Subjects
Sixteen healthy volunteers (9 females and 7 males), with a mean age of 24 years (range 20–31), were enrolled in the study. All subjects were non-smokers with normal lung function and negative skin prick tests against a standard panel of airborne allergens – see supplemental table s3. They were all free from respiratory tract infections at least six weeks prior to, and during, the study period. The study was approved by the local Ethical Review Board at Umeå University and performed in accordance with the Declaration of Helsinki, with written informed consent of all participating volunteers.
DE exposure
All subjects were exposed on two different occasions, once to filtered air and once to diesel engine exhaust, in a randomized order, at least three weeks apart. Each exposure lasted for one hour, during which the subjects alternated between fifteen-minute intervals of rest and exercise on a bicycle ergometer, with the workload adjusted to achieve a minute ventilation of 20 L/min/m2 body surface. Diesel exhaust was generated by an idling Volvo diesel engine Volvo (TD45, 4.5 L, 4 Cylinders, 1991, 680 rpm) running on Gasoil E10 (Preem, Sweden). The majority of the exhaust was shunted away, while the remaining part was diluted with filtered air and fed into the exposure chamber where air pollution parameters were continuously monitored. The mean concentration of particulates with a mass median diameter smaller than 10 µm (PM10) was 290 ± 27 µg/m3 during the diesel exhaust exposures. This was associated with concentrations of nitric oxide (NO) of 2.9 ± 0.37 ppm, nitrogen dioxide (NO2) of 0.84 ± 0.10 ppm, total hydrocarbons (HC) of 1.2 ± 0.15 ppm and carbon monoxide (CO) of 2.4 ± 0.53 ppm. Chemical characterization of the exposure emissions has been published elsewhere (33, 37).
Bronchoscopy and processing of samples
Bronchoscopy was performed six hours after both exposures using a flexible video bronchoscope (Olympus BF IT160, Tokyo, Japan). Endobronchial mucosal biopsies were taken either from the anterior aspect of the main carina and the subcarinae of the 3rd and 4th generation airways of the right side or from the posterior aspect of the main carina and the corresponding subcarinae on the left side. Bronchial wash (BW, 2 x 20 ml) and bronchoalveolar lavage (BAL, 3 x 60 ml) with saline were carried out on the contra-lateral side, in a pre-determined randomized way. The aspirates recovered from the first and second 20 ml instillations of the BW and the pooled BAL were collected into separate siliconized containers placed on ice. All lavage samples were filtered through a nylon filter (pore diameter 100 µm) and centrifuged at 400 g for 15 minutes. Cell pellets were re-suspended in PBS at a cell concentration of 106 cells/ml. Differential cell counts were performed on slides made by cyto-centrifuge preparation and stained with May-Grünwald Giemsa and 400 cells per slide were counted. Biopsies were fixed overnight (16–20 h) at -20°C in chilled acetone, containing protease inhibitors (20 mM iodoacetamide and 2mM phenylmethylsulphonyl fluoride). After fixation the biopsies were processed into glycolmethacrylate (GMA) resins, as described earlier (38).
Immunohistochemistry
Antibodies for detection of inflammatory cells, including neutrophils and mast cells, were purchased from Dako (Glostrup, Denmark). Human anti CD3, which is a T cell marker, and human anti ECP (EG2), an eosinophil marker, were purchased from Serotec (Oxford, UK) and Diagnostic development (Uppsala, Sweden), respectively. Antibodies used to detect transcription factors and detoxification enzymes, including p-c-Jun, NQO1, c-Fos (mouse monoclonal antibodies), Nrf2 and AHR (rabbit polyclonal antibodies), were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The supplier for the antibodies against Cyp1A1, Cyp1B1, EPHX (mouse monoclonal antibodies) and Aldo-keto reductases (AKR1A1, AKR1C1 and AKR1C3) was Abcam (www.abcam.com). Biotinylated rabbit anti-mouse and swine anti-rabbit antibodies were purchased from Dako.
The staining procedure has been previously described in detail (4, 11). Briefly, the GMA-embedded biopsies were cut in 2-µm thin slices and floated onto ammonia water (1:500). They were collected onto 0.01% poly-L-lysine-coated glass slides and dried at room temperature for one hour. For staining of inflammatory cells, the sections were treated to block endogenous peroxidases and nonspecific antibody binding, and the primary antibody was applied and incubated at room temperature overnight. After rinsing, biotinylated rabbit anti-mouse antibodies against the monoclonal antibodies, and swine anti-rabbit antibodies against polyclonal antibodies (Dako), were applied for two hours, followed by VECTASTAIN Elite ABC kit (Vector Laboratories, Burlingame, USA) for another two hours. Sections for submucosal analysis were developed with aminoethyl carbazole (AEC) as substrate. All sections were counterstained with Mayer´s hematoxylin. Sections where the primary antibody was omitted served as negative controls. The staining procedure was partially modified for transcription factors and detoxification enzymes. Supplementary steps were added in order to increase the permeability of the cells. Sections for epithelial measurements were developed as a brown colour with 3,3-diaminobenzidine (DAB). A detailed description of the staining procedure has previously been published (11).
When quantifying the immunohistochemical staining, a light microscope was used to count inflammatory cells according to their immunoreactivity with specific antibodies in the epithelium and the submucosa respectively, excluding mucosal glands, blood vessels and smooth muscle. Cell counts were expressed as cells/mm2 in the submucosa and cells/mm in the epithelium. A computer-assisted image analysis program (Leica Q500IW, Leica Cambridge UK) was used to calculate the length of the epithelium and the area of the submucosa. For quantification of transcription factors in the bronchial epithelium (Fig. 3), total staining (cytoplasmic and nuclear) was expressed as the percentage of the total epithelial area showing positive immunostaining, using the image analysis equipment. It was possible to distinguish between nuclear and cytoplasmic staining using a light microscope. Positive staining of the nucleus was expressed as the number of positive nuclei/mm2 of epithelium.
Due to lack of double staining or sequential sections for co-localization, we were not able to quantify nuclear translocated AhR in specific cells in the submucosa. Therefore, AhR activation in the submucosal leukocytes was expressed as the number of nuclear translocated AhR in the leukocytes/mm2 submucosa area.
Antioxidant analysis
Cell-free BW and BAL supernatants were analysed for total protein, GSH, GSSG, vitamin C (AA and DHA), and UA concentration, as previously described (39). Briefly, total glutathione concentrations were measured using the GSSG-reductase-DTNB recycling method. AA and UA were measured simultaneously by reverse phase HPLC with electrochemical detection, as previously described (39). Total vitamin C (DHA + AA) was measured by pre-treating samples with 50 mM Tris(2-carboxylethyl) phosphine for 15 minutes to reduce DHA and then performing the lipid extraction and HPLC analysis as described above. The DHA concentration was then calculated by subtracting the AA concentration from the total vitamin C concentration.
Cytokine analyses
Cytokine concentrations were determined in untreated lavage fluids. IL-17A, IL-17F, IL-23, and TGF-β1 concentrations were measured with commercial ELISA kits (Gen-Probe Diaclone, France) and IL-17E concentrations was determined by using Human IL-17E ELISA Construction Kit (Antigenix America). IL-6 and IL-10 were measured using Quantikine HS ELISA kit, R&D Systems (Abingdon United Kingdom). All analyses were performed according to manufacturer’s instructions.
Statistical analysis
The Wilcoxon nonparametric signed-rank test for paired observations was employed. A p-value of < 0.05 was considered significant. Correlation analyses were carried out using Spearman's rank-order correlation and a p-value of < 0.01 was considered significant. All statistical analyses were performed using SPSS, version 18.0 (SPSS inc., Chicago, US).