SUBJECTS

One hundred and eleven asthmatic volunteers took part in the study. Airway endobronchial biopsy specimens were obtained from 80 subjects and BAL cells from 89 (table 1). BAL cells were obtained from a number of subjects from which biopsy specimens were not available and vice versa, hence the total number of asthmatics in the study was 111 rather than closer to 89. Asthmatic subjects fulfilled the American Thoracic Society criteria for asthma14 with documented airway reversibility to inhaled bronchodilators and increased responsiveness to methacholine. They all used albuterol for relief of symptoms as needed, with 93 patients using regular ICS. Salmeterol (50 μg twice daily) was used by 12 patients from whom BAL cells were obtained and in five of those from whom biopsy specimens were taken. No patient was taking any other anti-asthma medication. Volunteers who had suffered from acute respiratory tract infection during the previous 4 weeks, who had any change in asthma medication, or had been admitted to hospital with airway disease in the 4 weeks before the study were excluded.

Forty five normal volunteer control subjects were also recruited. They had no history of asthma or other respiratory diseases, were using no medication, had no bronchodilator response to albuterol, and a negative challenge test to 3.2 mg inhaled methacholine. All asthmatic and control subjects were non-smokers and gave informed written consent before commencing the study, which was approved by the Alfred Hospital ethics committee.

Atopic status was assessed by skin prick testing to a panel of seven common aeroallergens and was defined by a response of ⩾3 mm diameter to one or more of them. Forced expiratory volume in one second (FEV₁) and bronchial responsiveness to methacholine were measured in the morning and after a period of at least 8 hours without bronchodilators (24 hours for long acting β agonists). A rolling seal spirometer (SensorMedics Corporation, Yorba Linda, CA, USA) was used for measurement of FEV₁ according to American Thoracic Society guidelines. Bronchial responsiveness to methacholine was assessed by a previously established dosimeter technique15and expressed as the cumulative dose required to provoke a 20% decrease in FEV₁ (PD₂₀) using linear interpolation from a dose-response plot.

BRONCHOSCOPY

Bronchoscopy was performed within 2 days of methacholine challenge. Subjects were premedicated with 0.4 mg intravenous atropine and 5–15 mg midazolam. Inhaled albuterol (200 μg) was given to all subjects 15 minutes before bronchoscopy. Lignocaine spray (4%) was applied to the pharynx and larynx and 2% lignocaine was administered below the vocal cords in 2 ml aliquots as required up to a maximum dose of 6 ml. Subjects were monitored using pulse oximetry and oxygen was administered at 4 l/min during the procedure. BAL of the right middle lobe was performed by instilling three aliquots of 60 ml phosphate buffered saline with immediate aspiration into siliconised glass containers. Endobronchial biopsy specimens were taken from the segmental subcarinae of the right lower lobe of each patient using alligator forceps (Olympus, Tokyo, Japan). Samples were transported on ice to the laboratory and two biopsy specimens were snap frozen in liquid nitrogen within 15 minutes and stored at –80°C. BAL cells in aliquots of 1–4 × 10⁶ cells were rapidly pelleted by centrifugation at 1200 rpm for 15 minutes, snap frozen, and stored at –80°C.

RNA EXTRACTION

RNA from BAL cells and endobronchial biopsy specimens were extracted with RNeasy total RNA kit (Qiagen, Hilden, Germany). Tissue was homogenised in 700 μl lysis buffer RLT using a Polytron rotor-stator (Kinematica, Luzern, Switzerland) followed by aspiration 10 times through a 21 gauge syringe needle to shear chromosomal DNA. Cell pellets were resuspended in 700 μl lysis buffer RLT by aspiration 10 times through a 21 gauge syringe needle. RNA was eluted from spin columns with 40 μl diethylpyrocarbonate treated water. Aliquots of 4 μl of RNA were quantitated by capillary spectrophotometry using an absorbance capillary adaptor cell (Helix, San Diego, USA) and a Cary 1 spectrophotometer (Varian, Melbourne, VIC, Australia) with background correction at 320 nm. Samples with (A₂₆₀–A₃₂₀)/(A₂₈₀–A₃₂₀) ratios less than 1.7 and/or yields less than 0.5 μg total RNA were excluded from subsequent analysis. BAL cells gave a success rate of 90% with a yield of 2.9 μg (range 0.6–12.2) while biopsy specimens were extracted with a success rate of 70% and a median yield of 1.0 μg (range 0.5–6.5).

COMPETITOR TEMPLATES AND PRIMERS

An IL-5 competitor was synthesised by PCR mediated mutagenesis as previously described.16 The 42 base composite primer (table 2) was used with the lower IL-5 primer in 30 cycles of PCR with 250 ng of human chorionic male DNA as the template. The PCR product was diluted 1:30 and 2 μl aliquots were amplified in five secondary reactions as for samples for 35 cycles. The resulting PCR product was purified by phenol-chloroform extraction and ethanol precipitation.17

DNA competitor templates for IL-4 and IFNγ PCR were obtained from Clontech (Palo Alto, CA, USA). IL-2 PCR used a synthetic mRNA competitor, pAW109 cRNA (Perkin-Elmer, Foster City, CA, USA). Primers (synthesised by GeneWorks, Adelaide, SA, Australia) were redesigned using oligo 5.0 primer analysis software (National Biosciences, Plymouth, MN, USA) to improve their specificity and annealing profiles (table 2). All competitors were diluted to working concentrations with 50 μg/ml nuclease-free glycogen (Roche Diagnostics Australia, Castle Hill, NSW, Australia) in 10 mM Tris HCl, pH 7.5.

VALIDATION OF COMPETITIVE PCR

The simultaneous quantitation of IL-4 and IL-4δ2 by dual mRNA species competitive PCR was conducted as previously.8 To establish that mRNA and competitor templates amplify equally, a “changing input-output ratio” series of reactions was set up for each gene. To test the validity of IL-2 RT-PCR, a serial titration of pAW109 cRNA from 2 × 10³ to 5.12 × 10⁵copies per reaction was established in 15 μl cDNA synthesis reactions containing 180 ng phytohaemagglutinin (PHA) stimulated peripheral blood mononuclear cell (PBMC) RNA.17Aliquots of 2 μl cDNA were added to 100 μl PCRs which were then split into duplicates and amplified for 35 and 40 cycles. For IL-5 competitive PCR the competitor was titrated in twofold steps from 5.12 × 10⁴ copies to 200 copies against PHA stimulated PBMC cDNA and duplicate reactions were amplified for 35 and 40 cycles. IFNγ competitive PCR was similarly tested, titrating from 8 × 10⁴ copies to 3.13 × 10² copies competitor and amplifying for 30 and 35 cycles.

REVERSE TRANSCRIPTION AND COMPETITIVE PCR

cDNA was synthesised from 0.27–3.86 μg RNA (mean 0.95 and 2.03 μg for biopsy specimens and BAL cells, respectively) in a volume of 57 μl consisting of 1 × GeneAmp PCR buffer, 5 mM MgCl₂, 1 mM deoxyribonucleotide triphosphate, 0.8 U/μl RNase inhibitor, 2 μM oligo d(T)₁₆ and 2 U/μl MuLV reverse transcriptase (all from Perkin-Elmer). For cDNA synthesis from BAL cell RNA, 3 × 10⁵ copies of pAW109 cRNA was added to each reaction while, for biopsy RNA, 4 × 10³ copies of pAW109 RNA was used. Reactions were incubated for 1 minute at 15°C and then the temperature was increased to 42°C over 9 minutes followed by 1 hour at 42°C, 5 minutes at 85°C and 1 minute at 4°C.

For IL-4 and IL-5 competitive PCR, 20 μl of cDNA was added to a 100 μl reaction consisting of 2 U AmpliTaq Gold, 1 × GeneAmp PCR buffer, 1.5 mM MgCl₂, 200 μM deoxyribonucleotide triphosphate (all from Perkin-Elmer), 0.2 μM upper and lower primers, and 40 copies of competitor. Thermal cycling was conducted in an MJ Research PTC-200 (Watertown, MA, USA) with the temperature profile commencing with an initial denaturation of 8 minutes at 94°C followed by 50 cycles of 94°C for 1 minute, annealing temperature (table 2) for 1 minute, and 72°C for 1 minute, concluding with 7 minutes at 72°C and 1 minute at 4°C. IL-2 and IFNγ PCR was conducted in 50 μl reactions as above except that 5 μl and 3 μl of cDNA, respectively, and 45 cycles of amplification were used. For IFNγ PCR of BAL cDNA 600 copies of competitor was used, while 200 copies were used with biopsy cDNA.

The PCR products were resolved by electrophoresis through 1.5 mm 4% 19:1 PAGE gels (acrylamide:bisacrylamide; Bio-Rad Laboratories, Hercules, CA, USA) in 2 × TAE17 using active cooling to maintain the temperature at 16–18°C. PAGE gels were stained by overlay with 1:10 000 SYBR Green I (Molecular Probes, Eugene, OR, USA) in 2 × TAE for 45 minutes and then scanned directly in a laser based fluorescence scanner (FluorImager 575, Molecular Dynamics, Sunnyvale, CA, USA). PCR product band volumes were quantitated with ImageQuaNT software (Molecular Dynamics). Native:competitor product ratios were calculated and the levels of gene expression reported as copies of mRNA per μg total RNA.

STATISTICAL ANALYSIS

The differences between atopic and non-atopic asthmatics and controls were analysed using a generalised linear model with an interaction between atopy and asthma being fitted to the model. Similarly, other interaction terms such as ICS usage were fitted where necessary. Age, sex, use of long acting β₂ agonists, and RT-PCR batch factors were included in all models as potential confounding variables and then post hoc comparisons were made between groups after fitting the overall model. The results are presented as least squares mean (SE) values with differences between groups calculated in the order written. Given that some outcome variables followed a log-normal distribution, the relevant data were calculated as log_e so these comparisons are presented as ratios with 95% confidence intervals (CI) rather than differences. The Pearson correlation was used to test associations between IL-4 and IL-4δ2 and the Mann-Whitney U test was used for intergroup comparisons for IL-4/β-actin ratios. Statistical analyses were performed using SAS version 6.12 software (SAS institute, Cary, NC, USA).18