Temporal correlation of measurements of airway
hyperresponsiveness in ovalbumin-sensitized mice
Kurt H.
Albertine1,2,
Lu
Wang2,
Suetaro
Watanabe2,
Gopal K.
Marathe3,
Guy A.
Zimmerman2,3, and
Thomas M.
McIntyre2,3
Departments of 1 Pediatrics and
2 Medicine and 3 The Eccles Institute
for Human Genetics, University of Utah, Salt Lake City, Utah 84132-2202
 |
ABSTRACT |
Airway hyperresponsiveness, airway
inflammation, and reversible airway obstruction are physiological
hallmarks of asthma. These responses are increasingly being studied in
murine models of antigen exposure and challenge, using whole body
plethysmography to noninvasively assess airway hyperresponsiveness.
This approach infrequently has been correlated with indexes of airway
hyperresponsiveness measured by invasive means. Furthermore,
correlation with quantitative histological data for tissue infiltration
by inflammatory and immune cells, particularly in the wall of airways,
during daily airway challenge is lacking. To address these
uncertainties, we used C57BL/6 mice that were immunized with ovalbumin
or vehicle (saline) and sensitized to aerosolized ovalbumin or vehicle
8 days later. The mice were subsequently exposed to aerosolized ovalbumin or vehicle, respectively, on days 14-22. We
assessed airway hyperresponsiveness to methacholine noninvasively on
days 14, 15, 18, or 22; we
studied the same mice 24 h later while they were anesthetized for
invasive analyses of airway hyperresponsiveness. Plasma total IgE
concentration was significantly higher in the ovalbumin-treated mice
compared with the vehicle-treated mice, but this did not correlate with
eosinophil number. Peak airway hyperresponsiveness measured by either
approach correlated early during daily antigen challenge (days
14 and 15), but this correlation was lost later during
subsequent daily antigen challenges (days 18 and
22). On days 14 and 15, peak airway
hyperresponsiveness correlated with transmigration of neutrophils and
macrophages, but not lymphocytes, in the peribronchovascular connective
tissue sheaths. This extravascular accumulation was found to be focal by three-dimensional microscopy. We conclude that, although ovalbumin treatment changed lung function in mice, correlation between
noninvasive and invasive measures of peak airway hyperresponsiveness
was inconsistent.
allergic asthma; murine model of asthma; pulmonary resistance; pulmonary dynamic compliance; inspiratory and expiratory times; whole
body plethysmography; lung histopathology; quantitative histology; image analysis
 |
INTRODUCTION |
AIRWAY HYPERRESPONSIVENESS, airway
inflammation, and reversible airway obstruction are physiological
hallmarks of asthma (22), yet the mechanisms that govern
these pathophysiological responses are not fully understood. These
hallmarks of asthma are being examined in murine models of allergic
asthma, where manipulation can be applied to identify components of the
underlying responses (18, 20, 21, 25). The marriage
between the genetically manipulatable murine system and induced airway
hyperresponsiveness has defined roles for inflammatory and immune
regulatory molecules, but fundamental issues remain unresolved. These
include the requirement and contribution for influx of specific
immune-effector cell subtypes (7, 9, 15, 21), the basis
for different responses among mouse strains (21), and the
contributions of route and sequence of antigen exposure (9,
24).
Increasingly, murine models of antigen exposure and challenge are being
evaluated physiologically, using a recording system (whole body
plethysmography) that noninvasively assesses airway hyperresponsiveness
to methacholine (4, 5, 8, 14). This noninvasive
physiological approach offers the advantages of eliminating the effects
of anesthesia and surgical trauma and permitting repeated assessment of
the same mice while they breathe spontaneously. The noninvasive index
of airway hyperresponsiveness, enhanced pause (Penh), is an
empirically derived, unitless value based on the pressure waveform in
the plethysmograph box (8). However, the physiological
meaning of Penh compared with conventional methods of
measuring lung resistance (RL) and compliance
(16) has been incompletely investigated (8,
17). One question that has not been addressed is whether
Penh reliably detects changes in airway hyperresponsiveness
as the number of days of allergen exposure is increased.
Furthermore, there has been no correlation of airway
hyperresponsiveness with quantitative histological analysis of
tissue infiltration of inflammatory and immune cells, particularly in
the walls of airways, at various times during allergen exposure.
In the present study, we used a murine model of airway
hyperresponsiveness (3) to ask two questions. First, we
asked whether the noninvasive method for assessing changes in airway
hyperresponsiveness in mice is reliable during repeated exposure to
aerosolized allergen. To this end, we immunized and sensitized C57BL/6
mice to ovalbumin before exposing them daily to aerosolized ovalbumin
and noninvasively assessing airway hyperresponsiveness to methacholine
while the mice were awake and unrestrained. Twenty-four hours later, we measured RL and compliance invasively in the same mice
while they were anesthetized. Control mice were treated with saline,
the vehicle for ovalbumin. The second question we asked was if peak airway responsiveness correlated with transmigration of specific inflammatory and immune cells in the walls of airways. To address this
question, we assessed airway hyperreactivity to methacholine while the
mice were awake and unrestrained. Four hours later, we fixed the lung
of these mice to identify and quantify leukocyte transmigration in the
peribronchovascular connective tissue sheaths. We found that, while
ovalbumin immunization and sensitization changed all of the
airway parameters that we measured, correlation was inconsistent
between a commonly employed noninvasive method and a widely
accepted invasive method of assessing pulmonary mechanics. We
also found that neutrophils and macrophages transmigrated into the
peribronchovascular connective tissue sheaths in the allergen-treated mice, and this correlated well with changes in RL.
 |
MATERIALS AND METHODS |
Animals.
Pathogen-free, adult male C57BL/6 mice, 30-35 g body wt, were
purchased from B&L Universal (Fremont, CA). Upon delivery, the mice
were kept in a pathogen-free rodent facility and were provided food and
water ad libitum. The animal experiments were approved by the
Institutional Animal Care and Use Committee at the University of Utah.
Ovalbumin immunization and airway challenge.
Mice were immunized, on day 0, by intraperitoneal injection
of 100 µg of ovalbumin (Sigma Chemical, St. Louis, MO) adsorbed to 1 mg of alum (Sigma Chemical) in a total volume of 0.5 ml of sterile PBS
(Sigma Chemical). Later (8 days), we sensitized the airways to
ovalbumin by exposing the mice to aerosolized 2% ovalbumin in sterile
PBS for 30 min in a chamber (dimensions: 38 × 20 × 20 cm).
This immunization and sensitization regimen emulated that used by
Brusselle and colleagues (3). The efficacy of this immunization/sensitization protocol is shown in Plasma IgE
concentration. Aerosolization was done using a DeVilbiss nebulizer
(model 99; UltraNeb, Somerset, PA) driven by compressed air. Output of
the nebulizer was 1 ml/min, with a mean particle diameter of 3.5 µm (manufacturer's specification). On day 14 and daily
thereafter for 8 days (days 15-22), the
ovalbumin-immunized and -sensitized mice were exposed to aerosolized
2% ovalbumin in sterile PBS for 30 min.
We initially used the bias flow supply and its tubing to deliver the
nebulized ovalbumin to the mouse plethysmograph chambers. However, this
delivery route clogged the flow valves in the bias flow supply,
particularly when nebulized albumin was used. This challenge was
circumvented by delivering the nebulized ovalbumin in a separate
exposure chamber, which enabled us to expose all the mice to the same
concentration and duration of ovalbumin. The mice were then transferred
to the plethysmograph chambers. These mice are designated "ovalbumin treated."
Control mice were given an intraperitoneal injection of 0.5 ml of
sterile PBS with 1 mg of alum, the vehicle for ovalbumin, on day
0. On days 8 and 14 and daily thereafter for
8 days (days 15-22), the control mice were exposed to
aerosolized sterile PBS for 30 min in a chamber, as described above.
These mice are designated "vehicle treated."
Respiratory system responses to methacholine provocation.
Respiratory system variables were assessed by two sequential
approaches. The first approach used awake, unrestrained mice that were
placed in a Plexiglas whole body barometric plethysmograph (Buxco
Electronics, Sharon, CT; see Ref. 8). The same mice were
then restudied 24 h later. At that time, the mice were
anesthetized, a tracheostomy was made, their trachea was cannulated,
their pleural spaces were opened, and the mice were placed in a rodent
body box (16). The interval between each day's
nebulization of ovalbumin or saline and measurement of lung function
was 4-6 h.
The Buxco system is composed of sealable, clear, cylindrical Lucite
chambers (9 cm × 9 cm in length and width and 6 cm in height;
~500 cm3 in volume) connected to a gas flow and pressure
control unit. Each Lucite chamber held one mouse. Four chambers were
connected in parallel to the control unit so that simultaneous
measurements were made for two ovalbumin-treated mice and two
vehicle-treated (control) mice. The Buxco whole body barometric
plethysmograph measured pressure changes within each chamber
continuously as room air flowed through the chambers. Continuous
recordings of Penh were gathered on-line. This is a
calculated parameter of airway obstruction (8). Among
other respiratory system parameters that are measured are inspiratory
time, expiratory time, and respiratory frequency.
Respiratory system variables were measured before (baseline) and after
10 min of methacholine nebulization (15 and 30 mg/kg). Measurements
were recorded continuously for 15 min. Results are reported at the time
when Penh was at its peak. The time to peak Penh response among the mice ranged between 3 and 8 min
after nebulization was stopped, regardless of the two dosages of
methacholine. Nebulized methacholine was delivered to a distribution
reservoir (made by the manufacturer at our request), from which
parallel hoses of identical length and diameter delivered the nebulized methacholine to four Lucite chambers simultaneously. The distribution reservoir ensured that all of the mice received the same concentration and duration of methacholine. The 10-min nebulization interval was
chosen after a time-response curve was established (2, 5, and 10 min of
nebulization). The two doses of methacholine were chosen after a
dose-response curve was established (5, 10, 15, 30, and 50 mg/ml
methacholine in sterile PBS). We selected two dosages of methacholine
for the vehicle-treated (control) mice that, at the lower dosage, did
not elicit airway hyperreactivity and at the higher dosage elicited
minimal airway hyperreactivity. Reproducible, dose-dependent elevation
of Penh occurred with 15 and 30 mg/ml of methacholine in
the ovalbumin-treated mice. Thus the two doses failed to induce changes
in airway function by themselves but elicited airway hyperreactivity in
the immunized and sensitized mice. The mice were allowed to recover for
45 min between the two doses of methacholine. Recovery was verified by
the respiratory system variables returning to each mouse's baseline values.
We also determined that it was critical to monitor and alter parameters
for the rejected breath algorithm, without which the default algorithm
rejected many breaths, thereby causing the tracings of respiratory
system parameters for the ovalbumin-treated mice to appear to be
shorter than control mice. This quality control issue was resolved with
assistance from the manufacturer. Resolution involved lowering the
default threshold for rejecting breaths until the length of the tracing
was equivalent for the two groups of mice. This is a selectable option
in the software and thus is easy to change. Once the threshold for
rejected breaths was established, the threshold was saved and used for
the entire study.
Respiratory system variables were recorded at the following intervals
4-6 h after daily nebulization of ovalbumin (or vehicle) on
day 14 (4-6 h), day 15 (24 h), day
18 (4 days), and day 22 (8 days). The experimental
design is shown in Fig. 1. The same times
were used to ascertain recruitment of leukocytes to the lung (see
Leukocyte accumulation in lung tissue).

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 1.
Experimental design. Mice were intraperitoneally injected
with ovalbumin or saline (vehicle control) on day 0.
Nebulization of ovalbumin or saline was begun on day 8 and
was repeated daily on days 14-22. Noninvasive
measurement of pulmonary mechanics was performed on days 14,
15, 18, and 22 (labeled "Pulmonary
Mechanics N"). Later (24 h and 4-6 h after a final nebulization
procedure; day 23), invasive measurement of pulmonary
mechanics was performed on the same mice (labeled "Pulmonary
Mechanics I"). Separate groups of mice were studied on days
14/15, 18/19, and 22/23 (n = 4-10 mice/group, as indicated in Figs. 3-5 by the
and ). Pulmonary histology was
assessed on days 14, 15, 18, and
22 in another set of ovalbumin-treated and saline-treated
mice (n = 6-7 mice each), after noninvasive
measurement of airway parameters (labeled "Histology X").
|
|
The assessment of airway physiology was then repeated in the same mice
24 h later to correlate Penh to RL and
dynamic compliance (Cdyn) measured directly. RL
and Cdyn were measured by a standard and widely accepted
method (16). Briefly, the mice were treated with nebulized
ovalbumin or saline as described for the noninvasive measurements.
Later (4 h), the mice were anesthetized with pentobarbital sodium
(60-70 mg/kg ip). An anterior, midcervical skin incision was made
to expose the trachea, which was cannulated with an 18-gauge blunt
needle. An opening was made in each side of the chest wall so that
pleural pressure (Ppl) equaled body surface pressure. The
tracheostomy catheter was passed through a hole in a plethysmograph box
(clear Plexiglas cylinder 3.5 cm ID; 11.5 cm long; 0.35 cm wall
thickness; 110.6 ml volume). One end (head end) of the cylinder was
made of a Plexiglas plate, machined for an air-tight fit with an O-ring
seal. The center of the end plate was perforated by a 16-gauge blunt
needle, to which the tracheal cannula was attached. A tray was bonded
to the end plate so that the mouse's face could be placed against the
end plate without disturbing the tracheal catheter's connection. Mice
were ventilated (Mouse Ventilator, model 687; Harvard Apparatus,
Holliston, MA) initially with a tidal volume (Vt) of
5-6 ml/kg at 120 breaths/min and 3 cmH2O of positive
end-expiratory pressure. The dead space of the system was 0.03 ml. The
mice were paralyzed with pancuronium bromide (0.3 mg/kg ip) to prevent
respiratory movements. We used a 1-liter glass bottle filled with
copper sponge to ensure isothermal conditions during measurements. Two
small ports in the mouse-compartment end of the cylinder were used to
monitor box pressure (very low range differential transducer, model
DP45; Validyne Engineering, Northridge, CA). The reference side of that
transducer was connected through a three-way stopcock to a second
1-liter glass bottle, also filled with copper sponge, to prevent
atmospheric pressure swings. Changes in the box pressure provided
volume (V) information. Airflow (
) was derived from
differentiation of the volume signal. The delay between the volume and
flow signals was <0.5 ms. Transpulmonary pressure (Ptp)
was measured with a second pressure transducer as the difference
between airway pressure (Paw), taken from the opening of
the tracheal cannula, and the box pressure, which was equal to
Ppl; Ptp = Paw
Ppl.
Measurements of RL and Cdyn were made before
(baseline) and after 30 tidal breaths of 15 and 30 ml/ml nebulized
methacholine, delivered through the ventilator at 60 breaths/min.
Forty-five minutes were allowed between the two doses of methacholine
for the mice to recover to their baseline values. Peak responses to either dose were reached between 1 and 2 min of delivery of
methacholine, regardless of the two dosages of methacholine. We report
peak results. We analyzed the tracings of V,
, and
Ptp at a respiratory rate of 120 breaths/min.
RL was calculated as an average value for inspiration
(insp) and expiration (exp) at isovolumes in the midtidal breathing as
follows
Cdyn is calculated as the ratio of Vt to
the change in Ptp at the instant of zero flow
Values of both RL and Cdyn are reported
as the average of five consecutive breaths.
Leukocyte accumulation in lung tissue.
We also determined the distribution and number of leukocytes that
accumulated in the lungs of ovalbumin-treated mice compared with
vehicle-treated mice on days 14, 15,
18, and 22 of daily nebulization. This
morphological analysis was done on replicate groups of
ovalbumin-treated and vehicle-treated mice, as described above,
including noninvasive assessment of airway hyperresponsiveness to
nebulized methacholine (n = 6-7 mice each/day of
study). We measured respiratory system variables to be certain that the
group of mice used for the morphological analysis behaved
physiologically as the mice used for the functional studies. After
completing the measurements of respiratory system variables for a given
interval (e.g., day 14), we killed the mice with
pentobarbital sodium (100 mg/kg ip). Their tracheas were cannulated,
their rib cages were opened, and their lungs were inflated with air to
~75% total lung capacity. The hilum of the left lung was
cross-clamped to maintain its inflation and to trap vascular contents
in its blood vessels. The left lung was removed, with the clamp
attached to its hilum, and immersed in 10% buffered neutral formalin
at 4°C overnight. The next day, the left lung was processed and
embedded whole in paraffin wax. The right lung was allowed to collapse
and then insufflated with Tissue-Tek optimum-cutting temperature (OCT) compound (VWR, Media, PA). The right lung was immersed in OCT compound
and frozen.
Three-dimensional stereomicroscopy was used to assess accumulation of
leukocytes in the lung tissue. For this analysis, we cut 60- to
100-µm-thick slabs of lung tissue, stained them with hematoxylin and
eosin (H&E), and observed the slabs with the aid of an Edge
three-dimensional microscope (Edge Scientific Instrument, Santa Monica,
CA) equipped with fluorescence illumination. Stereopair photographs
were taken of the autofluorescent stain.
Immunohistochemistry was performed on paraffin-embedded sections
(26). Briefly, tissue sections (4-5 µm) were
collected on PLUS slides (VWR). The sections were treated with 3%
H2O2 in methanol for 10 min at 37°C to remove
endogenous peroxidase. The sections were washed with PBS, blocked with
normal goat serum, and then incubated with purified rat anti-mouse
monoclonal primary antibodies. The primary antibodies were directed
against neutrophils (rat anti-mouse neutrophil antibody; Caltag
Laboratories, Burlingame, CA; see Ref. 11), activated
macrophages (rat anti-Mac-3 antibody; PharMingen, San Diego, CA; see
Ref. 6), and T lymphocytes and some B lymphocytes (rat
anti-CD5 antibody; PharMingen; see Ref. 13). Optimal
dilutions were 1:200 for the rat anti-mouse neutrophil antibody,
1:1,000 for the anti-Mac-3 antibody, and 1:200 for the anti-CD5
antibody at 4°C overnight. Staining controls included omission of the
primary antibody, omission of the secondary antibody (biotinylated
IgG), and substitution of the primary antibody with a species-matched,
isotype-matched irrelevant antibody (insulin). Furthermore, we
immunostained smears of mouse peripheral blood to confirm cell-specific
labeling. For the rat anti-mouse neutrophil and CD5 primary antibodies,
antigen detection was done by the tyramide signal amplification method
(NEL 700 kit; NEN Research Products, Boston, MA). For the rat anti-Mac3
primary antibody, antigen detection was done by the
avidin-biotin-horseradish peroxidase method (ABC Elite kit; Vector
Laboratories, Burlingame, CA). Both antigen detection methods were used
for the anti-insulin antibody. We used Gill's no. 3 hematoxylin to
counterstain the immunostained tissue sections. Photography was done
with the aid of a Zeiss Axiophot light microscope. We expressed the
results as the number of extravascular leukocytes for each leukocyte
type per millimeter of airway basal lamina length, which was measured
by tracing the basal lamina in calibrated digital images (Bioquant True
Color Image Analysis System; R & M Biometrics, Nashville, TN). We also traced the outside perimeter of the peribronchovascular connective tissue sheaths. The subtended area surrounding each bronchiole was the
extravascular tissue space in which leukocytes were counted by
electronic touch count. This quantitative morphological analysis of
extravasated leukocytes in the peribronchovascular connective tissue
sheaths was performed on one tissue section that was cut from each
mouse's entire left lung on days 15, 18, and
22. The number of peribronchovascular connective tissue
sheaths that were analyzed per left lung ranged between 15 and 20. We
did not perform this analysis on lung tissue sections cut from the mice
that were killed on day 14 because leukocytes had
insufficient time to transmigrate in the peribronchovascular connective
tissue sheaths 4 h after nebulization of ovalbumin, which is the
time when these mice were killed.
Bronchoalveolar lavage cell counts.
Replicate experiments, including whole body plethysmography using the
Buxco system, were performed using ovalbumin-treated and
vehicle-treated mice. After methacholine challenge and measurement of
airway hyperresponsiveness, the mice were anesthetized with pentobarbital sodium (60-70 mg/kg ip). Their tracheas were
cannulated, and their chests were opened. Bronchoalveolar lavage (BAL)
was performed five times (0.8 ml PBS/lavage) through the tracheal cannula. The retrieved lavage aliquots were pooled and centrifuged, from which the cell pellet was resuspended in PBS and counted using a
hemocytometer. Slide smears were treated with Wright's stain (Sigma
Chemical) for differential cell counts.
Plasma IgE concentration.
The ovalbumin-treated and vehicle-treated mice that were used to
measure leukocytes in BAL fluid were also used to measure plasma total
IgE concentration. Blood was withdrawn from the heart in a heparinized
syringe. The blood was centrifuged to obtain the plasma layer, which
was analyzed by enzyme-linked immunosorbent assay for plasma IgE
concentration, using a rat anti-mouse IgE monoclonal antibody (clone
R35-7; PharMingen) and the manufacturer's protocol.
Measurements were made in triplicate, the average for which is reported.
Statistical analysis.
The results are shown as means ± SD (n = 4-10 mice/group, as described in RESULTS and legends
for Figs. 1-11). Unpaired t-test was used to detect
differences between the ovalbumin-treated and vehicle-treated mice
(23). Simple linear regression and correlation tests were
used to identify relationships among airway responsiveness parameters
and between leukocyte subtypes in the walls of airways and airway
responsiveness parameters (23). Fisher's
r-to-z test was used to identify statistically
significant correlations (23). We used StatView 5.0 (Abacus Concepts, Berkeley, CA) and accepted P < 0.05 as indicating statistical significance.

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 2.
Summary histograms showing peak airway responses to two doses of
nebulized methacholine (15 and 30 mg/ml) on days 14,
15, 18, and 22 in ovalbumin-treated
mice (filled bars; n = 4-10 mice/day, see Fig. 3
for "n" each day) and vehicle-treated mice (open bars;
n = 4-10 mice/day, see Fig. 3 for n
each day). Data are means ± SD. "Enhanced pause"
(Penh) and the ratio of inspiratory time-to-expiratory
(Ti/Te ratio) were
assessed noninvasively, using whole body plethysmography (Buxco
Electronics). Lung resistance (RL) and pulmonary dynamic
compliance (Cdyn) were assessed invasively, using a rodent
body box (16). When Penh was at its peak, both
Penh and RL were statistically higher in the
ovalbumin-treated mice compared with the vehicle-treated mice on the
same day (*P < 0.05 by unpaired t-test).
When Penh was at its peak, Cdyn and the
Ti/Te ratio were
statistically lower in the ovalbumin-treated mice compared with the
vehicle-treated mice on the same day (*P < 0.05 by
unpaired t-test).
|
|

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 3.
Regression plots comparing peak Penh (independent
parameter) and RL (dependent parameter) in response to 2 doses of nebulized methacholine (15 and 30 mg/ml) on days
14, 15, 18, and 22 in
ovalbumin-treated mice ( ) and vehicle-treated mice
( ). Penh was assessed noninvasively, using
whole body plethysmography. RL was assessed invasively
24 h later, using a rodent body box. When Penh was at
its peak, both Penh and RL were consistently
greater in the ovalbumin-treated mice compared with the vehicle-treated
mice on the same day. Correlation coefficients ("C" in each
graph) were significant (*P < 0.05 by Fisher's
r-to-z test) on days 14 and
15 in response to 15 mg/ml methacholine and on days
14, 15, and 18 in response to 30 mg/ml
methacholine. The circles represent results for individual mice. Some
open circles (vehicle-treated mice) overlap.
|
|

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 4.
Regression plots comparing peak Penh (independent
parameter) and compliance of the lung (Cdyn) in response to
2 doses of nebulized methacholine (15 and 30 mg/ml) on days
14, 15, 18, and 22 in
ovalbumin-treated mice ( ) and vehicle-treated mice
( ). Penh was assessed noninvasively, using
whole body plethysmography. Cdyn was assessed invasively
24 h later, using a rodent body box. When Penh was at
its peak, both Penh and Cdyn were consistently
greater in the ovalbumin-treated mice compared with vehicle-treated
mice on the same day. Correlation coefficients were significant
(*P < 0.05 by Fisher's r-to-z
test) on days 14 and 15 in response to 15 mg/ml
methacholine and on days 14, 15, and
18 in response to 30 mg/ml methacholine. The circles
represent results for individual mice. Some open circles
(vehicle-treated mice) overlap.
|
|

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 5.
Regression plots comparing peak Penh (independent
parameter) and the Ti/Te
ratio (dependent parameter) in response to 2 doses of nebulized
methacholine (15 and 30 mg/ml) on days 14, 15,
18, and 22 in ovalbumin-treated mice
( ) and vehicle-treated mice ( ).
Penh and the
Ti/Te ratio were assessed
noninvasively, using whole body plethysmography. When Penh
was at its peak, Penh was consistently greater, and the
Ti/Te ratio was
consistently lower in the ovalbumin-treated mice compared with the
vehicle-treated mice on the same day. Correlation coefficients were
significant (*P < 0.05 by Fisher's
r-to-z test) on days 14 and
15 in response to 15 mg/ml methacholine and on day
14 in response to 30 mg/ml methacholine. The circles represent
results for individual mice. Some open circles (vehicle-treated mice)
overlap.
|
|

View larger version (96K):
[in this window]
[in a new window]
|
Fig. 6.
Lung histology showing inflammatory responses in the
peribronchovascular connective tissue sheaths surrounding
intrapulmonary arteries (PA) and airways (AW) of ovalbumin-treated mice
(a-d; n = 6-7 mice) and
vehicle-treated mice (e-h; n = 6-7
mice) on days 14, 15, 18, and
22. On each day, the lungs were fixed for histology 4 h
after ovalbumin nebulization and noninvasive assessment of pulmonary
mechanics. The tissue sections are stained with hematoxylin and eosin.
For the ovalbumin-treated mice, leukocytes (arrows) are marginated in
the lumen of intrapulmonary arteries early (day 14) and in
the peribronchovascular connective tissue later (days 15,
18, and 22). a-h are the same
magnification (scale bar is 20 µm).
|
|

View larger version (114K):
[in this window]
[in a new window]
|
Fig. 7.
Lung histology demonstrating the inflammatory responses in
the perivascular connective tissue sheaths surrounding
intrapulmonary veins (PV) of ovalbumin-treated mice (a-c;
n = 6-7 mice) and vehicle-treated mice
(d-f; n = 6-7 mice) on days
14, 15, and 22. On each day, the lungs were
fixed for histology 4 h after ovalbumin nebulization and
noninvasive assessment of pulmonary mechanics. The tissue sections are
stained with hematoxylin and eosin. For the ovalbumin-treated mice,
leukocytes (arrows) are marginated in the lumen of intrapulmonary veins
early (day 14) and in the walls of intrapulmonary veins
later (days 15, 18, and 22). Histology
on day 18 is not shown because it was the same as on
day 22. a-f are the same magnification (scale
bar is 20 µm).
|
|

View larger version (104K):
[in this window]
[in a new window]
|
Fig. 8.
Stereo pair micrographs of lung tissue from a mouse on day
15 of ovalbumin exposure. Thick tissue sections (60 µm) were
stained with hematoxylin and eosin and observed with a 3-dimensional
microscope equipped with fluorescence illumination. Yellow
autofluorescence is emitted from cytoplasm (eosin stain), whereas red
autofluorescence is emitted from nuclei (hematoxylin stain). The
aggregates of red dots (arrows) in the lumen of the intrapulmonary
artery and between it and the neighboring airway are infiltrates of
leukocytes. Note that the leukocyte infiltrate is focal and that it is
located on the half of the intrapulmonary artery that faces its
neighboring airway. Original magnification was ×10. The airway is 1 mm
in diameter.
|
|

View larger version (69K):
[in this window]
[in a new window]
|
Fig. 9.
Immunohistochemical differentiation of leukocytes in the
peribronchovascular connective tissue sheaths surrounding airways of
ovalbumin-treated mice (a-c; n = 6-7
mice) and vehicle-treated mice (d-f; n = 6-7 mice) on days 15 and 22. The paired
panel for each type of leukocyte was chosen to show the results on the
day when leukocyte subtype was near maximum or maximum (see Fig. 10).
Monoclonal antibodies were used for neutrophils (anti-neutrophil
antibody; a and d), Mac3-positive macrophages
(b and e), and CD5-positive (T and some B)
lymphocytes (c and f). The brown immunoperoxidase
stain reveals each cell type. a-f are the same
magnification (scale bar is 20 µm).
|
|

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 10.
Summary histogram showing accumulation of leukocyte
subtypes in the peribronchovascular connective tissue sheaths
surrounding bronchioles on days 15, 18, and
22 in ovalbumin-treated mice (solid bars; n = 7/day) and vehicle-treated mice (open bars; n = 7/day). Data are means ± SD. Quantitative histology was used to
estimate the number of neutrophils (PMN), Mac3-positive macrophages,
and CD5-positive lymphocytes/mm airway basal lamina. The three types of
leukocytes were identified immunohistochemically (see Fig. 8). The
number of each type of leukocyte/mm distal airway basal lamina was
determined by image analysis. The number of immunopositive cells/type
of leukocyte in the peribronchovascular connective tissue sheaths
surrounding each bronchiole was divided by the length of that airway's
basal lamina, measured by calibrated tracing. Neutrophil and
Mac3-positive macrophage accumulation was statistically greater in the
ovalbumin-treated mice compared with the vehicle-treated mice on
days 15 and 18 (*P < 0.05 by
unpaired t-test). On day 22, neutrophil
accumulation decreased in the ovalbumin-treated mice to a level that
was equivalent to that in the vehicle-treated mice. On the same day,
Mac3-positive macrophage accumulation increased further in the
ovalbumin-treated mice compared with the vehicle-treated mice.
CD5-positive lymphocytes (T and some B lymphocytes) were equivalent
between ovalbumin-treated and vehicle-treated mice on days
15 and 18. Only on day 22 was accumulation
of CD5-positive lymphocytes statistically greater in the
ovalbumin-treated mice compared with the vehicle-treated mice.
|
|

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 11.
Summary histogram showing retrieval of leukocyte
subtypes by bronchoalveolar lavage (BAL) on days 15,
18, and 22 in ovalbumin-treated mice (filled
bars; n = 4/day) and vehicle-treated mice (open bars;
n = 4/day). Data are means ± SD. The 3 types of
leukocytes were identified by cytological characteristics in stained
slide smears (see Fig. 8). Neutrophil, macrophage, and lymphocyte
accumulation peaked on day 18. Because of variability,
however, few statistical differences were detected between the
ovalbumin-treated mice and vehicle-treated mice. The number of
neutrophils retrieved from ovalbumin-treated mice was significantly
greater compared with vehicle-treated mice on days 15 and
22 (*P < 0.05 by unpaired
t-test). No differences were detected statistically for
macrophages between the two groups of mice, regardless of day of study.
The number of lymphocytes retrieved from ovalbumin-treated mice was
significantly greater compared with vehicle-treated mice on day
22 only (*P < 0.05 by unpaired
t-test).
|
|
 |
RESULTS |
Plasma IgE concentration.
Plasma total IgE concentration transiently increased in the
ovalbumin-immunized and -sensitized mice. For example, plasma total IgE
concentration was 3.0 ± 1.3 µg/ml (mean ± SD;
n = 4) on day 15, 21.3 ± 3.7 µg/ml
on day 18 (n = 4), and 4.7 ± 7.2 µg/ml (n = 4) on day 22. The concentration
of total plasma IgE was statistically different on day 18 compared with days 15 and 22 (P < 0.05). IgE was not detected in the plasma of the vehicle-treated mice.
Peak Penh and RL are increased, whereas
Cdyn and the ratio of inspiratory to expiratory time are
decreased, in ovalbumin-treated mice.
Penh is commonly used to investigate alterations in airway
responsiveness in awake, unrestrained mice (8), but it is
an empirical parameter that may reflect changes in the respiratory system in addition to changes in airway responsiveness. For this reason, we assessed Penh and the ratio of inspiratory time
to expiratory time (Ti/Te
ratio), both analyzed by the noninvasive method, and compared the
results with directly measured parameters of airway responsiveness
(RL and Cdyn). The latter two measurements were
made 24 h after the noninvasive measurements (see MATERIALS AND METHODS and Fig. 1). The daily physiological results for
ovalbumin-treated and vehicle-treated mice are summarized in Fig.
2.
Peak Penh and RL were significantly higher in
the ovalbumin-treated mice compared with the matched vehicle-treated
mice, regardless of the dose of methacholine or day of study (Fig. 2).
The only exception occurred on day 18 for RL at
30 mg/ml methacholine, because of variability among the
ovalbumin-treated mice. In general, the Penh and
RL results were higher during airway provocation with 30 mg/ml methacholine compared with 15 mg/ml methacholine. These
differences are expected physiological indicators of airway obstruction.
When Penh was at its peak, peak Cdyn and the
Ti/Te ratio were lower in
the ovalbumin-treated mice compared with the matched vehicle-treated mice, regardless of the dose of methacholine or day of
study (Fig. 2). The only exceptions occurred on day 22 for
Cdyn at both doses of methacholine and on day 22 for the Ti/Te ratio at 30 mg/ml methacholine, again because of variability among the mice.
The Ti/Te ratio was
lower because the inspiratory time was decreased, whereas the
expiratory time was increased (data not shown). These differences also
are expected physiological indicators of airway obstruction.
Peak Penh inconsistently correlated with
RL, Cdyn, and the Ti/Te
ratio in ovalbumin-treated mice.
The first question that our study addressed was whether
Penh reliably identified changes in airway
hyperresponsiveness during eight consecutive days of allergen exposure
and methacholine provocation. Early in the course of daily exposure to
nebulized ovalbumin (days 14 and 15, regardless
of methacholine dose), peak Penh correlated with peak
RL (Fig. 3), Cdyn
(Fig. 4), and the
Ti/Te ratio (Fig. 5). Later in the course of daily exposure
to nebulized ovalbumin (days 18 and 22), however,
peak Penh did not correlate well with peak RL,
Cdyn, or the
Ti/Te ratio (Figs.
3-5).
Leukocytes accumulated in the walls of arteries, airways, and veins
in ovalbumin-treated mice.
We also sought to correlate the changes in airway hyperresponsiveness
after exposure to nebulized ovalbumin with the accumulation of
inflammatory and immune cells in the peribronchovascular connective tissue sheaths. We first examined Wright-stained sections of lung tissue. This analysis revealed that neutrophils and mononuclear cells
accumulated in the walls and surrounding interstitium of intrapulmonary
arteries and airways (Fig. 6) and veins
(Fig. 7) in the lungs of the
ovalbumin-treated mice compared with the vehicle-treated mice.
Particularly notable was the margination and transmigration of
leukocytes in as little as 4 h (day 14) after exposure
to nebulized ovalbumin. Accumulation increased over the succeeding
24 h. The inflammatory infiltrates remained in the walls and
surrounding interstitium of arteries, airways, and veins throughout the
8 days of ovalbumin exposure (Figs. 6 and 7).
We obtained a broader perspective on the tissue distribution of the
transmigrated cells by examining the autofluorescence of H&E-stained
thick sections (60 µm) of lung, using three-dimensional fluorescence
microscopy that allows imaging of thick sections to provide a depth
perspective. This technique showed focal aggregation inside and outside
pulmonary arteries (Fig. 8; stereopair)
and in the wall of the neighboring airways (Fig. 8) and veins (data not
shown). Imaging the vascular and airway structures in this fashion
resulted in a novel observation. Margination of leukocytes in and
transmigration across pulmonary arteries occurred around the half of
the pulmonary arteries adjacent to the neighboring airway.
Neutrophils and macrophages comprised the tissue infiltrates in
ovalbumin-treated mice.
We used immunohistochemistry as the second step to test for correlation
between changes in airway hyperresponsiveness after exposure to
nebulized ovalbumin with an inflammatory and immune cell infiltrate.
Immunohistochemistry was used to differentiate among the leukocytes
that accumulated in the peribronchovascular connective tissue sheaths
surrounding intrapulmonary airways and arteries, according to the
expression of surface antigens specific for neutrophils (neutrophil
antibody positive), activated macrophages (Mac3 antibody positive), and
lymphocytes (CD5 antibody positive; T lymphocytes and some B
lymphocytes). Representative micrographs of the immunopositive cells in
the ovalbumin-treated and vehicle-treated mice are shown in Fig.
9. Quantitative histology was then used to estimate the number of leukocytes per millimeter of airway basal
lamina for both groups of mice (Fig.
10). We focused on the leukocytes that
accumulated in the peribronchovascular connective tissue sheaths,
because inflammatory mediators released from those infiltrating
leukocytes may affect airway smooth muscle reactivity. Among the
ovalbumin-treated mice, neutrophils and activated macrophages were the
first and predominant types of leukocyte that accumulated in the
peribronchovascular connective tissue sheaths. Neutrophil infiltration
increased from day 15 to day 18 and then
diminished on day 22 (Fig. 10). Activated macrophage
infiltration increased from day 15 to day 22 (Fig. 10). CD5-positive lymphocytes also infiltrated the walls of
airways of the ovalbumin-treated mice. However, accumulation of
CD5-positive lymphocytes was modest compared with neutrophils and
activated macrophages (Fig. 10).
Eosinophils, identified by characteristic staining of their
granules in the H&E-stained sections, were seen in the
peribronchovascular connective tissue sheaths of the ovalbumin-treated
mice. However, they were seen the least among the infiltrating
leukocytes. The small number of tissue eosinophils in the
ovalbumin-treated mice occurred despite a robust elevation in plasma
total IgE concentration, as described above.
Neutrophil and macrophage accumulation around airways is correlated
to lung function variables in ovalbumin-treated mice.
We used the quantitative immunohistochemical results to test for
correlation between airway hyperresponsiveness and inflammatory cell
accumulation in the peribronchovascular connective tissue sheaths after
exposure to nebulized ovalbumin (Table
1). Neutrophil and activated macrophage
accumulation correlated with Penh, RL, and
Cdyn on days 15, 18, and
22 (Table 1). Lymphocyte accumulation, however, did not
correlate well or consistently with Penh, RL, and Cdyn (Table 1). Eosinophil accumulation was
insufficient to analyze statistically.
Increased BAL cell counts in ovalbumin-treated mice did not reflect
interstitial infiltrates early in the time course.
We found that BAL fluid from the ovalbumin-treated mice had more
leukocytes per microliter than the vehicle-treated mice. On day
15, lavage leukocyte number was 2.4 ± 1.5 vs. 3.5 ± 3.3/µl for the ovalbumin-treated and vehicle-treated mice,
respectively (not significant). On day 18, the number of
leukocytes in lavage fluid was 39.1 ± 23.8 vs. 5.4 ± 1.4/µl for the ovalbumin-treated and vehicle-treated mice,
respectively (P < 0.05). On day 22, lavage
leukocyte number was 11.1 ± 5.3 vs. 3.9 ± 0.4/µl for the ovalbumin-treated and vehicle-treated mice, respectively
(P < 0.05). The lavage fluid contained more
neutrophils, alveolar macrophages, and lymphocytes retrieved from the
ovalbumin-treated mice than the vehicle-treated mice (Fig.
11). Neutrophils appeared first in the
lavage fluid (day 15; P < 0.05). Variability was such, however, that few statistically significant differences were detected, despite having seven mice per group per day. Eosinophils were infrequently observed in the BAL fluid, so their number was too small
to analyze statistically.
The ratio of tissue leukocytes (Fig. 9) to lavage leukocytes
(Fig. 11) for neutrophils, macrophages, or lymphocytes in the ovalbumin-treated mice was skewed early in the course in favor of more
tissue leukocytes than lavage leukocytes (Table
2). This disproportionate distribution of
leukocytes early in the course indicates that the abundance of
leukocytes in lavage fluid underestimated leukocyte abundance in
tissue.
 |
DISCUSSION |
The purposes of our study were to use a murine model of
allergen-induced airway inflammation (3) to test the
reliability of a noninvasive method for assessing changes in airway
hyperresponsiveness and to identify whether early changes in airway
hyperresponsiveness are related to pulmonary infiltration of
inflammatory and immune cells. The noninvasive method that we used for
assessing changes in airway responsiveness was barometric whole body
plethysmography, using the empirical and unitless parameter
Penh and the
Ti/Te ratio. An invasive
method for measuring airway responsiveness (RL and
Cdyn) was used to isolate airway function from other
respiratory system influences in the same mice. Peak Penh
and RL were increased, whereas the
Ti/Te ratio and
Cdyn were decreased, in the ovalbumin-treated mice compared
with the vehicle-treated mice. Peak Penh correlated with
peak RL, the
Ti/Te ratio, and
Cdyn in the first 2 days only (days 14 and
15). Importantly, these correlations were lost at later
times (days 18 and 22) and were lost for unknown
reasons. We conclude that the noninvasive method for assessing changes in airway hyperresponsiveness in ovalbumin-treated mice provides an
inconsistent indication of airway hyperresponsiveness to methacholine.
Coincident with the rapid airway functional changes were rapid
margination and transmigration of inflammatory and immune cells in the
peribronchovascular connective tissue sheaths. Accumulation of
neutrophils and activated macrophages around airways correlated well
with peak Penh, RL, and Cdyn early
(day 15), but the correlations disappeared at later times
(days 18 and 22). Accumulation of
lymphocytes correlated poorly with peak Penh,
RL, and Cdyn, regardless of the day of study.
Eosinophil accumulation in the tissue was insufficient to test for
correlation with indexes of airway hyperresponsiveness. Finally,
leukocyte number and differential percentage in BAL fluid did not
accurately reflect the temporal accumulation of inflammatory and immune
cells in lung tissue sections. We conclude that airway hyperresponsiveness in mice exposed to ovalbumin is related, in part,
to accumulation of neutrophils and activated macrophages in the wall of airways.
Eosinophils in BAL fluid is typically a hallmark of asthma models in
immunologically intact mice (2, 3, 7, 8, 14, 25). Our
study showed that eosinophils accumulated in the lung but that their
accumulation was less than neutrophils, activated macrophages, and
CD5-positive lymphocytes. Minimal accumulation of eosinophils in lung
tissue or BAL in allergen-induced lung inflammation in mice is not a
new observation, however. Several studies have shown a paucity of
eosinophils in either lung tissue or BAL fluid of allergen-exposed mice
(2, 4, 10, 21). Our results provide another example of
airway hyperreactivity in mice that occurs without eosinophils.
Why our mouse model of ovalbumin-induced airway inflammation and
hyperresponsiveness failed to elicit large numbers of eosinophils remains unclear. This observation was surprising, given the robust increase in total plasma IgE concentration that occurred in the ovalbumin-treated mice. One possible explanation is our protocol used
only one intraperitoneal injection of ovalbumin, whereas studies that
detected increased numbers of eosinophils in blood and BAL used two or
more intraperitoneal (or subcutaneous) injections of ovalbumin before
aerosol sensitization (8, 20, 25). On the other hand,
Brusselle and colleagues (3) used a single intraperitoneal
injection of ovalbumin, followed by repeated exposure to aerosolized
ovalbumin, and observed accumulation of eosinophils. We followed their
protocol. Another possibility is the strain of mouse appears to be a
determinant of eosinophil recruitment and airway hyperresponsiveness
(4, 5, 21, 25). In this regard, we and Brusselle and
coworkers (3) used C57BL/6 mice.
We compared two standard approaches to measure lung function in the
same mice. The first, and commonly used today, is Penh. The
second is to directly measure RL and Cdyn. The
first approach is noninvasive and readily determined, whereas the
second approach is invasive and technically more challenging. We found
that correlation between the two approaches was inconsistent, in that
correlation occurred during the first days of daily aerosolization of
ovalbumin, but correlation did not occur during later days of daily
aerosolization. This outcome is not suspect because Penh
does not directly measure the function assessed by the invasive
approach (17). However, it is clear that such comparisons
can be unreliable and that Penh cannot simply stand in for
RL. Until other noninvasive measurements are available,
however, a reasonable design is to compare the noninvasive and invasive
results for the same mice. Another group of investigators has found
inconsistent results when Penh measurements are compared
with lung mechanics measurements (19). Combined, their
results and our results suggest that Penh is unreliable for
characterizing lung mechanics.
Penh is an empiric parameter that changes as a consequence
of bronchoconstriction (8). It is derived from analysis of
the waveform of the plethysmograph box pressure and pressure in a reference chamber. Penh is calculated from the formula
as described by Hamelmann and associates (8). The
unitless Penh parameter reflects changes in the waveform of
the box pressure from both peak inspiration (peak inspiratory pressure) and peak expiration (peak expiratory pressure) and combines the waveform with the timing comparison of early and late expiration (also
called "pause"). Thus Penh is an arbitrary mathematical function of the proportion of the box pressure signal from inspiration and expiration and the timing of expiration. Although a number of
studies, including the present study, have established that bronchoconstriction increases Penh, the opposite question
of whether increased Penh is a consequence of changes in
the mechanical properties of the respiratory system has not been
established. Other potential contributors to the plethysmograph box
pressure signal are differences in the temperature and humidity of
inspired and expired air, as well as changes in respiratory rate,
Vt, or phase lags between nasal and thoracic flow
(compression; see Ref. 12). Hamelmann and colleagues
(8) showed that these other potential contributors changed
asynchronously with Penh and therefore concluded that Penh is a valid indicator of bronchoconstriction in mice.
Our results and those recently reported by Peták and colleagues
(19) suggest that increased Penh may not be a
consequence of changes in the mechanical properties of the respiratory
system because correlation is inconsistent between Penh and
more widely accepted measurements of lung mechanics.
The regression plots (Figs. 3-5) for the ovalbumin-treated and
vehicle-treated groups of mice were clustered later in the time course
(days 18 and 22). This clustering was associated
with poor correlation between peak Penh and direct measures
of allergen-induced airway obstruction. This is a new observation. An
explanation for this observation relates to experimental design. We
measured airway responsiveness on 4 of 8 days of daily nebulization of ovalbumin. Other investigators who have used whole body plethysmography and direct measures of airway obstruction in mice evaluated changes in
airway responsiveness just one time after two or three repetitions of
antigen exposure (4, 5, 8). Therefore, the number of daily
repetitions of antigen exposure appears to influence indexes of airway
obstruction. Other explanations are possible for the difference between
our results and those reported by other investigators. For example, no
two studies, including ours, have used the same allergen exposure
regimen. The regimens have used different types, sources, and
concentrations of allergen; different routes of sensitization;
different intervals of time between sensitization and airway challenge;
different numbers of airway challenges and intervals between repeated
challenges; and different concentrations of methacholine (4, 5,
8, 18, 21). Therefore, translating the results from one
physiological study to another is difficult. Another explanation may be
genetic background. Several studies have shown that BALB/c mice
demonstrate greater airway responsiveness after allergen exposure than
C57BL/6 mice (4, 21, 25). We used C57BL/6 mice because we
are using this strain for the genetic background of transgenic and
knockout mice. We have not evaluated other strains of mice.
A novel observation provided by our study is the focal nature of
leukocyte margination in and transmigration across pulmonary arteries
and veins (Fig. 8). This observation was made possible by the use of a
three-dimensional microscope to evaluate thick sections (60-100
µm) of lung tissue. The advantage of this technique is it enables
depth to be appreciated in a single thick slab of tissue, whereas
typically relatively thin tissue sections (5 µm) are used to evaluate
histopathology (Figs. 6 and 7). Although observation of 5-µm-thick
tissue sections has localized the site of leukocyte margination in and
transmigration across pulmonary arteries and veins, in addition to
alveolar capillaries, in the lungs of allergen-challenged mice
(2-5, 21, 25) and in the lungs of sheep during air
embolism-induced acute lung injury (1), appreciation that
the sites of margination and transmigration are focal and may occur in
locations besides capillaries has been lacking. An interesting
subjective impression that derived from observing the tissue slabs
three-dimensionally is that the focal spots of leukocyte margination in
and transmigration across pulmonary arteries were on the half of the
artery that faced the neighboring airway. Other investigators, using
thin sections, have not seen this association (2). Our
observation raises questions about the regulation of directed
margination and transmigration in the lung when an inflammatory
stimulus is delivered on the airway side of the air-blood barrier.
We also noted that accumulation of leukocytes in the
peribronchovascular connective tissue sheaths (Fig. 10) and retrieval of leukocytes in BAL fluid (Fig. 11) did not match. Accumulation of
leukocytes in lung tissue occurred sooner than revealed by retrieval of
leukocytes in BAL fluid. Although this observation makes sense
intuitively, the observation serves as a reminder that leukocyte counts
in BAL fluid are temporally delayed compared with tissue inflammatory responses.
We conclude that ovalbumin sensitization alters airway reactivity in
mice over at least an 8-day period during daily exposure to aerosolized
ovalbumin. The empiric, unitless parameter (Penh) was
statistically different between ovalbumin-treated and -untreated mice
throughout the 8-day study period. However, peak Penh
correlated with direct measurements of airway obstruction
(RL and Cdyn) only during the early days of the
experimental protocol. At later days of analysis, peak
Penh, RL, and Cdyn varied
independently. Thus Penh appears to have limitations as an
index of airway obstruction.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Derek A. Uchida and John R. Hoidal at the University
of Utah for expert advice during the experiments and critical review of
the manuscript. The microscopy analyses were performed in the Research
Microscopy Facility at the University of Utah Health Sciences Center.
 |
FOOTNOTES |
This work was supported by an Asthma Research Center grant from the
American Lung Association and resources from National Heart, Lung, and
Blood Institute Specialized Center of Research Grant in acute lung
injury HL-50153.
Address for reprint requests and other correspondence:
K. H. Albertine, Dept. of Pediatrics, Univ. of Utah Health
Sciences Center, 30 North 1900 East, Salt Lake City, Utah
84132-2202.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published March 22, 2002;10.1152/ajplung.00324.2001
Received 13 August 2001; accepted in final form 24 February 2002.
 |
REFERENCES |
1.
Albertine, KH,
Wiener-Kronish JP,
Koike K,
and
Staub NC.
Quantification of damage by air emboli to lung microvessels in anesthetized sheep.
J Appl Physiol
57:
1360-1368,
1984[Abstract/Free Full Text].
2.
Blyth, DI,
Pedrick MS,
Savage TJ,
Hessel EM,
and
Fattah D.
Lung inflammation and epithelial changes in a murine model of atopic asthma.
Am J Respir Cell Mol Biol
14:
425-438,
1996[Abstract].
3.
Brusselle, GG,
Kips JC,
Tavernier JH,
van der Heyden JG,
Cuvelier CA,
Pauwels RA,
and
Bluethmann H.
Attenuation of allergic airway inflammation in IL-4 deficient mice.
Clin Exp Allergy
24:
73-80,
1994[ISI][Medline].
4.
Corry, DB,
Folkesson HG,
Warnock ML,
Erle DJ,
Matthay MA,
Wiener-Kronish JP,
and
Locksley RM.
Interleukin 4, but not interleukin 5 or eosinophils, is required in a murine model of acute airway reactivity.
J Exp Med
183:
109-117,
1996[Abstract].
5.
Eum, SY,
Haile S,
Lefort J,
Huerre M,
and
Vargaftig BB.
Eosinophil recruitment into the respiratory epithelium following antigenic challenge in hyper-IgE mice is accompanied by interleukin 5-dependent bronchial hyperresponsiveness.
Proc Natl Acad Sci USA
92:
12290-12294,
1995[Abstract].
6.
Flotte, TJ,
Springer TA,
and
Throbecke GJ.
Dendritic cell and macrophage staining by monoclonal antibodies in tissue sections and epidermal sheets.
Am J Pathol
111:
112-124,
1983[Abstract].
7.
Gonzalo, JA,
Lloyd CM,
Kremer L,
Finger E,
Martinez AC,
Siegelman MH,
Cybulsky M,
and
Gutierrez-Ramos JC.
Eosinophil recruitment to the lung in a murine model of allergic inflammation. The role of T cells, chemokines, and adhesion receptors.
J Clin Invest
98:
2332-2345,
1996[Abstract/Free Full Text].
8.
Hamelmann, E,
Schwarze J,
Takeda K,
Oshiba A,
Larsen GL,
Irvin CG,
and
Gelfand EW.
Noninvasive measurement of airway responsiveness in allergic mice using barometric plethysmography.
Am J Respir Crit Care Med
156:
766-775,
1997[Abstract/Free Full Text].
9.
Hamelmann, E,
Takeda K,
Oshiba A,
and
Gelfand EW.
Role of IgE in the development of allergic airway inflammation and airway responsiveness-a murine model.
Allergy
54:
297-305,
1999[ISI][Medline].
10.
Henderson, WR,
Chi EY,
Albert RK,
Chu SJ,
Lamm WJE,
Rochon Y,
Jonas M,
Christie PE,
and
Harlan JM.
Blockade of CD49d (4 integrin) on intrapulmonary but not circulating leukocytes inhibits airway inflammation and hyperresponsiveness in a mouse model of asthma.
J Clin Invest
100:
3083-3092,
1997[Abstract/Free Full Text].
11.
Hirch, S,
and
SG
Polymorphic expression of a neutrophil differentiation antigen revealed by a monoclonal antibody 7/4.
Immunogenetics
18:
229-239,
1983[ISI][Medline].
12.
Ingram, RH, Jr,
and
Schilder DP.
Effects of gas compression on pulmonary pressure, flow, and volume relationship.
J Appl Physiol
21:
1821-1826,
1966[Free Full Text].
13.
Ledbetter, JA,
Rouse RV,
Micklem HS,
and
Herzenberg LA.
T Cell subsets defined by expression of Lyt-1,2,3 and Thy-1 antigens. Two parameter immunofluorescence and cytotoxicity analysis with monoclonal antibodies modifies current views.
J Exp Med
152:
280-295,
1980[Abstract].
14.
Lee, JJ,
McGarry MP,
Farmer SC,
Denzler KL,
Larson KA,
Carrigan PE,
Brenneise IE,
Horton MA,
Haczku A,
Gelfand EW,
Leikauf GD,
and
Lee NA.
Interleukin-5 expression in the lung epithelium of transgenic mice leads to pulmonary changes pathognomonic of asthma.
J Exp Med
185:
2143-2156,
1997[Abstract/Free Full Text].
15.
MacLean, JA,
Sauty A,
Luster AD,
Drazen JM,
and
DeSanctis GT.
Antigen-induced airway hyperresponsiveness, pulmonary eosinophilia, and chemokine expression in B cell-deficient mice.
Am J Respir Cell Mol Biol
20:
379-387,
1999[Abstract/Free Full Text].
16.
Martin, TR,
Gerard NP,
Galli SJ,
and
Drazen JM.
Pulmonary responses to bronchoconstrictor agonists in the mouse.
J Appl Physiol
64:
2318-2323,
1988[Abstract/Free Full Text].
17.
Mitzner, W,
and
Tankersley C.
Correspondence: noninvasive measurement of airway responsiveness in allergic mice using barometric plethysmography.
Am J Respir Crit Care Med
158:
340-342,
1998[Free Full Text].
18.
Peebles, RS, Jr,
Sheller JR,
Johnson JE,
Mitchel DB,
and
Graham BS.
Respiratory syncytial virus infection prolongs methacholine-induced airway hyperresponsiveness in ovalbumin-sensitized mice.
J Med Virol
57:
186-192,
1999[ISI][Medline].
19.
Peták, F,
Habre W,
Donati YR,
Hantos Z,
and
Barazzone-Argiroffo C.
Hyperoxia-induced changes in mouse lung mechanics: forced oscillations vs barometric plethysmography.
J Appl Physiol
90:
2221-2230,
2001[Abstract/Free Full Text].
20.
Schramm, CM,
Puddington L,
Yiamouyiannis CA,
Lingenheld EG,
Whiteley HE,
Wolyniec WW,
Noonan TC,
and
Thrall RS.
Proinflammatory roles of T-Cell receptor (TCR)
and TCR
lymphocytes in a murine model of asthma.
Am J Respir Cell Mol Biol
22:
218-225,
2000[Abstract/Free Full Text].
21.
Wilder, JA,
Collie DD,
Wilson BS,
Bice DE,
Lyons CR,
and
Lipscomb MF.
Dissociation of airway hyperresponsiveness from immunoglobulin E and airway eosinophilia in a murine model of allergic asthma.
Am J Respir Cell Mol Biol
20:
1326-1334,
1999[Abstract/Free Full Text].
22.
Woolcock, AJ.
Asthma.
In: Textbook of Respiratory Medicine, edited by Murray JF,
and Nadel JA.. Philadelphia, PA: Saunders, 1994, p. 1288-1330.
23.
Zar, JH.
Biostatistical Analysis (2nd ed.). Englewood Cliffs, NJ: Prentice-Hall, 1984.
24.
Zhang, K,
Flanders KC,
and
Phan SH.
Cellular localization of transforming growth factor-
expression in bleomycin-induced pulmonary fibrosis.
Am J Pathol
147:
352-361,
1995[Abstract].
25.
Zhang, Y,
Lamm WJ,
Albert RK,
Chi EY,
Henderson WR, Jr,
and
Lewis DB.
Influence of the route of allergen administration and genetic background on the murine allergic pulmonary.
Am J Respir Crit Care Med
155:
661-669,
1997[Abstract].
26.
Zimmerman, GA,
Albertine KH,
Carveth HJ,
Gill EA,
Grissom CK,
Hoidal JR,
Imaizumi T,
Maloney CG,
McIntyre TM,
Michael JR,
Orme JF,
Prescott SM,
and
Topham MS.
State of the art: endothelial activation in the acute respiratory distress syndrome (ARDS).
Chest
116:
18S-24S,
1999[Free Full Text].
Am J Physiol Lung Cell Mol Physiol 283(1):L219-L233
1040-0605/02 $5.00
Copyright © 2002 the American Physiological Society