Aryl Hydrocarbon Receptor-Deficient Mice Develop Heightened Inflammatory Responses to Cigarette Smoke and Endotoxin Associated with Rapid Loss of the Nuclear Factor-κB Component RelB
2007; Elsevier BV; Volume: 170; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2007.060391
ISSN1525-2191
AutoresThomas H. Thatcher, Sanjay B. Maggirwar, Carolyn J. Baglole, Heather F. Lakatos, Thomas A. Gasiewicz, Richard P. Phipps, Patricia J. Sime,
Tópico(s)Inflammatory mediators and NSAID effects
ResumoThe 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-α 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-α and interleukin-6 when treated with endotoxin in vitro. Nuclear factor-κB 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-κB so that in the absence of AhR RelB is prematurely degraded, resulting in heightened inflammatory responses to multiple proinflam-matory stimuli. 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-α 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-α and interleukin-6 when treated with endotoxin in vitro. Nuclear factor-κB 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-κB so that in the absence of AhR RelB is prematurely degraded, resulting in heightened inflammatory responses to multiple proinflam-matory stimuli. Cigarette smoking is widely recognized as a cause or leading risk factor for many chronic diseases including cancer, chronic obstructive pulmonary disease, hypertension, and cardiovascular disease. One common process involved in all these conditions is chronic inflammation. 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This hypothesis was tested by analyzing acute lung inflammation in wild-type and AhR knockout (KO) mice exposed to cigarette smoke or inhaled bacterial lipopolysaccharide (LPS).Materials and MethodsMiceAhR KO mice (AhR−/−, strain B6.129-Ahrtm1Bra) were purchased from The Jackson Laboratory (Bar Harbor, ME) and bred at the University of Rochester. This strain carries a targeted deletion of exon 2 of the AhR gene and was backcrossed for 12 generations onto C57BL/6.34Lahvis GP Bradfield CA AhR null alleles: distinctive or different?.Biochem Pharmacol. 1998; 56: 781-787Crossref PubMed Scopus (112) Google Scholar For some experiments, heterozygous colony littermates (AhR+/−) were used as controls; for other experiments, C57BL/6 mice were purchased from The Jackson Laboratory. No differences were observed between C57BL/6 and AhR+/− mice. All animal procedures were performed under the supervision of the University Committee on Animal Research.Cigarette Smoke ExposureAge- and gender-matched AhR−/− (KO) mice and C57BL/6 (B6) or heterozygous littermate (AhR+/−) controls were placed in individual compartments of a wire cage, which was placed inside a closed plastic box connected to the smoke source. Research cigarettes (1R3F; University of Kentucky, Lexington, KY) were smoked according to the Federal Trade Commission protocol (1 puff/minute/cigarette of 2 seconds duration and 35-ml volume) in a Baumgartner-Jaeger CSM2072i cigarette smoking machine (CH Technologies, Westwood, NJ). Mainstream cigarette smoke was diluted with filtered air and directed into the exposure chamber. The smoke exposure (total particulate matter per cubic meter of air, TPM) was monitored in real time with a MicroDust Pro aerosol monitor (Casella CEL, Bedford, UK) and verified by gravimetric sampling. The smoke concentration was set at a nominal value of 250 mg/m3 TPM by adjusting the flow rate of the dilution air. The average actual exposure for these experiments was 286 ± 45 mg/m3 TPM. Mice received two 1-hour exposures, 4 hours apart, on days 1 and 2, a single exposure on day 3, and were euthanized 4 or 24 hours after the final exposure. Control mice were exposed to filtered air in an identical chamber according to the same schedule.LPS Aerosol ExposureAge- and gender-matched AhR KO and B6 controls were exposed to an aerosol of saline alone (control) or saline containing Pseudomonas aeruginosa LPS (Sigma, St. Louis, MO) as described.35Johnston CJ Finkelstein JN Gelein R Oberdorster G Pulmonary cytokine and chemokine mRNA levels after inhalation of lipopolysaccharide in C57BL/6 mice.Toxicol Sci. 1998; 46: 300-307Crossref PubMed Google Scholar Total exposure time was 8 minutes, and the estimated deposition in the lungs was 5 endotoxin units (EUs) per animal. Four hours after LPS exposure, the mice were euthanized and the lungs were lavaged as described below.Tissue Harvest and Bronchoalveolar Lavage (BAL)Mice were anesthetized with 2,2,2-tribromoethanol (Avertin, 250 mg/kg i.p.; Sigma) and euthanized by exsanguination. The heart and lungs were removed en bloc, and the lungs were lavaged twice with 0.5 ml of phosphate-buffered saline (PBS). The lavage fluid was centrifuged, and the cell-free supernatants were frozen for later analysis. The BAL cell pellet was resuspended in PBS, and the total cell number was determined by counting on a hemocytometer. Differential cell counts (minimum of 300 cells per slide) were performed on Cytospin-prepared slides (Thermo Shandon, Pittsburgh, PA) stained with Diff-Quik (Dade Behring, Newark, DE). In some experiments, the remaining BAL cells were pelleted and frozen for electrophoretic mobility shift assays (EMSAs) (see below). The left and right lungs were frozen separately in liquid nitrogen for later analysis.Analysis of BAL FluidMouse IL-6, tumor necrosis factor (TNF)-α, MIP-2, and KC were measured in BAL samples by commercial enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN). PGE2 was measured by enzyme immunoassay using commercially available reagents (Cayman Chemical, Ann Arbor, MI) as described.36Koumas L King AE Critchley HO Kelly RW Phipps RP Fibroblast heterogeneity: existence of functionally distinct Thy 1(+) and Thy 1(−) human female reproductive tract fibroblasts.Am J Pathol. 2001; 159: 925-935Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar The limit of detection was 7 pg/ml for IL-6, MIP-2, and KC, 15 pg/ml for TNF-α, and 30 pg/ml for PGE2. To assay β-glucuronidase activity, the liberation of p-nitrophenol from 4-nitrophenol glucuronide (Sigma) was measured in fresh (not frozen) BAL samples as described.37Stahl PD Fishman WH Measurement of beta-glucuronidase activity.in: Bergmeyer HU Methods of Enzymatic Analysis, Enzymes 2. Deerfield Beach, FL, Weinheim1984: 246-256Google Scholar, 38Thatcher TH McHugh NA Egan RW Chapman RW Hey JA Turner CK Redonnet MR Seweryniak KE Sime PJ Phipps RP Role of CXCR2 in cigarette smoke-induced lung inflammation.Am J Physiol. 2005; 289: L322-L328Google Scholar Total protein in lavage fluid was measured by the bicinchoninic (BCA) colorimetric assay (Pierce, Rockford, IL).Histological Analysis and ImmunohistochemistryMouse lungs (which had not been lavaged) were fixed by inflation with 10% neutral buffered formalin at a pressure of 15 cm H2O. Tissues were embedded in paraffin, sectioned (5 μm), and stained with hematoxylin and eosin. To visualize tissue neutrophils, sections were stained with a rat monoclonal anti-mouse neutrophil antibody (MCA771GA, 1:25 dilution; Serotec, Oxford, UK), developed with NovaRed (Vector Laboratories, Burlingame, CA), and counterstained with hematoxylin.Myeloperoxidase AssayFrozen lung tissue was homogenized in 50 mmol/L potassium phosphate buffer, pH 6.0, containing a protease inhibitor cocktail and 0.5% hexadecyltrimethylammonium bromide (Sigma), sonicated with a probe sonicator (Branson Ultrasonics, Danbury, CT), and then subjected to one freeze-thaw cycle in a dry ice-ethanol bath. The suspension was centrifuged at 16,000 × g, and the resulting supernatant was assayed. Myeloperoxidase activity was determined by monitoring the oxidation of o-dianisidine dihydrochloride (Sigma) spectrophotometrically at 460 nm as described.39Bradley PP Priebat DA Christensen RD Rothstein G Measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker.J Invest Dermatol. 1982; 78: 206-209Crossref PubMed Scopus (3000) Google ScholarBAL Cell CulturesBAL cells were obtained from naive control and AhR−/− mice as described, except that the lungs were lavaged 10 times with Dulbecco's modified PBS (Invitrogen, Carlsbad, CA), containing 1% glucose, for a total volume of 5 ml. BAL cells from four to six mice per strain were pooled, washed twice with PBS, resuspended in RPMI medium containing 10% fetal calf serum and supplemented with l-glutamine (2 mmol/L) and gentamicin (50 μg/ml) (Invitrogen), and counted with a hemocytometer. Quadruplicate cultures were established containing 1 × 105 cells in 0.2 ml of medium per well in 96-well plates and incubated at 37°C. After 2 hours, nonadherent cells were removed by gentle washing with serum-free medium, and the adherent cells were cultured in medium containing 1% fetal calf serum, with or without the addition of LPS (10 EU/ml P. aeruginosa endotoxin; Sigma). In some experiments, the cells were pretreated for 1 hour with the NF-κB inhibitors SN50 (25 μg/ml) or helenalin (5 μmol/L) (Biomol, Plymouth Meeting, PA). After 18 hours, the culture medium was harvested and assayed for inflammatory cytokines by ELISA, as described.EMSANuclear extracts were prepared from frozen lung tissue or BAL cell pellets as described.40Schreiber E Matthias P Muller MM Schaffner W Rapid detection of octamer binding proteins with ‘mini-extracts’, prepared from a small number of cells.Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3908) Google Scholar Nuclear extracts were incubated with radiolabeled oligonucleotide probes for NF-κB and OCT-1 and analyzed by autoradiography of polyacrylamide gels as described.41Ramirez SH Sanchez JF Dimitri CA Gelbard HA Dewhurst S Maggirwar SB Neurotrophins prevent HIV Tat-induced neuronal apoptosis via a nuclear factor-kappaB (NF-kappaB)-dependent mechanism.J Neurochem. 2001; 78: 874-889Crossref PubMed Scopus (82) Google ScholarWestern Blot AnalysisTo analyze the immediate effect of smoke and LPS exposure on NF-κB, mice were exposed to mainstream smoke for 1 hour as described and sacrificed 30 minutes and 4 hours later or exposed to LPS as described and sacrificed 30 minutes and 3 hours later. The lungs were lavaged to obtain BAL cells and then homogenized to prepare total and nuclear lung cell protein extracts using a commercially available kit according to the supplier's instructions (Active Motif, Carlsbad, CA). BAL cells from three mice per group were pooled, and protein extracts were prepared. Lung and BAL proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by Western blot using antibodies specific for NF-κB p65 and RelB (Santa Cruz Biotechnology, Santa Cruz, CA) and total and phosphorylated IκBα (Cell Signaling, Danvers, MA).Statistical AnalysisAll results are reported as the mean ± SEM. The differences between air- and smoke-exposed AhR KO and control mice on differential cell counts, lavage cytokines, and biochemical markers were assessed by one-factor analysis of variance and the Student's t-test. A P value <0.05 was considered significant.ResultsElevated Inflammatory Cells in BAL of AhR KO Mice Exposed to Cigarette SmokeTo investigate the potential role of the AhR in regulating the inflammatory response to cigarette smoke, wild-type and AhR KO mice were exposed to cigarette smoke for 1 hour, twice a day, for 3 days and sacrificed 4 or 24 hours after the final smoke exposure. This exposure protocol elicits an acute inflammatory response characterized by a twofold to threefold increase in the total number of cells and a greater than 100-fold increase in the number of neutrophils recovered by BAL (Figure 1). Compared with wild-type B6 mice, AhR KO mice developed significantly greater neutrophilic influx into the lungs, with twofold to threefold more neutrophils recovered in BAL at both 4 and 24 hours after exposure (Figure 1, B and C). The total number of cells recovered by BAL is also significantly elevated in the AhR KO mice 24 hours after exposure (Figure 1A). Cigarette smoke exposure resulted in increased numbers of BAL lymphocytes that were not significant (Figure 1D), but no changes in BAL eosinophils (data not shown). The protein content of BAL fluid, a marker of inflammation, was significantly increased by cigarette smoke exposure, but the differences between wild-type and AhR KO mice were not significant (Figure 1E). β-Glucuronidase, a hydrolytic enzyme found in phagocytic cells and thought to be involved in tissue breakdown in inflammatory lung disease,42Pérez-Arellano JL Barrios MN Martin T Sanchez ML Jimenez A Gonzalez-Buitrago JM Hydrolytic enzyme of the alveolar macrophage in diffuse pulmonary interstitial disease.Respir Med. 1996; 90: 159-166Abstract Full Text PDF PubMed Scopus (13) Google Scholar is also elevated in BAL fluid of smoke-exposed animals, with enzyme activity 60% higher in smoke-exposed AhR KO mice compared with controls (Figure 1F). Similar results were observed in experiments comparing AhR heterozygote and KO mice (data not shown).AhR KO Mice Exposed to Cigarette Smoke Exhibit Increased Lung Tissue InflammationAcute cigarette smoke exposure at the dose used in this study results in a mild inflammation marked by perivascular accumulation of inflammatory cells, including neutrophils, and increased numbers of alveolar macrophages and neutrophils (Figure 2C). Perivascular infiltrates are more pronounced in the AhR KO mice, as is the appearance of neutrophils in alveolar capillaries (Figure 2D). Immunohistochemical staining using an antibody specific for mouse neutrophils confirms these observations, with increased numbers of neutrophils in the airspaces and perivascular infiltrates in AhR KO mice (Figure 2, E and F).Figure 2Lung 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Lung neutrophilia in the AhR KO− mice was confirmed biochemically by assaying whole lung tissue for myeloperoxidase (MPO), an enzyme found in neutrophil granules that can be used to estimate the level of neutrophilic inflammation in tissues.39Bradley PP Priebat DA Christensen RD Rothstein G Measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker.J Invest Dermatol. 1982; 78: 206-209Crossref PubMed Scopus (3000) Google Scholar MPO activity is significantly higher in naive AhR KO mice than B6 controls and is further increased in these mice by cigarette smoke exposure to levels threefold higher than smoke-exposed B6 controls (Figure 3).Figure 3AhR 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).View Large Image Figure ViewerDownload Hi-res image Download (PPT)AhR KO Mice Produce Elevated Levels of Proinflammatory CytokinesAcute cigarette smoke exposure induces several proinflammatory cytokines in the BAL of wild-type mice, including IL-6, TNF-α, and PGE2 (Figure 4, A, B, and E). The neutrophil chemoattractant CXC chemokines MIP-2 and KC are also significantly elevated by cigarette smoke (Figure 4, C and D). Interestingly, air-exposed AhR KO mice express threefold to fivefold higher levels of proinflammatory cytokines and chemokines than untreated controls. In the case of IL-6, MIP-2, TNF-α, and PGE2, the basal levels in AhR KO mice are as high or higher than in smoke-exposed B6 mice. AhR KO mice exposed to smoke express higher levels of TNF-α, MIP-2, and PGE2 than control mice at 4 ho
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