Increased susceptibility of Nrf2^–/– mice to CS-induced emphysema. Shown are H&E-stained lung sections from Nrf2^+/+ and Nrf2^–/– mice exposed to air alone (A and B, E and F, and I and J) and to CS (C and D, G and H, and K and L) at the indicated times. Sections from the air-exposed Nrf2^+/+ and Nrf2^–/– mice show normal alveolar structure (n = 5 per group). Lung sections from the CS-treated (6 months) Nrf2^–/– mice show increased air space enlargement when compared with the lung sections from the CS-treated Nrf2^+/+ mice. Original magnification, ×20.

CS treatment leads to activation of caspase-3 in Nrf2^–/– lungs. (A) Active caspase-3 expression in lung sections from CS-exposed (6 months) Nrf2^+/+ and Nrf2^–/– mice. CS-exposed Nrf2 ^–/– mice show increased numbers of caspase-3–positive cells in the alveolar septa (n = 5 per group). Magnification, ×40. (B) Number of caspase-3–positive cells in the lungs of air- and CS-exposed mice. Caspase-3–positive cells were significantly higher in the lungs of CS-exposed Nrf2^–/– mice. (C) Increased expression of the 18-kDa active form of caspase-3 in lungs of CS-exposed (6 months) Nrf2^–/– mice (Western blot; lanes 1 and 3: air- and CS-exposed Nrf2^+/+ mice, respectively; lanes 2 and 4: air- and CS-exposed Nrf2^–/– mice, respectively). (D) Quantification of procaspase-3 and active caspase-3 obtained in Western blots of air- or CS-exposed Nrf2^+/+ and Nrf2^–/– lungs. Values are represented as mean ± SEM. (E) Caspase-3 activity in the lungs of air- or CS-exposed (6 months) Nrf2^+/+ and Nrf2^–/– mice. Caspase-3 activity was significantly higher in the lungs of CS-exposed Nrf2^–/– mice than in the lungs of their wild-type counterparts (n = 3 per group). Values (relative fluorescence units [RFU]) are represented as mean ± SEM. *P – 0.05 vs. CS-exposed Nrf2^+/+ mice.

Increased inflammation in the lungs of CS-exposed Nrf2^–/– mice. (A) Lavaged inflammatory cells from control and CS-exposed mice. The number of macrophages in BAL fluid collected from the CS-exposed (both 1.5 months and 6 months) Nrf2^–/– mice was significantly higher than in the BAL fluid from CS-exposed Nrf2^+/+ mice and the respective age-matched control mice. Values represent mean ± SEM (n = 8). PMNs, polymorphonuclear leukocytes. *P – 0.05 vs. control of the same genotype; P – 0.05 across the genotypes in CS-exposed group. (B) Immunohistochemical detection of macrophages (arrows) in lungs of Nrf2^+/+ and Nrf2^–/– mice exposed to CS for 6 months. Magnification, ×40. Scale bars: 25 μm. (C) Quantification of macrophages in lungs after 6 months of CS exposure. The lung sections from the CS-exposed Nrf2^–/– mice showed significantly more macrophages than did those from wild-type counterparts exposed to CS (**P – 0.025). However, there was no significant difference in the number of alveolar macrophages between the air-exposed Nrf2^+/+ and Nrf2^–/– mice (P > 0.9).

Validation of microarray data by Northern blot and enzyme assays. (A) Analysis of mRNA levels of NQO1, GCLm, GST-α1, HO-1, TrxR, Prx-1, GSR, and G6PDH in the lungs of Nrf2^+/+ and Nrf2^–/– mice exposed to either air or CS (n = 3 per group). (B) Effect of CS on the specific activities of selected enzymes in the lungs of Nrf2^+/+ and Nrf2^–/– mice. Values represent mean ± SE (n = 3 per group). *P – 0.05 vs. control of the same genotype.

Cigarette smoke exposure causes lung cell apoptosis as assessed by TUNEL in Nrf2^–/– lungs. (A) Lung sections (n = 5 per group) of room air– or CS-exposed (6 months) Nrf2^+/+ or Nrf2^–/– mice were subjected to TUNEL (left column) and DAPI stain (middle column). Merged images are shown in the right column. CS-exposed Nrf2^–/– mice show abundant TUNEL-positive cells (arrows) in the alveolar septa. Magnification, ×20. (B) Quantification of TUNEL-positive cells (per 1,000 DAPI-stained cells). The number of TUNEL-positive cells was significantly higher in the CS-exposed Nrf2^–/– mice as compared with their wild-type counterparts (*P – 0.05). Values represent mean ± SEM. (C) Identification of apoptotic (TUNEL-positive) type II epithelial cells (left column), endothelial cells (middle column), and alveolar macrophages (right column) in the lungs of CS-exposed (6 months) Nrf2^+/+ and Nrf2^–/– mice. Type II epithelial cells, endothelial cells, and alveolar macrophages were detected with anti-SpC, anti-CD34, and anti–Mac-3 antibodies, respectively, as outlined in Methods. Nuclei were detected with DAPI (blue). Shown are the merged images, with colocalization (yellow arrows) of cell-specific markers (cytoplasmic red signal) and apoptosis (nuclear green + blue DAPI signal, resulting in a lavender-like signal); non-apoptotic (TUNEL-negative) cells with positive cell specific marker (red signal) are highlighted with a red arrow. TUNEL-positive apoptotic cells lacking a cell-specific marker are highlighted by white arrowheads. The majority of TUNEL-positive cells consisted of endothelial and type II epithelial cells, whereas most alveolar macrophages were TUNEL negative. Scale bars: 5 μm.

Increased sensitivity of Nrf2^–/– mice to oxidative stress after CS exposure. (A) Immunohistochemical staining for 8-oxo-dG in lung sections from the mice exposed to CS (6 months) (n = 5 per group). Lung sections from the CS-exposed Nrf2^–/– mice show increased staining for 8-oxo-dG (indicated by arrows) when compared with lung sections from CS-exposed Nrf2^+/+ mice and the respective air-exposed control mice. Magnification, ×40. (B) Quantification of 8-oxo-dG–positive alveolar septal cells in lungs after 6 months of CS exposure. The number of cells that reacted with anti–8-oxo-dG antibody was significantly higher in the lung tissues of the CS-exposed Nrf2^–/– mice than in the lung tissues of the CS-exposed Nrf2^+/+ mice and air-exposed control mice. Values (positive cells/mm alveolar length) represent mean ± SEM. *P – 0.05 vs. CS-exposed Nrf2^+/+ mice. (C) Immunohistochemical staining with normal mouse-IgG1 antibody in lung sections from air- or CS-exposed Nrf2^+/+ and Nrf2^–/– mice. Magnification, ×40. Scale bars: 25 μm.

Activation of Nrf2 in CS-exposed Nrf2^+/+ lungs. (A) EMSA to determine the DNA binding activity of Nrf2. For gel shift analysis, 10 μg of nuclear protein from the lungs of air-and CS-exposed mice was incubated with the labeled human NQO1 ARE sequence and analyzed on a 5% non-denaturing polyacrylamide gel. For supershift assays, the labeled NQO1 ARE was first incubated with 10 μg of nuclear extract and then with 4 μg of anti-Nrf2 antibody for 2 hours. Nuclear protein of Nrf2^+/+ lungs showed increased binding to the ARE-containing sequence (lower arrow) after CS exposure, with a supershifted band caused by preincubation with anti-Nrf2 antibody, thus confirming the binding of Nrf2 to the ARE sequence (upper arrow). Ra–IgG1, rabbit IgG1. (B) Nuclear accumulation of Nrf2. Western blot analysis with anti-Nrf2 antibody showed the nuclear accumulation of the transcription factor Nrf2 in the lungs of Nrf2^+/+ mice in response to CS exposure (lanes 1 and 3: air-exposed Nrf2^–/– and Nrf2^+/+ mice, respectively; lanes 2 and 4: CS-exposed Nrf2^–/– and Nrf2^+/+ mice, respectively; lamin B1 was used as the loading control). Western blot analysis was carried out 3 times with the nuclear proteins isolated from the lungs of 3 different air- or CS-exposed Nrf2^+/+ and Nrf2^–/– mice.