BALB/c female mice, age 6 to10 weeks and free of specific pathogens, were obtained from M&B, Denmark. STAT4-/- and STAT6-/- mice, both of Balb/c background, were purchased from Jackson Laboratory (Bar Harbor, Maine, USA). All the mice were housed under specific pathogen-free conditions and maintained on an ovalbumin-free diet at the Finnish Occupational Health Institute. All the experiments were approved by the State Provincial Office of Southern Finland.
Sensitisation and airway challenge
On days 0 and 14, all mice were sensitised with an intraperitoneal (i.p) injection of 50 μg of ovalbumin (OVA) (Grade V; Sigma, St. Louis, MO, USA) emulsified in 2 mg of aluminium hydroxide in a total volume of 100 μl. The mice were intranasally challenged with 50 μl of PBS (controls) or with 50 μg of OVA diluted in 50 μl of PBS for three days (days 28, 29 and 30) under isoflurane anaesthesia with spontaneous breathing. Eight mice were assigned to each group.
Determination of airway responsiveness
Airway responsiveness was assessed 24 hours after the last OVA airway challenge. A single-chamber whole-body plethysmograph system obtained from Buxco Technologies (Troy, NY, USA) was used as described [22
]. Briefly, mice were placed unrestrained into a chamber and exposed for five min to nebulised PBS and subsequently to increasing concentrations of methacholine (MCh) (Sigma-Aldrich, UK) in PBS using an AeroSonic 5000 D ultrasonic nebuliser (DeVilbiss, Somerset, PA). Recordings were taken after each nebulisation for five min. The enhanced pause (Penh) values for each five-min sequence were evaluated and expressed for each MCh concentration. Baseline Penh values did not differ significantly between groups.
Sample collections and lung preparations
The mice were killed by isoflurane overdose and the blood was drained from the hepatic vein. The chest cavity was opened, and the lungs were lavaged with 800 μl of PBS via the tracheal tube. The bronchoalveolar lavage (BAL) sample was cytospun on a slide, the cells were stained with MayGrünwald-Giemsa (MGG) stain, and the cell differentials were counted under a light microscope. The left lung was removed, quick-frozen and stored at -70°C for RNA isolation. For the immunohistological examination, half of the right lung was embedded in TissueTek OCT Compound (Sakura Finetek Europe B.V., The Netherlands), quick-frozen and stored at -70°C. The other half was fixed in paraformaldehyde and embedded in paraffin.
Real-time quantitative RT-PCR assay
Total RNA from the lungs and cells was extracted using Eurozol Reagent (EuroClone, Italy) according to the manufacturer's instructions. Total RNA was quantified with spectrophotometry (Nanodrop, Nanodrop Technologies, Wilmington, DE, USA). The RNA was reverse-transcribed into cDNA with a High Capacity cDNA Reverse Transcription Kit (Applied Biosytems, Foster City, CA, USA). The real-time quantitative polymerase chain reaction (PCR) was performed with an Applied Biosystems 7500 Fast Real-Time PCR System, according to the manufacturer's instructions. PCR primers and probes were obtained as predeveloped assay reagents (cytokines and 18 S rRNA) or were generated (tenascin-C) with PrimerExpress version 1.5 software and ordered from Applied Biosystems. The primer and probe sequences for tenascin-C were as follows: forward primer 5'-ACC ATG CTG AGA TAG ATG TTC CAA A-3', reverse primer 5'-CTT GAC AGC AGA AAC ACC AAT CC-3', and probe 5'-ACC ACA CTC ACA GGT CTA AGG CCC GG-3'. The accumulation of PCR products was detected directly by monitoring the increase in fluorescence of the reporter dye. The signals were standardised to the internal passive reference ROX to exclude non-PCR-related fluctuations in the fluorescence. In addition, FAM signals were standardised to the endogenous reference rRNA 18S to normalise the quantification of mRNA targets for differences in total RNA.
Quick-frozen samples were used for immunofluoresence. Monoclonal rat anti-mouse antibody clone MTn-12 from Sigma-Aldrich (1:200) served to detect tenascin-C and fluorescein (FITC)-conjugated anti-rat IgG (Jackson ImmunoResearch Laboratories Europe, UK) (1:100) served as a secondary antibody. The intensity of immunoreactivity for tenascin-C was graded in random order slide by slide. All the groups were graded at the same time. This resulted in grades from 0-4 (0 = no reactivity, 1 = weak reactivity, 2 = intermediate reactivity, 3 = strong reactivity, 4 = very strong reactivity). All samples were coded, and the codes were broken after completion of all the tests.
MRC-9 cells were obtained from ATCC (Manassas, VA, USA). The cells were cultured in MEM supplemented with 10% FBS, 2 mM glutamine, 0.1 mM nonessential amino acids, 1.5 g/L of sodium pyruvate and antibiotics (Gibco, Invitrogen, Life Technologies, Carlsbad, CA, USA) at 37°C in 5% CO2. Cells were stimulated with TNF-α (10 ng/ml) (Biosource International, Camarillo, CA, USA) at 37°C for three and nine hours.
Primary human dermal fibroblasts isolated from adult skin were purchased from Gibco and cultured in Medium 106 (Gibco) supplemented with LSGS (Low serum growth supplement) and antibiotics (Gibco) at 37°C in 5% CO2. Cells were stimulated with TNF-α (20 ng/ml), IFN-γ (500 IU/ml, Immuno Tools, Friesoythe, Germany) and with a combination of TNF-α and IFN-γ for 2, 6 and 18 hours.
Tenascin-C protein was analyzed by Western blot from whole-cell extracts and concentrated cell supernatants. Human primary fibroblasts stimulated with the combination of TNF-α and IFN-γ for 18 h were lysed in protein lysis buffer containing 10 mM Tris (pH7.4), 150 mM NaCl, and 25% ethylene glycol supplemented with complete mini protease inhibitor mixture (Roche Diagnostic, Indianapolis, IN). The cell extract was homogenized with ultrasound sonicator (Sanyo Electronics, San Diego, CA). Total protein concentrations were determined with Bio-Rad Dc Protein Assay (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer's instructions. 20 μg of protein from lysed cell extracts and 7,5 μl of concentrated cell culture supernatants were separated with SDS-PAGE on 12% gels and transferred onto Immobilon-P Transfer Membranes (Millipore). Membranes were blocked with 5% non-fat milk in PBS and exposed to monoclonal mouse antihuman antibody 100EB2 recognizing tenascin-C [23
]. After this, membrane was incubated with HRP-conjugated polyclonal goat anti-mouse immunoglobulins (Dako Cytomation). Proteins were visualized by Luminescent Image Analyzer (Image Quant LAS4000mini, GE Healthcare, Sweden). Total protein in the membrane was stained with SYPRO Ruby Protein Blot Stain (Bio-Rad Laboratories).
The data were analysed with GraphPadPrism Software (GraphPad Software, Inc., CA, USA). Single-group comparisons were performed with the Mann-Whitney U-test or Student's t-test when appropriate. Values for measurements are expressed as the mean ± the standard error of the mean (SEM). P values < 0.05 were considered significant.