| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Regular Articles |
From the Department of Biology,*
Capital Community
Technical College, Hartford, Connecticut; the Departments of
Pediatrics
and
Medicine,
University of Connecticut School of
Medicine, Farmington, Connecticut; Boehringer-Ingelheim
Pharmaceuticals, Inc.,¶
Ridgefield,
Connecticut; and Department of Pathobiology,||
University
of Connecticut, Storrs, Connecticut, and Eli Lilly and
Company,§
Lilly Research Laboratories,
Indianapolis, Indiana
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
cells in lung tissue, increased serum IgE
levels, and airway hyperresponsiveness to methacholine. In mice
subjected to chronic (6-week) aerosol challenge with ovalbumin,
airway inflammation and serum IgE levels were significantly attenuated
and airway hyperresponsiveness was absent. The marked increases in lung
B and T cell populations seen in the acute stage were also
significantly reduced in the chronic stage of this model. Thus,
acute ovalbumin challenge resulted in airway sensitization
characteristic of asthma, whereas chronic ovalbumin challenge
elicited a suppressed or tolerant state. The transition from antigenic
sensitization to tolerance was accompanied by shifts in lymphocyte
profiles in the lung and bronchoalveolar lavage fluid.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. IFN- has been shown to stimulate low-level IgG production
and to potently inhibit IL-4-mediated IgE responses both in
vivo and in vitro.7
The mechanisms that
control CD4+ T lymphocyte polarization into either Th1 or
Th2 phenotypes are incompletely understood but appear to involve
genetic predispositions, local factors such as existing cytokine
concentrations and inflammation, and antigenic factors such as the
potency, dose, and duration of exposure of the eliciting antigen. In
susceptible individuals, antigen sensitization results in specific
local and systemic IgE production and airway eosinophilia, which in
turn induce the airway inflammation, airway hyperresponsiveness, and
reversible airway obstruction characteristic of asthma. The factors influencing antigen sensitization or tolerance can be better studied in mice, given their well defined immune system, the creation of genetically altered animals, and the availability of reagents to various cellular receptors and cytokines. Furthermore, the mouse is a suitable model for allergic responses in the lungs because the major antibody in murine allergic pulmonary inflammation is IgE.8 Thus, several investigators have employed mouse models to investigate the mechanisms of allergic airway sensitization.10-15 In addition, mice have been used to study the chronic tolerant state to airway exposure to antigen.16, 17 Nevertheless, murine models have not been used to assess the natural history of antigen sensitization and the re-establishment of the tolerant state in allergic airway disease. In this study, we have characterized the inflammatory and physiological responses to chronic inhaled ovalbumin (OVA) administration in C57BL/6J mice. With initial immunization and aerosol challenge, we have developed a murine model of allergic sensitization with similar characteristics to human asthma, such as increased IgE production, increased pulmonary eosinophils, increased pulmonary B and T lymphocytes, and airway hyperresponsiveness. We also have found that chronically challenged mice can develop antigenic tolerance, as characterized by diminished IgE levels and loss of airway eosinophilia and hyperreactivity. These pathophysiological changes were accompanied by profound alterations in lymphocyte profiles within the airways.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Male and female C57BL/6J mice were purchased from Jackson Laboratory, Bar Harbor, ME, and housed conventionally in plastic cages with corncob bedding. The animal room was maintained at 22 to 24°C with a daily light/dark cycle (light from 0600 to 1800 hours). Chow and water were supplied ad libitum. Mice were 3 to 6 months of age and weighed 18 to 30 g. All animal manipulations were approved by the Animal Care Committee at the University of Connecticut Health Center.
Ovalbumin Exposure Protocol
Mice were initially immunized with three weekly intraperitoneal (i.p.) injections of a suspension containing 8 µg of OVA (grade V, Sigma Chemical Co., St. Louis, MO) and 2 mg of aluminum hydroxide (alum) in 0.5 ml of saline. One week after the last injection the mice were exposed either to 1% aerosolized OVA, 1 hour/day, for 1 to 10 days (acute stage) or to 3 to 6 weeks at 1 hour/day for 5 days/week (chronic stage). The mice were placed in plastic restraint tubes (Research and Consulting Co., Basel, Switzerland) for nose-only exposures. The aerosols were generated by a Lovelace nebulizer (In-Tox Products, Albuquerque, NM) into a 7.6-L inhalation exposure chamber to which restraint tubes were attached. Chamber airflow was 10 L/minute. Aerosol particle size of OVA was determined by gravimetric analysis, with a Mercer cascade impactor (In-Tox Products). The mass median aerodynamic diameter and geometric standard deviations (GSDs) were 1.4 and 1.6 µm, respectively. The estimated daily inhaled OVA dose approximated 80 µg/mouse. Twenty-four hours after the final aerosol exposure, the mice were sacrificed by ketamine/xylazine overdose and exsanguination, and analyses of bronchoalveolar lavage (BAL), lung tissue, and blood samples were performed.
BAL Analysis
The lungs from each animal were lavaged in situ with five 1-ml aliquots of sterile saline, with 3 to 4 ml of BAL fluid recovered from each animal. The BAL was centrifuged, and resulting cell pellets were resuspended in 250 µl of saline. Total leukocytes were counted with a hemocytometer using trypan blue dye exclusion as a measure of viability. Cytospin slides were made and stained with May-Grunwald/Giemsa to determine the BAL cell differential. The remaining cells were analyzed by fluorescence flow cytometry. BAL protein concentrations were measured in the supernatants using bovine serum albumin as a standard.18
Flow Cytometry and Immunofluorescence
Monoclonal antibodies (MAbs) purchased from PharMingen (San Diego,
CA) were directed against the following antigens: CD45 (clone 30-F11),
TCRß (H57-597),19
TCR (GL3),20
CD3
(500A2), CD8 (53-6.7), and B220 (RA3-6B2). These were conjugated with
biotin, phycoerythrin (PE), fluorescein isothiocyanate (FITC),
allophycocyanine (APC), or Cychrome. Anti-CD4-PE (clone GK1.5) was
purchased from Becton-Dickinson Collaborative Technologies, Bedford,
MA. Anti-CD8
-FITC (clone 3.168) was conjugated to FITC in our
laboratory.21
Biotin-conjugated antibodies were detected
with streptavidin-PE or -Cy5 (Jackson ImmunoResearch Laboratories, West
Grove, PA) or -Cychrome (PharMingen). For fluorescence flow cytometry,
BAL samples were washed in PBS containing 0.2% bovine serum albumin
and 0.1% NaN3. Aliquots containing 104
to
105
cells were incubated with 100 µl of appropriately
diluted antibodies for 30 minutes at 4°C. After staining, the cells
were washed twice with the above PBS solution, and relative
fluorescence intensities were determined on a 4-decade log scale by
flow cytometric analysis using a FACScan or FACScalibur (Becton
Dickinson, San Jose, CA).
Tissue immunofluorescence was assessed for distribution of cells using
the above stated antibodies to B cells and to TCRß and TCR
lymphocytes and was correlated with standard hematoxylin and eosin
(H&E) histological assessment. Unmanipulated lungs (not exposed to BAL
or methacholine) were excised, cut into small pieces, and rapidly
frozen in optimal cutting temperature embedding media (OCT). The pieces
were then cut into 5-µm frozen sections using a Hacker cryostat,
mounted onto microscope slides (Clay Adams Gold Seal), and stored at
-20°C. For immunofluorescence staining, the slides were fixed in
acetone (-20°C) for 5 minutes, dried, and blocked with 1% ChromPure
IgG solution (Jackson ImmunoResearch) for 30 minutes at room
temperature. After two washes with PBS plus 0.1% NaN3,
specific PE-mouse antibody was added to the tissue and incubated for 1
hour in a humidity chamber. Slides were then washed twice with PBS plus
0.1% NaN3. Sections were mounted in PBS/glycerol (1:1) and
viewed with a Zeiss LSM 410 confocal microscope.
Histology
After sacrifice, the unmanipulated lungs were removed, fixed with 10% buffered formalin, and processed in a standard manner. Tissue sections were stained with H&E.22
Measurement of Airway Hyperreactivity: Lung Resistance
Measurements of pulmonary resistance (RL) were obtained in anesthetized, mechanically ventilated mice via standard protocol.23 OVA-immunized mice were exposed to 1% aerosolized OVA, 1 hour/day, for 1 to 10 days. Mice were studied 24 hours after the last inhalation and compared with naive mice. After anesthetizing the animals with pentobarbital (75 mg/kg i.p. injection), the abdominal inferior vena cava was cannulated, and a tracheostomy catheter was placed. The chest was opened by a small anterior incision, and the animal was placed in a whole-body plethysmograph. Mechanical ventilation was established with a small rodent respirator (model 683, Harvard Apparatus, Natick, MA) delivering a 10 ml/kg tidal volume at 140 breaths/minute, with a positive end-expiratory pressure (PEEP) of 3 cm H2O. Values for RL were calculated by analysis of electrical signals proportional to lung volume, airflow, and transpulmonary pressure. Changes in lung volume were determined from the measured changes in plethysmographic pressure and were differentiated over time to obtain flow measurements. Transpulmonary pressure was obtained from the difference between measured pressures at the airway opening and within the plethysmograph. After the establishment of baseline lung function, the animal received sequentially increasing intravenous doses of methacholine (Sigma; 3 to 3000 µg/ml in 1 ml/kg body weight increments). Maximal RL responses were determined from measurements averaged over 6-second intervals. Pulmonary function was allowed to return to baseline before each subsequent dose.
Measurement of Airway Hyperreactivity: Excised Lung Gas Volume
In a separate series of experiments, air trapping in response to aerosolized methacholine was measured by excised lung gas volume (ELGV). This technique is based on Archimedes' principle and is a sensitive index of airway obstruction in small rodents.24-26 Twenty-four hours after the last OVA aerosol challenge, acute-stage and chronic-stage mice were exposed to aerosolized methacholine using the same chamber and nebulizer as in the sensitization protocol. Airflow was 10 L/minute, and nebulizer methacholine concentration ranged from 1 to 300 mg/ml. After an 8-minute exposure, mice were sacrificed via exsanguination after an i.p. ketamine/xylazine injection. The lungs and trachea were removed and trimmed of nonpulmonary tissue. ELGV was measured as previously described26 and compared with values in control, unexposed mice.
IgE Analysis
Blood was obtained by cardiac puncture just before sacrifice, and serum IgE levels were measured by ELISA. Total IgE was captured using Immunlon 2 microtiter plates (Dynatech Laboratories, Chantilly, VA) coated with anti-mouse IgE (clone R35-72 at 2 µg/ml in PBS). Duplicate twofold dilutions of serum (1:10, 1:20, and 1:40) were added and incubated for 1 hour at room temperature. Detection was with biotinylated anti-mouse IgE (clone R35-92 at 2 µg/ml; antibodies from PharMingen, San Diego, CA) and avidin-conjugated horseradish peroxidase (1:2000 dilution; Zymed Laboratories, San Francisco, CA). Development was typically for 10 minutes (the time when the A405 was less than 1.0 for the 400 µg/ml IgE standard).
Statistical Analysis
Student's unpaired t-test and Bonferronis' adjustment for multiple comparisons were used for data analysis. Dose-response data were compared by repeated-measures analysis of variance (ANOVA) using StatView 4.5 (Abacus Concepts, Berkeley, CA).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Mice immunized with three weekly injections of i.p. OVA/alum and
then acutely challenged to OVA aerosol displayed significant increases
in the numbers of BAL macrophages, eosinophils, and lymphocytes
compared with control (unexposed) mice (Table 1)
. The numbers of BAL cells began to
increase by day 3 of aerosol challenge and peaked at 7 to 10 days of
continued exposure. Total leukocytes per mouse increased from 3.7
x 104
in control, unexposed animals to a maximum of
133 x 104
in acute OVA challenged mice at day 10. The
ratios of cell types changed dramatically during the acute OVA
challenge. In the unexposed mice, over 99% of the BAL cells were
macrophages, but this fraction dropped to 8% after 10 days of OVA
aerosol. Eosinophils and lymphocytes increased from less than 1% in
unexposed mice to 73% and 19%, respectively. BAL protein content was
significantly higher at days 5, 7, and 10 versus control
protein BAL (P < 0.05). There were no
differences in total and differential cell counts or protein content
among control unexposed mice, mice that received only 3 weeks of i.p.
OVA/alum immunizations without aerosol exposure, or mice that received
the 10-consecutive-day OVA aerosols without preceding i.p. immunization
(data not shown). The combination of i.p. immunization and aerosol
challenge was necessary to see the inflammatory response.
|
Mice immunized with three weekly i.p. injections of OVA/alum and
then exposed to daily OVA aerosol for 6 weeks had no significant
difference in total BAL leukocytes as compared with control mice (Table 1)
. There was a significant increase in the percentage of lymphocytes
(29%) versus the acute challenged state at day 10 (19%),
but the total number of lymphocytes was much less in the chronic stage
than in the 10-day acute stage (P < 0.0001).
The remaining BAL cells in the chronically challenged animals were
macrophages. Eosinophils were completely absent in these chronic mice.
Immunized mice exposed to 3 weeks of OVA aerosol had total and
differential BAL cell counts that were intermediate between those of
the 10-day acute allergic and 6-week chronic tolerant stages. Of note,
although the 3-week OVA aerosol-exposed mice still had a significant
increase in BAL eosinophils, these cells were markedly decreased as
compared with the 10-day acute-stage animals.
Relative Distributions of BAL B and T Lymphocytes Differed between the Acute and Chronic Stages
Along with the 17-fold decrease in total lymphocytes in the
chronic- versus acute-stage animals, the proportions of
lymphocytes in BAL also changed dramatically (Table 2
; Figure 1
). In the 10-day acute OVA challenge
stage, 53% of BAL lymphocytes were B cells, compared with 11% in the
6-week chronic OVA challenged mice. Regarding the T lymphocytes, in the
acute OVA challenge stage, 34% of total BAL lymphocytes were TCRß
cells and 12% were TCR cells. These fractions changed to 82%
and 7%, respectively, in the chronic OVA challenged animals. Thus,
TCR cells comprised 26% of the T lymphocytes in the acute stage
but only 7% of T cells in the chronic stage. In addition, the ratio of
CD4/CD8 ß T cells shifted in BAL fluid in acute- versus
chronic-stage animals (Table 3
; Figure 1
). In acute OVA challenged mice, the ratio of CD4+ to
CD8+ TCRß cells was 2.4, in contrast to the ratio of
0.6 in chronic OVA challenged animals. This change was due to a lesser
reduction of CD8+ TCRß cells relative to the decreases
in CD4+ TCRß cells and B cells in the airways of
chronic-stage animals.
|
|
|
Cells and B Cells in the Lungs of Acute-Stage Mice
In contrast to the absence of detectable TCR cells in
unexposed mice (Figure 2a)
, clusters of
positive staining TCR cells (Figure 2b)
and B cells (Figure 2c)
were regularly found in the lungs of acute OVA challenged mice. These
clusters of cells were localized to peribronchial and perivascular
areas of inflammation. Such clusters of TCR or B lymphocytes were
not seen in the lungs of control animals that received either the i.p.
immunizations alone or the 10-consecutive-day inhalations alone. Of
interest, we were unable to detect clusters of TCR cells or B
cells in the chronic OVA challenged animals (not shown).
|
No histological evidence of lung damage was found in unexposed
control animals, animals that received only the i.p. immunization
protocol, or animals that received only the 10-consecutive-day
inhalations (Figure 3
, top panel). In
contrast, the acute OVA challenge stage was characterized by dense
peribronchial inflammation consisting primarily of lymphoplasmacytic
cells and eosinophils (Figure 3
, middle panel). There were also areas
of perivascular inflammation and slight peribronchial muscle
hypertrophy noted in the acute-stage animals. These histological
findings are consistent with the pathophysiology of allergic asthma.
The chronic-stage animals had qualitatively less peribronchial
lymphoplasmacytic inflammation, mild peribronchial muscle hypertrophy,
and slight to mild bronchial lining cell hyperplasia. Eosinophils were
notably absent in lungs from these chronic-stage animals (Figure 3
,
lower panel).
|
Intravenous administration of methacholine elicited dose-dependent
increases in RL measurements (Figure 4)
over the dose range of 30 to 3000
µg/kg. This response was significantly potentiated by acute OVA
challenge (P = 0.002 by repeated-measures
ANOVA). Baseline resistance measurements did not vary among naive mice
and mice exposed to OVA aerosols for 3, 5, 7, or 10 days, and
maximal RL responses were similar in naive,
3-day, and 5-day OVA challenged animals. In contrast, the maximal
RL response to methacholine was significantly
increased after 7 and 10 days of exposure (12.5 ± 1.6 and
9.5 ± 0.7 cm H2O/ml/second, respectively;
P < 0.01 each) compared with the control response in
naive animals (6.7 ± 0.9 cm H2O/ml/second; Figure 4
).
Sensitivity to methacholine, as depicted by the interpolated
concentration associated with RL equaling 270%
of baseline,23
increased nearly threefold with acute OVA
challenge; however, sensitivity increased earlier than maximal
responsiveness and returned to control values by 10 days of OVA aerosol
challenge (Figure 5)
.
|
|
. In
unexposed mice, the ELGV value was significantly increased at nebulized
methacholine concentrations of 30 mg/ml and 300 mg/ml versus
deionized water alone. There was no significant difference in baseline
lung volumes between the OVA exposed and unexposed mice. The 10-day OVA
challenged mice had significant airway hyperreactivity relative to
control animals, as evidenced by increased ELGV values at methacholine
concentrations of 3 and 10 mg/ml (Figure 7)
. The maximal ELGV response was
observed at 10 mg/ml methacholine in the 10-day OVA challenged mice
versus 30 mg/ml in unexposed mice. Sensitivity to
methacholine, as characterized by the interpolated concentration of
methacholine associated with an ELGV value 200% of baseline (an
increase of >3 SDs), was increased over fivefold in 10-day OVA
challenged mice (3.3 ± 0.6 mg/ml) compared with unexposed mice
(17.5 ± 4.9 mg/ml; P < 0.05). An absence of
airway hyperreactivity was noted in the chronic OVA challenged mice, in
which gas trapping was not significantly different from control mice.
|
|
Serum IgE levels in nonimmunized control mice were <400 ng/ml and
were significantly increased by the i.p. OVA immunization. An
additional increase in serum IgE occurred during the acute OVA
challenge period (Figure 8)
. When
compared with 1 day of aerosol exposure, significant differences in IgE
concentrations were noted at days 5, 7, and 10 of acute OVA aerosol
exposures. In contrast, total serum IgE levels in mice subjected to
chronic exposure (35 days) was not significantly different from mice
exposed to OVA for 1 day.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
lymphocytes around
airways. These histological findings were accompanied by elevations in
serum IgE levels and the development of nonspecific airway
hyperreactivity, as assessed both by lung resistance responses to
intravenous methacholine and air trapping responses to aerosolized
methacholine. Our findings are consistent with observations in other
acute allergic murine models.10-15
Levels of IL-4, IL-5,
and IFN- were generally below detectable limits in unconcentrated
BAL samples from our mice, as assayed by specific ELISA methods. Such
allergen-induced eosinophilic inflammation and airway hyperreactivity
have been shown to depend upon allergen-specific IgE,13, 27
CD4+ T cells,8, 9, 28
and the cytokines
IL-429, 30
and IL-5.8, 15, 31
However, this
acute inflammatory response diminished over 3 to 6 weeks of continuous
inhaled OVA exposure in our model. At 3 weeks, there were still
significant BAL eosinophilia and IgE elevations. By 6 weeks, the BAL
eosinophilia and airway hyperreactivity had resolved. Serum IgE was
significantly decreased and approached naive levels in 75% of
the mice. Thus, whereas acute OVA challenge elicited allergic
inflammatory responses, chronic OVA challenge induced a suppressed or
tolerant state in the mice.
The mechanisms underlying this progression of allergic to tolerant
states are unknown. Our data show that the progression is associated
with shifts in lymphocyte populations in the lung. Tissue
immunofluorescence revealed clustering of both B and TCR
lymphocytes within peribronchial inflammatory lesions in the lungs of
acute-stage animals. Although the contribution of B cells to allergic
responses is well appreciated, this is the first direct demonstration
that lung TCR cells acutely increase upon allergen challenge. We
were unable to identify either B or TCR lymphocytes in
inflammatory sites in chronic-stage animals. Based on their
epitheliotropism, it has been suggested that TCR cells may act as
first responders in the lung to inhaled antigens.32
Intraepithelial TCR lymphocytes have been observed in increased
number in the nasal mucosa of humans with allergic
rhinitis33
and in BAL fluid of severe asthmatic
patients.34
As TCR cells have been shown to
synthesize either Th2 or Th1 cytokines,35
it is unclear
whether they are playing a pro-inflammatory or suppressor role in these
mice or in individuals with allergic airway disease. Recent
observations of attenuated allergic airway inflammation in TCR
cell-deficient mice would support a pro-inflammatory role for
these lymphocytes in the acute stage of this model.36, 37
In contrast, Holt and colleagues16
have demonstrated
that TCR lymphocytes isolated from the spleens of tolerant
animals can suppress serum IgE responses to intraperitoneal antigen
when transferred to naive recipient animals. Their studies suggest a
suppressive role for TCR cells in allergic diseases. At present,
the specific contributions of TCR lymphocytes in the acute
allergic and chronic tolerant stages of inhaled antigen exposure remain
to be elucidated.
In correlation with the tissue immunofluorescence, BAL lymphocyte
profiles shifted between the acute allergic and chronic tolerant
stages. In acute-stage (10-day) BAL fluid, the lymphocyte profile was
53% B cells, 12% TCR cells, 21% CD4+ TCRß
cells, and 9% CD8+ TCRß cells. Our
CD4+:CD8+ T cell ratio of 2.3:1 is similar to
the 3:1 to 4:1 ratio observed by others in acute OVA challenged
C57BL/6J mice.11
These BAL profiles changed dramatically
with chronic (6-week) OVA challenge, such that the lymphocyte profile
was 11% B cells, 7% TCR cells, 28% CD4+ TCRß
cells, and 46% CD8+ TCRß cells. It should be noted
that the total number of lymphocytes was 17-fold less in the chronic
than acute animals, reflecting the lesser degree of airway inflammation
in these mice. Nevertheless, the establishment of chronic tolerance was
associated with a dramatic change in the distribution of lymphocytes,
with a fivefold increase in CD8+ TCRß cells and a
fivefold decrease in B cells. These shifts occurred concurrently with a
decrease in serum IgE levels.
The attenuation of serum IgE with chronic aerosol exposure in immunized
mice is comparable to what has been observed in naive mice, in which
repeated exposure to low levels of aerosolized OVA abrogates an IgE
response to subsequent systemic OVA challenge.16, 17
The
mechanisms underlying this inhalational tolerance are unclear, as the
process has been found to be both dependent16,38
and
independent17
of IFN-, CD8+ lymphocytes, and
TCR cells. Genetic factors are likely involved, related to the
ability of differing animal strains to mount IgE
responses.39
Far more work has been done regarding the
analogous process of oral tolerance to ingested antigens. Oral
tolerance probably occurs via multiple distinct
mechanisms,40
but at least one of these involves
preferential stimulation of enteric CD8+ T cells.41, 42
Our demonstration of an increase in the relative
proportion of CD8+ TCRß lymphocytes in BAL of chronic
OVA challenged animals suggest that these cells may also mediate the
down-regulation of allergic airway responses. It may be that the
persistence of TCR cells in chronic BAL samples was associated
with similar shifts in their CD4+:CD8+ ratios.
Additional studies with mice lacking CD8+ or TCR
lymphocytes will further define the factors of airway inflammation and
tolerance to repeated OVA exposure.
It should be emphasized that our mouse model, and specifically the chronic stage, is fundamentally different from those previously used to assess inhalational tolerance. Previous models were designed to promote tolerance and assessed the ability of inhaled antigen exposure to inhibit systemic IgE responses to subsequently administered intraperitoneal antigen.16, 17 In contrast, our murine model characterizes the initial development of allergic airway sensitization to inhaled antigen in the presence of previous IgE generation with intraperitoneal antigen. It then goes on to determine the kinetics of this sensitization and the redevelopment of antigen tolerance with chronic inhalational exposure. Previous studies have relied solely upon immunoglobulin responses to detect sensitization or tolerance. We have extended these observations and have correlated IgE levels to airway eosinophil and lymphocyte profiles, lung inflammation, and airway hyperreactivity in an allergic pulmonary process similar to asthma.
Thus, we have followed the natural history of allergic airway
inflammation in a murine model of asthma. We found that systemic
immunization followed by aerosolized antigen challenge resulted in a
breaking of normal homeostasis and the creation of allergic airway
inflammation and airway hyperreactivity. This process persisted but was
subsiding at 3 weeks of aerosol exposure. By 6 weeks, mice appeared to
have regained their normal, homeostatic tolerant state, as defined by
the disappearance of airway eosinophilia, a marked reduction in
histological peribronchial inflammation, a decrease in serum IgE
levels, and the resolution of airway hyperreactivity despite continued
antigen exposure. This observation suggests that local regulatory
mechanisms and/or other factors such as antigen redistribution can
influence systemic sensitization to re-establish airway homeostasis.
Coinciding with the acute inflammatory changes and their resolution was
the sequential appearance and disappearance of peribronchial clusters
of B cells and TCR cells. We speculate that these clusters
represent clonal expansion of B cells and pro-inflammatory TCR
cells with Th2-like cytokine characteristics. As the model progresses
to the chronic stage, these lymphocytes disappear, perhaps through
apoptotic mechanisms, leaving the lung with primarily suppressive
lymphocyte populations. The persistence of CD8+ TCRß
cells and TCR cells in BAL of chronic challenged animals
implicates these lymphocytes as mediators of the re-established airway
homeostasis.
This study represents the first comprehensive characterization of the sequential progression of acute allergic airway inflammation and its subsequent resolution to a chronic antigen-tolerant state. This murine model allows us to study the mechanisms of the natural history of asthma, including the initial breaking of normal homeostasis, the peak allergic response, and the redevelopment of the normal tolerant state. Our observations provide an essential framework for additional, more mechanistic investigations focused on the association of various cells with pathophysiological events in the development and resolution of asthma. Most human subjects sensitized to an inhalational antigen remain sensitized for many years, often for life. Current asthma therapies are aimed at controlling airway inflammation and hyperresponsiveness, but at present we lack the ability to re-establish normal antigen homeostasis in asthmatic patients. Insights into the mechanisms of airway sensitization and tolerance gained with further use of this murine model may lead to the development of novel treatment strategies for the prevention or eradication of allergic airway disease and asthma.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Supported by Career Investigator Award, American Lung Association (C.M. Schramm), Faculty Research Grant, University of Connecticut Health Center (C.M. Schramm), and grant DK51505 from the National Institutes of Diabetes and Digestive Diseases (L. Puddington).
Preliminary findings were presented at the American Thoracic Society International Conference and published in abstract form: Puddington L, Schramm CM, Yiamouyiannis CA, Thrall RS: Changes in gamma/delta T cell distributions in the acute allergic and chronic tolerant stages of an ovalbumin mouse model of asthma. Am J Respir Crit Care Med 1998, 157:A832.
Accepted for publication March 13, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. J Immunol 1986, 136:949-954[Abstract]
T cells. Science 1994, 265:1869-1871+ T cells or interferon (IFN)- in a murine model of allergic sensitization. J Exp Med 1998, 187:721-731ß T cell receptors. J Immunol 1989, 142:2736-2742[Abstract]
T cells, allergen recognition and airway inflammation. Immunol Today 1998, 19:22-26[Medline]
/ T cells in allergic and infectious rhinitis. Am J Respir Crit Care Med 1996, 153:1655-1665[Abstract]
/ T cells in bronchoalveolar lavage from patients with asthma. Ann Intern Med 1996, 124:223-227 and interleukin-4 in response to Th1 and Th2 stimulating pathogens by / T cells in vivo. Nature 1995, 373:255-257[Medline]
-T cell knock-out mice exhibit reduced airway eosinophilia in an asthma model. Am J Respir Crit Care Med 1997, 155:A736
T cells in allergic airway inflammation. Science 1998, 280:1265-1267 T cells down-regulate primary IgE responses in rats to inhaled soluble protein antigens. J Immunol 1995, 154:4390-4394[Abstract]
This article has been cited by other articles:
 |
|
 |
|
  J. H. T. Bates, M. Rincon, and C. G. Irvin Animal models of asthma Am J Physiol Lung Cell Mol Physiol, September 1, 2009; 297(3): L401 - L410. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Bogaert, K. G. Tournoy, T. Naessens, and J. Grooten Where Asthma and Hypersensitivity Pneumonitis Meet and Differ: Noneosinophilic Severe Asthma Am. J. Pathol., January 1, 2009; 174(1): 3 - 13. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Singh, W. F. Carson IV, E. R. Secor Jr, L. A. Guernsey, R. A. Flavell, R. B. Clark, R. S. Thrall, and C. M. Schramm Regulatory Role of B Cells in a Murine Model of Allergic Airway Disease J. Immunol., June 1, 2008; 180(11): 7318 - 7326. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. R. Secor Jr, W. F. Carson IV, A. Singh, M. Pensa, L. A. Guernsey, C. M. Schramm, and R. S. Thrall Oral Bromelain Attenuates Inflammation in an Ovalbumin-induced Murine Model of Asthma Evid. Based Complement. Altern. Med., March 1, 2008; 5(1): 61 - 69. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. P. Matson, L. Zhu, E. G. Lingenheld, C. M. Schramm, R. B. Clark, D. M. Selander, R. S. Thrall, E. Breen, and L. Puddington Maternal Transmission of Resistance to Development of Allergic Airway Disease J. Immunol., July 15, 2007; 179(2): 1282 - 1291. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Niu and L. Cohn Response to Comment on "A Novel Pathway That Regulates Inflammatory Disease in the Respiratory Tract" J. Immunol., June 15, 2007; 178(12): 7501 - 7502. [Full Text] [PDF]
|
|
|
|
|
|
N. Niu, M. K. Le Goff, F. Li, M. Rahman, R. J. Homer, and L. Cohn A Novel Pathway That Regulates Inflammatory Disease in the Respiratory Tract J. Immunol., March 15, 2007; 178(6): 3846 - 3855. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Pierce, J. Rir-Sima-Ah, I. Estrada, J. Wilder, A. Strasser, and Y. Tesfaigzi Loss of pro-apoptotic Bim promotes accumulation of pulmonary T lymphocytes and enhances allergen-induced goblet cell metaplasia Am J Physiol Lung Cell Mol Physiol, November 1, 2006; 291(5): L862 - L870. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. W. Hollingsworth, G. S. Whitehead, K. L. Lin, H. Nakano, M. D. Gunn, D. A. Schwartz, and D. N. Cook TLR4 Signaling Attenuates Ongoing Allergic Inflammation J. Immunol., May 15, 2006; 176(10): 5856 - 5862. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Tesfaigzi Roles of Apoptosis in Airway Epithelia Am. J. Respir. Cell Mol. Biol., May 1, 2006; 34(5): 537 - 547. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Wands, C. L. Roark, M. K. Aydintug, N. Jin, Y.-S. Hahn, L. Cook, X. Yin, J. Dal Porto, M. Lahn, D. M. Hyde, et al. Distribution and leukocyte contacts of {gamma}{delta} T cells in the lung J. Leukoc. Biol., November 1, 2005; 78(5): 1086 - 1096. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. G. Xisto, L. L. Farias, H. C. Ferreira, M. R. Picanco, D. Amitrano, J. R. Lapa e Silva, E. M. Negri, T. Mauad, D. Carnielli, L. F. F. Silva, et al. Lung Parenchyma Remodeling in a Murine Model of Chronic Allergic Inflammation Am. J. Respir. Crit. Care Med., April 15, 2005; 171(8): 829 - 837. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. M. Cloutier, L. Guernsey, C. A. Wu, and R. S. Thrall Electrophysiological Properties of the Airway: Epithelium in the Murine, Ovalbumin Model of Allergic Airway Disease Am. J. Pathol., May 1, 2004; 164(5): 1849 - 1856. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. M. Schramm, L. Puddington, C. Wu, L. Guernsey, M. Gharaee-Kermani, S. H. Phan, and R. S. Thrall Chronic Inhaled Ovalbumin Exposure Induces Antigen-Dependent but Not Antigen-Specific Inhalational Tolerance in a Murine Model of Allergic Airway Disease Am. J. Pathol., January 1, 2004; 164(1): 295 - 304. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. A. Shore Modeling Airway Remodeling: The Winner by a Nose? Am. J. Respir. Crit. Care Med., October 15, 2003; 168(8): 910 - 911. [Full Text] [PDF]
|
|
|
|
|
|
K. Shinagawa and M. Kojima Mouse Model of Airway Remodeling: Strain Differences Am. J. Respir. Crit. Care Med., October 15, 2003; 168(8): 959 - 967. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. K. Kumar and P. S. Foster Modeling Allergic Asthma in Mice: Pitfalls and Opportunities Am. J. Respir. Cell Mol. Biol., September 1, 2002; 27(3): 267 - 272. [Abstract] [Full Text]
|
|
|
|
|
|
J. L. Curtis, J. Sonstein, R. A. Craig, J. C. Todt, R. N. Knibbs, T. Polak, D. C. Bullard, and L. M. Stoolman3 Subset-Specific Reductions in Lung Lymphocyte Accumulation Following Intratracheal Antigen Challenge in Endothelial Selectin-Deficient Mice J. Immunol., September 1, 2002; 169(5): 2570 - 2579. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Lahn, A. Kanehiro, K. Takeda, J. Terry, Y.-S. Hahn, M. K. Aydintug, A. Konowal, K. Ikuta, R. L. O'Brien, E. W. Gelfand, et al. MHC class I-dependent Vgamma 4+ pulmonary T cells regulate alpha beta T cell-independent airway responsiveness PNAS, June 25, 2002; 99(13): 8850 - 8855. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. O-Quan Shi, M. J. Fischer, G. T. De Sanctis, M. R. Schuyler, and Y. Tesfaigzi IFN-{gamma}, But Not Fas, Mediates Reduction of Allergen-Induced Mucous Cell Metaplasia by Inducing Apoptosis J. Immunol., May 1, 2002; 168(9): 4764 - 4771. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Wu, L. Puddington, H. E. Whiteley, C. A. Yiamouyiannis, C. M. Schramm, F. Mohammadu, and R. S. Thrall Murine Cytomegalovirus Infection Alters Th1/Th2 Cytokine Expression, Decreases Airway Eosinophilia, and Enhances Mucus Production in Allergic Airway Disease J. Immunol., September 1, 2001; 167(5): 2798 - 2807. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. A. Volgyesi, L. N. Tremblay, P. Webster, N. Zamel, and A. S. Slutsky A new ventilator for monitoring lung mechanics in small animals J Appl Physiol, August 1, 2000; 89(2): 413 - 421. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Todt, J. Sonstein, T. Polak, G. D. Seitzman, B. Hu, and J. L. Curtis Repeated Intratracheal Challenge with Particulate Antigen Modulates Murine Lung Cytokines ,2 J. Immunol., April 15, 2000; 164(8): 4037 - 4047. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) and 10-day OVA
challenged mice (
)
relative to naive control mice
(
). Data represent
mean ± SEM values, with four to seven mice in each group.
0.005;
*P