Division of Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, Ohio 45229-3039
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ABSTRACT |
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Clara cell secretory protein
(CCSP) is a 16-kDa homodimeric polypeptide secreted by respiratory
epithelial cells in the conducting airways of the lung. To assess the
role of CCSP in bacterial inflammation and to discern whether CCSP
expression is influenced by bacterial infection, CCSP-deficient
[(/
)] gene-targeted mice and wild-type mice were given
Pseudomonas aeruginosa intratracheally. Infiltration by
polymorphonuclear cells was significantly increased in the lungs of
CCSP(
/
) mice 6 and 24 h after the administration of the
bacteria. The number of viable bacteria isolated from the lungs in
CCSP(
/
) mice was decreased compared with that in wild-type mice.
Concentrations of the proinflammatory cytokines interleukin-1
and
tumor necrosis factor-
were modestly increased after 6 and 24 h, respectively, in CCSP(
/
) mice. The concentration of CCSP protein
in lung homogenates decreased for 1-5 days after infection and
recovered by 14 days after infection. Likewise, CCSP mRNA and
immunostaining for CCSP markedly decreased in respiratory epithelial
cells after infection. CCSP deficiency was associated with enhanced
pulmonary inflammation and improved killing of bacteria after acute
pulmonary infection with P. aeruginosa. The finding that
Pseudomonas infection inhibited CCSP expression provides further support for the concept that CCSP plays a role in the modulation of pulmonary inflammation during infection and recovery.
Clara cell secretory protein; mucosal immunity; bacterial infection
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INTRODUCTION |
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CLARA CELL SECRETORY PROTEIN (CCSP; also called CC10, CC16, and uteroglobin) is a 16-kDa homodimeric protein produced by nonciliated bronchiolar cells in the lung (20). CCSP mRNA is also expressed to a lesser extent in the prostate, thyroid, mammary, and pituitary glands and in the uterus during pregnancy (16, 17). In the lung, CCSP is one of the most abundant soluble proteins in the extracellular lining fluid of airways (20). Despite its abundance in the airways of mammals, the physiological function of CCSP in the lung has not been elucidated.
In in vitro studies, CCSP inhibited the secretory phospholipase
A2 enzyme (12) and inhibited chemotaxis of
macrophages and neutrophils in uterine tissue (24).
Likewise, in vitro studies (6, 27) with
airway epithelial cells have suggested that CCSP may modulate the
activity of various cytokines, including interferon- (IFN-
) and
tumor necrosis factor (TNF)-
. In vivo, CCSP-deficient [(
/
)]
mice generated by gene targeting (22) showed increased
inflammatory response after lung injury and viral infection. After
hyperoxic exposure, CCSP(
/
) mice had reduced survival time and
increased proinflammatory cytokine production in the lung
(10). They also had increased sensitivity to lung injury
induced by ozone (14). CCSP also plays a role in immune modulation that follows pulmonary infection. After administration of
adenovirus, inflammation, neutrophil migration, and proinflammatory cytokine production were increased in the lungs of CCSP(
/
) mice (9). Collectively, these studies suggest a role for CCSP
as an important constitutive protein that modulates inflammatory responses after lung injury.
Pseudomonas aeruginosa is a common gram-negative pathogen and causes a variety of infections in compromised hosts and patients experiencing prolonged hospitalization. Ventilator-associated pneumonia caused by P. aeruginosa has a mortality rate of 40-68% (4, 23). Mucoid P. aeruginosa is a major bacterial pathogen in the airways during cystic fibrosis-related disease and is commonly associated with a decline in lung function in cystic fibrosis patients (8). The respiratory epithelium produces a number of polypeptides that influence host defense and lung inflammation, including collectins [surfactant protein (SP) A and SP-D] (5), defensins (15), lysozyme (11), and other proteins that may influence the pathogenesis of pulmonary P. aeruginosa infection. For example, SP-A, an abundant host defense protein produced by the lung epithelium, increases bacterial clearance and decreases the inflammatory response to mucoid P. aeruginosa in vivo (13).
The present study was designed to discern the potential role of
CCSP in the host immune response and to determine whether acute
Pseudomonas pneumonia influences the expression of CCSP by
respiratory epithelial cells. CCSP(/
) gene-targeted and wild-type mice were infected with a mucoid strain of P. aeruginosa.
CCSP deficiency was associated with increased influx of
polymorphonuclear cells and improved killing of P. aeruginosa in vivo. Pulmonary infection with P. aeruginosa markedly decreased CCSP mRNA and protein in vivo.
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MATERIALS AND METHODS |
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Mice.
CCSP(/
) (129J Ola/129J hybrid) and wild-type (129J; Taconic Farms,
Germantown, NY) mice were housed under pathogen-free conditions in the
Children's Hospital Research Foundation vivarium following American
Association for Accreditation of Laboratory Animal Care guidelines.
Preparation of bacteria.
A stock culture of a mucoid strain of P. aeruginosa from a
clinical isolate was kindly provided by Dr. J. R. Wright (Duke University, Durham, NC). Bacteria were suspended in 2× yeast tryptone (2×YT) agar with 20% glycerol and were frozen at 80°C. Before each experiment, an aliquot was thawed and plated on 2×YT agar. It was
then inoculated into 5 ml of 2×YT broth and grown for 15 h at
37°C with continuous shaking. The broth was centrifuged and resuspended in 1 or 2 ml of PBS. The concentration of the suspension was determined by quantitative culture on 2×YT agar; concentration was
then adjusted by dilution with sterile PBS to 1 × 109
colony-forming units (CFU)/ml.
Intratracheal administration of P. aeruginosa.
Eight- to twelve-week-old CCSP(/
) and wild-type control mice
(n = 6-12 mice/group) were used. Mice were
anesthetized with isofluorane vapor, and a ventral midline incision was
made to expose the trachea. Intratracheal inoculation of 1 × 108 CFU of P. aeruginosa in 100 µl of PBS was
performed with the use of a bent, 27-gauge tuberculin syringe. The
incision was closed with one drop of Nexaband. At a predetermined time
for each analysis, mice were killed by a lethal injection of
pentobarbital sodium. A midline incision was made in the abdomen. Mice
were exsanguinated by transection of the inferior vena cava to reduce
pulmonary hemorrhage.
Bronchoalveolar lavage. Mice were killed 6 and 24 h after bacterial administration as described in Intratracheal administration of P. aeruginosa. The lungs were lavaged three times with 1 ml of sterile PBS. Bronchoalveolar lavage fluid (BALF) was centrifuged at 2,000 rpm for 10 min and was then resuspended in 0.5-2 ml of PBS. Cell suspension (100 µl) was mixed with 100 µl of 0.4% trypan blue (GIBCO BRL, Life Technologies, Grand Island, NY), and total cell counts were determined with a hemocytometer and trypan blue exclusion. Differential cell counts were made on cytospin preparations stained with Diff-Quik (Baxter Healthcare, Miami, FL). Photographs of randomly selected fields in light microscopy were taken for each sample. In the photographs, each of which contains 500~1000 cells, the percentage of neutrophils and macrophages was determined. The number of each cell type was calculated from this percentage and the total cell count.
Bacterial killing. Mice were killed 6 and 24 h after infection. Whole lungs were removed and weighed, then homogenized in 1 ml of sterile PBS. Serial dilutions of lung homogenates were plated on 2×YT agars to determine the number of CFU of P. aeruginosa.
Cytokine and chemokine production.
Lung homogenates were centrifuged at 2,000 rpm for 20 min, and the
supernatants were stored at 20°C. Interleukin (IL)-1
, TNF-
,
and IL-6 were quantitated in supernatants with ELISA kits (Endogen,
Woburn, MA) according to the manufacturer's directions. The
neutrophilic chemokines marcrophage inflammatory protein (MIP)-1
and
MIP-2 were also quantitated with ELISA kits (R&D systems, Minneapolis,
MN). All plates were read on a microplate reader (Dynatech, Chantilly,
VA), and data were analyzed for significance with Excel 98 (Microsoft,
Seattle, WA).
Western blot analysis. Wild-type mice were killed 6, 24, and 48 h and 5 and 14 days after bacterial infection. Right lobes were clamped with a hemostat. Right middle lobes and half of the right lower lobes were removed and homogenized in 1 ml of PBS. Lung homogenates were centrifuged at 2,000 rpm for 20 min, and total protein concentration of the supernatants was measured by the Bradford assay (Bio-Rad, Richmond, CA). Ten micrograms of protein from each sample were electrophoresed on 16% SDS-polyacrylamide gels (Novex, San Diego, CA). Western transfer to a nitrocellulose filter was performed at 60 V for 1 h. The membrane was incubated with rabbit anti-rat CCSP polyclonal antibody for 15 h at room temperature. Anti-rat CCSP antibody was a generous gift from Gurmukh Singh and Sikandar Katyal (Veterans Affairs Medical Center, Pittsburgh, PA). After being washed with Tris-buffered saline, the membrane was incubated with peroxidase-conjugated goat anti-rabbit IgG antibody for 3 h at room temperature. Detection of CCSP was performed with Western blotting detection reagents (Amersham Pharmacia Biotech, Piscataway, NJ).
Immunohistochemical analysis. At each predetermined time point after Pseudomonas administration, left lobes of wild-type mice were inflated with 4% paraformaldehyde (Electron Microscopy Sciences, Ft. Washington, PA) at 20 cmH2O pressure for 1 min and were used for histological examination and immunohistochemical analysis. Inflation-fixed lungs were washed in PBS and divided in half for paraffin embedding. Paraffin-embedded lungs were sectioned at 5 µm. The sections were deparaffinized, blocked with 2% normal goat serum, and incubated with rabbit anti-rat CCSP polyclonal antibody diluted 1:100,000 for 15 h at 4°C. The sections were washed and incubated with biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA) diluted 1:200 for 30 min at room temperature. CCSP was detected with Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA), then counterstained with 0.1% nuclear fast red. Immunohistochemical staining was analyzed by light microscopy with a Nikon microscope.
S1 nuclease protection assay.
S1 nuclease protection assay was performed as described previously
(1, 18). At each set time point after
Pseudomonas administration, right upper lobes and the other
half of the right lower lobes of wild-type mice were homogenized in 1 ml of TRIzol reagent (Life Technologies, Grand Island, NY) and used for
RNA isolation. Total RNA was isolated from lung homogenates according to the manufacturer's directions. The RNA was quantified by absorbance at 260 nm. Radiolabeled cDNA probes for CCSP, SP-A, SP-B, SP-C, and L32
were generated by end labeling with T4 kinase according to
manufacturer's instructions (GIBCO BRL, Life Technologies, Gaithersburg, MD). Labeled cDNA was purified by centrifugation in
Sephadex G-50 spin columns (Boehringer Mannheim, Indianapolis, IN).
Briefly, 2 µg of lung total RNA were incubated overnight at 55°C
with radiolabeled cDNA probes. S1 nuclease digestion of unhybridized
probe was performed at room temperature for 1 h. Labeled
transcripts were separated by 6% PAGE for 1.5 h at 30 W, and the
gel was dried at 80°C under vacuum. After 3-15 h, transcript abundance was detected by autoradiography at 70°C with Kodak film
(Rochester, NY).
Statistical analysis. Significance was determined by Student's t-test with the use of Microsoft Excel software.
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RESULTS |
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Cell counts and cell differential analysis in BALF in CCSP(/
)
mice after bacterial infection.
To assess the role of CCSP in modulating inflammatory cell migration
into the air space, CCSP(
/
) gene-targeted mice and wild-type mice
(8-12 wk of age) were injected with 1 × 108 CFU
of a mucoid strain of P. aeruginosa. Mice recovered within 1 h after administration, with no lethality observed in either group. Total cell counts in BALF were assessed 6 and 24 h after infection. Total cell counts in BALF were increased after 6 h in
lungs from both CCSP(
/
) mice and wild-type mice compared with those
in uninfected mice. In both groups of animals, total cell counts were
further increased 24 h after administration of bacteria. At both 6 and 24 h after bacterial administration, total cell counts were
significantly greater in the BALF from the lungs of CCSP(
/
) mice
compared with those in wild-type mice (Fig. 1).
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Bacterial killing in CCSP(/
) mice.
To discern the role of CCSP in lung bacteria killing after
Pseudomonas infection, lung homogenates from CCSP(
/
)
mice and wild-type mice were assessed for bacterial CFU 6 and 24 h
after P. aeruginosa infection. Bacterial CFU in lung
homogenates from CCSP(
/
) mice were significantly decreased compared
with those in wild-type mice 24 h (Fig.
3). Although bacterial CFU in the lungs
of CCSP(
/
) mice at 6 h were also decreased compared with those
in wild-type mice, this difference did not reach significance.
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Cytokine and chemokine concentrations in lungs from CCSP(/
)
mice after bacterial infection.
To assess the possible mechanism by which CCSP deficiency causes
increased neutrophilic recruitment after infection, cytokines (IL-1
,
TNF-
, and IL-6) and chemokines (MIP-1
and MIP-2) were measured in
lung homogenates from CCSP(
/
) mice and wild-type mice by ELISA.
After administration of P. aeruginosa, IL-1
, TNF-
, IL-6, MIP-1
, and MIP-2 concentrations were markedly elevated in both
CCSP(
/
) mice and wild-type mice compared with those in uninfected
mice (Fig. 4). Compared with wild-type
mice, IL-1
and TNF-
concentrations were modestly but
significantly increased in the lungs of CCSP(
/
) mice 6 and 24 h, respectively, after infection. MIP-1
, MIP-2, and IL-6
concentrations were similar in CCSP(
/
) mice and wild-type mice 6 or
24 h after infection.
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Decreased CCSP expression after acute Pseudomonas infection.
To determine whether CCSP expression was influenced by bacterial
infection of the lung, CCSP protein was estimated in lung homogenates
from wild-type mice by Western blot analysis 6, 24, and 48 h and 5 and 14 days after administration of P. aeruginosa. CCSP was
readily detected in lung homogenates from uninfected wild-type mice.
CCSP protein was decreased 6, 24, and 48 h after infection (Fig. 5), returning to
the levels seen in uninfected mice 14 days after infection.
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Decreased CCSP mRNA after acute Pseudomonas infection.
To determine whether the decrease in CCSP protein seen after bacterial
infection was regulated by changes in CCSP mRNA, an S1 nuclease
protection assay of total RNA from lung homogenates was performed at
various time points after bacterial infection of wild-type mice. In
addition, SP-A, SP-B, and SP-C mRNAs were determined as markers of lung
epithelium-specific gene expression. Whereas the abundance of mRNA for
the housekeeping gene L32 was unchanged after bacterial infection, CCSP
mRNA was decreased 24 and 48 h after bacterial infection (Fig.
7). Likewise, SP-B and SP-C mRNAs were
decreased 24 and 48 h after infection. In contrast, P. aeruginosa infection did not alter pulmonary SP-A mRNA at any time. Thus CCSP, SP-B, and SP-C mRNAs were reduced 24 and 48 h after P. aeruginosa infection of the lung.
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DISCUSSION |
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Neutrophil recruitment into the air spaces of CCSP(/
) mice was
increased compared with that in wild-type mice 6 and 24 h after
administration of P. aeruginosa. Surprisingly, bacteria were
killed faster in CCSP(
/
) mice 24 h after infection. This, perhaps, was mediated by the increased inflammatory response. The
current studies in CCSP(
/
) mice suggest that CCSP modulates lung
inflammation during pulmonary bacterial infections. Likewise, the
marked decrease in CCSP expression in wild-type mice after bacterial
infection suggests that changes in CCSP concentration may play a role
in initiation or resolution of lung inflammation.
In the present study, CCSP(/
) mice killed P. aeruginosa
similarly or slightly faster than wild-type mice, suggesting that CCSP
does not play a direct role in host defense against P. aeruginosa. Findings in the CCSP(
/
) mice are distinct from
recent studies in SP-A(
/
) mice. Increased lung inflammation and
decreased P. aeruginosa killing were observed in the
SP-A(
/
) mice in vivo (13). As seen in SP-A(
/
)
mice, the current study demonstrated that CCSP(
/
) mice also have
increased inflammation in response to P. aeruginosa, yet in
contrast to the findings in SP-A(
/
) mice, bacterial
killing was enhanced in CCSP(
/
) mice. The increase in bacterial
killing in the lungs of CCSP(
/
) mice may be explained in part by
the increased recruitment of inflammatory cells in the lung. Indeed,
both alveolar macrophages and neutrophils have been shown to influence
the clearance of P. aeruginosa from the lungs
(7, 21, 25). However, the
current work supports the concept that CCSP limits inflammation in the
lung during bacterial infection through a mechanism that is independent
of bacterial killing. The observed increase in lung inflammation in
CCSP(
/
) mice after bacterial infection is consistent with a
previous study (9) performed by this laboratory in which
adenovirus-mediated lung inflammation was increased in CCSP(
/
)
mice. Intratracheal administration of adenovirus to CCSP(
/
) mice
caused increased inflammation, production of proinflammatory cytokines,
and enhanced clearance of the virus (9).
Although IL-1 and TNF-
concentrations in lung homogenates were
modestly increased in CCSP(
/
) mice after infection, these differences were small, and it is unclear whether differences in
cytokine production mediated the increase in neutrophils observed after
infection. Although the neutrophilic chemokines MIP-1
and MIP-2 were
not increased in CCSP(
/
) mice after bacterial infection, pulmonary
infiltration with polymorphonuclear cells was consistently more severe
in CCSP(
/
) mice after infection. Vasanthakumar et al.
(24) demonstrated that uteroglobin (CCSP) inhibited
chemotaxis of macrophages and neutrophils in vitro. Dierynck et al.
(6) demonstrated that CCSP inhibited IFN-
production
from mononuclear cells and that the biological activity of IFN-
was
diminished by CCSP. On the other hand, Yao and colleagues
(26, 27) found that IFN-
and TNF-
stimulated CCSP production from human airway epithelial cells in vitro.
Taken together, this evidence shows that CCSP plays an
anti-inflammatory role in the lung; however, the mechanisms by which
CCSP influences neutrophil recruitment into the lung remain unclear.
In a previous study (22) performed by this laboratory, the
ultrastructure of Clara cells was found to be altered in CCSP(/
) mice. The importance of this observation to the present findings regarding bacterial killing and inflammation is unclear at present. The
concentrations of SP-A and SP-B mRNA were unchanged in CCSP(
/
) mice, suggesting no direct effect of CCSP deficiency on these SPs
expressed with CCSP in Clara cells. Nevertheless, the current study
does not exclude the possibility that a general alteration in Clara
cell function secondary to CCSP deficiency may influence the host
response after Pseudomonas infection. The current studies, however, strongly support a role for CCSP, whether directly or indirectly, in modulating lung inflammation after infection.
The concentrations of CCSP in lung homogenates and CCSP mRNA were
decreased in wild-type mice after intratracheal administration of
P. aeruginosa. Whereas CCSP was present at high
concentrations in the lung homogenates from wild-type mice, it was
nearly undetectable in the lungs of mice after infection. Decreased
CCSP was noted as early as 6 h after infection, coincident with
the early influx of inflammatory cells into peribronchiolar and
alveolar regions of the lung. It is unclear whether the loss of CCSP
that occurred in the lungs of wild-type mice early in the course of
infection enhanced pulmonary inflammation, perhaps in a manner similar
to that seen in the CCSP(/
) mice.
A number of mechanisms may be involved in the observed decrease in CCSP during lung inflammation, including changes in gene transcription or mRNA and protein stability. CCSP mRNA was also decreased after P. aeruginosa infection; however, the decrease in CCSP preceded the changes in CCSP mRNA, suggesting that CCSP clearance was also influenced by the infection. The Clara cell is thought to be a progenitor cell for the epithelium in injured bronchi and bronchioles in vivo (20). Cell injury may also be involved in the decrease in CCSP protein and mRNA. However, histological evaluation of the tissue did not demonstrate cell necrosis, and L32 and SP-A mRNA were not decreased, suggesting that the effects of infection on CCSP expression were not caused by cell loss.
P. aeruginosa is a common pathogen in immunocompromised
hosts and a frequent cause of nosocomial infection of the lung
(2). Colonization and infection of the respiratory tract
by P. aeruginosa is a major cause of morbidity and mortality
in patients with cystic fibrosis (8).
Ventilator-associated pneumonia caused by P. aeruginosa has
a mortality rate of 40-68% (4, 23).
Despite advances in therapeutic practices and the advent of useful
antibiotics, P. aeruginosa pneumonia is still associated
with high mortality. The present findings support the concept that CCSP
serves a role in the regulation of pulmonary inflammation but is not
critical for clearance of the bacteria. The finding that TNF-
production and associated lung inflammation are required for
Pseudomonas clearance (3) supports the concept
that recruitment of polymorphonuclear neutrophils to the lung is
critical for bacterial clearance. However, administration of the
anti-inflammatory cytokine IL-10 improved survival rates and
decreased lung inflammation in P. aeruginosa pneumonia
in mice (19). The present study demonstrated that deficiency of CCSP increased the clearance of P. aeruginosa
but was also associated with increased neutrophil recruitment after infection. CCSP inhibited neutrophilic recruitment after P. aeruginosa infection in the lung. The timing of the changes in
CCSP expression after P. aeruginosa infection support its
role in modulating inflammation after acute pneumonia and during recovery.
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ACKNOWLEDGEMENTS |
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This study was partially funded by the Cystic Fibrosis Foundation; the Research Development Program; National Heart, Lung, and Blood Institute Grants HL-28623 and HL-61646 (to J. A. Whitsett) and Program of Excellence in Molecular Biology Grant HL-41496; and by the Parker B. Francis Foundation (K. S. Harrod).
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FOOTNOTES |
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Present address of K. S. Harrod: Asthma and Immunology Program, Lovelace Respiratory Research Institute, PO Box 5890, Albuquerque, NM 87185.
Address for reprint requests and other correspondence: J. A. Whitsett, Children's Hospital Medical Center, Division of Neonatology and Pulmonary Biology, 3333 Burnet Ave., Cincinnati, OH 45229-3039 (E-mail: jeff.whitsett{at}chmcc.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. §1734 solely to indicate this fact.
Received 18 August 1999; accepted in final form 28 March 2000.
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