METHODS Methacholine inhalation challenge Allergen inhalation

METHODS
Methacholine inhalation challenge
Subjects inhaled through a mouthpiece attached to a Wright
nebulizer (Roxon Medi-Tech, Montreal, Quebec, Canada). Normal
saline, then doubling concentration increases in methacholine were
nebulized for 2 minutes each. The FEV1 was measured at 30, 90, 180,
and 300 seconds after each inhalation using a Collins water-sealed
spirometer (Warren E. Collins, Braintree, Mass) and kymograph. The
test was terminated when FEV1 had fallen to a level at least 20%
below the postsaline measurement.
Allergen inhalation challenge
The allergen producing the largest skin wheal diameter after skin
prick testing was diluted in sterile normal saline. The concentration of
allergen extract for inhalation was determined using a formula
derived by Cockcroft et al24 using the results from skin test titrations
and the methacholine PC20. During the screening allergen challenge,
the starting concentration of allergen extract for inhalation was 2
doubling concentrations below that predicted to cause a 20% decrease
in FEV1. Doubling increases in allergen concentration were inhaled
every 10 minutes until a 15% reduction in FEV1 was achieved. FEV1
was then measured at 10, 20, 30, 45, 60, 90, and 120 minutes after
allergen inhalation, then each hour until 7 hours after allergen
inhalation. The early bronchoconstrictor response was taken to be the
largest percent fall in FEV1 within 2 hours after allergen inhalation,
and the late response was taken to be the largest percent fall in FEV1
in the period beginning 3 hours and ending 7 hours after allergen
inhalation.
Sputum analysis
Subjects inhaled 3%, 4%, and then 5% saline for 7 minutes each.
The induction was stopped when an adequate sample was obtained or
if the FEV1 dropped 20% from baseline. Cell plugs with little or no
squamous epithelial cells were selected from the sample using an
inverted microscope, separated from saliva, and weighed. Samples
were aspirated in 4 times their volume of 0.1% dithiothreitol
(Sputolysin; Calbiochem Corp, San Diego, Calif) and 4 times their
volume of Dulbecco PBS (Gibco BRL, Life Technologies, Grand
Island, NY). The cell suspension was filtered through a 52-mm nylon
gauze (BNSH Thompson, Scarborough, Ontario, Canada) to remove
debris, then centrifuged at 1500 rpm for 10 minutes. The total cell
count was determined using a hemocytometer (Neubauer Chamber;
Hausser Scientific, Blue Bell, Pa) and expressed as the number of
cells per milliliter sputum. Samples were divided into 2 portions for
differential cell counts and flow cytometry analysis. For differential
counts, cells were resuspended in Dulbecco PBS at 0.75 3 106/mL to
1.0 3 106/mL. Cytospins were prepared on glass slides using 50 mL
cell suspension and a Shandon III Cytocentrifuge (Shandon Southern
Instruments, Sewickly, Pa) at 300 rpm for 5 minutes. Differential cell
counts were obtained from the mean of 2 slides with 400 cells counted
per slide stained with Diff-Quik (American Scientific Products,
McGaw Park, Ill). The percentage of eosinophils was used as an index
of eosinophilic airway inflammation.
Cell cultures
Twenty milliliters of heparinized peripheral blood was obtained
from each subject. PBMCs were isolated by Lymphoprep (Nycomed,
Oslo, Norway) density gradient separation and washed twice with
RPMI-1640 (GibcoBRL, Gaithersburg, Md). Induced sputum cells
were also washed twice with RPMI-1640. Cells were suspended in
RPMI-1640 with 10% FCS (GibcoBRL) and 25 mmol/L HEPES
(GibcoBRL) at a density of 2 3 106 cells/mL. Cells were then
cultured in 6-well culture plates (Becton Dickinson Labware,
Franklin Lakes, NJ) in a volume of 4 mL in the presence of phorbol
12-myristate13-acetate, 20 ng/mL) and ionomycin (2 mmol/L) for 4
hours at 37°C in 5% CO2 in an incubator with 85% humidity.
Monensin (GolgiStop; PharMingen, 0.2 mL/mL) was added over the
incubation time to inhibit cytokine secretion, resulting intracellular
accumulation of cytokines. At harvest, cells were gently removed
from the plate with a cell scraper (Becton Dickinson Labware,
Franklin, NJ). The cell viability was assessed by trypan blue dye
(GibcoBRL) method. The contents of each well were transferred to
5-mL polystyrene tubes (Becton Dickinson Labware, Franklin, NJ) at
1 3 106 cells/tube in ice-cold PBS with 1% BSA, 0.2% EDTA, and
0.2 % sodium azide, and then processed for staining.
FIG E1. Flow-cytometric analysis of lymphocytes in induced sputum. A, Forward and side scatter characteristics of induced sputum samples. B, Fluorescence characteristics of lymphocytes stained with mAbs to CD4
and CD8. C, Fluorescence characteristics of lymphocytes, gated by CD41 or CD81, stained with isotype control
mouse IgG1 or mAbs to human IFN-g. SSC, Side scatter; FITC, fluorescein isothiocyanate; PE, phycoerythrin.
TABLE E1. Subject characteristics
Subject number
IER
1
2
3
4
5
6
7
8
9
10
11
12
Mean 6 SEM
DR
1
2
3
4
5
6
7
8
9
10
11
12
Mean 6 SEM
Age (y)
Sex
FEV1 (% predicted)
Methacholine PC20 (mg/mL)
Inhaled allergen
Allergen dilution*
25
26
24
22
21
25
24
23
19
20
27
40
24.7 6 1.5
F
M
M
F
F
F
M
M
F
F
F
F
82.5
90.2
73.7
79.5
88.8
77.8
84.1
81.2
82.0
82.3
86.6
73.2
81.8 6 5.1
13.45
5.76
6.59
13.45
4.86
2.76
4.00
16.00
0.18
7.46
10.89
21.11
5.91 (1.42 GSEM) Cat
Ragweed
Cat
HDM
Cat
Ragweed
Ragweed
HDM
HDM
Ragweed
Grass
HDM
1:16
1:32
1:64
1:32
1:16
1:32
1:1024
1:256
1:8192
1:32
1:128
1:4
23
24
22
22
21
22
52
19
19
51
56
19
29.2 6 4.0
M
M
F
F
F
F
M
F
F
F
F
M
89.6
69.6
72.7
74.3
70.1
85.1
84.8
72.6
86.8
74.3
79.7
67.5
77.3 6 2.1
9.55
2.26
1.69
0.91
1.49
0.16
2.13
0.55
4.09
0.91
15.06
0.39
1.56 (1.43 GSEM) Cat
Ragweed
Cat
Alternaria
Ragweed
Ragweed
Cat
HDM
Ragweed
Cat
Cat
HDM
1:32
1:128
1:256
1:16
1:1024
1:256
1:64
1:128
1:64
1:16
1:8
1:1024
HDM, House dust mite.
*The dilution of allergen extract used for the allergen challenges.
Expressed as geometric mean and geometric SEM (GSEM).

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