METHODS
Subjects
Sixteen mild asthmatic subjects were screened for this study and twelve went on to
complete bronchial provocation challenge with AMP and bronchoscopy. All subjects
had mild asthma (forced expiratory volume in 1 second (FEV1) > 70% of predicted)
and a history of atopy.
Each subject had infrequent symptoms, controlled with
occasional inhaled short-acting ß2-agonists alone. No subject was taking any regular
anti-asthma therapy and none had taken inhaled corticosteroids for at least 3 months
prior to entry. None of the subjects had an exacerbation of asthma or respiratory
infection in the preceding 6 weeks and all subjects were non-smokers.
demographics are summarised in Table I.
Patient
For comparison, biopsies from eight
healthy age-matched non-smoking subjects obtained in a separate but recent previous
study under the same conditions were also analysed. The demographics from these
subjects are described in Table 1. Written informed consent was obtained from each
subject and the study was approved by the Ethics Committee of King's College
Hospital.
Study design
Procedures at Visit 1 for all subjects included medical history and physical
examination, an asthma characterisation questionaire, intradermal skin prick tests,
spirometry and methacholine challenge to determine provocative concentration (PC)20
values. After approximately 1 week, asthmatic subjects returned for Visit 2 where
they underwent AMP challenge to determine the PC20 to AMP. All subjects refrained
from using rescue medication and caffeinated beverages for at least 8 hours before
each visit. After a period of 3-4 weeks, all subjects returned for Visit 3 at which
bronchoscopy was performed.
Intradermal Skin Prick Tests
Stock solutions of allergen extract (10,000 U/ml) for Dermatophagoides
pteronyssinus, mixed grass pollen or cat dander (ALK Abello, Horsholm, Denmark)
and saline control were applied intradermally to the volar aspect of the forearm. The
wheal and flare response was measured after 15 minutes and results recorded. A skin
response greater than 3 mm in comparison to saline control was considered to be a
positive reaction.
Bronchial provocation challenge with AMP/methacholine and pulmonary
function measurement
On each challenge day a fresh stock solution of 800 mg ml-1 AMP (Sigma
Biochemicals, Poole, U.K) was prepared using 0.9% saline as a diluent. Doubling
dilutions from 800 mg ml-1 to 0.39 mg ml-1 were then prepared by sequential 2-fold
dilution with saline. In a similar manner a fresh solution of 32 mg ml-1 methacholine
(Sigma Biochemicals, Poole, U.K) was made up in 0.9% saline and doubling dilutions
from 32 mg ml-1 to 0.0625 mg ml-1 were prepared.
Each solution was administered from a hand-held nebuliser attached to a breathactivated dosimeter (Mefar, Brescia, Italy) with a delivery time of 1s per breath. The
nebuliser delivers particles with an aerodynamic mass median diameter of 3.5-4.0 m
and an output of 14 l per breath. Spirometry was assessed by measurement of FEV1
with a dry wedge spirometer (Vitalograph, Buckingham, U.K.). A standard protocol
was followed for all AMP/methacholine provocational challenges. Following a 15minute rest period, subjects wearing a nose-clip, performed three FEV1 measurements
one minute apart, the best of which was taken as baseline. Subjects then inhaled 5
breaths of saline as a control with an inhalation time of one second from functional
residual capacity (FRC) to total lung capacity (TLC) and a breath-hold time of 6
seconds.
Provided FEV1 remained within 10% baseline, subjects continued by
inhaling 5 breaths of the lowest concentration of AMP/methacholine followed by
doubling increments, at 3-minute intervals. FEV1 was measured at 1.5 and 2.5 minute
intervals following inhalation of each of each dilution and the highest value was
recorded for analysis. Challenges were terminated when at least a 20% fall in FEV 1
from post-saline was recorded or the maximum concentration had been given. A
logarithmic concentration-response curve was conducted and the PC20 calculated by
linear interpolation.
Fiberoptic bronchoscopy
All subjects underwent bronchoscopy in the same manner.
Supplemental nasal
oxygenation and intravenous sedation with alfentanil (0.2-1 mg) (Janssen-Cilag Ltd,
High Wycombe, U.K.) and midazolam (1-10 mg) (CP Pharmaceuticals, Wrexham,
U.K.) was given to each subject.
Topical anaesthesia was achieved with the
application of 4% lignocaine (Astra-Zeneca, Sweden) to the nasal passages, pharynx,
vocal chords and trachea followed by 2% lignocaine to the carina, right main
bronchus and right middle lobe and lower lobe bronchi. The bronchoscope was
passed transnasally or orally with the subject at 45 degrees. Following inspection of
the airways and administration of any additional topical anaesthetic (lignocaine gel,
Bio-rex, Enfield, U.K.) to control cough, biopsies were taken under direct
bronchoscopic vision from the right middle, upper and lower lobe segmental and/or
subsegmental carinae.
Immunohistochemistry
Using the methods described below to validate the adenosine receptor subtype
specificity of an antibody, we found that the specificity of commercially available
antibodies against all adenosine receptor subtypes was unsatisfactory, hence an
antibody against the adenosine A1 receptor was created specifically for the present
study (the costs involved in obtaining a custom-made antibody was also a contributing
factor when deciding not to concurrently profile expression of the other adenosine
receptor subtypes). Thus, the custom-made affinity purified A1 receptor polyclonal
antibody was raised in the rabbit against a synthetic peptide corresponding to the
amino acid sequence 309-326 (CQPAPPIDEDLPEERPDD) of the COOH-terminus of
the human A1 receptor (Cambridge Research Biochemicals, Cleveland, U.K.).
Biopsies were lightly fixed in 4% paraformaldehyde for 1-2 hours and then
cryoprotected in 15% w/v sucrose solution overnight at 4ºC. Biopsies were then
embedded in Optimal Cutting Temperature Compound (OCT, Agar Scientific Ltd,
Stansted, U.K.), snap frozen in isopentane cooled by immersion in dry ice and then
stored at -80ºC until required for sectioning.
In addition to immunohistochemical analysis of healthy and asthmatic bronchial
biopsies with the A1 receptor antibody, brain and cardiac tissue sections were
concurrently analysed as a positive control, as it is well established that these tissues
highly express A1 receptors. Cadaveric brain cortex and cardiac tissue samples were
obtained from the Institute of Psychiatry Brain Bank and Peterborough Tissue Bank
respectively, following GlaxoSmithKline and United Kingdom guidelines for the
acquisition and use of human tissues, including ethical approval and the use of
appropriate consent forms. Brain cortex and cardiac tissue biopsies from a total of 3
subjects were analysed.
A Shandon cryotome cryostat was used to section biopsies at 6 µm. Sections were
placed on Vectabond™ (Vector Laboratories, Peterborough, UK) coated slides and
the brain cortex and cardiac tissue sections post-fixed in ice-cold 4% w/v
paraformaldehyde. Sections were then immunostained for the A1 receptor using an
Optimax automatic immunostaining machine (Biogenex, San Ramon, CA, USA). In
summary, non-specific protein staining was blocked by treatment with Power
Block™ prior to incubation with the A1 receptor antibody (1 µg ml-1) or rabbit IgG
isotype control antibody (1 μg ml-1) at room temperature for 1 hour. After primary
antibody incubation, sections were then incubated with a goat anti-rabbit secondary
antibody labelled with horseradish peroxidase-streptavidin and positive staining
detected with diaminobenzidine. With the exception of the adenosine A1 receptor
antibody, all immunohistochemistry antibodies and substrates were obtained from
Vector Laboratories, Peterborough, U.K. The sections were also counterstained with
Mayer’s haematoxylin (Pioneer Research Chemicals Ltd, Colchester, U.K.).
Image analysis
Fully automated densitometry of A1 receptor expression was developed with the
Zeisss Vision KS400 system (Carl Zeiss, Gottingen, Germany). After a 30-minute
warming up of the microscope (X1000 magnification) the images were digitised with
a three-chip colour camera, which was connected to a personal computer with a 2.66
GHz Pentium 4 processor and frame grabber with appropriate corrections for nonuniform illumination (shading correction) and settings of the black and white camera
signal (white balance). Descriptive data for each compounded image were stored in a
corresponding image database. The image processing was based on the red-greenblue (RGB) colour model.
Custom macros were written in KS300 interpreter
language to separate the brown-red staining from the blue counter-stain. Four images
from each endobronchial biopsy section (1 section per subject) were randomly
selected in epithelial and smooth muscle regions. Positive staining colour intensity
data were stored in a corresponding database.
Stain intensity was described in
arbitrary units (A.U.). All image analysis and measurements were performed blind by
one observer.
Antibody validation-generation of adenosine receptor transfectant cells
In addition to immunohistochemical analysis of brain and cardiac tissue, further
antibody validation experiments were to be performed through comparing the binding
of the A1 receptor antibody to each adenosine receptor subtype expressed on Chinese
hamster ovary (CHO)-K1 cells (which lack any known subtype of adenosine receptor)
transfected with the human recombinant A1, A2A, A2B or A3 receptor. These cell lines
were also to be used in a separate study and so for that purpose, were first stably
transfected with a reporter plasmid designated CRE-SPAP-Hyg. For this reason,
hygromycin B was present in the medium. Therefore, in order to further transfect the
CRE-SPAP-Hyg host with the human recombinant A1, A2A, A2B or A3 receptor, the
host cells were cultured to 50% confluence in 6-well 9cm2 plates in M1 medium
(Dulbecco's modified Eagle's medium/Hams F-12 (1:1) mix (Sigma Biochemicals,
Poole, U.K) supplemented with 2 mM Glutamax, 10% foetal calf serum) containing
500 µg ml-1 hygromycin B (all Gibco, Paisley, U.K.). Cultures were maintained at
37°C in a 5% CO2/humidified air atmosphere). The cells were then washed twice in
DMEM/Ham’s F-12 and left in the same medium for 3-4 hours. Each DNA construct
was linearised with SspI in Boerhinger Buffer H, and then sterilised with
Phenol:Chloroform:Isoamyl alcohol (PCI) (25:24:1) and washed in CI (24:1) using
phase lock tubes (CP laboratories, Saffron Walden, U.K.). 8 µl of each construct was
placed in a sterile microcentrifuge tube with 42 µl of DOTAP (Roche Diagnostics,
Lewes, U.K.):Hepes buffered saline (HBS) (300:950). After very gentle mixing, the
tubes were left for 15 minutes at room temperature. While the tubes were incubating,
most of the medium on the cells was removed to leave to 1 ml. Each DNA construct
was then added to one well. The cells were incubated for 5 hours after which time
3ml of M1 medium containing 500 µg ml-1 hygromycin B was added. The cells were
then incubated for 24 hr before each well before applying further selection pressure.
After this period the cells were passaged into M1 and 500 µg ml-1 hygromycin B and
1 mg ml-1 G418. The cells transfected with the A1 receptor were scaled up in 175 cm2
and then 500 cm2 flasks. The expression level of the receptor within this pool
remained consistent over several months as judged by flow cytometric analysis of the
HA tag on the N-terminus of the receptor. The cells transfected with the A 2A, A2B or
A3 receptor did not have an HA tag. These recombinants were cloned as follows: The
transfected cells were passaged and diluted and plated into microtitre plates at 0.3 and
1 cell per well. The medium was changed after 5 days. After 10-12 days selection
pressure, wells under the microscope that had 1 colony per well were identified.
These were maintained until confluent and then scaled up into 9 cm2 wells. Once
confluent 90% of the cells were passaged off, spun down and made into small
membrane preparations to identify high expressors via binding assays (data not
shown). Those identified as such were scaled up further into 175 cm2 or 500 cm2
flasks for banking. For experiments, all the cell lines were cultured with M1 medium
supplemented with 500 µg ml-1 hygromycin B and 500 µg ml-1 G418 and harvested
when approximately 70% confluent.
Extraction of total RNA
In order to compare expression of the transfected receptor, total RNA was purified from
the cell preparations using a RNeasy midi kit (Qiagen, U.K.) according to the
manufacturer’s instructions. The RNA was then dissolved in nuclease free water and
quantified using a spectrophotometer, the quality of RNA was assessed by running the
samples in an agarose gel (1%).
Reverse transcription and specific amplification by Polymerase Chain Reaction
(PCR)
cDNA was generated from 1µg of total RNA per sample using a SuperScript FirstStrand Synthesis System for RT-PCR kit (Invitrogen, Paisley, U.K.) according to the
manufacturer’s instructions. The samples were directly amplified by quantitative
real-time RT-PCR using a 7900HT Sequence Detector (Applied Biosystems,
Warrington, U.K.). Primer pairs (Sigma Genosys Haverhill, U.K.) and internal probe
sequences (Applied Biosystems, Warrington, U.K) are described in Table II. DNA
contamination of RNA samples was checked for by including samples to which no
reverse transcriptase had been added. The resulting data were analysed using the
sequence detector system software (Applied Biosystems, Warrington, U.K.) with
TAMRA as the reference dye. Data are expressed as gene copy number per 50 ng
cDNA.
Flow cytometry
A comparison of the A1 receptor antibody binding to the A1, A2A, A2B and A3
receptors expressed on the transfected CHO-K1 cells was performed by using flow
cytometric analysis. Cells were cultured as described above until approximately 70%
confluent and then fixed and permeabilised using Fix and Perm® (Caltag Laboratories,
Burlingame, CA, USA), according to the manufacturer’s instructions (1 x 105 cells
per sample). Cells were then incubated at room temperature with the 1 µg of the A 1
receptor antibody in 100 µl or pre-immune serum for 15 min. Cells were washed with
PBS containing 3% heat-inactivated foetal calf serum and incubated at room
temperature with an R-phycoerythrin (PE)-conjugated goat anti-rabbit polyclonal
antibody (2 µg per sample) (Sigma Biochemicals, Poole, U.K) for a further 15 min.
After washing, cells were finally resuspended in Cell Fix™ (BD Biosciences,
Erembodegem, Belgium) and analyzed by appropriate gating for immunofluorescence
using a FACS Calibur flow cytometer (Beckton Dickinson, Oxford, U.K.) after
excitation at 488 nm. At least 3,000 events were collected and mean fluorescence
intensity values recorded. Values obtained following labelling with the A1 receptor
antibody were corrected for non-specific staining with values obtained with control
cells incubated with pre-immune serum.
Statistical analysis
Data were analysed by unpaired Student’s t-test, and unless stated otherwise, are
expressed as mean  standard error of the mean (SEM).
Table II Primer pairs and internal probe sequences for human adenosine receptors
Gene
Sequences
A1 receptor
Sense
Antisense
Probe
5’-CTGCCTTTGCACATCCTCAAC-3’
5’-GATGGCAATGTAGGTAAGGATGCT-3’
FAM-TGCCCGTCCTGCCACAAGCC
A2A receptor
Sense
Antisense
Probe
5’-GGGTGTCTATTTGCGGATCTTC-3’
5’-CAGAGGCTGGCTCTCCATCT-3’
FAM-CGGCGCGACGACAGCTGAAG
A2B receptor
Sense
Antisense
Probe
5’-GAACCGAGACTTCCGCTACACTT-3’
3’-CCTGACCATTCCCACTCTTGA-3’
FAM-TCACAAAATTATCTCCAGGTATCTTCTCTGCCAA
A3 receptor
Sense
Antisense
Probe
5’-TTCGGAACAAACTCAGTCTGAACT-3’
5’-AACAAGGACTTAGCCGTCTTGAAC-3’
FAM-CAAAGAGACAGGTGCATTTTATGGACGGG