Allergen-induced IL-9 directly stimulates mucin transcription in respiratory epithelial cells (original) (raw)

Cell culture. All tissue culture media and supplies were obtained from GIBCO BRL (Grand Island, New York, USA) or Clonetics (San Diego, California, USA). A human pulmonary mucoepidermoid carcinoma cell line, NCI-H292, was purchased from the American Type Culture Collection (Rockville, Maryland, USA) and maintained in RPMI-1640 with 2 g/L glucose, 0.3 g/L L-glutamine, 2 g/L NaHCO3, 10% fetal calf serum, 100 U/mL penicillin, and 100 mg/mL streptomycin at 37°C in 5% CO2. Human primary bronchial epithelial cells (Clonetics) were maintained in bronchial epithelial basal medium (BEBM) with added growth factors according to the recommendations of the supplier. Both cell types express receptors for IL-5, IL-9, and IL-13 as determined by RT-PCR of whole-cell RNA (see below).

For function-blocking antibody experiments, antibodies to cytokine receptors were purchased from R&D Systems Inc. (Minneapolis, Minnesota, USA) or Chemicon International (Temecula, California, USA) and used at concentrations recommended by the manufacturers (1–3 μg/mL). We immunoprecipitated cytokines from experimental medium using standard methods and cytokine- or receptor-specific antibodies (R&D Systems Inc.).

Human tracheal aspirates. Tracheal aspirates were obtained from subjects recruited from Moffitt-Long and San Francisco General Hospital (University of California–San Francisco, San Francisco, California, USA). Medical consent was obtained, and a medical questionnaire was filled out for each patient. Subjects who had smoked cigarettes or used any other recreational inhalant in the past 10 years were excluded from the study. Also excluded were subjects who had evidence of congestive heart failure or who had a history of lung disease other than asthma. Five patients (4 women, 1 man; mean age 39.8 ± 6.9 SEM) diagnosed as having acute severe asthma by intensive care physicians, and who had been intubated for the management of their asthma, and 5 patients (2 women, 3 men; mean age 32.8 ± 5.4 SEM) undergoing delivery of general anesthesia for nonpulmonary surgical procedures all had tracheal aspirates collected 0–12 hours after intubation. Standard clinical methods were used for collecting tracheal aspirates from healthy and asthmatic subjects in this study. A 30-cm thin plastic catheter (Suction Catheter Kit; Kendall Health Care Products Company, Mansfield, Massachusetts, USA) was passed through the endotracheal tube and into the airway. Mucus was aspirated into a clean plastic container. Aspirate volume was determined and mixed with an equal volume of sterile saline. This was then diluted with saline containing DTT (10% sputolysin [0.01% final concentration]; Behring Diagnostics Inc., Somerville, New Jersey, USA). The aspirated sample (now diluted 1:4 from the original sample) was incubated in a shaking water bath at 37°C for 15 minutes. The samples were then centrifuged at 1,037 g for 5 minutes, and the supernatants were stored at –70°C. We exposed NCI-H292 cells to human tracheal aspirates diluted 1:6 with culture medium for 8 hours. We harvested cultured cell RNA and monitored it for the presence of MUC5AC mRNA by RT-PCR. MUC5AC transcriptional activity was evaluated after 16 hours by measuring luciferase activity (see below).

Allergic dog lavage. Dogs were sensitized as pups with ragweed antigen and 1 lung in each of 5 adult dogs was challenged with ragweed. See Collie et al. (21) for detailed sensitization, challenge, and lavage procedures. Challenges were followed by timed bronchoalveolar lavages. For the present study, we focused on the “3-day pre-challenge” and the “4-day post-challenge” samples as these showed the greatest difference in in vitro mucin-inducing activity in preliminary screens. NCI-H292 cells were transfected with MUC5AC luciferase (see below) and treated with dog lavage samples diluted 1:3 with serum-free RPMI. Macrosep centrifugal concentrators (Pall Filtron Corp., Northborough, Massachusetts, USA) were used for fractionation of lavage fluids by molecular weight (100kDa, 30kDa, 10kDa, and 3kDa) according to manufacturer’s recommendations. The Macrosep device was rinsed with buffer, loaded with biological fluid, and then centrifuged at 3,000–5,000 g for 60–90 minutes. All retentates were then volume corrected with PBS to maintain original concentration of the retained molecules. Some lavage samples were incubated with proteinase K (1:20, proteinase/protein ratio) at 60°C for 1 hour followed by 15 minutes of boiling to determine whether the active factor was a protein. Negative controls consisting of PBS with proteinase K alone were used to determine possible effects of proteinase K on mucin transcription.

RT-PCR. Reverse transcription of RNA to cDNA was carried out using the following reaction mixture: 5 μg total RNA from each sample in 8 μL diethyl cyanophosphate–treated (DEPC) water, 4 μL 5× first strand buffer (GIBCO BRL), 2 μL 10 mM deoxynucleotide triphosphate (dNTP) (GIBCO BRL), 2 μL 0.1 M DTT (GIBCO BRL), 1 μL RNase inhibitor (40 U; Roche Molecular Biochemicals, Indianapolis, Indiana, USA), 2 μL 10× hexonucleotides (Roche Molecular Biochemicals), and 1 μL SuperScript II (GIBCO BRL). The reaction mixture was incubated at 37°C for 1 hour, then at 75°C for 15 minutes, and stored at 4°C. PCR was carried out using Ready-To-Go PCR Beads (Pharmacia Biotech Inc., Piscataway, New Jersey, USA) in a 25-μL reaction mixture containing: 1.5 U Taq polymerase, 10 mM Tris-HCl (pH 9.0 at room temperature), 50 mM KCl, 1.5 mM MgCl2, 200 μM of each dNTP and stabilizers, including BSA, 0.2 μM of each primer, and 1 μL of RT-PCR reaction product. Custom primers were obtained from Life Technologies (Gaithersburg, Maryland, USA). Human GAPDH primers were 3′: GGG GTC TAC ATG GCA ACT GTG AGG AGG GGA and 5′: AAA CCT GCC AAA TAT GAT GAC ATC AAG AAG; human MUC5AC primers 3′: TCA CAG CCG GGT ACG CGT TGG CAC AAG TGG and 5′: TGC TAT TAT GCC CTG TGT AGC CAG GAC TGC; human IL-9 receptor (IL-9R) α primers were 3′: TGC CTG ACA AAC TCA GTG CCA CAC TGG and 5′: CCT GTG TGA ACC TGT CGT GCA AAG CTCA; human IL-13Rα primers were 3′: GCC CTT GTT AAG ATC AAA CCC ATC and 5′: TGA GTC TAA CGG TCT TCC GGA TGAA; human IL-5Rα primers were 3′: CAA CAA GCC AGG TGC AGT GAAG and 5′: CCA CTG AGA TAC TGC AAG CTG AC. We used a standard PCR protocol: 5 minutes at 95°C followed by 35 cycles of 30 seconds at 95°C, 30 seconds at either 60° or 55°C (depending on the annealing temperature of the primers), and 60 seconds at 72°C. After cycling, the samples were kept at 72°C for 20 minutes and stored at 4°C. Samples were run on a 1.2% agarose gel containing ethidium bromide for visualization under an ultraviolet lamp.

Luciferase assay. Approximately 3.7 kb of the 5′ flanking region of the human MUC5AC gene (22) was cloned into pGL3-Basic luciferase vector (Promega Corp., Madison, Wisconsin, USA). NCI-H292 cells were seeded in 6-well tissue culture plates (106 cells/well) and left for 18 hours in complete growth medium. Lipofectamine (GIBCO BRL) transfection of cells was done in accordance with the instructions of the manufacturer. Briefly, 1 μg DNA, 0.8 μg histone, and 2 μg Lipofectamine in 1 mL OPTIMEM (GIBCO BRL) reduced serum medium was added to each well. After 4 hours, the OPTIMEM medium was replaced with complete growth medium and the cells allowed to recover overnight. Luciferase activity was measured according to manufacturer’s recommendations (Promega Corp.). Briefly, 250 μL of lysis buffer (Promega Corp.) was added to each well and plates were agitated for 1 hour at room temperature. The wells were then scraped with a rubber cell scraper, and the contents of the wells were transferred into 1.5-mL tubes and briefly centrifuged to remove cell debris. Twenty microliters of supernatant was added to 100 μL luciferase assay reagent and light units measured for 30 seconds on a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, California, USA). All transfections were carried out in triplicate. Luciferase activity was normalized with respect to relative light units emitted from serum-free, medium-treated controls.

ELISA. Serial dilutions of each dog lavage or human aspirate sample were plated in duplicate on 96-well microtiter plates (Immulon; PGC Scientific, Gaithersburg, Maryland, USA). Wells containing no sample served as negative controls, and wells containing recombinant human IL-9, IL-5, or IL-13 (R&D Systems Inc.) served as positive controls and concentration standards on each plate. Antigen was dried onto the plates overnight at 40°C. Plates were then treated with 1% normal rabbit serum/PBS/Tween 20 (RPT) for 1 hour before incubation with primary antibody for 2 hours at room temperature. Antigen-coated wells were incubated with 1 μg/mL goat anti-human IL-9, IL-5, or IL-13 antibody (R&D Systems Inc.). The plates were then washed 3 times with RPT. Plates were then incubated with a 1:200 dilution of biotinylated rabbit anti-goat IgG (Vector Laboratories, Burlingame, California, USA) for 2 hours at room temperature. All plates were then washed 3 times with RPT and incubated (1 hour at room temperature) with an avidin/alkaline phosphatase conjugate (ABC-AP; Vector Laboratories). After washing, wells were incubated (15 minutes, 37°C) with 1 mg/mL _para_-nitrophenyl phosphate (Sigma Chemical Co., St. Louis, Missouri, USA) in Tris buffer (pH 7.2). Absorbance at 492 nm was determined on a Titertek Multiskan MC microplate reader (Eflab Oy, Helsinki, Finland). IL-9 concentrations were determined from a standard curve. The lower detection limits for ELISAs were between 8 and 16 pg/mL for IL-9; 31 and 63 pg/mL for IL-13; and 63 and 125 pg/mL for IL-5.

Ribonuclease protection assay (RPA). A PCRII plasmid (Invitrogen Corp., San Diego, California, USA) containing MUC5AC (clone 298 BP) or cyclophilin was linearized with the appropriate restriction enzyme in a 100-μL volume overnight at 37°C to give the antisense strand. The enzyme was then digested with the addition of 5 μL of 10% SDS and 1 μL proteinase K (10 mg/mL) at 65°C for 15 minutes before phenol/chloroform/isoamyl alcohol extraction. The extract was precipitated with 50 μL of 5 M ammonium acetate and 450 μL ethanol for 15 minutes at –80°C. The pellet was redissolved in 20 μL DEPC water. Riboprobe was synthesized with 5 μL 5× transcription buffer, 10 μM cold NTPs, 10 μM DTT, 40 U RNase inhibitor, 50 μCi α32P-labeled UTP (400 Ci/mM), DEPC water, 2 μL linearized and purified DNA template, and 1 μL of T7 polymerase at 37°C for 1 hour. RNase-free DNase I was then added at 37°C for 30 minutes. The probe was then ethanol precipitated twice. The pellet was resuspended in 8 μL of gel-loading buffer. To purify the probe, it was run on a 0.75-mm-thick 6% polyacrylamide/8 M urea gel at 1,000 V for 30 min. The target band on the gel was located by exposing the gel on film for 15 seconds. The band was excised and incubated in elution buffer (RPA II kit; Ambion Inc., Austin, Texas, USA) at 37°C overnight. The RNA samples were reprecipitated and redissolved in 20 μL of 1× hybridization buffer (80% formamide/20% 5× hybridization buffer). The purified probe was diluted in 1× hybridization buffer so that each sample would have 500,000 cpm. After 10 μL of the probe was added, the samples were heated at 95°C for 5 minutes before hybridizing at 45°C overnight. After hybridization, 200 μL of diluted RNase A and T (RPA II kit; Ambion Inc.) was added to each sample, and they were incubated at 37°C for 30 min. The samples were precipitated and redissolved in 4 μL gel-loading buffer. The samples were then denatured at 95°C for 5 minutes before running on a 0.5-mm-thick 6% polyacrylamide/8 M urea gel at 1,500 V for 2 hours. The gel was dried and exposed to X-omat AR film (Eastman Kodak Co., Rochester, New York, USA) at –80°C for 2–7 days.

Bronchial biopsies. All biopsies were performed with informed consent of the study participants by S.W. (National Jewish Hospital, Denver, Colorado, USA). Five asthmatic individuals and 5 normal controls participated. The bronchoscope was passed nasally, and 6 endobronchial biopsies were taken from the first and second subcarinae of the right or left lower lobes. Endobronchial tissue was fixed overnight at –20°C in acetone, and then embedded in glycol methacrylate resin. Tissue blocks were stored at –20°C until 2-μm sections were cut using a Reichert Ultracut E ultramicrotome (Leica Microsystems Inc., Deerfield, Illinois, USA). Tissue sections were treated with trypsin at 37°C for 10 minutes and rinsed in PBS containing 0.05% Tween 20. The tissue was blocked with normal horse serum for 20 minutes and incubated in either anti–IL-9R (R&D Systems Inc.) or isotype control (anti-trinitrophenol; PharMingen, San Diego, California, USA) for 2 hours. After washing in PBS/Tween 20, the tissue was treated with a secondary biotinylated anti-mouse IgG (Vector Laboratories) for 1 hour and washed again. The tissue was then incubated with avidin-HRP conjugate (Vector Laboratories) for 30 minutes and washed thoroughly. The slides were then developed with diaminobenzidine tetrahydrochloride (DAB) (Vector Laboratories) for 4 minutes at room temperature and counterstained with hematoxylin (Sigma Chemical Co.).

Intratracheal instillation of IL-9 in mice. Female (age 8 weeks, weighing 20–23 g) C57BL6/J mice were purchased from The Jackson Laboratory (Bar Harbor, Maine, USA). Mice were anesthetized with Avertin (2.5% solution, 0.15 mL/10 g intraperitoneally). The trachea was surgically exposed by making a 1.5-cm incision in the skin and carefully separating the thyroid gland and overlying strap muscles. A 100-μL volume of rmIL-9 (10 μg/mL in PBS; R&D Systems Inc.), or PBS was instilled into the trachea using a 27-gauge needle. The incision was closed with surgical silk, and the mice were observed until they recovered from anesthesia (usually 1 hour). The mice were returned to filter-topped, autoclaved cages for 1 week with free access to food and water.

The mice were sacrificed by cervical dislocation, and their lungs were excised and fixed by perfusion through the trachea with 4% paraformaldehyde at a constant pressure of 20 cm H20. After 2 hours perfusion-fixation, the lungs were immersed in a large volume of the same fixative overnight. Tissues were embedded in paraffin, sectioned (5 μm), and then stained with periodic acid/Schiff reagent (PAS) and hematoxylin.

The trachea and main stem bronchi were the focus of study as these airways have a quantifiable population of mucin-producing goblet cells, and these airway structures are easily identifiable in section. We obtained measurements from 17–19 cross sections of the main stem bronchi from each mouse and 14–16 longitudinal sections of each trachea taken at 20-μm intervals. Using ×400 magnification, all PAS-positive cells lining the trachea and main stem bronchi were counted. The length of the basal lamina for each sectioned airway was determined using a computerized sterology system that used software capable of making calibrated linear measurements from contour tracings (Stereoinvestigator; Microbrightfield Inc., Colchester, Vermont, USA). The data for each group were expressed as the mean number of PAS-positive cells per millimeter of basal lamina.

Statistical analysis. Experimental data were evaluated by one- or two-way analysis of variance (23). Student-Newman-Keuls post-hoc comparison of means (23) was used to further assess significance. Significance was defined as P < 0.05.