Bax is Crucial for IFN-γ-Induced Resolution
of Allergen- Induced Mucus Cell Metaplasia
This information is current as
of June 18, 2017.
Yohannes Tesfaigzi, Mark J. Fischer, Massoud Daheshia,
Francis H. Y. Green, George T. De Sanctis and Julie A.
Wilder
J Immunol 2002; 169:5919-5925; ;
doi: 10.4049/jimmunol.169.10.5919
http://www.jimmunol.org/content/169/10/5919
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References
The Journal of Immunology
Bax is Crucial for IFN-␥-Induced Resolution of AllergenInduced Mucus Cell Metaplasia1
Yohannes Tesfaigzi,2* Mark J. Fischer,* Massoud Daheshia,† Francis H. Y. Green,‡
George T. De Sanctis,† and Julie A. Wilder*
I
Received for publication June 19, 2002. Accepted for publication September 6, 2002.
In rats, exposure to LPS or allergen causes MCM in airway
epithelia (6, 7), and 20 –30% of the resulting metaplastic mucus
cells express Bcl-2, an inhibitor of the cell death program (8). The
reduction of Bcl-2 expression precedes the resolution of MCM,
thus indicating an antiapoptotic role for Bcl-2 in this system (8, 9).
Bcl-2 and related cytoplasmic proteins are key regulators of the
cell suicide program (10).
Bcl-2 is the founding member of a family of proteins characterized by at least one of four conserved motifs known as Bcl-2
homology domains BH1 to BH4 (11). This family is subdivided
into the BH4 proteins that contain all four homology regions and
the BH3 domain-only-containing proteins. Bax is a proapoptotic
protein that contains all BH4 domains and can heterodimerize with
Bcl-2 or other antiapoptotic family members to register diverse
forms of intracellular damage, gauge whether a positive or negative death stimulus is present, and determine the progression or
inhibition of the suicide program (11–13).
P-selectin is both necessary and sufficient for the recruitment of
eosinophils, because this protein is critical for the interaction of
leukocytes and endothelial cells (14) before the migration through
the vasculature to the site of inflammation (15). Previous studies
have shown that allergic airway responses are reduced in sensitized and allergen-challenged P-selectin-deficient (⫺/⫺) mice
(16). The purpose of the current study was to investigate whether
reduced inflammation affects the development and resolution of
MCM during prolonged exposure of mice to allergen and whether
regulators of apoptosis may play a role in the resolution process.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
Materials and Methods
1
Animals
nflammatory conditions, injury, or genotoxic stress cause epithelial cells lining the skin, and the reproductive, digestive,
or respiratory tracts to undergo proliferation. During recovery, cell numbers are reduced by regulated cell death processes (1,
2). Disruption of these homeostatic mechanisms can lead to increased residual cells in epithelia, abnormal proportions of cell
types that constitute the epithelia, or precancerous lesions. Despite
the importance of these regulatory processes, the molecular mechanisms underlying apoptosis that maintain constant numbers of
epithelial cells in airways have been poorly studied.
In airway epithelia, pre-existing and proliferating epithelial cells
can differentiate into mucin-producing cells and establish mucus
cell metaplasia (MCM)3 following inflammatory responses (3).
MCM resolves during the recovery process, and the original proportions of cell types in the airway epithelium are restored (3).
Resolution of MCM involves down-regulation of mucin biosynthesis and reduction of cell numbers (4). Therefore, disruption of
the mechanisms involved in the reduction of mucus cells may at
least in part be responsible for the pathological conditions in
asthma and chronic bronchitis, in which MCM persists and contributes to increased mucus secretions and airway obstruction (5).
*Lovelace Respiratory Research Institute, Albuquerque, NM 87108; †Respiratory Research Group, University of Calgary, Calgary, Alberta, Canada; and ‡Aventis Pharmaceutical, Bridgewater, NH 08870
This study was supported by National Institutes of Health Grant ES09237.
2
Address correspondence and reprint requests to Dr. Yohannes Tesfaigzi, Lovelace
Respiratory Research Institute, 2425 Ridgecrest Drive, SE, Albuquerque, NM 87108.
E-mail address: [email protected]
3
Abbreviations used in this paper: MCM, mucus cell metaplasia; AB, Alcian blue;
BALF, bronchoalveolar lavage fluid; DAB, diaminobenzidine; PAS, periodic
acid-Schiff.
Copyright © 2002 by The American Association of Immunologists, Inc.
Male pathogen-free, wild-type (⫹/⫹) C57BL/6J mice and P-selectin-deficient (⫺/⫺) mice on C57BL/6 background were purchased from The Jackson Laboratory (Bar Harbor, ME). Dr. S. J. Korsmeyer (Dana-Farber Institute, Boston, MA) provided Bax ⫹/⫺ C57BL/6 mice, and we bred and
genotyped by PCR the Bax ⫺/⫺ and ⫹/⫹ littermates, as described previously (17). Mice, 6 – 8 wk of age, were housed in isolated cages under
0022-1767/02/$02.00
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Allergic airway responses cause proliferation of epithelial cells and mucus cell metaplasia (MCM), and the resolution of MCM
involves reduction of cell numbers. The role of inflammation and apoptosis on this process was investigated in P-selectin ⴙ/ⴙ and
ⴚ/ⴚ mice sensitized and challenged with OVA by analyzing the expression and the role of regulators of apoptosis in metaplastic
mucus cells. No differences were observed in MCM at 5 days of allergen exposure between ⴙ/ⴙ and ⴚ/ⴚ mice, despite reduced
IL-13 levels in ⴚ/ⴚ mice. Although IL-4 levels were similar in both ⴚ/ⴚ and ⴙ/ⴙ mice, IL-13 and IL-5 levels had decreased and
IFN-␥ levels were increased earlier in ⴚ/ⴚ compared with ⴙ/ⴙ mice. MCM levels were decreased 4-fold at 7 days of allergen
exposure in ⴚ/ⴚ mice and at 15 days in ⴙ/ⴙ mice. The percentage of Bax-expressing mucus cells increased significantly at 7 days
in ⴚ/ⴚ mice and at 10 days in ⴙ/ⴙ mice. The Bax-positive mucus cells exhibited caspase-specific cleavage of cytokeratin 18. IFN-␥
caused Bax expression in IL-13-induced MCM in microdissected airway cultures. MCM remained significantly elevated in Bax
ⴚ/ⴚ mice following 15 days of allergen exposure compared with ⴙ/ⴙ mice, while the number of eosinophils was reduced in both
Bax ⴙ/ⴙ and ⴚ/ⴚ mice at 15 days. Together, these data demonstrate that reduced IL-13 levels were sufficient to elicit maximum
MCM, that IFN-␥ induces Bax in metaplastic mucus cells, and that Bax plays a critical role in the resolution of MCM, but not
in the resolution of eosinophils. The Journal of Immunology, 2002, 169: 5919 –5925.
5920
specific pathogen-free conditions. Mice were acclimatized for 12 days after
arrival, then entered into the experimental protocol at 8 –10 wk of age.
Mice were sensitized by i.p. injection with 1 ␮g OVA/100 ␮g Al(OH)3
(grade III; Sigma-Aldrich, St. Louis, MO) in 0.5 ml of saline. A booster
injection was given on day 7, using the identical reagents. Seven days later,
mice were exposed 5 h/day to OVA aerosol at a concentration of 2.3 mg/
m3. Mice were exposed to allergen for 5, 7, 10, or 15 consecutive days and
euthanized immediately after the end of the last exposure.
Histological evaluation
Quantification of macrophages, eosinophils, lymphocytes, and
neutrophils
The cells recovered by lavage were enumerated using a hemacytometer.
Cytological preparations were made and stained with Wright Giemsa stain
(American Scientific Products, McGraw Park, IL) to determine the different types of cells present in the BALF. At least 400 cells were counted from
each slide, and the distribution of macrophages, neutrophils, eosinophils,
and lymphocytes was determined.
Cytokine detection
Lungs of mice were lavaged three times with 0.5 ml of PBS via the tracheal
tube, and BALF was stored at ⫺80°C until use. IL-4, IL-5, IL-13, and
IFN-␥ were measured using Quantikine M Murine (R&D Systems, Minneapolis, MN). A total of 35 ␮l of BALF was added to each anticytokinecoated plate and incubated at 4°C overnight. The cytokine concentrations
in the BALF were determined by standard ELISA techniques, as described
in the manufacturer’s manual. Detection limits were 3.9 pg/ml for IL-4 and
IL-13, 4.7 pg/ml for IFN-␥, and 7.8 pg/ml for IL-5.
Immunohistochemistry
Endogenous peroxidase activity was blocked by incubating sections in 2%
hydrogen peroxide/methanol for 1 min. Slides were washed in deionized
water, and all subsequent washes consisted of 0.05% Brij/Dulbecco’s PBS
(pH 7.4). Proteins were unmasked by incubating tissue sections with a
trypsin solution (Zymed, San Francisco, CA) at 37°C for 10 min, as described by the manufacturer. Slides were first incubated for blocking in 1%
normal goat serum in 2% BSA/0.1% Triton X-100 before the primary Ab
(polyclonal rabbit anti-mouse Bax, Bcl-2, Bcl-x, Bak, or normal rabbit
serum; BD PharMingen, San Diego, CA; at 1/500 dilution) was applied.
After an overnight incubation at room temperature, the immunoreaction
was visualized using biotinylated goat anti-rabbit Ab, VECTASTAIN ABC
reagent, and the peroxidase substrate diaminobenzidine (DAB; Vector Laboratories, Burlingame, CA), as described by the manufacturer. The following procedures verified the specificity of the Bax Ab reaction: the immunohistochemical reactions using rabbit nonimmune IgG showed no
staining; the reaction of the Bax Ab was inhibited when the antigenic
peptide (19) was added to the reaction. Positive immunoreaction of Abs to
Bcl-2, Bcl-x, and Bak was tested on lung tissues from mice and rats instilled with LPS. Epithelial cells with mucosubstances were detected by
counterstaining tissue sections with AB (20). The percentage of Bax-positive mucus cells was determined by counting all AB-positive cells
throughout the airway epithelia of the lung sections (from 400 to 700
AB-positive cells/mouse).
Detection of caspase cleavage product of cytokeratin 18
Caulin et al. (21) showed that activation of caspases in epithelial cells leads
to cleavage of cytokeratins, in particular cytokeratin 18. Cytokeratins 8 and
18 are major intermediate filaments of single-layer epithelial tissues (22),
so we investigated their cleavage in mucus cells. M30CytoDEATH (Roche
Diagnostic, Indianapolis, IN) recognizes a specific caspase cleavage site
within cytokeratin 18 not detectable in native cytokeratin 18 of normal
cells (23), and was used essentially as described by the manufacturer, except for the steps described below. The immunoreaction of the Ab was
detected using 1.5 mg/ml (1/200 dilution) of the anti-mouse Ig biotin,
VECTASTAIN ABC reagent, and DAB (Vector Laboratories, Inc.). The
reaction was terminated by extensive rinsing in water, and mucus cells
were visualized by staining with AB. Finally, the sections were counterstained with hematoxylin and mounted with Permount (Sigma-Aldrich).
Immunoblot detection of proteins
Protein extracts were prepared from the entire right lung of mice by homogenizing in radioimmunoprecipitation assay buffer (10 mM Tris, pH
7.4, 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 5 mM
EDTA) supplemented with the protease inhibitors PMSF (1 mM), pepstatin
(10 ␮g/ml), aprotinin (2 ␮g/ml), and benzamidine (2 ␮g/ml), and analyzed
by Western blotting, as described (8). The Bax Ab was either at 1/3000
dilution (Upstate Biotechnology, Lake Placid, NY) or 1/500 dilution (BD
PharMingen).
Microdissected airway cultures
Distal airway bronchioles were removed by microdissection, essentially as
described (24). After removal from mice, lungs were inflated with 1%
SeaPlaque low-melting-temperature agarose (FMC Bioproducts, Rockland,
ME) in 2⫻ DMEM (Life Technologies, Grand Island, NY) and immersed
in ice-cold F12 medium. The lung lobes were isolated, and distal airway
branches were dissected starting from the branch point. Up to 15 bronchioles were obtained from each mouse, and 3 bronchioles were used for each
treatment. Bronchioles from at least three mice were prepared for each
treatment to normalize for mouse-to-mouse variability. Distal airways (3–
5/well) were transferred to 0.4-␮m-pore-size Transwell (Costar, Pleasanton, CA) mesh inserts in 12-well plates. A total of 400 ␮l of medium was
added to the bottom of the Transwell system and 60 ␮l to the top chamber
with the bronchioles. They were placed in the center of the mesh inserts,
with the long axis of the cylindrical bronchiole fully extended in the plane
of the insert membrane. Culture medium, previously described (24), was
changed 24 h after placing the organ cultures and every other day thereafter. Bronchioles were kept in culture at the air-liquid interface for 7 days,
and were treated with nothing as control. IL-13 (20 ng/ml) or IFN-␥ (50
ng/ml) was added to the IL-13 treatment for the last 2 days in culture.
Experiments with these treatments were repeated at least three times. The
volume density of mucosubstances was quantified in these tissues in a
blinded fashion. Explants were fixed in zinc Formalin for 1–3 days and
pre-embedded in 1% agarose before processing for paraffin embedding.
Tissue sections (5 mm) were then prepared and subjected to AB/periodic
acid-Schiff (PAS)-staining or immunostaining procedures. Mice that
showed mucus cells in control medium without IL-13 were excluded from
analyses.
Statistical analyses
Grouped results from at least four different mice were expressed as mean ⫾
SEM, and differences between groups were assessed for significance by
Student’s t test, when data were available in only two groups. When data
were available in more than two groups, ANOVA was used to perform
pairwise comparisons, and Fisher’s least significant difference test was
used to determine differences between groups. A p value of ⬍0.05 was
considered to indicate statistical significance.
Results
Reduced inflammation and earlier immune deviation in
P-selectin ⫺/⫺ mice
Overall, P-selectin ⫺/⫺ mice had fewer lymphocytes (Fig. 1A)
and eosinophils (Fig. 1B) in their BALF after 10 days of repeated
allergen exposure. Although the number of lymphocytes was similar in both ⫹/⫹ and ⫺/⫺ mice after 5 days of exposure, the
number of lymphocytes increased significantly by ⬃2-fold in ⫹/⫹
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Mice were euthanized by i.p. injection of 15 mg sodium pentobarbital
(Abbott Laboratories, Chicago, IL) and exsanguinated via the renal artery.
The thoracic contents were exposed, and the lungs were perfused by cardiac puncture with 0.9% saline (w/v; McGaw, Irvine, CA). The trachea was
cannulated with a 23-gauge blunt needle tipped with surgical tubing, the
lungs were lavaged three times with 0.5 ml ice-cold PBS containing 1%
FBS, and the bronchoalveolar lavage fluid (BALF) was collected. For histopathological examinations, lungs from another set of mice were inflated
with 10% zinc Formalin (Stephens Scientific, Riverdale, NJ) at a constant
pressure of 25 cm for 3 h and immersed in a large volume of the same
fixative for 2–3 days, as described previously (18).
A stratified, random-sampling scheme was used to cut the fixed lung
lobes into cross-sectional slices, each ⬃0.3 cm thick. Depending on the
size of the lung, five or six slices were prepared, embedded in paraffin, and
sectioned at 5 ␮m thickness. Sections were used for Alcian blue (AB) or
immunohistochemical staining. The number of AB-positive cells/mm basal
lamina was quantified using a Zeiss microscope equipped with the morphometry software system from Intelligent Imaging (Denver, CO).
Tissue sections were stained with H&E and scored for eosinophils in the
periarterial, perivenular, and peribronchial regions. The scores for the three
regions were summed to show the total lung score. The scores of each lung
slice were averaged to produce one score for each lung. An immunologist
who was blinded to their identity scored each slide by grading from 0 (0%
area involved) to 4 (⬎50% are involved).
Bax IN ALLERGEN-INDUCED MUCUS CELLS
The Journal of Immunology
5921
mice, while it remained unchanged in ⫺/⫺ mice at 10 days of
allergen exposure (Fig. 1A). The number of eosinphils was ⬃2fold higher in ⫹/⫹ mice compared with ⫺/⫺ mice at both 5 and
10 days of allergen exposure (Fig. 1B). No differences were observed in the number of macrophages and neutrophils between
⫹/⫹ and ⫺/⫺ mice at these time points (data not shown).
IL-4 levels in the BALF were similar in ⫹/⫹ and ⫺/⫺ mice,
and did not decrease significantly over the 10-day exposure period
(Fig. 2A). IL-5 levels were decreased to almost undetectable levels
after 7 and 10 days in ⫺/⫺ mice, while their levels were still
elevated after 7 days in ⫹/⫹ mice and decreased to background
levels after 15 days (Fig. 2B). IL-13 levels were ⬃4-fold higher in
⫹/⫹ compared with ⫺/⫺ mice at 5 and 7 days of exposure and
decreased to background levels in both groups at 15 days (Fig.
2C). IFN-␥ was only detected in ⫺/⫺ mice at 5 days of exposure.
In ⫹/⫹ mice, IFN-␥ levels were at background levels at 5 and 7
days, but were significantly increased at 10 days of allergen exposure compared with ⫺/⫺ mice (Fig. 2D).
FIGURE 2. The levels of IL-4 (A), IL-5 (B), IL-13 (C), and IFN-␥ (D)
in BALF of P-selectin ⫹/⫹ and ⫺/⫺ mice following 5, 7, and 10 days of
repeated exposure to allergen. Bars ⫽ group means ⫾ SEM (n ⫽ 3– 4
mice/group). ⴱ, Significantly different from 5 days.
days of allergen exposure (Fig. 4A). We demonstrated immunospecificity for the Bax protein by inhibition with the antigenic
peptide (Fig. 4, B and C). Furthermore, Abs raised to two different
MCM and Bax expression
Despite reduced inflammatory cells in ⫺/⫺ mice, the number of
mucus cells/mm basal lamina was increased from essentially 0 in
nonexposed mice to similarly high levels in both ⫹/⫹ and ⫺/⫺
mice after 5 days of allergen exposure (Fig. 3). However, while
MCM levels remained high in ⫹/⫹ mice after 7 and 10 days of
exposure, MCM was significantly decreased in P-selectin ⫺/⫺
mice at 7 and 10 days of allergen exposure (Fig. 3). In ⫹/⫹ mice,
MCM did not decline until 15 days of allergen exposure.
During this 15-day exposure period, the percentage of Bax-positive mucus cells increased from ⬃2 to 21% in ⫹/⫹ mice and to
⬃34% in ⫺/⫺ mice (Fig. 4A). A higher percentage of mucus cells
was immunopositive for Bax in P-selectin ⫺/⫺ compared with
⫹/⫹ mice at 7 days of exposure; the difference between P-selectin
⫺/⫺ and ⫹/⫹ mice was statistically not significant at 10 and 15
FIGURE 3. Continuous exposure to allergen causes MCM to decrease
at 7 days in P-selectin ⫺/⫺ mice and at 15 days in ⫹/⫹ mice. The left
lungs were inflated under constant pressure and processed for histochemical staining with AB/PAS, and the number of mucus cells/mm basal lamina was quantified. Bars ⫽ group means ⫾ SEM (n ⫽ 3– 4 mice/group). ⴱ,
Significantly different from 5 days.
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FIGURE 1. Continuous exposure of sensitized mice to allergen for 5 and
10 days causes less eosinophilic and lymphocytic infiltration of the lungs in
P-selectin ⫺/⫺ compared with ⫹/⫹ mice. Mice were immunized with OVA
and challenged with OVA aerosols for 5 or 10 days, and the leukocytes in the
BALF were determined. The number of lymphocytes (A) and eosinophils (B)
in the BALF of P-selectin ⫹/⫹ and ⫺/⫺ mice was quantified following an
exposure to allergen for 5 and 10 days. Bars ⫽ group means ⫾ SEM (n ⫽ 3– 4
mice/group). ⴱ, Significantly different from 5 days (for lymphocytes) and significantly different from knockout (for eosinophils).
5922
Bax IN ALLERGEN-INDUCED MUCUS CELLS
epitopes of the Bax peptide (aa 1–24 and 43– 61) showed similar
immunoreaction in mucus cells (data not shown). Bcl-2, Bcl-x, and
Bak were not detected.
M30CytoDEATH recognizes a specific caspase cleavage site
within cytokeratin 18 not detectable in native cytokeratin 18 of
normal cells (23). This Ab specifically immunostained the mucus
FIGURE 5. IL-13 induced MCM in microdissected airway explant cultures, and the addition of IFN-␥ causes Bax
expression in these mucus cells. Microdissected airway
bronchioles were placed in culture in an air-liquid interface
and treated with IL-13 or vehicle for 7 days; IFN-␥ was
added to the IL-13-treated explant cultures for the last 2
days. A, Tissues were processed for AB/PAS staining, and
mucus cells/mm basal lamina were quantified by morphometry. Bars ⫽ group means ⫾ SEM (n ⫽ 6 – 8 mice/
group). ⴱ, Significantly different from untreated controls.
Tissue sections from distal airway cultures were also immunostained with Bax Abs, as described in Materials and
Methods. Immunoreaction for Bax was not detected in explant cultures treated with IL-13 only (B), but was only
detected in those treated with IL-13 plus IFN-␥ (C).
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FIGURE 4. A continuous 15-day exposure to allergen
causes the percentages of Bax-positive mucus cells to increase (A). Tissue sections from the left lung lobe from
mice sensitized and challenged with OVA aerosols for 5,
7, 10, and 15 days were immunostained for Bax and AB to
detect the mucus cells, and the percentage of Bax-positive
mucus cells was quantified. Bars ⫽ group means ⫾ SEM
(n ⫽ 3– 4/group). ⴱ, Significantly different from 5 days.
Immunoreaction of the Bax Abs was detected with DAB
and shows brown (B). Tissue sections were also stained
with AB to identify mucus secretory cells. Bax immunostaining (B) could be inhibited (C) when the Abs were
preincubated with the antigenic peptide (⫻420). Detection
of cleaved cytokeratin 18 at the caspase-recognition site in
Bax-positive mucus cells. Two serial tissue sections were
immunostained with Bax Abs (D) and M30CytoDEATH
(E), as described in Materials and Methods. Many of the
mucus cells expressing Bax showed the presence of cytokeratin 18 cleaved at the caspase recognition sequence,
DEVD (⫻420). Photomicrographs are from tissue sections
from P-selectin ⫺/⫺ mice exposed to allergen for 7 days.
The Journal of Immunology
cells expressing Bax in serial tissue sections (Fig. 4, D and E).
Bax-negative mucus cells showed no immunostaining with
M30CytoDEATH (data not shown). These observations suggested
that overexpression of Bax in mucus cells was involved in the
apoptotic process through activation of caspases.
IFN-␥ reduces MCM and induces Bax expression in
microdissected airway cultures
We had previously shown that IFN-␥ is essential for the resolution
of MCM during prolonged exposure to allergen (4). To confirm
that IFN-␥ acts directly on airway epithelial cells through a Baxmediated pathway, microdissected distal airways were treated for
7 days with IL-13 only, or IFN-␥ was added for the last 2 days.
The number of mucus cells (Fig. 5A) and the volume density of
mucosubstances (data not shown) per mm basal lamina were increased significantly when IL-13 was present, but not when IFN-␥
was added for the last 2 days of culture (Fig. 5A). Furthermore, im-
5923
munostaining for Bax was only detected in explants treated with
IFN-␥, but not in explants treated only with IL-13 (Fig. 5, B and C).
Bax is essential for resolution of MCM, but not for eosinophilia
Discussion
FIGURE 6. A, Western blot analysis of proteins extracted from lungs of
Bax ⫺/⫺ and ⫹/⫹ mice sensitized to OVA/Alum at 1 and 7 days and
exposed to OVA aerosols for 15 days, as described in Materials and Methods. The 21-kDa Bax protein was detected in Bax ⫹/⫹, but not in ⫺/⫺
mice. Equal loading is shown by immunoreaction with the actin Ab, which
appears in all lanes. B, After repeated exposure to allergen for 15 days, Bax
⫺/⫺ showed increased numbers of metaplastic mucus cells compared with
⫹/⫹ mice. In this experiment, mice were 5 wk of age, which may explain
the overall lower numbers of mucus cells. Mice were sacrificed, and lungs
were processed for AB/PAS staining and morphometry. Bars ⫽ group
means ⫾ SEM (n ⫽ 5/group). ⴱ, Significantly different from wild-type
controls. C, Both Bax ⫹/⫹ and ⫺/⫺ mice showed extensive eosinophils in
the lung at 5 days, but not 15 days postexposure.
The increase of mucus-producing cells during exposure to allergen
involves epithelial cell proliferation (25), and the resolution of
MCM involves reduction of mucus cell numbers (4). Our previous
study (4) shows that IFN-␥ is increased in the BALF of mice
during prolonged exposure to allergen and mediates cell death in
metaplastic mucus cells. The present study demonstrates that the
proapoptotic regulator Bax is an essential component of this resolution process.
The presence of IL-4 in both P-selectin ⫹/⫹ and ⫺/⫺ mice at
5, 10, and 15 days of allergen exposure shows that the production
of this Th2 cytokine was not inhibited in these mice even after the
coincident appearance of the Th1 cytokine, IFN-␥. However, IL-5
and IL-13 were reduced in ⫺/⫺ mice as early as 7 days of allergen
exposure, while the levels of these cytokines had decreased to
background levels in ⫹/⫹ mice only after 10 days of exposure.
Whereas P-selectin ⫺/⫺ mice had low IL-13 and detectable IFN-␥
levels at 5 days of exposure, it took 10 days of exposure to increase
IFN-␥ and decrease IL-13 to undetectable levels in ⫹/⫹ mice.
Thus, the levels of IL-5 and IFN-␥ were kinetically altered in
P-selectin ⫺/⫺ mice over the exposure period of 10 days. These
data suggest that synthesis of IL-13 and IFN-␥ is inversely correlated; however, this inverse correlation was not observed between
IL-4 and IFN-␥.
The simultaneous presence of IL-4, a cytokine produced by Th2
cells, and IFN-␥, a cytokine produced by Th1 cells, suggests that
Th2 cells were not eliminated when IFN-␥-producing Th1 cells
were present. Low IL-13 levels as a result of P-selectin deficiency
may have been responsible for the earlier increase of IFN-␥ levels.
The presence of IFN-␥ at 5 days, but not at later time points in
P-selectin ⫺/⫺ mice suggests that only a transient increase in
IFN-␥ is sufficient to reduce MCM.
No differences were observed in MCM at 5 days of allergen
exposure in P-selectin ⫹/⫹ and ⫺/⫺ mice despite reduced inflammatory cell numbers and IL-13 levels in ⫺/⫺ compared with ⫹/⫹
mice. These data suggest that maximum levels of MCM were
reached by the lower levels of IL-13 in ⫺/⫺ mice, and increased
IL-13 levels in ⫹/⫹ mice did not further increase MCM. Our
previous studies show that instillation of IL-13, which induces
MCM by directly affecting airway epithelial cells (26 –28), into
mice that were exposed to allergen for 5 days does not further
increase MCM (4). These observations suggest that 60 – 80 mucus
cells/mm basal lamina is the maximum number of mucus cells that
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To test the hypothesis that Bax is important for the decrease of
MCM during prolonged and repeated allergen challenge, we immunized and challenged Bax ⫹/⫹ and ⫺/⫺ mice with OVA. Expression of Bax in ⫹/⫹ and ⫺/⫺ mouse lungs was assessed by
Western blotting. The 21-kDa Bax was absent from the Bax ⫺/⫺
mouse lungs, but was detected in ⫹/⫹ mice and further confirmed
the specificity of the Ab (Fig. 6A). Although nonchallenged Bax
⫹/⫹ and ⫺/⫺ mice had little mucus in airway epithelia, MCM
was increased to similar levels in both ⫹/⫹ and ⫺/⫺ mice following 5 days of allergen exposure (data not shown). However,
after 15 days, Bax ⫺/⫺ mice still had elevated MCM compared
with ⫹/⫹ mice (Fig. 6B). Furthermore, eosinophils were present in
both Bax ⫹/⫹ and ⫺/⫺ mice at 5 days of allergen challenge, but
were essentially absent in lung tissues of both ⫹/⫹ and ⫺/⫺ mice
at 15 days of allergen exposure, indicating that Bax was not essential for the elimination of eosinophils (Fig. 6C).
5924
whether pre-existing cells also undergo apoptosis during this resolution process.
The lungs of both Bax ⫹/⫹ and ⫺/⫺ mice were cleared of
eosinophils following exposure to allergen for 15 days, indicating
that Bax was not essential for reducing eosinophil numbers. This
finding agrees with previous reports that eosinophils are cleared by
apoptosis through the Fas/Fas ligand-dependent pathway (30).
Ochiai et al. (31) have presented evidence in vitro that IL-5 inhibits
eosinophil apoptosis by up-regulating Bcl-2 expression.
In summary, our results demonstrate that a reduced inflammatory response is sufficient to elicit maximum MCM; immune deviation occurs earlier in P-selectin ⫺/⫺ mice; and Bax is crucial
for the resulting IFN-␥-induced decrease of MCM by causing apoptosis in metaplastic mucus cells.
Acknowledgments
We thank Yoneko Knighton (Lovelace Respiratory Research Institute) for
the preparation of tissue samples. Lovelace Respiratory Research Institute
is fully accredited by the Association for the Assessment and Accreditation
of Laboratory Animal Care International.
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inflammatory responses are sufficient to cause maximum MCM.
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Mucus cells from P-selectin ⫹/⫹ and ⫺/⫺ mice that were exposed to allergen under a different protocol (16) showed Bax expression at percentages similar to those reported in this study,
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The difference in the percentage of Bax-positive mucus cells
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Bax IN ALLERGEN-INDUCED MUCUS CELLS
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