Asthma and Pulmonary Immunology Program, Lovelace Respiratory Research Institute, Albuquerque, New Mexico 87185
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ABSTRACT |
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Clara cell secretory protein (CCSP)
is synthesized by nonciliated bronchiolar cells in the lung and
modulates lung inflammation to infection. To determine the role of CCSP
in the host response to allergic airway disease, CCSP-deficient
[(/
)] mice were immunized twice with ovalbumin (Ova) and
challenged by Ova (2 or 5 mg/m3) aerosol. After 2, 3, and 5 days of Ova aerosol challenge (6 h/day), airway reactivity was
increased in CCSP(
/
) mice compared with wild-type [CCSP(+/+)]
mice. Neutrophils were markedly increased in the bronchoalveolar lavage
fluid of CCSP(
/
) Ova mice, coinciding with increased
myeloperoxidase activity and macrophage inflammatory protein-2 levels.
Lung histopathology and inflammation were increased in CCSP(
/
)
compared with wild-type mice after Ova challenge. Mucus production, as
assessed by histological staining, was increased in the airway
epithelium of CCSP(
/
) Ova mice compared with that in CCSP(+/+) Ova
mice. These data suggest a role for CCSP in airway reactivity and the
host response to allergic airway inflammation and provide further
evidence for the role of the airway epithelium in regulating airway
responses in allergic disease.
Clara cell secretory protein; lung epithelium; asthma; ovalbumin
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INTRODUCTION |
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CLARA CELL SECRETORY
PROTEIN (CCSP; also called CC10, CC16, or uteroglobin) is
expressed in and secreted from nonciliated bronchial epithelial cells
(Clara cells) of the conducting airways and distal bronchioles
(26, 27). CCSP mRNA is also expressed to a lesser extent
in the prostate, thyroid, mammary, and pituitary glands and in the
uterus during pregnancy (18, 19). In the lungs, CCSP is
one of the most abundant proteins in the extracellular lining fluid of
the airways (27). Despite its abundance in the airways of
mammals, the physiological function of CCSP has not been elucidated. In
vivo, CCSP gene-targeted [(/
)] mice have increased inflammatory
responses to hyperoxia and ozone exposure (12, 15).
Likewise, lung inflammation and neutrophil migration are increased in
CCSP(
/
) mice after acute viral and bacterial infection (6, 7,
10). Taken together, studies in CCSP-deficient mice strongly
suggest that CCSP modulates inflammatory responses after lung injury or infection.
The role of the Clara cell and its secretory products in allergic airway disease and asthma is unclear. A recent study (24) indicated that CCSP-positive epithelial cells were decreased in the small airways in patients with asthma. Likewise, CCSP is decreased in the bronchoalveolar lavage fluid (BALF) (32) and serum of patients with asthma (25, 33). Interestingly, a polymorphism in the CCSP gene is associated with reduced plasma CCSP levels and increased asthmatic episodes in patients with asthma (13, 14). In addition, production of inflammatory mediators by Clara cells in transgenic models is sufficient to induce pulmonary inflammation and lung responses characteristic of asthma (20, 30, 36). These studies suggest that Clara cells and CCSP may play an important role in the pathophysiology of asthma.
The present study examines the role of CCSP in the development of
airway hyperreactivity (AHR) and airway inflammation in a mouse model
of allergic airway disease. CCSP(/
) and wild-type (WT) mice were
preimmunized with ovalbumin (Ova) twice and subsequently challenged
with aerosolized Ova by inhalation. CCSP deficiency was associated with
increased AHR, increased neutrophil migration into the lungs, and
increased levels of Alcian blue (AB)-positive mucosubstances. These
findings suggest that CCSP modulates the host response in the lungs of
Ova-challenged mice in vivo and provide further evidence for the role
of the lung epithelium per se in allergic airway inflammation and reactivity.
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MATERIALS AND METHODS |
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Mice.
Female CCSP(/
) mice (129J Ola/129J hybrid; kindly provided by Dr.
Jeffrey A. Whitsett, Children's Hospital, Cincinnati, OH) and WT
(129J; Jackson Laboratory, Bar Harbor, ME) mice were housed under
pathogen-free conditions in the Lovelace Respiratory Research Institute
vivarium following Association for the Assessment and Accreditation of
Laboratory Animal Care international guidelines.
Ova-challenge mouse model. Ova exposure of mice was modified according to a protocol described previously (32, 35). Briefly, the mice were immunized twice by an intraperitoneal injection of heat-aggregated Ova (10 µg chicken egg, grade V, Sigma, St. Louis, MO) and 2 mg of alum in a total volume of 0.5 ml in endotoxin-free water on days 1 and 8. Preliminary studies indicated that Ova aerosol exposure (5 mg/m3, 6 h/day) for 5 days induced significant airway inflammation in Ova-immunized mice. Therefore, the Ova-immunized mice were then challenged with Ova aerosol (2 or 5 mg/m3, 6 h/day) for 2, 3, or 5 days in whole body exposure chambers (H1000 or H2000; Hazleton Systems, Aberdeen, MD) beginning on day 15. Food and water were available ad libitum.
Measurement of airway reactivity. Airway reactivity (AR) was measured with a whole body plethysmograph (Buxco Electronics, Sharon, CT). As previously described (23), AR has been expressed as enhanced pause, a calculated value showing a strong correlation with airway resistance measured with standard procedures (4). Individual mice were placed in parallel chambers connected to a Hudson nebulizer (Micro Mist, Hudson Respiratory Care, Temecula, CA) and a recording system. The nebulizer was driven by stable airflow at a level of 4.42 l/min. The baseline AR of the mice was recorded for 5 min. Subsequently, the mice were challenged for 1 min to nebulized saline and increasing concentrations (6, 12, 25, and 50 mg/ml) of nebulized methacholine (MCh; ICN Biomedicals, Aurora, OH). After each nebulization, recordings were taken for 10 min. The enhanced pause values measured during the first 5 min were averaged and expressed as means ± SE (n = 10 mice/group).
Cell counts and cytology in BALF. BALF (n = 10 mice/group) was obtained by three serial intratracheal instillations of 1 ml of PBS into the lung, and the samples were pooled for each animal. Cells in the BALF were isolated by centrifugation at 1,500 rpm and resuspended in 200 µl of PBS. Viable cells were counted by hemacytometer in a 1:1 solution of BALF suspension to 0.4% trypan blue (GIBCO BRL, Grand Island, NY). To determine inflammatory cell types in BALF, 5 × 104 cells were mounted on slides by cytospin centrifugation (400 rpm for 4 min) in 100 µl of PBS. Lung cytology was identified and counted by differential staining microscopy with a Hema-Tek stain pack, a modified Wright-Giemsa stain (Bayer, Elkhart, IN). Inflammatory cell populations were determined by counting 100 cells, and a percentage was calculated based on two sample sets from 10 animals/group. The absolute cell numbers for each cell type were calculated according to the total leukocyte counts in BALF and the percentage of each cell type for every sample.
Pulmonary histopathology. Lung inflammation was assessed histopathologically as described (6). Blood was collected by right ventricular puncture. Left lungs were inflated via a tracheal cannula at 20 cmH2O of pressure with 4% paraformaldehyde and removed en bloc from the thorax. Inflation-fixed lungs were washed in PBS three times and bisected transversely in the dorsoventral direction, just caudal to the entry of the mainstream bronchus, for paraffin embedding. Paraffin-embedded lungs were sectioned at 5 µm and stained with hematoxylin and eosin for histological analysis. For each animal, slides of the left lung, with two sections at 100-µm intervals, were graded on a scale of minimal, mild, moderate, and marked, corresponding to numbers 1 (minimal) to 4 (marked) for the following criteria: septal infiltrates, perivascular infiltrates, peribronchiolar infiltrates, and epithelial cell hyperplasia/hypertrophy. The score for each animal is the average score of the above four criteria. All slides were scored blindly by a trained pathologist, and a score was determined for the mean (±SE) of 6-8 animals/group.
Macrophage inflammatory protein-2 production. The apical and intermediate lobes of the right lungs (6-8 mice/group) were homogenized in 1 ml of PBS and centrifuged at 2,000 rpm for 7 min at 4°C. The supernatants were collected for macrophage inflammatory protein (MIP)-2 production analysis with ELISA by volume of homogenate supernatants according to the manufacturer's recommendations (R&D Systems, Minneapolis, MN). Standard curves were calculated for known standards to verify linearity of analysis and for calculation of MIP-2 concentration.
Myeloperoxidase activity. Myeloperoxidase (MPO) activity in the lung homogenates (6-8 mice/group) and BALF was determined as described (28). Briefly, the lung homogenate supernatants, original BALF, and BALF concentrated 10-fold were mixed with reagent 1 (including acetate buffer, hexadecyltrimethylammonium bromide, and EDTA). Reagent 2 (tetramethylbenzidine and H2O2) was added to the wells, and the absorbance at 650 nm was measured over 4 min. MPO activity was calculated and expressed as the rate of MPO activity per lung lobe.
Mucous cell staining.
Lung sections (5 µm) of the left lung lobe (6-8 mice/group)
100-200 µm from the reference point were stained by AB-periodic acid-Schiff (PAS) stain to identify mucus-secreting cells as described (31). Briefly, the lung sections were deparaffinized in
xylene and hydrated in decreasing concentrations of ethanol. The slides were then stained in AB for 30 min, washed in running water for 5 min,
oxidated in 1% periodic acid for 10 min, and washed in running water
for another 5 min. After staining in Schiff's reagent for 10 min,
slides were rinsed three times in sodium metabisulfite, washed in
running water for 10 min, and after dehydration, were mounted in
ethanol and xylene. Photomicrographs (×415) showed the mucus-producing
cells with the distinctive colors for PAS positivity (pink) and AB-PAS
positivity (purple). In all mice, only the mucous cells in the
epithelia lining the main airway were analyzed. The length of the basal
lamina lining the large airways of WT and CCSP(/
) mice and the
volume density of the AB-PAS-stained mucosubstances in the surface
epithelium were determined with an Everest microscope system equipped
with slidebook software (Intelligent Imaging Innovations, Denver, CO)
as described previously (5).
Statistical analysis. Statistical significance between groups was determined by t-test for parametric data or Mann-Whitney test for nonparametric data. Statistical analysis for multiple groups was determined by ANOVA. All data are presented as means ± SE. Differences were considered significant at P < 0.05.
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RESULTS |
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AR is increased in CCSP(/
) mice
after Ova exposure.
To assess the role of CCSP in modulating the development of AHR induced
by Ova, CCSP(
/
) and WT mice (8-9 wk of age at the time of AR
measurement) were immunized twice by a weekly intraperitoneal injection
of Ova-alum. On day 15, the mice were challenged with Ova
aerosol (2 or 5 mg/m3) for 2, 3, or 5 days (6 h/day). The
WT air control mice responded very poorly to all doses of MCh (Fig.
1). The AR in CCSP(
/
) air control
mice was not different from that in WT air control mice at all doses of
MCh challenge. The AR in WT mice challenged with 25 mg/ml of MCh was
only increased after 3 days of Ova exposure at 2 mg/m3 and
increased at 50 mg/ml of MCh challenge after 2 or 3 days of Ova
exposure at 2 mg/m3 compared with that in WT air control
mice (Fig. 1). In contrast, the AR of CCSP(
/
) mice challenged at
both 25 and 50 mg/ml of MCh was increased after 2, 3, and 5 days of Ova
exposure at 2 mg/m3 compared with that of WT mice exposed
to Ova at 2 mg/m3 for 2, 3, and 5 days, respectively. The
AR of both WT and CCSP(
/
) mice exposed to 5 mg/m3 of
Ova (data not shown) was similar to that of mice exposed to 2 mg/m3 of Ova. Overall, the AR of CCSP(
/
) mice after Ova
exposure was increased compared with that in WT mice after 2, 3, and 5 days of Ova exposure at either 2 (Fig. 1) or 5 mg/m3 of Ova
aerosol.
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Inflammation is increased in the lungs of
CCSP(/
) mice challenged with Ova.
To determine the role of CCSP in modulating inflammatory responses in
lung tissues, pathological assessment of lung inflammation and injury
was scored blindly according to septal infiltrates, perivascular
infiltrates, peribronchiolar infiltrates, and epithelial cell
hyperplasia/hypertrophy in CCSP(
/
) mice and WT mice after Ova
challenge. The mean pathological scores were markedly increased in both
WT and CCSP(
/
) mice challenged with 2 mg/m3 of Ova
compared with scores in air control mice. The mean pathological scores
of CCSP(
/
) mice were increased compared with those of WT mice after
Ova challenge at 2 mg/m3 for 2 and 3 days (Fig.
2A). No statistical difference
was observed between CCSP(
/
) and WT mice after Ova exposure at 5 mg/m3 for 2 and 3 days, but lung histopathology was
increased in CCSP(
/
) mice exposed to Ova for 5 days. Representative
photomicrographs show the histopathological changes in the lungs of WT
and CCSP(
/
) mice challenged with 2 mg/m3 of Ova for 3 days (Fig. 2B).
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Cell differential analysis in BALF of
CCSP(/
) mice after Ova challenge.
Differential cell analysis was assessed in BALF of CCSP(
/
) mice
after Ova exposure to determine the role of CCSP in modulating inflammatory cell populations in the lung. Eosinophils were the predominant inflammatory cells in the BALF of all mice challenged with
Ova even after 2 days of exposure. Eosinophils in BALF from CCSP(
/
)
mice were increased compared with WT mice after Ova challenge at 5 mg/m3 for 5 days (Fig. 3) but
were not altered at 2 or 3 days or in 2 mg/m3 Ova-exposed
CCSP(
/
) mice at 2, 3, or 5 days. The numbers of neutrophils in BALF
from WT mice were increased 2 and 3 days after Ova challenge compared
with numbers in WT air control mice. Neutrophils in BALF from
CCSP(
/
) mice were further increased compared with those in WT mice
after 2 and 3 days of Ova challenge at both 2 and 5 mg/m3
(Fig. 4). In contrast to eosinophils,
which increased later in CCSP(
/
) mice after Ova exposure compared
with WT mice, neutrophils increased early during the course of Ova
exposure in CCSP(
/
) mice and were decreased after 5 days of
exposure. Lymphocytes in BALF from both WT and CCSP(
/
) mice after 2 or 5 mg/m3 of Ova exposure were increased compared with
levels in air control mice. The numbers of lymphocytes in BALF from
CCSP(
/
) mice were increased compared with those in WT mice after 2 or 5 mg/m3 of Ova exposure for 5 days (Fig.
5). Macrophages were increased in BALF of
both WT and CCSP(
/
) mice exposed to Ova at 2 mg/m3 for
2 and 3 days and 5 mg/m3 for 2, 3, and 5 days compared with
macrophages in air control mice. However, there was no statistical
difference between macrophage counts in BALF of WT and CCSP(
/
) mice
at all dose and time points (data not shown). Overall, neutrophils were
increased in the BALF of CCSP(
/
) mice after 2 and 3 days of Ova
exposure at either 2 or 5 mg/m3, whereas eosinophils were
increased in the BALF from CCSP(
/
) mice only after 5 days of Ova
challenge at 5 mg/m3. Lymphocytes were increased in the
BALF from CCSP(
/
) mice after 5 days of Ova exposure at both 2 and 5 mg/m3.
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MPO activity is increased in the lungs of
CCSP(/
) mice after Ova exposure.
As a marker of neutrophil activation during Ova-induced lung
inflammation, MPO activity was measured in lung homogenates and BALF of
CCSP(
/
) and WT mice after Ova challenge. MPO activity was
undetectable in the original BALF and the concentrated BALF from WT and
CCSP(
/
) mice. MPO activity was also undetectable in both WT and
CCSP(
/
) air control mice. MPO activity was increased in WT mice
after Ova challenge compared with that in WT air control mice (Fig.
6). MPO activity was highest in the lungs
of mice after 2 days of Ova exposure and declined with time. MPO
activity was only mildly apparent after 5 days of Ova exposure at 2 or
5 mg/m3. MPO activity was further increased in the lungs of
CCSP(
/
) mice compared with that in WT mice after 2 or 3 days of Ova
challenge at 2 mg/m3 of Ova aerosol, consistent with the
increased numbers of neutrophils in the BALF from CCSP(
/
) mice.
After 5 mg/m3 of Ova challenge, MPO activity was increased
in the lung homogenates of CCSP(
/
) mice compared with that in WT
mice after 2 days of Ova exposure. Interestingly, MPO activity was
similar in the lung homogenates from CCSP(
/
) and WT mice after 3 days of exposure at 5 mg/m3 of Ova aerosol. Collectively,
MPO activity was increased in the lungs of CCSP(
/
) mice early
during the course of Ova-induced lung inflammation and coincided with
increased neutrophils in the lungs of CCSP(
/
) mice after Ova
challenge.
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Increased MIP-2 concentration in lungs from
CCSP(/
) mice exposed to Ova.
To determine a possible mechanism by which CCSP deficiency causes
increased neutrophilic recruitment in BALF and increased lung
infiltration, the neutrophil chemokine MIP-2 was assessed in lung
homogenates of CCSP(
/
) and WT mice. MIP-2 levels were not readily
detectable in either WT or CCSP(
/
) air control mice. MIP-2 levels
were increased in the lungs of WT mice after 2, 3, or 5 days of Ova
challenge compared with levels in WT air control mice (Fig.
7, A and B). In the
lungs of CCSP(
/
) mice, MIP-2 levels were increased twofold compared
with those in WT mice after 2 and 3 days of Ova challenge with 2 mg/m3 of Ova aerosol (Fig. 7A). Furthermore, the
MIP-2 levels were increased in the lung homogenates of CCSP(
/
) mice
at all time points after Ova exposure at 5 mg/m3 (Fig.
7B). Overall, MIP-2 production was increased in the lungs of
CCSP(
/
) mice after Ova challenge, in accordance with increased neutrophil migration in the lung.
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Increased AB-PAS-positive mucosubstances in
CCSP(/
) mice challenged with Ova.
The role of CCSP in modulating Ova-induced mucous cell
metaplasia was examined by AB-PAS staining of the airways in
CCSP(
/
) and WT mice. Mucus staining was not apparent in the airway
epithelium of WT or CCSP(
/
) air-challenged mice (Fig.
8A). Very few AB-PAS-positive cells were visible in CCSP(
/
) and WT mice after 2 or 3 days of challenge with both 2 and 5 mg/m3 of Ova aerosol (data
not shown). The intensity and the number of AB-PAS-stained cells
increased after 5 days of challenge with 2 and 5 mg/m3 of
Ova aerosol compared with results at 2 or 3 days of challenge. Many of
the mucous cells in the WT mice showed only PAS positivity, whereas
many mucous cells in CCSP(
/
) mice displayed AB-PAS-positive staining (Fig. 8A). Although the numbers of AB-PAS-positive
cells per millimeter of basal lamina were similar in WT and CCSP(
/
) mice (data not shown), the volume density of AB-positive material was
significantly higher in CCSP(
/
) mice compared with WT mice after
Ova challenge at 5 mg/m3 for 5 days (Fig. 8B).
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DISCUSSION |
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The role of CCSP and Clara cells in the pathogenesis of allergic
airway disease has not been elucidated. In the present study, AR was
increased in CCSP(/
) mice after Ova aerosol challenge. Lung
inflammation was increased in the lungs of CCSP(
/
) mice concurrent
with increased AR. Neutrophils appeared earlier and were increased
during the pathogenesis of Ova-induced lung disease in CCSP(
/
)
mice. Likewise, eosinophils and lymphocytes were also increased at
later time points. MPO activity and MIP-2 levels were increased in
CCSP(
/
) mice after Ova challenge, coinciding with increased
neutrophils in the lungs of CCSP(
/
) mice. Furthermore, the amount
of AB-PAS-positive mucosubstances was increased in the airways of
CCSP(
/
) mice after Ova exposure. These results indicate that CCSP
modulates AHR and lung inflammation induced by Ova exposure and are
consistent with the concept that CCSP limits the pathogenesis of asthma
and allergic airway disease.
Herein, AR was markedly higher in CCSP(/
) mice than in WT mice
after Ova challenge. The role of CCSP in regulating airway responses
during airway disease has not been investigated. Previous studies
indicate that CCSP levels are decreased in BALF of patients with
chronic bronchitis (9) and in animal models of acute
bacterial infection (7). In a recent clinical study
(24), CCSP-positive epithelial cells were reduced in the
small airways of patients with asthma, and CCSP-positive epithelial
cell proportions were inversely correlated with numbers of T cells and
mast cells in the small airways of these patients. Likewise, CCSP
levels in BALF (33) and serum (25) are also
decreased in asthma patients. Taken together, these studies suggest
that CCSP is decreased during chronic airway diseases, particularly
asthma. The present study supports the concept that CCSP deficiency in
the lung can contribute to the development of AHR and the pathogenesis
of asthma.
In the present study, either 2 or 5 mg/m3 of Ova exposure
for 6 h/day caused increased AR and lung inflammation characteristic of
asthma. Lung inflammation and AR were increased in the lungs of
CCSP(/
) mice compared with WT mice. A previous study
(2) with 129J strain mice and acute Ova exposure (either
20 or 60 min/day) reported poor host responses to Ova challenge. In the current study, a 6-h exposure of WT 129J strain mice to Ova aerosol induced mild AHR at high concentrations (25 or 50 mg/ml) of MCh challenge. Importantly, the AR was further increased in CCSP(
/
) mice exposed to Ova for 2, 3, or 5 days at either 25 or 50 mg/ml of MCh
challenge compared with that in WT mice, indicating that CCSP
deficiency can render 129J strain mice susceptible to Ova challenge.
The measurement of Ova concentrations in the exposure chambers
strengthens the experimental protocol used herein and limits variations
between experimental studies.
Neutrophils were increased early during the course of Ova-induced lung
disease in the lungs of CCSP(/
) mice compared with numbers in WT
mice. The role of CCSP in modulating neutrophil migration into the lung
has been shown. Neutrophils in BALF from CCSP(
/
) mice are markedly
increased after acute adenovirus or Pseudomonas aeruginosa
infection (6, 7). Likewise, lung neutrophils are increased
in CCSP(
/
) mice after hyperoxic exposure (12).
Neutrophilia in the airways has been associated with severe asthma
(22), and neutrophils can damage airway epithelial cells in airway inflammation (34). Thus increased neutrophils
early during Ova-induced airway disease in CCSP(
/
) mice may, in
part, account for the increased AR observed in CCSP(
/
) mice after Ova exposure. In the present study, MPO activity, an important effector
of neutrophil-induced cytotoxicity, was also increased in the lungs of
CCSP(
/
) mice during the allergic response to Ova. Importantly, MPO
activity was measured in total lung homogenates as an index of
neutrophils in the lungs after Ova exposure. Thus extracellular MPO
activity cannot be distinguished from intracellular stores of MPO. The
contribution of intravascular MPO within lung blood vessels is likely
small due to exsanguination of tissues during preparation. Therefore,
the increased MPO activity in the lung homogenates of CCSP(
/
) mice
after Ova challenge suggests that MPO-containing leukocytes accumulated
more in the lungs of CCSP(
/
) than in WT mice after Ova challenge.
Collectively, these results strongly suggest a role for CCSP in
modulating neutrophil or MPO-containing leukocyte responses in the lung
after allergic challenge.
In contrast to neutrophils, which increased earlier during the course
of Ova challenge, lymphocytes increased in the BALF of CCSP(/
) mice
after 5 days of Ova exposure at both 2 and 5 mg/m3.
However, eosinophils increased in the BALF of CCSP(
/
) mice after 5 days of Ova exposure only at 5 mg/m3, not at 2 mg/m3. The findings in the current study suggest an Ova
dosage-dependent infiltration of eosinophils into the airways of
sensitized mice, indicating that a larger dose of allergen challenge
could induce more severe leukocyte, especially eosinophil, infiltrates
in the lung. This finding is similar to the observations from a study in human patients with atopic asthma in which only subjects challenged with a high dose of antigen recruited significant eosinophils to the
lung (3).
Lung histopathology was increased earlier in CCSP(/
) mice during
the course of Ova-induced lung pathogenesis. Increased lung
infiltration coincided with increased neutrophils, increased MIP-2
production, and MPO activity in the lungs of CCSP(
/
) mice after Ova
challenge. Likewise, the increased lung inflammation coincided with
increased AR in CCSP(
/
) mice. In previous studies, lung
inflammation was increased in CCSP(
/
) mice after acute respiratory
infections (6, 7, 10). Lung inflammation and injury also
increased in CCSP(
/
) mice exposed to ozone and hyperoxia (12,
15). Inflammation of the lung is closely associated with AHR in
both experimental animal models and clinical studies. Undoubtedly, inflammatory cells in the lung secrete a number of mediators that are
involved in AR to allergens and are strongly associated with the
development of asthmatic responses. Taken together, these studies
suggest that CCSP modulation of lung inflammation may reduce AR in
Ova-induced lung disease in vivo.
The migration of specific inflammatory cells into the lung is a complex
mechanism involving, in part, chemotaxis by various effector molecules.
MIP-2 levels were increased in the lungs of CCSP(/
) mice compared
with those in WT mice after Ova challenge. In the current study, MIP-2
levels were measured in total lung homogenates. As with the
measurements of MPO, intravascular MIP-2 could not be distinguished
from MIP-2 in lung interstitium in the present study. In mice, MIP-2 is
an important chemoattractant for neutrophils, both in vitro and in vivo
(1, 17). In the present study, increased MIP-2 levels in
the lung coincided with increased neutrophil migration in the airways
and lung parenchyma. In a previous study (6) of CCSP
deficiency, MIP-2 was increased in the lungs of CCSP(
/
) mice after
acute viral infection. Increased lung MIP-2 levels in virally infected
CCSP(
/
) mice strongly coincided with increased neutrophils in the
lung as is the case herein. Likewise, various chemokines, in particular
MIP-2, are increased in the lungs of CCSP(
/
) mice during oxygen- or
ozone-induced lung injury (11). These findings suggest
that CCSP may play an important role in modulating chemokine production
in the lung and may provide the basis for the presence of increased
neutrophils in the lungs of CCSP-deficient mice.
The role of CCSP in mucus production has not been previously
investigated. After Ova exposure, both CCSP(/
) and WT mice exhibited similar numbers of AB-PAS-positive cells. However, more AB-positive material was detected in the airways of CCSP(
/
) than in
WT mice. The change in staining pattern denotes a change in the content
from neutral (PAS-positive) to acidic (AB-positive) glycoprotein
(21). Clara cells have been shown to differentiate into
mucus-containing cells in the airways of mice (16, 36). It
is possible that after allergen exposure, the changes in inflammatory responses in CCSP(
/
) mice led to the expression of different glycosylating mechanisms and thereby to a different pattern of glycosylation.
In the current study, CCSP deficiency was associated with increased
lung pathology and inflammation after Ova challenge. A previous study
(29) in CCSP(/
) mice showed that Clara cell ultrastructures are also altered in this animal model. The significance of this observation after Ova aerosol challenge herein is unclear. Importantly, CCSP replacement with recombinant CCSP in the
lungs of CCSP(
/
) mice attenuates lung inflammation and cytokine
production in the lung after lipopolysaccharide challenge
(8), suggesting that the absence of CCSP was, in part,
involved in the increased host response to lipopolysaccharide in
CCSP(
/
) mice. Surfactant mRNA production is not altered in
CCSP(
/
) mice (29), suggesting that Clara cells retain
normal surfactant production in CCSP(
/
) mice. Likewise, surfactant
homeostasis is not altered in the lungs of uninfected or
adenovirus-infected CCSP(
/
) mice (10). The current
study does not exclude other functions of the Clara cells secondary to
CCSP deficiency that contribute to the present findings. Importantly,
the dosimetry of aerosol deposition is not altered in the lungs of
CCSP(
/
) mice (Stripp BR, personal communication). The current
studies strongly suggest a role for CCSP, whether direct or indirect,
in modulating lung inflammation and AHR in response to allergic airway inflammation.
CCSP in vivo modulates lung inflammation during environmental injury and acute infection. The present study supports the concept that CCSP modulates lung inflammation and airway responsiveness to inhaled allergens in vivo and suggests a role for Clara cells in the pathogenesis of asthma. CCSP is decreased in the airways of patients with asthma (23, 32). Thus decreased CCSP may have important implications in the development of chronic lung inflammation in asthmatic and atopic diseases.
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ACKNOWLEDGEMENTS |
---|
We acknowledge Monique Nysus for help with immunizing the animals and staining slides, Dr. Thomas H. March for assistance with histopathological scoring and microphotography, and Justin E. Kubatko for assistance with statistical analysis of the data.
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FOOTNOTES |
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This study was supported in part by National Heart, Lung, and Blood Institute Grant HL-66964 and grants from the American Lung Association-Asthma Research Center (to S.-Z. Wang) and the Parker B. Francis Foundation (to K. S. Harrod) under Cooperative Agreement DE-FC04-96AL76406 in facilities fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care.
Address for reprint requests and other correspondence: K. S. Harrod, Lovelace Respiratory Research Institute, Albuquerque, NM 87185 (E-mail: kharrod{at}lrri.org).
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.
Received 17 January 2001; accepted in final form 5 July 2001.
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