Carbon nanoparticles (CNP, Printex 90) were obtained from Degussa (Germany), and carbon particles (CP) were from H. Haeffner (Chepstow, UK; as Huber 990). Stock suspensions [1 mg/ml] of particles were prepared in PBS by sonication for 60 min. Particles and particle suspensions were characterized by (i) scanning electron microscopy (JSM 7000 F, JEOL Ltd., Japan), (ii) BET using FlowSorb II 2300 analyzer (Micromeritics, Norcross, USA), and (iii) light scattering using ZetratracTM NPA152 (Microtrac, Montgomeryville, PA, USA). Particle properties are listed in Table 1S (see Additional file 1).
Cell culture and exposure
The rat lung epithelial cell line RLE-6TN was purchased from ATCC (Manassas, VA) and grown at 37°C, 5% CO2 in supplemented Ham's F-12 medium . For experiments, cells were grown to 80 – 90% confluence, then starved for 20 h in serum reduced medium [0.5% FCS] prior to particle exposure [10 μg/cm2. Cells were treated with CNP and CP for up to 1 h in the absence or presence of inhibitors. Inhibitors were added to the cells 18 h (NAC), 4 h (ectoine), or 60 min (compound 32, alpha-tocopherol) prior to treatment with CNP or C6 ceramide (N-hexanoyl-D-erythro-shingosine, Calbiochem, Schwalbach, Germany). Alpha-tocopherol [50 μM] was solubilized in ethanol (final ethanol concentration 0.03%). C6 ceramide was solubilized in DMSO (final DMSO concentration 0.1%). In experiments using these compounds, respective vehicle controls were performed. The effect of DMSO on lipid raft composition was investigated in one control experiment. DMSO treated samples showed no difference to PBS treated samples.
Cell viability was evaluated by WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) assay (Roche diagnostics, Mannheim, Germany) according to the manufacturers’ instructions after 1 h exposure as described above. Eight-fold measurements were performed in three independent (n=3) experiments. Viability was estimated relative to medium (negative) controls and exposure to NaN3 [1 M] as positive control.
Female C57BL/6JRj mice (8 weeks old, Janvier, France) were treated via pharyngeal aspiration with a volume of 50 μl suspension, under inhalation anaesthesia (isoflurane, 5%, 1–2 min). Animals were sacrificed by exsanguination under anaesthesia 24 h after treatment. After broncho-alveolar lavage (4 x 1 ml PBS), lung tissues were minced, shock frozen and stored at −80°C until further use. Differential cell counts were performed from Giemsa/May-Grünwald stainings of lavage cells. Cell free lavage fluids were subjected to solid-phase ELISA in order to determine KC (R&D systems, Minneapolis, MN). All animal experiments were performed after relevant permission according to German animal protection laws.
Isolation of detergent resistant membrane raft fractions
Membrane fractions were isolated by density gradient ultracentrifugation as described (22). Exposed cells were harvested in 1 ml TNE buffer [50 mM Tris/HCl pH 7.4, 150 mM NaCl, 2 mM EDTA]. After Dounce homogenization (25 strokes in 1 ml TNE), Triton X-100 was added (1% w/v final concentration). After 30 min on ice the lysate was subjected to density centrifugation (5-30-50% OptiPrep™, 40 000 rpm, rotor 50.2-Ti, 4 h at 4°C). Fractions (7 × 600 μl) were collected starting at the top of each gradient.
Identification of raft fractions was performed by detection of the raft marker ganglioside GM1 in 2 μl of each fraction spotted on nitrocellulose by HRP-labelled cholera toxin subunit B (1 h; Sigma, Germany). Signal strength was detected using the ECL Plus Western Blotting Detection System (GE Healthcare, Buckinghamshire, UK).
Lipid extraction and high-performance thin layer chromatography (HPTLC)
Both methods were performed according to Grether-Beck et al. . Briefly, quantification of lipids was carried out using 500 mg protein for Folch extraction. Ceramides need to be extracted from the biological sample by alkaline hydrolysis, whereas sphingomyelin and glycosphingolipids are extracted without the hydrolysation. After extraction, samples and standards were separated on silica gel HPTLC plates using a stepwise elution gradient with methanol, dichloromethane, and n-hexane. Visualization of separated bands was carried out by post-chromatic derivatisation after dipping in a manganese chloride solution.
Cells: After exposure, cells were washed twice with ice-cold PBS and lysed in modified RIPA buffer [25 mM Tris/HCl, pH 7.4, 150 mM NaCl, 0.1 mM EDTA, 1% Nonidet P-40, 0.1% SDS, 1% deoxycholate, 0.025% NaN3, 1% protease inhibitor cocktail, and 1% phosphatase inhibitor cocktail]. Lung tissue: Minced tissue was incubated with RIPA buffer and protein was isolated using a pebble mill. After determination of protein contents, equal amounts of total cell protein [5 – 40 μg] were separated by SDS-PAGE [7.5 or 10%] and transferred onto PVDF membranes (Hybond-P, Amersham Biosciences, Little Chalfont, UK). The following antibodies were used: phospho-EGFR (Tyr1173), phospho SFK (Tyr416), and phospho-p44/42 MAPK (Thr202/Tyr204) (all from Cell Signalling Technology, Danvers, MA), total EGFR (Upstate Biotechnology, Lake Placid, NY), total SFK (Cell Signalling Technology), and GAPDH (Imgenex Corp., San Diego, CA). Signal strength was detected using the ECL Plus Western Blotting Detection System. Band intensities from X-ray films (immunosignal) were used for statistical calculations. The depicted graphs show immunosignals realtive to the respective controls.
Cells were fixed with 4% paraformaldehyde for 15 min at room temperature. After permeabilisation and blocking (in 3% bovine serum albumin, 0.3% Triton X-100 in PBS), cells were incubated with anti-EGFR antibody (1:50; Cell Signalling Technology) overnight at 4°C. After incubation with anti-rabbit Alexa Fluor 594-coupled antibody (1 h, 1:500; Invitrogen, Darmstadt, Germany), nuclei were counterstained with 4′,6-diamidino-2-phenylindole (1:2000, Invitrogen). Cells were visualized using an Axiovert 40 microscope (Zeiss, Jena, Germany). As control for the specificity of the reactions, mock immunostainings without primary antibody were performed.
Cells were incubated for dye uptake with 20 μM of the fluorogenic probe H2DCF-DA (2', 7'-Dichlorofluorescein diacetate, Calbiochem, Schwalbach, Germany). Generation of ROS was determined by flow cytometry according to Weissenberg et al. .
In vitro experiments were repeated independently at least three times; animal experiments were performed with 5 animals per group. Unless otherwise stated, results were analyzed by analysis of variance followed by post-hoc testing. Differences between groups were considered as significant when p < 0.05.