Aryl hydrocarbon receptor-deficient mice develop heightened inflammatory responses to cigarette smoke and endotoxin associated with rapid loss of the nuclear factor-kappaB component RelB

Abstract

The transcription factor aryl hydrocarbon receptor (AhR) plays an important role in the response to environmental pollutants. However, its role in normal physiology is unclear. To investigate the role of AhR in acute lung inflammation, control and AhR knockout (KO) mice were exposed to inhaled cigarette smoke or bacterial endotoxin. Smoke-induced lung inflammation was twofold to threefold more severe in AhR KO mice than controls. Intriguingly, levels of tumor necrosis factor-alpha and interleukin-6 in the bronchoalveolar lavage of air-exposed KO mice were equal to the levels seen in smoke-exposed controls, suggesting that AhR-deficient mice are inflammation prone. AhR KO mice challenged with inhaled endotoxin, which does not contain AhR ligands, also developed greater lung neutrophilia than controls, and bronchoalveolar lavage cells from AhR KO mice produced elevated levels of tumor necrosis factor-alpha and interleukin-6 when treated with endotoxin in vitro. Nuclear factor-kappaB DNA-binding activity was elevated in smoke-exposed AhR KO mice compared with controls and was associated with a rapid loss of RelB only in the KO mice. We propose that AhR is a previously unrecognized regulator of inflammation that interacts with nuclear factor-kappaB so that in the absence of AhR RelB is prematurely degraded, resulting in heightened inflammatory responses to multiple proinflam-matory stimuli.

Figure 1

AhR KO mice exhibit increased lung inflammation markers in BAL after cigarette smoke exposure. Wild-type (B6, gray bars) and AhR KO (black bars) mice were exposed to air or cigarette smoke (CS) as described and sacrificed 4 or 24 hours after the final exposure. Differential counts were performed on BAL cells, and the total cell number (A), number of neutrophils (B), percentage of neutrophils (C), and number of lymphocytes (D) are reported. E: Total protein in BAL fluid was measured by BCA assay. F: β-Glucuronidase activity was measured in fresh BAL fluid and is expressed as units of activity per mg total protein. Results shown are the means ± SE for n = 6 mice per group from a single experiment and are representative of three independent experiments. *Significant increase over air-exposed wild-type mice; ^†significant increase over smoke-exposed, wild-type mice (P < 0.05).

Figure 2

Lung inflammation is increased in AhR KO mice exposed to cigarette smoke. Mice were exposed to air or smoke as described and sacrificed 24 hours after the final smoke exposure. Lungs were inflated and fixed with formalin, and sections were stained with H&E. A and B: Air-exposed mice have normal alveoli and blood vessels. C: Smoke-exposed mice exhibit perivascular inflammation with extravasating monocytes and neutrophils and monocytes and neutrophils in the alveolar capillaries. D: Perivascular infiltrations and alveolar neutrophils are more prominent in smoke-exposed AhR KO mice. Sections were immunostained with an antibody against mouse neutrophils (red) and counterstained with hematoxylin. Tissue-infiltrating neutrophils are much more prominent in AhR KO mice (F) than wild-type (E). White arrows, monocyte; black arrows, neutrophil. Scale bars = 25 μm.

Figure 3

AhR KO mice exposed to cigarette smoke have elevated levels of myeloperoxidase activity in lung tissue compared with wild-type controls. Wild-type (B6, gray bars) and AhR KO (black bars) mice (n = 4 to 5 per group) were exposed to air or cigarette smoke (CS) and sacrificed 24 hours after the final exposure as described. Myeloperoxidase activity in the left lung was measured as described in Materials and Methods. Results shown are the means ± SE for n = 6 mice per group from a single experiment and are representative of two independent experiments. *Significant increase compared with air-exposed wild-type mice; ^†significant increase compared with smoke-exposed, wild-type mice (P < 0.05).

Figure 4

Proinflammatory cytokine levels are elevated in the BAL of smoke-exposed AhR KO mice. Wild-type (B6, gray bars) and AhR KO (black bars) mice (n = 6 per group) were exposed to air or cigarette smoke (CS) and sacrificed 4 or 24 hours after the final exposure. IL-6 (A), TNF-α (B), MIP-2 (C), KC (D), and PGE₂ (E) were measured in BAL fluid by commercial ELISA or enzyme immunoassay, as described. Results shown are the means ± SE for n = 6 mice per group from a single experiment and are representative of three independent experiments. *Significant difference from air-exposed wild-type mice; ^†significant difference from smoke-exposed, wild-type mice (P < 0.05).

Figure 5

AhR KO mice exhibit increased neutrophils in BAL after exposure to bacterial endotoxin. Wild-type (B6, gray bars) and AhR KO (black bars) mice were exposed to an aerosolized suspension of bacterial endotoxin (LPS) in saline, or saline aerosol alone, and sacrificed 4 hours later. Differential cell counts were performed on BAL cells, and the number (A) and percentage (B) of neutrophils in the BAL is reported. Values shown are the mean ± SE of n = 4 mice per group and are representative of two independent experiments. *Significant increase compared with air-exposed wild-type mice; ^†significant increase compared with smoke-exposed, wild-type mice (P < 0.05).

Figure 6

BAL cells from AhR KO mice treated in vitro with bacterial endotoxin produce elevated levels of proinflammatory cytokines. Lung cells were obtained by lavage from six naive AhR heterozygous (Het) and KO mice, pooled by strain, and cultured in vitro with or without 3.3 ng/ml bacterial endotoxin (LPS), 5 μmol/L helenalin, or 25 μg/ml SN50. After 18 hours, culture supernatants were harvested and assayed for IL-6 (A) and TNF-α (B) by commercial ELISA. The results shown are the mean ± SEM of quadruplicate cultures and are representative of three independent experiments performed using both AhR Het littermates and B6 mice as controls. *Significant increase compared with control cells (P < 0.05).

Figure 7

AhR KO mice exhibit elevated levels of NF-κB DNA-binding activity in lung tissue with and without cigarette smoke exposure. Nuclear extracts were made from lung tissue of wild-type (B6) and AhR KO mice exposed to air or cigarette smoke as described. DNA-binding activity to radiolabeled oligonucleotide probes containing NF-κB (left) or OCT-1 (right) consensus binding sequences was analyzed by EMSA. Arrows indicate nonspecific (NS), NF-κB-specific, and OCT-1-specific DNA binding activity. A total of 20 mice were analyzed (n = 4 per air-exposed group and 6 per smoke-exposed group). A: One representative animal per group; B: NF-κB activity relative to air-exposed B6 mice, determined by densitometric analysis of all 20 mice, normalized to OCT-1. *Significant increase over air-exposed B6 mice; ^†significant exposure over both air-exposed KO and smoke-exposed B6, P < 0.05. C: BAL cells harvested from air- and smoke-exposed AhR heterozygous (Het) and KO mice were pooled in groups of two to obtain sufficient nuclear extract for EMSA (a total of four Het and six KO mice were exposed in this experiment). NF-κB and OCT-1 binding activity was determined as described.

Figure 8

AhR KO mice exhibit rapid loss of RelB after smoke and LPS exposure. Control (Het) and AhR KO mice were exposed to cigarette smoke for 1 hour, and BAL cells and lung tissue were harvested 30 minutes and 4 hours later. Control mice were exposed to air only. A: Nuclear extracts were prepared from lung tissue and analyzed by Western blot. One representative mouse per group is shown. Equal amounts of protein were loaded in each lane. The blots were probed with antibodies to RelB and actin (a loading control). NS, a nonspecific band sometimes observed with this antibody. Data are representative of two independent experiments with three mice per group in each experiment. B: The BAL cells retrieved from all three mice in each group were pooled and total protein extracts were prepared and analyzed by Western blot for RelB. Equal amounts of protein were loaded in each lane. The results are representative of two independent experiments. C: Control (Het) and AhR KO mice were exposed to LPS as described, and BAL cells were harvested after 30 minutes and 3 hours. Control mice were exposed to saline only. BAL cell protein extracts were analyzed by Western blot for RelB.