Study design
The study protocol was approved by the Ethics Committee at Karolinska Institute (Diary N. KI 95:347). Six healthy, non-smoking volunteers gave their written consent to participate in the study after receiving both written and oral information.
All the participants were non-atopic, non-asthmatic as determined by history and questionnaire. They were exposed to organic dust while weighing pigs in a swine confinement (ie. a barn) during three hours, as previously described [
14‐
16]. The procedure incorporates exposure to aerosolised organic dust, with a concentration of approximately 24 mg dust and 1.2 μg endotoxin per m
3, as assessed in a recent study [
13]. Normal lung function and normal airway responsiveness were ascertained in all subjects prior to exposure. The subjects underwent bronchoscopy with bronchoalveolar lavage (BAL-see below) 2 weeks before and 24 hours after the exposure.
Bronchoalveolar lavage
Bronchoscopy and bronchoalveolar lavage were performed according to standard procedures previously described [
13]. In short, after pre-medication with morphine-scopolamine, a flexible fibreoptic bronchoscope (Olympus Type 4B2; Olympus Optical Co. Ltd., Shinjukuku, Tokyo, Japan) was inserted through the nose under local anaesthesia with lidocaine (Xylocain R, Astra, Södertälje, Sweden). The bronchoscope was wedged in a middle lobe bronchus and a total of 250 ml of sterile saline at 37°C was instilled in 5 aliquots of 50 ml. After each instillation, the BAL suspension was gently aspirated and collected in a siliconized plastic bottle kept on ice.
After sterile filtration (70 μm filter), the BAL suspension was centrifuged (Hereus Omnifuge 300 g, 10 min, +4°C). The cell-free supernatant was kept frozen (-80°C) until analysis. The cell pellet was resuspended (phosphate-buffered saline [PBS]: 5 ml). Viability of cells was assessed using trypan blue exclusion and the total cell number (ie. concentration) was determined for each sample in a Bürker chamber. BAL cytospin samples were prepared and stained (May-Grünwald-Giemsa) and differential cell counts were performed using light microscopy (Zeiss Axiopaln 2, Carl Zeiss, Jena, Germany).
Messenger RNA for IL-17A
Remaining cells from the resuspended pellet were centrifuged (see above) and the supernatant was removed. The cell pellet was re-suspended (PBS: 50 μl plus RNAlater from Ambion Inc, Austin, USA: 250 μl per 3 × 106 cells), transferred into microtubes, (3 × 106 cells per tube) and incubated (24 hrs, +4°C). The RNeasy minikit was used according to the manufacturer's protocol with the following modification: The eluted RNA was treated with Dnase I (Rnase-Free Dnase set: 30 min, 37°C). After this, the steps from adjusting binding conditions to eluting RNA were repeated.
The eluted total RNA was measured for quantity and purity using spectrophotometry (SpectraMaxPlus®Molecular Devices Corporation Sunnyvale, CA, USA; absorbance at 260 and 260/280 nm respectively) and then stored frozen (-83°C) until purification of Total RNA.
The RT-PCR ELISA technique was employed to quantify relative changes in the IL-17A mRNA gene transcript. A one-step RT-PCR was utilized with a Gene Amp PCR system 2400 (Perkin Elmer, Wellesley, MA, USA) for amplification. Each RT-PCR of 50 μl was conducted using 100 ng of total cellular RNA and 30 pmol of each primer, 10 U RNase inhibitor (recombinant RNasin, Promega Corporation, Madison, WI, USA), 200 μM PCR DIG Labelling mix (20 μM dATP, dGTP, dCTP each plus 19 μM dTTP plus 1 μM DIG-dUTP), 5 mM DDT, 10 μl RT-PCR reaction buffer (1.5 mM Mg+), 1 μl Titan enzyme mix (AMV reverse transcriptase+Taq DNA polymerase+Pwo DNA polymerase) or 0,5 μl Expand high fidelity PCR system (Taq DNA polymerase+Pwo DNA polymerase) for DNA controls (all reagents from Roche Diagnostics). Reverse transcription was performed (30 min, 50°C). The IL-17A and the house-keeping gene HPRT were subsequently annealed (55°C). IL-17A and HPRT were then amplified (35 and 30 cycles, respectively) and the DIG labelled PCR product was stored frozen (-20°C) before the detection step. RT-PCRs were performed in duplicate. Gene sequences were accessed from NCBI Database and accession number used was for human (h) HPRT V00530 and for hIL-17 U32659. Scandinavian Gene Synthesis AB (Köping, Sweden) provided the oligonucleotides.
The gene primer sequences used for RT-PCR and ELISA detection:
hHPRT
Sense (5') 5'CGT CGT GAT TAG TGA TGA TGA AC3'
Antisense (3') 5'GCA AAG TCT GCA TTG TTT TGC CA3'
Internal probe 5'GAG GCC ATC ACA TTG TAG CCC TCT GTG3'
hIL-17
Sense (5') 5' GTG AAG GCA GGA ATC ACA ATC 3'
Antisense (5') 5' ACC AGG ATC TCT TGC TGG AT 3'
Internal probe 5' CAG AGT TCA TGT GGT AGT CCA CGT TCC CA 3'
The DIG labelled PCR products were denaturated and hybridized with the biotinylated internal probe specifically designed to hybridize with each gene PCR product, and immobilized on streptavidin coated microtiter plates (3 hrs, 42°C), utilising the PCR ELISA DIG-detection kit (Roche). After washing, the bound PCR-products were incubated (30 min, 37°C) with an anti-DIG-horseradish peroxidase conjugated antibody followed by reaction (20 min) with the substrate 2,2'-azino-di(3-ethyl benzthiazoline sulfonate) (ABTS). The absorbance was then measured using spectrophotometry (see above, at 405/492 nm) in an ELISA plate reader (Labsystems Multiscan Multisoft, Vanda, Finland). The expression of transcripts for IL-17 mRNA was normalized to the expression of HPRT transcripts and shown as percent of the house-keeping gene (% HPRT). In parallel with tested samples, control samples for PCR, probe specificity, DNA contamination, hybridisation and sample dilution were run.
Immunocytochemistry (ICC)
General procedure
BAL cells were fixed with formaldehyde (2%, 30 min, on ice) and washed twice in buffer prior to making cytospin preparations. Air-dried samples were stored frozen (-80°C) until further use. After thawing, samples were treated with donkey serum (10%) to avoid unspecific binding. Endogenous biotin was blocked using the Biotin Blocking System (DAKO corporation, Glostrup, Denmark).
IL-17A immunoreactivity (IR)
The intracellular expression of IL-17A protein was assessed utilising cytospin slides incubated with a polyclonal goat anti-human IL-17A antibody (R&D: 1 hr). As secondary antibody, a biotinylated F(ab')2 fragment donkey anti-goat IgG (Jackson ImmunoResearch laboratories Inc) was used, followed by alkaline phosphatase-conjugated streptavidin (DAKO). All the solutions above were supplemented with saponin (Sigma: 0.1 %), to permeabilise cell membrane. Bound antibodies were visualised with Vector Red Alkaline Substrate Kit (Vector Laboratories, Inc. Burlingame, CA, USA). Mayer's Hematoxylin (Sigma) was used for counterstaining.
MMP-9 immunoreactivity
The intracellular expression of MMP-9 protein was assessed utilising cytospin slides incubated with a polyclonal goat anti-human MMP-9 (R&D: 1 hr). The secondary antibody and the detection system were the same as for IL-17A (see above).