macrophages are the primary defense against inhaled particles; they phagocytose deposited particles and detoxify inhaled organic fractions through metabolic activation by phase I and II enzymes (11). It has been reported that macrophages produce many Nrf2-regulated antioxidants (4, 8, 12–17). Indeed, intratracheal elastase treatment has been shown to up-regulate some Nrf2 target genes, primarily in alveolar macrophages in mice. Moreover, transplantation of bone marrow derived from wild-type mice into Nrf2-deficient mice has been shown to rescue severe elastase-induced emphysema; Nrf2-positive macrophages appear, which suggests that macrophages play a crucial role via the Nrf2-dependent system in protecting the lungs against the development of emphysema (9).
There have been no studies dealing with Nrf2 regulation in pulmonary macrophages exposed to cigarette smoke (CS) or with the relationship between Nrf2 regulation in human pulmonary macrophages and COPD. In the present study, Nrf2 regulation in response to CS exposure was assessed in vitro using human alveolar macrophages. Second, the effects of aging on CS-induced regulation of Nrf2 and its target genes in human and mouse alveolar macrophages were determined. Finally, the association between Nrf2 mRNA levels in the pulmonary macrophages, bronchiolar epithelium, and alveolar septa and the presence of COPD was investigated using site-specific gene expression analysis using dissection techniques.
Human alveolar macrophages were collected from healthy volunteers as described previously (18). The bronchoalveolar lavage fluid (BALF) was centrifuged, and the cell-free supernatants were stored at −80°C until use. Cell pellets were counted in a hematocytometer, and smears stained with Diff-Quik (Sysmex International Reagents, Kobe, Japan) were used to identify differential profiles after cytospin preparation. Differential counts were performed by examining at least 300 cells using a standard light microscope. BAL cells were adjusted to 1 × 106 cells/ml and plated on plastic plates for 1 hour at 37°C in RPMI 1640 medium. After two washes in PBS, the adherent cells were used as alveolar macrophages for the following procedures. The alveolar macrophages were cultured in RPMI 1640 medium supplemented with 10% FCS and 100 U/ml penicillin/streptomycin.
Mouse alveolar macrophages were collected from male C57BL/6 mice (young and older groups; 3 mo and 20 mo of age, respectively) and male Imprinting Control Region (ICR) mice (adult and older groups; 8–10 mo and 19–20 mo of age, respectively) (CLEA Japan, Tokyo, Japan). The mice were killed by CO2 narcosis. The lungs were lavaged with 0.6 ml of saline five times through a tracheal cannula. The BAL cells were adjusted to 2 × 104 cells/ml (pooled from three mice for mRNA analysis) or 5 × 105 cells/ml (pooled from eight mice for protein analysis) and plated on plastic plates for 1 hour at 37°C in RPMI 1640 medium. After two washes in PBS, the adherent cells were used as alveolar macrophages for the following procedures. The alveolar macrophages were cultured in RPMI 1640 medium supplemented with 10% FCS and 100 U/ml penicillin/streptomycin.
The smoke of two cigarettes (12 mg of tar and 1.0 mg of nicotine) (Marlboro; Philip Morris, Richmond, VA) was bubbled through 15 ml of culture media using a 60-ml plastic syringe. Each cigarette was completely burned after six draws of the syringe, with each individual draw taking approximately 10 seconds to complete, and the smoke was bubbled into media at a speed of 1 ml/s. The resulting suspension was defined as 100% cigarette smoke extract (CSE) and was filtered through a 0.22-μm filter to remove bacteria and large particles. The pH of the CSE solution was between pH 7.1 and 7.4. Subsequently, the CSE was diluted to appropriate concentration with culture media. CSE concentrations of 10% for human macrophages and 2.5% for mouse macrophages were chosen to maintain cell viabilities at 80% after 24 hours of exposure to CSE. The cells were exposed to CSE within 15 minutes after the CSE was prepared. After incubation, the cells were harvested for RNA isolation or protein extraction.
For immunocytochemistry, the cells were grown on four-well Lab-TEK chamber slides (Nalge Nunc International Corp., Rochester, NY) and fixed in 4% paraformaldehyde for 10 minutes. After washing in PBS, the slides were processed using a catalyzed signal amplification kit (DakoCytomation, Glostrup, Denmark) according to the manufacturer's protocol. The slides were incubated for 15 minutes at room temperature with anti-Nrf2 rabbit polyclonal antibody (sc-13032; Santa Cruz Biotechnology Inc., Santa Cruz, CA) (19) diluted to 1:500 with PBS. The slides were counterstained with Mayer's hematoxylin (Muto Pure Chemicals, Tokyo, Japan).
For immunohistochemistry, the lung tissue was obtained from three lifelong nonsmokers, two Global Initiative for Obstructive Lung Disease (GOLD) stage I patients, and one GOLD patient with stage IV COPD who had lung resections to remove lung tumors. The tissue specimens were fixed in 10% phosphate-buffered formalin and embedded in paraffin; 5-μm sections were deparaffinized in xylene and rehydrated with graded alcohols. Antigenic activity was retrieved by incubating the specimens in 10 mM of citrate buffer (pH 6.0) in a microwave for 10 minutes. After washing in PBS, the sections were processed using a catalyzed signal amplification kit and prepared for immunocytochemistry. These sections were incubated for 15 minutes at room temperature with 1:500 anti-Nrf2 rabbit polyclonal antibody (Santa Cruz Biotechnology Inc.) and counterstained with Mayer's hematoxylin. Normal rabbit immunoglobulin fraction (DakoCytomation) was used as a negative control. To avoid run-to-run variations in the immunoreaction, all specimens were stained in the same run.
Nuclear extracts of human alveolar macrophages were made using ice-cold NE-PER Nuclear and Cytoplasmic Extraction Reagent (Pierce, Rockford, IL) according to the manufacturer's protocol. Whole-cell lysates of mouse alveolar macrophages were obtained with T-PER Tissue Extraction Reagent (Pierce). The protein concentrations were determined using the BCA Protein Assay (Pierce). For the Western blotting analysis, 3.5 μg of nuclear extracts or 10 μg of whole-cell lysates were resolved on 8% SDS-PAGE gels and transferred onto Immun-Blot PVDF membranes (Bio-Rad Laboratories, Hercules, CA). After blocking for 1 hour with 7.5% nonfat dried milk in TBS containing 0.5% Tween 20 at room temperature, the membranes were incubated overnight with 1:100 to 1:1,000 anti-Nrf2 rabbit polyclonal antibody (Santa Cruz Biotechnology Inc.) at 4°C, followed by incubation for 1 hour with 1:10,000 horseradish peroxidase–conjugated goat anti-rabbit secondary antibody (DakoCytomation). After washing, immunoreactive proteins were detected by chemiluminescence using Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA) and subsequent autoradiography.
Two sets of subjects were recruited: one set for the BAL study and the other set for the surgical tissue study. Some patients in each group had been subjects in our previous studies (18, 20). In all subjects, steady-state measurements of vital capacity and FEV1 were obtained (CHESTAC-55V; Chest Co., Tokyo, Japan). Current smokers were arbitrarily defined as individuals who had smoked up to at least 24 hours before the procedures. To objectively confirm the current smoking status, all subjects were examined for exhaled CO levels using a CO analyzer (Micro Smokerlyser; Bedfont, Rochester, UK). Written informed consent was obtained from each subject, and the Ethics Committee of Hokkaido University School of Medicine approved the study protocols.
Six or more blocks of peripheral lung tissue (1.0 × 1.0 × 0.5 cm) were collected and frozen as soon as possible after lung resection. The lung tissue was placed in the base of a cryomold (Sakura Finetek U.S.A., Torrance, CA), carefully overlaid with additional Tissue-Tek OCT (Sakura Finetek U.S.A.), and immediately frozen with dry ice. Sampling of pulmonary macrophages and bronchiolar epithelial cells was performed by laser capture microdissection (LCM) using a PixCell II System (Arcturus Engineering, Mountain View, CA) as described previously (20). Macrophages localized in the alveolar space and in the alveolar walls were harvested by immuno-LCM using CD-68 antibodies (DakoCytomation) as described previously (20). A total of 30,000 laser bursts was used to collect the macrophages from each subject. LCM of bronchiolar epithelial cells was performed on hematoxylin-stained peripheral lung sections; a total of 40,000 laser bursts was used to collect cells from each subject. Sampling of alveolar septa was performed using a manual dissection technique. Alveolar septa were identified, and adjacent unwanted tissues, such as airways, vessels, and pleurae, were manually dissected out under visual control using an 18-gauge, fine sterile needle, and the remaining tissue was harvested from hematoxylin-stained lung tissues. Four serial nonstained sections adjacent to the sections used for LCM were used for analysis as whole lung tissue specimens.
RNA extraction, reverse transcription, and PCR were performed as described previously (18, 20). Briefly, total RNA was extracted using an RNeasy Mini kit (Qiagen, Hilden, Germany). The quantity and quality of the RNA were determined using a LabChip kit (Agilent Technologies, Palo Alto, CA). The RNA was reverse transcribed using the TaqMan Reverse Transcription Reagents and the RT Reaction Mix (Applied Biosystems, Foster City, CA) on the ABI PRISM 7700 Sequence Detection System (Applied Biosystems). The resulting first-strand cDNA was used as a template for RT-PCR. 5′-exonuclease–based fluorogenic PCR was performed using an ABI Prism 7700 Sequence Detection System (Applied Biosystems). Taqman Gene Expression Assays probes (Applied Biosystems) were used for human glyceraldehyde-3-phosphatase-dehydrogenase (GAPDH) (assay ID: Hs99999905_m1); human Nrf2 (Hs00232352_m1); human heme oxygenase-1 (HO-1) (Hs00157965_m1); human NAD(P)H:quinone oxidoreductase-1 (NQO1) 1 (Hs00168547_m1); human glutamate-cysteine ligase, modifier subunit (GCLM) (Hs00157694_m1); human glutathione reductase (GSR) (Hs00167317_m1); mouse β2-microglobulin (Mm00437764_m1); mouse Nrf2 (Mm00477784_m1); mouse HO-1 (Mm00516007_m1), mouse NQO1 (Mm00500821_m1); mouse GCLM (Mm00514996_m1); and mouse GSR (Mm00833903_m1). The relative amount of each gene mRNA in the samples was assessed by interpolation of their threshold cycles from a standard curve, which was then normalized against human GAPDH mRNA or mouse β2-microglobulin mRNA.
Total glutathione and oxidized glutathione (glutathione disulfide [GSSG]) levels in BALF were measured using a glutathione assay kit (Cayman Chemical Co., Ann Arbor, MI), as described previously (21).
Oxidation of individual BALF proteins was measured using Western blotting analysis as described previously (21). BALF was derivatized with dinitrophenylhydrazine (DNP) using the OxyBlot Protein Oxidation Detection Kit (Chemicon International, Temecula, CA) with slight modification (21). Blots were performed using the anti-DNP antibody and scanned with a GT-9500 scanner (Epson, Nagano, Japan); the intensity of the bands was calculated using NIH Image software (version 1.62). Because a major carbonyl protein band for all of the subjects was a 68-kD protein corresponding to albumin (21), the DNP units of the 68-kD band were quantified by dividing by the result of a standard sample from a representative young nonsmoker. The value was normalized based on the BALF albumin concentration.
Data are expressed as the mean ± SE or the median and range. For the demographic data, differences between two groups were analyzed using an unpaired t test; more than two groups were compared using single-factor ANOVA followed by a post hoc Tukey-Kramer test. For other data, differences between the two groups were analyzed using the Mann-Whitney U test; more than two groups were compared using the Kruskal-Wallis test followed by the Mann-Whitney U test. Correlations were analyzed using Spearman's rank method. All tests were done using the StatView J 5.0 System (SAS Institute Inc., Cary, NC). Statistical significance was set at P < 0.05.
To examine whether Nrf2 is activated in response to CSE, nuclear accumulation of Nrf2 was evaluated using immunocytochemistry and Western blotting. On immunocytochemistry, Nrf2 was constitutively present in the cytoplasm of human alveolar macrophages isolated from young healthy volunteers (Figure 1A). In contrast, Nrf2 was strongly detected in the nuclei of human alveolar macrophages after exposure to 10% CSE for 1 hour (Figure 1A). Western blotting of nuclear extracts obtained from human alveolar macrophages after exposure to CSE for up to 2 hours confirmed the nuclear accumulation of Nrf2 protein (∼110 kD) (Figure 1B).
Figure 1. Regulation of Nrf2 in response to cigarette smoke extract (CSE) in human alveolar macrophages (A) Nrf2 immunocytochemistry. Left panel: Untreated human alveolar macrophages. Right panel: Human alveolar macrophages stimulated with 10% CSE for 1 hour. Positive immunostaining appears brown. Original magnification: ×400. HO-1 = human heme oxygenase-1. (B) Immunoblot analysis of nuclear extracts isolated from human alveolar macrophages with anti-Nrf2 antibody. Exposure to 10% CSE increased Nrf2 (∼110 kD) in the nuclear extracts. NQO1 = human NAD(P)H:quinone oxidoreductase-1. (C) Nrf2 mRNA expression in human alveolar macrophages in response to CSE. Alveolar macrophages were harvested by bronchoalveolar lavage (BAL) from three healthy volunteers. The data are shown as means ± SE of three experiments for each volunteer (n = 3). Values are corrected for GAPDH and expressed as fold increases against the value of the nontreatment control subjects at each time point. *P < 0.05 compared with nontreatment control subjects at each time point. GCLM = human glutamate-cysteine ligase, modifier subunit.
[More] [Minimize]Next, CSE-induced transcriptional regulation of Nrf2 and several Nrf2 target genes in human alveolar macrophages was investigated. We selected HO-1, NQO1, GCLM, and GSR genes because we recently found that these genes were primarily regulated by Nrf2 in response to CS in the immortalized mouse Clara cell line (C22 cells) (22) and because these genes were reported to be down-regulated in the lungs of CS-exposed, Nrf2-deficient mice (7). CSE exposure did not change Nrf2 expression over a 24-hour period, except for slight up-regulation at 2 hours (Figure 1C). On the other hand, Nrf2 target genes HO-1, NQO1, GCLM, and GSR were significantly up-regulated after CSE exposure (Figure 2). The results of these in vitro studies indicate that, in human alveolar macrophages, acute CSE exposure activates Nrf2 mainly via accumulation of its protein in the nucleus and then up-regulates several Nrf2 target genes.
Figure 2. mRNA expression of Nrf2 target genes in response to CSE in human alveolar macrophages (A) HO-1 mRNA. (B) NQO1 mRNA. (C) GCLM mRNA. (D) Human glutathione reductase (GSR) mRNA. Alveolar macrophages were harvested by BAL from three healthy volunteers. The data are shown as means ± SE of three experiments for each volunteer (n = 3). Values are corrected for GAPDH and expressed as fold increases against the value of the nontreatment control subjects at each time point. *P < 0.05 compared with nontreatment control subjects at each time point.
[More] [Minimize]The effects of aging and chronic smoking on Nrf2 expression levels in human pulmonary macrophages were determined. In this BAL study, 37 healthy volunteers were enrolled; the volunteers included seven young and seven older lifelong nonsmokers as well as nine young and 14 older current smokers (Table 1). All current smokers had stopped smoking cigarettes for at least 12 hours before to the BAL procedure. None of the subjects in the BAL study had a history of asthma or had a respiratory infection within the month before enrollment. Among the young volunteers, FEV1/FVC was significantly lower in current smokers than in lifelong nonsmokers. Among the older volunteers, the FEV1% predicted was significantly lower in current smokers than in lifelong nonsmokers, although none of the subjects had COPD. Regardless of age, the percentage recovery of instilled fluid was significantly lower in current smokers than in lifelong nonsmokers. Furthermore, in the young and older volunteers, the total numbers of BALF cells and the percentage of BALF macrophages were significantly increased in current smokers compared with lifelong nonsmokers.
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|
Young |
Older |
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|---|---|---|---|---|---|---|
|
|
Lifelong Nonsmokers
|
Current Smokers
|
Lifelong Nonsmokers
|
Current Smokers
|
||
| n | 7 | 7 | 9 | 14 | ||
| Sex, M/F | 7/0 | 7/0 | 9/0 | 12/2 | ||
| Age, yr | 23 ± 2 | 23 ± 1 | 68 ± 3 | 51 ± 2* | ||
| Pack-years | 0 | 5 ± 1 | 0 | 44 ± 5 | ||
| FEV1/FVC, % | 96 ± 2 | 86 ± 3† | 81 ± 3 | 81 ± 2 | ||
| FEV1, % predicted | 84 ± 5 | 89 ± 5 | 121 ± 7 | 103 ± 4* | ||
| BAL findings | ||||||
| Recovery rate, % | 80 ± 3 | 66 ± 4† | 67 ± 5 | 50 ± 4* | ||
| Total cells, ×104/ml | 9 ± 1 | 14 ± 2† | 12 ± 2 | 39 ± 9* | ||
| Macrophages, %
|
87 ± 2
|
94 ± 2†
|
84 ± 5
|
96 ± 1*
|
||
Among young subjects, Nrf2 mRNA expression in BALF alveolar macrophages did not differ between lifelong nonsmokers and current smokers (Figure 3A). Among older subjects, Nrf2 mRNA expression in BALF alveolar macrophages was significantly down-regulated in current smokers compared with lifelong nonsmokers (P < 0.001) (Figure 3A). Although there was no difference in the BALF macrophages' Nrf2 mRNA levels between young and older lifelong nonsmokers, Nrf2 mRNA expression was significantly down-regulated in older current smokers compared with young current smokers (P = 0.02) (Figure 3A). The issue of whether the basal transcriptional level of Nrf2 affects the levels of Nrf2 target genes in alveolar macrophages was then considered, and it was demonstrated that Nrf2 translocation into the nucleus is the key event in the induction of Nrf2 target genes in response to CS (see Figure 1). To address this issue, the expression of Nrf2 target genes in BALF alveolar macrophages was examined. In addition to Nrf2 mRNA, HO-1 mRNA in BALF alveolar macrophages was significantly down-regulated in older current smokers compared with older lifelong nonsmokers (P = 0.02) and young current smokers (P = 0.009) (Figure 3B). NQO1, GCLM, and GSR mRNAs were significantly up-regulated in young current smokers compared with young lifelong nonsmokers, whereas in the older subjects, NQO1, GCLM, and GSR mRNAs did not differ by smoking status (Figures 3C–3E).
Figure 3. mRNA expression of Nrf2 and Nrf2 target genes in human alveolar macrophages harvested by BAL. (A) Nrf2 mRNA. (B) HO-1 mRNA. (C) NQO1 mRNA. (D) GCLM mRNA. (E) GSR mRNA. Medians are indicated by horizontal lines. The values are corrected for GAPDH and are expressed as fold increases against the median value of lifelong nonsmokers.
We previously reported that oxidized glutathione (GSSG) and protein carbonyl levels were elevated in BALF obtained from older smokers with long smoking histories (21). This suggests that endogenous antioxidant defenses are overwhelmed in older current smokers. To investigate whether the down-regulation of Nrf2 mRNA in alveolar macrophages is related to the presence of excessive oxidative stress in the lungs, the relationships between macrophage Nrf2 mRNA levels and BALF GSSG and carbonylated albumin levels were examined. Among the older subjects, it was found that, in the BALF, macrophage Nrf2 mRNA levels were significantly correlated with the GSSG level (r = −0.57; P = 0.007) (Figure 4A) and the ratio of GSSG per total glutathione (r = −0.44; P = 0.03). Data on BALF GSSG levels and the ratio of GSSG per total glutathione of seven older lifelong nonsmokers and 14 older current smokers were obtained from a previous paper (21). BALF macrophage Nrf2 mRNA levels were significantly correlated with carbonylated albumin levels (r = −0.57; P = 0.008) (Figure 4B). Data on BALF carbonylated albumin levels of seven older lifelong nonsmokers and nine older current smokers were obtained from a previously published paper (21). These findings suggest that down-regulation of Nrf2 in alveolar macrophages obtained from older current smokers is associated with diminished expression of Nrf2 target genes, leading to the appearance of excessive oxidative stress markers in the epithelial lining fluid.
Figure 4. Correlations between Nrf2 mRNA levels in alveolar macrophages and oxidative stress markers in BAL fluid (BALF). (A) Correlation between BALF macrophage Nrf2 mRNA levels and BALF oxidized glutathione (GSSG) levels. (B) Correlation between BALF macrophage Nrf2 mRNA levels and BALF carbonylated albumin levels. These figures incorporate the data of 21 and 16 subjects from reference (17), respectively.
[More] [Minimize]The human BAL study showed that expression of Nrf2 and Nrf2 target genes in human alveolar macrophages differed between young and older current smokers. Thus, it was hypothesized that aging affects the regulation of alveolar macrophages' expression of Nrf2 and Nrf2 target genes in response to CS. To investigate this hypothesis, in vitro experiments were done using mouse alveolar macrophages. Alveolar macrophages were obtained from two age groups (young/adult and older groups) of C57BL/6 and ICR mice, and the alveolar macrophages were exposed to 2.5% CSE for 6 or 24 hours. Nrf2 was temporarily down-regulated in alveolar macrophages exposed to CSE for 6 hours in both age groups; the Nrf2 mRNA level was lower in older mice than in young mice (Figure 5A). CS-induced Nrf2 up-regulation was observed in alveolar macrophages at 24 hours only in young/adult mice but not in older mice independent of the strain (C57BL/6 or ICR) (Figures 5A and 5B). On Western blotting, the intensity of band corresponding to Nrf2 was weaker in the CSE-exposed alveolar macrophages of the older mice than of the young mice. Therefore, changes in the mRNA levels seem to mirror the Nrf2 protein levels (Figure 5C).
Figure 5. Nrf2 expression in response to CSE in mouse alveolar macrophages of different age groups. (A, B) Nrf2 mRNA expression in response to CSE in mouse alveolar macrophages. (A) C57BL/6 mice. (B) Imprinting Control Region (ICR) mice. Mouse alveolar macrophages stimulated with 2.5% CSE or medium only for 6 or 24 hours. The data are shown as means ±SE of three experiments (n = 3; each sample was pooled from three mice). The values are corrected for β2-microglobulin and expressed as fold increases against the median value of the nontreatment young/adult control mice. *P < 0.05 compared with the nontreatment control mice. †P < 0.05 compared with CSE-exposed alveolar macrophages of young/adult mice. (C) Immunoblot analysis of whole-cell lysates isolated from pooled alveolar macrophages of eight C57BL/6 mice exposed to CSE for 24 hours with anti-Nrf2 antibody. Nrf2 protein (∼110 kD) in CSE-exposed alveolar macrophages of older mice was less than in those of young mice.
[More] [Minimize]The effects of aging on the expression of Nrf2 target genes in mouse alveolar macrophages exposed to CSE for 6 hours were investigated. HO-1, GCLM, and GSR mRNAs were significantly decreased in CSE-exposed alveolar macrophages of older mice compared with those of young mice (Figure 6), whereas NQO1 mRNA did not change between the two age groups (data not shown). These results suggest that aging impairs induction of Nrf2 and its target genes in alveolar macrophages in response to CS in mice.
Figure 6. mRNA expression of Nrf2 target genes in response to CSE in mouse alveolar macrophages of different age groups (A) HO-1 mRNA. (B) GCLM mRNA. (C) GSR mRNA. Alveolar macrophages of C57BL/6 mice stimulated with 2.5% CSE or medium only for 6 hours. The data are shown as means ± SE of three experiments (n = 3; each sample was pooled from three mice). The values are corrected for β2-microglobulin and expressed as fold increases against the median value of the nontreatment young control mice. *P < 0.05 compared with the nontreatment control mice. †P < 0.05 compared with CSE-exposed alveolar macrophages of young mice.
[More] [Minimize]To investigate Nrf2 mRNA levels in macrophages and in other lung sites in association with COPD, a site-specific gene expression analysis using dissection techniques was done. For this surgical tissue study, 20 patients who had a lung resection for small peripheral tumors were recruited; 10 of these had normal respiratory functions (five lifelong nonsmokers and five former smokers) and served as control subjects, and 10 were former smokers who had COPD (Table 2). All former smokers in both groups had quit smoking at least 1 month before surgery. There was no difference in age between the two groups. The FEV1/FVC and FEV1% predicted values were significantly lower in subjects with COPD than in control subjects. None of the subjects in the surgical tissue study had a history of asthma or had a respiratory infection during the month before enrollment. The GOLD guideline was used to make the diagnosis and to grade COPD severity (23).
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Control Subjects |
Subjects with COPD |
|---|---|---|
| n | 10 | 10 |
| Sex, M/F | 4/6 | 10/0 |
| Former smokers | 5 | 10 |
| Age, yr | 61 ± 5 | 68 ± 3 |
| FEV1/FVC, % | 82 ± 2 | 58 ± 3* |
| FEV1, % predicted | 119 ± 5 | 87 ± 10* |
| GOLD stage, I/II/III/IV
|
—
|
8/1/0/1
|
Based on the whole lung analysis, subjects with COPD showed a significant decrease in Nrf2 mRNA expression compared with control subjects (P = 0.01) (Figure 7A). In particular, the Nrf2 mRNA of LCM-retrieved pulmonary macrophages was markedly down-regulated in subjects with COPD compared with control subjects (P = 0.001) (Figure 7B); it was significantly down-regulated in subjects with COPD compared with lifelong nonsmokers (P = 0.02) and in subjects with COPD compared with former smokers (P = 0.003). The Nrf2 mRNA level of LCM-retrieved bronchiolar epithelial cells also tended to be down-regulated in subjects with COPD compared with control subjects (P = 0.096) (Figure 7C); however, in the alveolar septa, there was no difference in the Nrf2 expression between the two groups (Figure 7D). When all of the subjects' data were analyzed together, there were significant correlations between FEV1/FVC and Nrf2 mRNA levels in the whole lung (r = 0.60; P = 0.009), pulmonary macrophages (r = 0.76; P < 0.001), and bronchiolar epithelial cells (r = 0.47; P = 0.04), whereas FEV1% predicted was significantly correlated only with Nrf2 mRNA levels in pulmonary macrophages (r = 0.60; P = 0.009).
Figure 7. Site-specific Nrf2 mRNA expression in the human lung. (A) Whole lung homogenates. (B) Pulmonary macrophages collected by immuno–laser capture microdissection (LCM). (C) Bronchiolar epithelial cells collected by LCM. (D) Alveolar septa collected by manual dissection. White circles, lifelong nonsmokers; gray circles, former smokers. Medians are indicated by horizontal lines. The values are corrected for GAPDH and expressed as fold increases against the median value of the control subjects.
[More] [Minimize]On immunohistochemistry, Nrf2 protein was predominantly located in the cytoplasm of alveolar macrophages and bronchiolar epithelial cells, whereas Nrf2-positive cells were sparsely seen within the alveolar septa in lung tissue obtained from lifelong nonsmokers (Figure 8A). In contrast, Nrf2 staining intensity was weak in alveolar macrophages and bronchiolar epithelial cells in the lung tissue of subjects with COPD (Figures 8B and 8C).
Figure 8. Nrf2 immunohistochemistry in the human lung. In lung tissue obtained from lifelong nonsmokers, Nrf2 has a cytoplasmic localization in alveolar macrophages (left panel, arrows), dome-shaped cells within alveolar septa (left panel, arrowheads), and bronchiolar epithelial cells (right panel) (A). Nrf2 staining intensity is weak in alveolar macrophages (left panel, arrows) and bronchiolar epithelial cells (right panel) in the lung tissue obtained from patients with GOLD stage I COPD (B) and stage IV COPD (C). Nrf2-positive cells within the alveolar septa are present in the lung tissue obtained from patients with COPD (B, C, left panels, arrowheads). Positive immunohistochemical staining appears brown. Original magnification: ×400 (left panels), ×100 (right panels).
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In the present study, CSE exposure to macrophages rapidly induced nuclear accumulation of Nrf2 and activated the transcription of Nrf2 target genes. This suggests that the Nrf2 present in macrophages plays a role in the human defense system against CS exposure. The basal Nrf2 mRNA levels and Nrf2 target gene expressions were significantly lower in alveolar macrophages obtained from older current smokers than from lifelong nonsmokers, and basal Nrf2 mRNA levels in pulmonary macrophages were significantly lower in patients with COPD than in nonsmokers and former smokers without COPD.
Under normal conditions, Nrf2 resides in the cytoplasm and is bound to its negative regulator, the Kelch-like erythroid cell-derived protein with CNC homology (ECH)-associated protein 1 (Keap1), which leads to the degradation of Nrf2. When cells are exposed to oxidative or xenobiotic stress, Nrf2 is released from Keap1 and rapidly translocates into the nucleus, where it activates its target genes (24–27). In macrophages, Nrf2 activation occurs in the presence of oxidative stress, such as 4-hydroxynonenal and diesel exhaust chemicals (15, 16). The present results show that macrophages also activate Nrf2-regulated genes in response to CS. Nrf2 activation seems to occur mainly by protein stabilization and translocation into the nucleus rather than by transcriptional up-regulation. However, Nrf2 mRNA was slightly up-regulated in human alveolar macrophages exposed to CSE for 2 hours (Figure 1C) and in alveolar macrophages obtained from young/adult mice after 24 hours of CSE exposure (Figures 5A and 5B). Because an ARE-like element is found in the Nrf2 gene promoter region (28, 29), Nrf2 transcription seems to be, at least in part, self-regulated.
No previous studies have analyzed basal Nrf2 transcriptional levels in human lungs. Therefore, to evaluate the effects of current smoking on in vivo Nrf2 expression in alveolar macrophages, a BAL study of healthy volunteers was done. The amount of time between the last exposure to cigarette smoke and the harvesting of macrophages in current smokers was strictly controlled. Nrf2 mRNA and Nrf2 target gene expressions were significantly lower in alveolar macrophages obtained from healthy older current smokers than in alveolar macrophages obtained from older lifelong nonsmokers or young current smokers (Figure 3). Taken together with the findings in the mouse experiments, it appears that, in alveolar macrophages, aging suppresses Nrf2 up-regulation and/or activation in response to CS exposure.
We previously reported that the potential antioxidant activity of alveolar macrophages was impaired and that oxidized glutathione and protein carbonyl levels were elevated in the BALF obtained from older smokers with long-term smoking histories (21, 30). Indeed, among older subjects, BALF macrophage Nrf2 mRNA levels were inversely correlated with oxidized glutathione and carbonylated albumin levels (Figures 5B and 5C). This implies that, with age, endogenous antioxidant defenses are overwhelmed in the lungs of subjects with long-term smoking histories. Based on our recent finding that lowering basal Nrf2 mRNA levels using Nrf2 siRNA abrogated CS-induced Nrf2 target gene expressions in C22 cells (the mouse Clara cell line) (22) and on similar findings found by others in various cells (31, 32), we wondered whether the expression of Nrf2 target genes might be lowered in alveolar macrophages whose Nrf2 mRNA level was down-regulated. As was the case for Nrf2 mRNA, HO-1 mRNA was significantly lower in macrophages from current smokers compared with never smokers only in older subjects. On the other hand, increased NQO1, GCLM, and GSR mRNA levels were observed only in young current smokers (see Figure 4). Therefore, the down-regulation of basal Nrf2 mRNA levels in alveolar macrophages might be related to impaired antioxidant capacity in older current smokers' lungs via impaired induction of Nrf2 target genes in those cells.
Next, in the surgical tissue study, site-specific Nrf2 mRNA levels in lung tissue obtained from patients with COPD and control subjects were determined. Among the cell types/sites that were examined, Nrf2 mRNA levels in pulmonary macrophages were markedly down-regulated in subjects with COPD compared with nonsmokers and former smokers without COPD (Figure 7B); in fact, they were significantly correlated with FEV1/FVC and FEV1% predicted values. These are the first human data supporting previous animal studies to suggest that Nrf2 deficiency is related to susceptibility for pulmonary emphysema (7–9) and that macrophages play a crucial role via the Nrf2-dependent system in protecting against the development of emphysema (9). This finding is also in line with previous reports showing that the expression of HO-1, one of the Nrf2 target genes, was diminished in pulmonary macrophages obtained from patients with COPD (33, 34). There is increasing evidence that there is an elevated oxidative stress level in the lungs of patients with COPD (35); the present data imply that decreased Nrf2 expression levels in pulmonary macrophages may contribute to the oxidant/antioxidant imbalance observed in COPD. It was found in the present study that, in subjects with COPD, down-regulation of Nrf2 mRNA in pulmonary macrophages is sustained despite smoking cessation. Recently, the Nrf2-ARE system was shown to play an important role in the innate immune response (36); this indicates that reduced Nrf2 levels might affect inflammatory responses in older current smokers and/or patients with COPD.
Some polymorphisms in the Nrf2 gene promoter region have been reported; a −617 C/A polymorphism in the ARE-like sequence is functionally related to a decrease in Nrf2 at the transcriptional level, and patients with this polymorphism had a significantly higher risk of developing acute lung injury after major trauma (29). The same polymorphism was found in a Japanese population, although it was not significantly associated with COPD risk (37). Although the mechanisms of decreased Nrf2 expression in alveolar macrophages associated with chronic smoking, aging, and/or COPD remain to be determined, some polymorphisms of the Nrf2 gene might affect basal Nrf2 mRNA levels.
There are some limitations to the present study. First, in the BAL study, among the older subjects, the current smokers were statistically significantly younger than lifelong nonsmokers. However, this is not likely to have affected the results because there was no evidence of an aging effect with respect to basal Nrf2 expression levels in alveolar macrophages when young and older lifelong nonsmokers were compared. Second, in the surgical tissue study, most of the subjects with COPD had only mild COPD (GOLD stage I). Nrf2 mRNA levels in the pulmonary macrophages were significantly correlated with FEV1/FVC and FEV1% predicted values. A larger sample of patients with more advanced disease should be assessed to further our understanding of the involvement of Nrf2 regulation in the mechanism of disease progression, given that the present study's findings indicate that decreased basal Nrf2 mRNA levels occur even with mild COPD. This supports the notion that decreased Nrf2 plays a role in the development of early COPD. Last, the results of the mouse experiments should be interpreted with caution because the alveolar macrophages' Nrf2 gene expression responses against CSE exposure differed between mice and humans. However, aging seems to be a common regulatory factor for the expression of Nrf2 and Nrf2 target genes in response to CS.
In summary, it was found that acute CSE exposure leads to Nrf2 activation in human alveolar macrophages. Nrf2 expression was attenuated in pulmonary macrophages obtained from older current smokers and patients with COPD. Although the mechanism of the down-regulation of Nrf2 mRNA remains to be elucidated, the pharmacologic activation of the Nrf2-ARE system might be a useful strategy for preventing and treating COPD.
The authors thank Ms. Yoko Suzuki for her invaluable technical assistance.
| 1. | Pauwels RA, Buist AS, Calverley PM, Jenkins CR, Hurd SS; GOLD Science Committee. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease. NHLBI/WHO Global Initiative for Chronic Obstructive Lung Disease (GOLD) Workshop summary. Am J Respir Crit Care Med 2001;163:1256–1276. |
| 2. | Pauwels RA, Rabe KF. Burden and clinical features of chronic obstructive pulmonary disease (COPD). Lancet 2004;364:613–620. |
| 3. | Itoh K, Chiba T, Takahashi S, Ishii T, Igarashi K, Katoh Y, Oyake T, Hayashi N, Satoh K, Hatayama I, et al. An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements. Biochem Biophys Res Commun 1997;236:313–322. |
| 4. | Ishii T, Itoh K, Takahashi S, Sato H, Yanagawa T, Katoh Y, Bannai S, Yamamoto M. Transcription factor Nrf2 coordinately regulates a group of oxidative stress-inducible genes in macrophages. J Biol Chem 2000;275:16023–16029. |
| 5. | Jaiswal AK. Nrf2 signaling in coordinated activation of antioxidant gene expression. Free Radic Biol Med 2004;36:1199–1207. |
| 6. | Cho HY, Reddy SP, Debiase A, Yamamoto M, Kleeberger SR. Gene expression profiling of NRF2-mediated protection against oxidative injury. Free Radic Biol Med 2005;38:325–343. |
| 7. | Rangasamy T, Cho HY, Thimmulappa RK, Zhen L, Srisuma SS, Kensler TW, Yamamoto M, Petrache I, Tuder RM, Biswal S. Genetic ablation of Nrf2 enhances susceptibility to cigarette smoke-induced emphysema in mice. J Clin Invest 2004;114:1248–1259. |
| 8. | Iizuka T, Ishii Y, Itoh K, Kiwamoto T, Kimura T, Matsuno Y, Morishima Y, Hegab AE, Homma S, Nomura A, et al. Nrf2-deficient mice are highly susceptible to cigarette smoke-induced emphysema. Genes Cells 2005;10:1113–1125. |
| 9. | Ishii Y, Itoh K, Morishima Y, Kimura T, Kiwamoto T, Iizuka T, Hegab AE, Hosoya T, Nomura A, Sakamoto T, et al. Transcription factor Nrf2 plays a pivotal role in protection against elastase-induced pulmonary inflammation and emphysema. J Immunol 2005;175:6968–6975. |
| 10. | O'Donnell R, Breen D, Wilson S, Djukanovic R. Inflammatory cells in the airways in COPD. Thorax 2006;61:448–454. |
| 11. | Castell JV, Donato MT, Gomez-Lechon MJ. Metabolism and bioactivation of toxicants in the lung: the in vitro cellular approach. Exp Toxicol Pathol 2005;57:189–204. |
| 12. | Jin FY, Nathan C, Radzioch D, Ding A. Secretory leukocyte protease inhibitor: a macrophage product induced by and antagonistic to bacterial lipopolysaccharide. Cell 1997;88:417–426. |
| 13. | Bea F, Hudson FN, Chait A, Kavanagh TJ, Rosenfeld ME. Induction of glutathione synthesis in macrophages by oxidized low-density lipoproteins is mediated by consensus antioxidant response elements. Circ Res 2003;92:386–393. |
| 14. | Itoh K, Mochizuki M, Ishii Y, Ishii T, Shibata T, Kawamoto Y, Kelly V, Sekizawa K, Uchida K, Yamamoto M. Transcription factor Nrf2 regulates inflammation by mediating the effect of 15-deoxy-Delta(12, 14)-prostaglandin j(2). Mol Cell Biol 2004;24:36–45. |
| 15. | Ishii T, Itoh K, Ruiz E, Leake DS, Unoki H, Yamamoto M, Mann GE. Role of Nrf2 in the regulation of CD36 and stress protein expression in murine macrophages: activation by oxidatively modified LDL and 4-hydroxynonenal. Circ Res 2004;94:609–616. |
| 16. | Li N, Alam J, Venkatesan MI, Eiguren-Fernandez A, Schmitz D, Di Stefano E, Slaughter N, Killeen E, Wang X, Huang A, et al. Nrf2 is a key transcription factor that regulates antioxidant defense in macrophages and epithelial cells: protecting against the proinflammatory and oxidizing effects of diesel exhaust chemicals. J Immunol 2004;173:3467–3481. |
| 17. | Saint-Georges F, Abbas I, Billet S, Verdin A, Gosset P, Mulliez P, Shirali P, Garcon G. Gene expression induction of volatile organic compound and/or polycyclic aromatic hydrocarbon-metabolizing enzymes in isolated human alveolar macrophages in response to airborne particulate matter (PM(2.5)). Toxicology 2008;244:220–230. |
| 18. | Nagai K, Betsuyaku T, Ito Y, Nasuhara Y, Nishimura M. Decrease of vascular endothelial growth factor in macrophages from long-term smokers. Eur Respir J 2005;25:626–633. |
| 19. | Singh A, Misra V, Thimmulappa RK, Lee H, Ames S, Hoque MO, Herman JG, Baylin SB, Sidransky D, Gabrielson E, et al. Dysfunctional KEAP1–NRF2 interaction in non-small-cell lung cancer. PLoS Med 2006;3:e420. |
| 20. | Fuke S, Betsuyaku T, Nasuhara Y, Morikawa T, Katoh H, Nishimura M. Chemokines in bronchiolar epithelium in the development of chronic obstructive pulmonary disease. Am J Respir Cell Mol Biol 2004;31:405–412. |
| 21. | Nagai K, Betsuyaku T, Kondo T, Nasuhara Y, Nishimura M. Long term smoking with age builds up excessive oxidative stress in bronchoalveolar lavage fluid. Thorax 2006;61:496–502. |
| 22. | Adair-Kirk TL, Atkinson JJ, Griffin GL, Watson MA, Kelley DG, DeMello D, Senior RM, Betsuyaku T. Distal airways in mice exposed to cigarette smoke: Nrf2-regulated genes are increased in Clara cells. Am J Respir Cell Mol Biol 2008;39:400–411. |
| 23. | Fabbri L, Pauwels RA, Hurd SS. Global strategy for the diagnosis, management and prevention of chronic obstructive pulmonary disease: GOLD executive summary updated 2003. J COPD 2004;1:105–141. |
| 24. | Itoh K, Wakabayashi N, Katoh T, Ishii T, O'Connor T, Yamamoto M. Keap1 regulates both cytoplasmic-nuclear shuttling and degradation of Nrf2 in response to electropiles. Genes Cells 2003;8:379–391. |
| 25. | McMahon M, Itoh K, Yamamoto M, Hayes JD. Keap1-dependent proteasomal degradation of transcription factor Nrf2 contributes to the negative regulation of antioxidant response element-driven gene expression. J Biol Chem 2003;278:21592–21600. |
| 26. | Itoh K, Tong KI, Yamamoto M. Molecular mechanism activating Nrf2-Keap1 pathway in regulation of adaptive response to electrophiles. Free Radic Biol Med 2004;36:1208–1213. |
| 27. | Kobayashi A, Ohta T, Yamamoto M. Unique function of the Nrf2-Keap1 pathway in the inducible expression of antioxidant and detoxifying enzymes. Methods Enzymol 2004;378:273–286. |
| 28. | Kwak MK, Itoh K, Yamamoto M, Kensler TW. Enhanced expression of the transcription factor Nrf2 by cancer chemopreventive agents: role of antioxidant response element-like sequences in the nrf2 promoter. Mol Cell Biol 2002;22:2883–2892. |
| 29. | Marzec JM, Christie JD, Reddy SP, Jedlicka AE, Vuong H, Lanken PN, Aplenc R, Yamamoto T, Yamamoto M, Cho HY, et al. Functional polymorphisms in the transcription factor NRF2 in humans increase the risk of acute lung injury. FASEB J 2007;21:2237–2246. |
| 30. | Kondo T, Tagami S, Yoshioka A, Nishimura M, Kawakami Y. Current smoking of elderly men reduces antioxidants in alveolar macrophages. Am J Respir Crit Care Med 1994;149:178–182. |
| 31. | Knorr-Wittmann C, Hengstermann A, Gebel S, Alam J, Muller T. Characterization of Nrf2 activation and heme oxygenase-1 expression in NIH3T3 cells exposed to aqueous extracts of cigarette smoke. Free Radic Biol Med 2005;39:1438–1448. |
| 32. | Singh A, Rangasamy T, Thimmulappa RK, Lee H, Osburn WO, Brigelius-Flohe R, Kensler TW, Yamamoto M, Biswal S. Glutathione peroxidase 2, the major cigarette smoke-inducible isoform of GPX in lungs, is regulated by Nrf2. Am J Respir Cell Mol Biol 2006;35:639–650. |
| 33. | Maestrelli P, Paska C, Saetta M, Turato G, Nowicki Y, Monti S, Formichi B, Miniati M, Fabbri LM. Decreased haem oxygenase-1 and increased inducible nitric oxide synthase in the lung of severe COPD patients. Eur Respir J 2003;21:971–976. |
| 34. | Slebos DJ, Kerstjens HA, Rutgers SR, Kauffman HF, Choi AM, Postma DS. Haem oxygenase-1 expression is diminished in alveolar macrophages of patients with COPD. Eur Respir J 2004;23:652–653. |
| 35. | MacNee W. Pulmonary and systemic oxidant/antioxidant imbalance in chronic obstructive pulmonary disease. Proc Am Thorac Soc 2005;2:50–60. |
| 36. | Thimmulappa RK, Lee H, Rangasamy T, Reddy SP, Yamamoto M, Kensler TW, Biswal S. Nrf2 is a critical regulator of the innate immune response and survival during experimental sepsis. J Clin Invest 2006;116:984–995. |
| 37. | Yamamoto T, Yoh K, Kobayashi A, Ishii Y, Kure S, Koyama A, Sakamoto T, Sekizawa K, Motohashi H, Yamamoto M. Identification of polymorphisms in the promoter region of the human NRF2 gene. Biochem Biophys Res Commun 2004;321:72–79. |
