Exposure of Differentiated Airway Epithelial Cells to Volatile Smoke in vitroBeisswenger C. · Platz J. · Seifart C. · Vogelmeier C. · Bals R.
Department of Internal Medicine, Division of Pulmonary Diseases, Philipps University of Marburg, Marburg, Germany
Background: Cigarette smoke (CS) is the predominant pathogenetic factor in the development of chronic bronchitis and chronic obstructive pulmonary disease. The knowledge about the cellular and molecular mechanisms underlying the smoke-induced inflammation in epithelial cells is limited. Objectives: The aim of this study was to develop an in vitro model to monitor the effects of volatile CS on differentiated airway epithelial cells. Methods: The airway epithelial cell line MM-39 and primary human bronchial epithelial cells were cultivated as air-liquid interface cultures and exposed directly to volatile CS. We used two types of exposure models, one using ambient air, the other using humidified and warm air. Cytokine levels were measured by quantitative PCR and ELISA. Phosphorylation of p38 MAP kinase was assessed by Western blot analysis. To reduce the smoke-induced inflammation, antisense oligonucleotides directed against the p65 subunit of NF-κB were applied. Results: Exposure of epithelia to cold and dry air resulted in a significant inflammatory response. In contrast, exposure to humidified warm air did not elicit a cellular response. Stimulation with CS resulted in upregulation of mRNA for IL-6 and IL-8 and protein release. Exposure to CS combined with heat-inactivated bacteria synergistically increased levels of the cytokines. Reactions of differentiated epithelial cells to smoke are mediated by the MAP kinase p38 and the transcription factor NF-κB. Conclusions: We developed an exposure model to examine the consequences of direct exposure of differentiated airway epithelial cells to volatile CS. The model enables to measure the cellular reactions to smoke exposure and to determine the outcome of therapeutic interventions.
Copyright © 2004 S. Karger AG, Basel
Exposure to cigarette smoke (CS) is the most important cause for chronic obstructive pulmonary disease (COPD) . Smoking increases the number and activity of inflammatory cells in the respiratory tract. In bronchoalveolar lavage studies, increased numbers of alveolar macrophages, neutrophils, and eosinophils are present in smokers [2, 3]. Although the relationship between smoke, airway inflammation, and COPD is obvious, little is known about the cellular and molecular mechanisms underlying the smoke-induced inflammation.
Several in vitro models have been used to study the effects of CS on airway epithelial cells. Most models use CS extracts (CSE) to initiate an inflammatory reaction in human bronchial epithelial cells grown in submersed cultures. Using this approach, it was demonstrated that CSE leads to the release of IL-8 from cultured human bronchial epithelial cells [4, 5, 6] and that CSE inhibits the ability of cultured human bronchial epithelial cells to participate in repair processes . Furthermore, CSE has cytotoxic effects on alveolar type II cells and induces apoptosis .
CS is a complex mixture of a large number of components, and many of them are toxic to epithelial cells . The exposure of cells grown under conventional conditions to aqueous extracts of CS likely reflects only a part of the spectrum of smoke-induced cellular reactions. Several groups developed models to directly expose epithelial cells to volatile smoke [10, 11, 12, 13]. The aim of the present study was to further characterize the experimental conditions to reproducibly measure cellular outcome parameters and to determine cellular mechanisms of smoke-induced inflammation. We found that cold and dry air causes a significant inflammatory response of the airway epithelial cells compared to warm and humidified air. Stimulation with CS resulted in a distinct inflammatory reaction that was mediated by MAP kinase p38 and the transcription factor NF-κB and was synergistically increased by exposure to microorganisms.
Materials and Methods
Human bronchial epithelial cells (HBE cells) were isolated from large airways resected during surgery and cultivated in air-liquid interface cultures as described previously . Differentiated HBE cell cultures were selected for experiments by measuring the transepithelial resistance (Rt) using an ohmmeter (EVOM, World Precision Instruments). Cultures were considered confluent and differentiated if the Rt was more than 500 Ω/cm2 and ciliary beating could be detected. MM-39 cells  were cultured in HAM-F12:DMEM = 1:1 supplemented with 10% adult bovine serum, 1% Ultroser G, penicilin G (100 IU/ml) and streptomycin sulfate (100 IU/ml) and cultivated in air-liquid interface cultures. MM-39 is a transformed human tracheal gland cell line, that retains serous secretory functions  and was used in air-liquid interface cultures for the experiments. All cultures were incubated at 37°C in a humidified 5% CO2 air atmosphere in the tissue culture incubator.
We used two different models to expose tissue cultures to CS. In both models, a continuous flow of CS was generated by burning commercially available Marlboro 100 cigarettes (11 mg tar, 0.9 mg nicotine) and guiding the smoke stream into an airtight exposure chamber (Billups Rothenberg, Del Mar, Calif., USA) with a capacity of 5.5 liters and containing the cultures. The continuous flow of 3,000 ml/min was generated by a pump located at the outlet of the exposure chamber. It took 5 min for a cigarette to burn with constant speed. A small ventilator inside the chamber distributed the CS equally. Tissue cultures were exposed to CS for 5 min (= 1 cigarette) or 15 min (= 3 cigarettes). After the exposure, the medium of the cultures was replaced immediately.
In the first exposure model, we used ambient air. In this model, the cultures were exposed to dry air at room temperature (fig. 1a). Reference cultures were kept in the tissue culture incubator for the same period in humidified air containing 5% CO2 at 37°C.
Fig. 1. Schematic drawing of the smoke chambers used to expose cultures to CS. A pump at the outlet of the exposure box generated a continuous air flow of CS from cigarettes positioned at the inlet of the system. A small ventilator inside the chamber distributes the CS equally. a In the first exposure model, unmodified ambient air was used. The cultures were exposed to dry air at room temperature. b In the second model, we used humidified air at 37°C originating from a tissue culture incubator. In addition, the exposure chamber was placed in a tissue culture incubator.
In the second model, we used humidified air at 37°C originating from a tissue culture incubator (fig. 1b). In addition, the exposure chamber was placed in a tissue culture incubator. Control cultures were incubated in the exposure chamber for the same time period without burning a cigarette. This model was used for all further experiments. To determine the inflammatory effects of CS in combination with bacteria, HBE cell cultures were stimulated with heat-inactivated Pseudomonas aeruginosa PAO 1 directly after exposure to CS. Bacteria were heat inactivated for 30 min at 96°C after they had grown to an OD650 of 0.8 in LB media. To study the role of the MAP kinase p38, we added the inhibitor SB-203580 (10 μM; Sigma, Taufkirchen, Germany) 1 h before CS exposure into the HBE cell cultures.
The HBE cell cultures were pretreated with NF-κB p65 antisense and sense oligonucleotides 12 h before exposure to CS (5 μM final concentration). The sense and antisense oligonucleotides were applied to the basolateral compartments of the transwell system. The nucleic acid sequence of the NF-κB p65 antisense phosphorothiate-modified oligodeoxynucleotide (MWG, Germany) was 5′-gccatggacgaactgttcccc-3′. The sequence of the NF-κB p65 sense phosphorothiate-modified oligodeoxynucleotide was 5′-gggaacagttcgtccatggc-3′ .
Levels of IL-6 and IL-8 in culture supernatants were determined by a commercially available sandwich-type enzyme-linked immunosorbent assay kit for IL-6 and IL-8, according to the manufacturer’s instructions (R&D Systems, Wiesbaden-Nordenstadt, Germany). Lactate dehydrogenase was measured using a cytotoxicity kit (Roche, Mannheim, Germany) according to the manufacturer’s instruction.
A total of 1.5 μg of total RNA preparation (Rneasy Mini Kit, Qiagen, Germany) was reverse transcribed using a cDNA synthesis kit (MBI Fermentas, St. Leon-Rot, Germany) applying oligo(dT)18. cDNA was diluted 1/5, and 5 μl were used as template in a 25-μl SYBR-Green-PCR mix, according to the manufacturer’s protocol (Bio-Rad, Munich, Germany). GAPDH primer (sense, 5′-GAAGGTGAAGGTCGGAGTC-3′; antisense, 5′-GAAGATGGTGATGGGATTTC-3′), IL-6 primer (sense, 5′-CCAGAGCTGTGCAGATGAGTACA-3′; antisense 5′′-CCTGCAGCTTCGTCAGCA-3′), and IL-8 primer (sense 5′′-GCTCTGTGTGAAGGTGCAGTT3′; antisense 5′-AAACTTCTCCACAACCCTCTG-3′) were purchased from TIB Molbiol (Berlin, Germany). Specificity of RT-PCR was controlled by omission of the template or the reverse transcription. Quantitative PCR results were obtained using the ΔΔCT method. Because PCR efficiencies for all four reactions were similar, threshold values were normalized to GAPDH and compared to unstimulated control cells.
For the measurement of the non-activated (non-phosphorylated) and activated (diphosphorylated) p38 MAP kinase, HBE cells were boiled for 10 min in Laemmli sample buffer (Bio-Rad). Protein concentrations were determined with the amido black method. Lysate concentrations were adjusted to ensure even protein loading and separated via electrophoresis (10–20% linear gradient acrylamide gels, Bio-Rad). After electrophoresis, proteins were transferred onto a nitrocellulose membrane. The monoclonal primary antibodies were against the diphosphorylated p38 MAP kinase (1:5,000, clone P38-TY, Sigma, ) and the non-phosphorylated, non-activated p38 MAP kinase (1:5,000, clone P38-YNP, Sigma). The detection was performed using a labeled secondary antibody and a chemiluminescent detection system (Pierce, Bonn, Germany). Blots were stripped and reprobed with antibody against the non-phosphorylated p38 kinase. Coomassie blue staining of the gel was used to control for equal loading.
Values are displayed as means ± SEM. Comparisons between groups were analyzed by t test (two sided), or ANOVA for experiments with more than two subgroups. Post hoc range tests were performed with the t test (two sided) with Bonferroni adjustment. Results were considered statistically significant for p < 0.05.
To characterize the effect of CS on respiratory epithelial cells, we exposed MM-39 cells in air-liquid interface culture in a first exposure model applying ambient air (fig. 1a). After exposure to CS or air, the cultures were kept for 8 h in the incubator. Incubator control cultures were kept in the tissue culture incubator. Unstimulated MM-39 cells constitutively released detectable amounts of IL-6 and IL-8 (fig. 2a). Ambient air caused increased IL-6 and IL-8 release compared to the incubator controls (fig. 2a). Secretion of both mediators was significantly increased by CS compared to air alone (fig. 2a). To further examine the influence of cold dry air on epithelial cytokine release, MM-39 cell cultures were exposed for 0, 5, 30, or 60 min to environmental air. Increased levels of IL-6 were detectable in a time-dependent manner (fig. 2b). Based on these results, we modified the exposure system and applied warm and humidified air from a tissue incubator (fig. 1b). MM-39 cell cultures were incubated for 5 min with or without CS. Exposure to warm and humidified air did not alter levels of IL-6 and IL-8 as compared to the incubator cultures (data not shown).
Fig. 2. Effects of cold dry air and CS on cytokine release from MM-39 cells. a MM-39 cells were incubated for 5 min with dry air at room temperature with (CS) or without CS (reference). Control cultures were incubated in the incubator (incubator control). * p < 0.05, n = 4. b MM-39 cells were incubated for 0, 5, 30, or 60 min in cold dry air. Control cultures were kept in the incubator (0 min).
Air-liquid interface cultures of MM-39 cells and well-differentiated HBE cells were incubated with CS or with warm humidified air in the modified incubation system for 5 or 15 min. CS led to an upregulation of IL-6 and IL-8 mRNA in MM-39 cells (fig. 3a, b). Highest mRNA levels were measured after 1 h. After 8 h, mRNA levels were almost equivalent to the reference. CS significantly increased the release of IL-6 and IL-8 protein from MM-39 and HBE cells (fig. 4a–c). Additionally, we characterized the effect of heat-inactivated P. aeruginosa PAO 1 in combination with CS. HBE cell cultures were stimulated with bacteria directly after incubation with CS. Heat-inactivated bacteria caused an increased inflammatory reaction in HBE cells compared to CS alone (fig. 4b–c). To assay cell viability, we measured LDH release into the medium. Incubation with CS did not lead to increased LDH release of MM-39 cultures and HBE cell cultures (data not shown). The transepithelial resistance of the cultures did not change 5 or 15 min after CS exposure in MM-39 and HBE cell cultures (data not shown).
Fig. 3. Effects of warm and humidified air and CS on induction of IL-6 and IL-8 mRNA in cultivated MM-39 cells. MM-39 cells were incubated with humidified air at 37°C with (CS) or without CS (reference) for 5 min. RNA was isolated after 1, 4 and 8 h. The induction of IL-6 (a) and IL-8 (b) was measured by quantitative RT-PCR. * p < 0.05, n = 4.
Fig. 4. Effect of warm and humidified air and CS on cytokine release from cultivated airway epithelia. a MM-39 cells were incubated with humidified air at 37°C with (CS) or without CS (reference) for 5 min. Cytokine release was measured 8 h after smoke exposure. b, c Coincubation of differentiated HBE cell cultures with heat-inactivated P. aeruginosa PAO1 (CS + P.a.) results in significantly increased levels of IL-6 (b) or IL-8 (c) as compared to the exposure with CS or to the reference (R). HBE cell cultures were incubated for 15 min with CS and subsequently stimulated with bacteria (P.a.). * p < 0.05, n = 4.
Next we tested whether the smoke exposure model is suitable to study signal transduction events initiated by CS. Activation of p38 kinase was assayed using an antibody specific for the phosphorylated, active form of the kinase. As a control, we detected the non-phosphorylated form of p38 (fig. 5a). Additionally, we showed equal loading by staining the gel with Coomassie blue (data not shown). After stimulation with heat-inactivated P. aeruginosa PAO 1, maximal phosphorylation was measured after 15 min (fig. 5a). CS-induced activation of p38 was only detectable after 15 min. Combination of CS with P. aeruginosa PAO 1 led to a stronger phosphorylation. To investigate whether the release of IL-6 after CS exposure is mediated by activation of p38 MAP kinase, the effect of the p38 MAP kinase inhibitor SB-203580 was assessed. SB-203580 completely blocked the CS-induced release of IL-6 (fig 5b).
Fig. 5. The MAP kinase p38 mediated smoke-induced epithelial activation. HBE cell cultures were incubated with P. aeruginosa PAO1 (P.a.), with CS (15 min), and with CS combined with P. aeruginosa PAO1 (CS + P.a.) and compared to the reference (R). All extracts were immunoblotted with antibodies specific for the phosphorylated and non-phosphorylated form of p38 (a). b Effect of the MAP kinase p38 inhibitor SB-203580 on CS-induced IL-6 release. HBE cell cultures were incubated with SB-203580 (10 μM) 1 h prior to CS exposure (15 min). IL-6 release was measured 8 h after CS exposure. SB-203580 significantly reduced the release of IL-6. * p < 0.05, n = 4.
To test whether the smoke exposure model is applicable to interventional studies, we determined whether NF-κB p65 antisense oligonucleotides have an effect on the release of IL-6 and IL-8 after CS exposure. MM-39 cultures and HBE cell cultures were treated with antisense and sense oligonucleotides before incubation with CS. The release of IL-6 and IL-8 from cultures treated with antisense oligonucleotides was significantly reduced compared to the sense reference (fig. 6a–d).
Fig. 6. Inhibition of epithelial inflammation by antisense oligonucleotides against p65. MM-39 cultures (a, b) and well-differentiated HBE cell cultures (c, d) were treated with single-stranded sense and antisense oligodeoxynucleotides. After 12 h of treatment with oligodeoxynucleotides, the cultures were exposed to CS. IL-6 (a, c) and IL-8 (b, d) levels were measured 8 h after smoke exposure. * p < 0.05, n = 4.
It was the aim of the present study to develop an in vitro exposure model to study effects of volatile CS on differentiated airway epithelium. The main finding is that exposure of airway epithelium to volatile CS results in a significant upregulation and secretion of proinflammatory cytokines. The epithelial inflammatory response to CS was synergistically increased by coincubation with bacteria. The MAP kinase p38 was activated by CS and inhibition of this signaling pathway blocked the CS-induced release of IL-6. Finally, we showed that the exposure model is suitable to test therapeutic approaches in vitro.
Many studies have evaluated the epithelial response to aqueous extracts of CS and found a variety of effects. Most of these studies use cell lines or undifferentiated epithelial cells. It has been described earlier that responses of airway epithelia are correlated with the differentiation status [17, 18]. In the present study we used two cellular systems. First, we applied the cell line MM-39. The variability in cell line cultures is small, and therefore the standardization of novel procedures is more practical. Second, we used primary cells grown as differentiated air-liquid interface cultures. Primary cell cultures are more variable, however, their biological properties may be more closely related to the in vivo situation. As outcome measurements we used the secretion of the proinflammatory cytokines IL-6 and IL-8. Both are known to be upregulated in airway epithelial cells from COPD patients  and likely represent key mediators in this disease. The responses of the two cellular systems used in the present study were comparable in a qualitative sense. We used an air-liquid interface culture system to allow the application of volatile CS directly to the apical side of the polarized epithelial cells. Volatile CS likely resembles the in vivo situation more closely as compared to aqueous smoke extracts. One focus of the study was to establish conditions that enable to perform reproducible experiments and to keep various parameters (temperature and humidity of the air, smoke dose) constant. One main finding was that cold and dry air causes an inflammatory response by airway epithelial cells. These results emphasize the need to use humidified and warm air in exposure models of cultivated cells.
Exposure of airway epithelial cells to CS results in the activation of different signaling molecules such as the MAP kinases ERK1/2 and MEK, NF-κB , and protein kinase C . Tobacco-smoke-induced lung cell proliferation appears to be mediated by tumor necrosis factor-α-converting enzyme and amphiregulin. This finally results in the activation of the epidermal growth factor receptor system . The MAP kinase p38 in alveolar macrophages of smokers is more activated by LPS as compared to alveolar macrophages of nonsmokers . In the present study, we found that CS activates p38 in airway epithelia. Coincubation with bacteria resulted in increased activation of p38 MAP kinase. Inhibition of p38 completely blocked CS-induced IL-6 release. These results emphasize the role of p38 in CS-induced inflammatory reactions. We also showed that NF-κB is involved in the activation of epithelial cells by CS: Application of antisense-oligonucleotides against p65 results in significantly decreased secretion of IL-6 and IL-8. Antisense technology has been used to influence inflammatory processes . It has been shown that CS condensate activates NF-κB via phosphorylation and degradation of IkBα . IL-6 expression is directly regulated by NF-κB involving p38 MAP kinase activation . In the present model system, CS results in phosphorylation of the MAP kinase p38 which subsequently activates NF-κB. This finally results in the increased expression and secretion of proinflammatory mediators. Here we show for the first time that inhibition of the p65 subunit of NF-κB is a suitable approach to inhibit smoke-induced epithelial inflammation. The targeting of signalling pathways is a possible strategy to develop future treatments of COPD .
The results obtained with the exposure model emphasize the role of the airway epithelium in the induction of pulmonary inflammation in response to smoke exposure. It is now accepted that the epithelium is actively involved in the secretion of proinflammatory cytokines and the chemoattraction of inflammatory cells [26, 27]. In fact, the epithelial cell is the first cell type of the human body to be in contact with inhaled smoke.
In conclusion, we developed an exposure model appropriate to examine short- and long-term consequences of direct exposure of differentiated airway epithelium to CS. The use of warm and humidified air and constant conditions are essential preconditions to ensure reproductive and valid results. The model will be useful to study molecular mechanisms of smoke-induced epithelial inflammation and to develop therapeutic approaches targeting the epithelium.
The authors thank Mrs. Annette Püchner and Mr. Thomas Damm for excellent technical support. This study was supported by grants from the ‘Bundesministerium für Bildung und Forschung’ (BMBF 01 GC 0103) to R.B. and C.S. and by the ‘Wilhelm-Sander Stiftung’ (R.B.).
Robert Bals, MD, PhD
Department of Internal Medicine, Division of Pulmonology
Hospital of the University of Marburg
Baldingerstrasse 1, DE–35043 Marburg (Germany)
Tel. +49 6421 286 4994, Fax +49 6421 286 8987, E-Mail email@example.com
Received: September 9, 2003
Accepted after revision: February 19, 2004
Number of Print Pages : 8
Number of Figures : 6, Number of Tables : 0, Number of References : 27
Respiration (International Review of Thoracic Diseases)
Founded 1944 as ‘Schweizerische Zeitschrift für Tuberkulose und Pneumonologie’ by E. Bachmann, M. Gilbert, F. Häberlin, W. Löffler, P. Steiner and E. Uehlinger, continued 1962–1967 as ‘Medicina Thoracalis’ as of 1968 as ‘Respiration’, H. Herzog (1962–1997)
Official Journal of the European Association for Bronchology and Interventional Pulmonology
Vol. 71, No. 4, Year 2004 (Cover Date: July-August 2004)
Journal Editor: C.T. Bolliger, Cape Town
ISSN: 0025–7931 (print), 1423–0356 (Online)
For additional information: http://www.karger.com/journals/res