1 Division of Respiratory Biology and Toxicology, Department of Environmental Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642; 2 Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15260; and 3 Department of Cell Biology and Anatomy, University of Bergen, Bergen, N5009 Norway
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ABSTRACT |
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Clara cell secretory
protein (CCSP) is one of the most abundant proteins present in airway
lining fluid of mammals. In an effort to elucidate the function of
CCSP, we established CCSP-null [CCSP(/
)] mice and demonstrated
altered sensitivity to various environmental agents including oxidant
pollutants and microorganisms. Although CCSP deficiency itself may be
central to the observed changes in environmental susceptibility,
altered lung gene expression associated with CCSP deficiency may
contribute to the observed phenotype. To determine whether CCSP
deficiency results in altered lung gene expression, high-density cDNA
microarrays were used to profile gene expression in the total lung RNA
of wild-type and CCSP(
/
) mice. Genes that were differentially
expressed between wild-type and CCSP(
/
) mice included a previously
nonannotated expressed sequence tag (EST W82219) and immunoglobulin A
(IgA), both of which were elevated with CCSP deficiency. mRNA
expression of EST W82219 and IgA was localized in the lungs of
wild-type and CCSP(
/
) mice to airway Clara cells and peribronchial
lymphoid tissues, respectively. We conclude that CCSP deficiency is
associated with 1) altered gene expression in Clara cells of
the conducting airway epithelium and 2) alterations to
peribronchial B lymphocytes. These findings identify new roles for
Clara cells and their secretions in airway homeostasis.
10-kDa Clara cell secretory protein; secretoglobin; uteroglobin; hyperoxia; oxidant injury
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INTRODUCTION |
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CLARA CELL SECRETORY PROTEIN (CCSP), otherwise commonly known as uteroglobin, is the prototypical member of a family of secretory proteins that have recently been termed secretoglobins (16). The predominant site of CCSP expression is in the nonciliated airway epithelial (Clara) cell of the conducting airway (3, 17). However, CCSP expression has been observed among other secretory cell types in the epithelia of both the respiratory and genitourinary tracts in certain species (6, 17). Sequence polymorphisms associated with the human CCSP gene are associated with variability in the levels of CCSP expression and have been associated with susceptibility to asthma and glomerulonephropathy (18, 42). Moreover, extensive studies of human patients have shown a relationship between chronic lung disease, such as that observed among asthmatic patients and individuals with chronic obstructive pulmonary disease (COPD), and reductions in lung and serum CCSP levels (reviewed in Ref. 10).
Many potential functions have been ascribed to CCSP, with a role as an
anti-inflammatory mediator the most frequently cited (25, 27,
28). This assertion is based on a combination of in vitro and in
vivo studies, including the findings that CCSP is a potential regulator
of secretory phospholipase A2, that it has anticytokine
activities, and that peptides derived from the CCSP sequence exhibit
anti-inflammatory properties (21, 26, 29). However,
despite the numerous properties described for CCSP, many remain
controversial (1, 36). Stripp et al.
(39) and others (48) have established
CCSP-null [CCSP(/
)] mice to define in vivo functions for CCSP.
The pulmonary phenotype of CCSP(
/
) mice includes alterations in
Clara cell ultrastructure (39, 41), susceptibility to
oxidants (13, 23) and resistance to microorganisms
(9), all of which are associated with changes in
inflammatory responses after challenge. However, the finding that
CCSP(
/
) mice show identical inflammatory responses to agents such
as endotoxin (12) argues against a direct role for CCSP in
the regulation of pulmonary inflammation per se.
Interestingly, the virtually undetectable steady-state phenotype
of CCSP(/
) mice generated by Stripp et al. (39) and
further characterized by Reynolds et al. (32) is in stark
contrast to the severe phenotype, including wasting, multiorgan damage,
and premature mortality, observed in an independently generated line of
CCSP(
/
) mice (48). The phenotype of CCSP(
/
) mice
generated by Zhang et al. (48) was of variable penetrance,
yet kidney defects observed in these mice could be reproduced with an
antisense strategy to reduce levels of CCSP expression
(49). Variable phenotypes observed among different mouse
models of CCSP deficiency highlight the importance of other genetic and
epigenetic factors in dictating the phenotypic outcome.
Phenotypic differences between mouse models of CCSP deficiency,
coupled with loose associations between CCSP deficiency and human
disease, have led us to hypothesize that CCSP deficiency among
CCSP(/
) mice is associated with the altered expression of other
genes in the lung. If correct, understanding the identity and
localization of differentially expressed genes would provide new
insights into cellular and molecular changes resulting from CCSP
deficiency and the impact of these genes on susceptibility to
environmental agents. This hypothesis was tested in the present study
through screening high-density cDNA microarrays to profile and compare
gene expression between steady-state wild-type (WT) and CCSP(
/
)
mice. We demonstrate that CCSP deficiency is associated with changes in
the abundance of mRNA species in the lung. In this study, we
demonstrate that CCSP deficiency is associated with altered gene
expression in Clara cells and bronchus-associated lymphoid tissues (BALT).
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MATERIALS AND METHODS |
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Animals.
Strain 129 CCSP(/
) mice (32, 39) and strain-matched WT
control mice were maintained as specific pathogen-free in-house colonies. All animals were housed in humidity- and
temperature-controlled rooms on a 12:12-h light-dark cycle and were
allowed food and water ad libitum. Representative mice were screened
quarterly for pathogens represented on a 16-agent serological panel
(Microbiological Associates, Rockville, MD). Animals used in this study
were 8-12 wk of age.
RNA isolation and gene microarray.
Six WT and six CCSP(/
) mice were killed with 100 mg/kg of
pentobarbital sodium. The animals were perfused with saline, and the
lungs were removed and homogenized in 4 M guanidinium. RNA was
extracted as previously described (5). Polyadenylated RNA was selected from 100 µg of total lung RNA from six WT and six CCSP(
/
) mice and used to probe a mouse GEM1 cDNA microarray representing 8,700 cDNAs (Incyte Pharmaceuticals, Palo Alto, CA). Standard labeling and hybridization conditions established by the
manufacturer were employed. Differences in probe-labeling efficiency
were accounted for through derivation of balanced coefficients, and the
WT fluorescence intensity and balanced CCSP(
/
) fluorescence intensity were reported. These values were used to calculate the ratio
of signal intensities between CCSP(
/
) and WT samples.
Northern hybridization.
Two criteria were used to select clones for validation by Northern blot
analysis. Clones with a balanced differential expression equal to or
greater than 1.9 or equal to or less than 1.9 were identified, and
the 20 clones with the highest P1 or balanced P2 intensities were
selected for further analysis. RNA was prepared as described for the
gene microarray except that the tissue was not perfused. Five
micrograms of total lung RNA from one WT or CCSP(
/
) mouse were
separated by electrophoresis through formaldehyde gels
(34) and transferred to Durolon membranes (Stratagene, La
Jolla, CA). Plasmids were prepared by standard methodology, and inserts
were isolated after double digestion with NotI and EcoR1. Inserts were labeled with [32P]dCTP
with a random-primer labeling kit (GIBCO BRL, Rockville, MD).
Hybridization was carried out at the calculated melting temperature (Tm) of
25°C, and blots were washed at
Tm
10°C, as previously described (32).
Hybridized membranes were exposed to film (Kodak, Rochester, NY), and
band intensities were analyzed with a phosphorimager and ImageQuant
software (Molecular Dynamics, Sunnyvale, CA). Blots were rehybridized
with
-actin or L32 for normalization. Differential expression of two
clones, expressed sequence tag (EST) W82219 and immunoglobulin A (IgA)
heavy chain constant region was further evaluated in six WT and
CCSP(
/
) mice by Northern blot hybridization as described above.
Expression of EST W82219 in WT and CCSP(
/
) heart, intestine,
kidney, liver, lung, salivary gland, spleen, testis, and thymus was
assessed by Northern blot hybridization as described above.
In situ hybridization.
Mice were killed with 100 mg/kg of pentobarbital sodium and
exsanguinated. The lungs were inflation fixed with neutral buffered formalin through a tracheal cannula for 10 min at 10 cmH2O
pressure. Lungs were then immersed in formalin for 16 h, immersed
in PBS for 24 h, dehydrated, and paraffin embedded.
Paraffin-embedded lung tissue was cut at 5 µm and placed onto
Superfrost slides. 35S-labeled sense and antisense
riboprobes (6 × 108 dpm/µg) were generated from rat
CCSP (38) or mouse cytochrome P-450 2F2
(CYP2F2) cDNA (40) cloned into pGEM3Z and from EST W82219
and IgA cDNA cloned into pT3T7 with a riboprobe transcription kit
(Promega, Madison, WI). Conditions and solutions for hybridization were
essentially as previously described (43). Hybridization was carried out overnight at 54°C, and the slides were washed under
high-stringency conditions. The slides were dipped in Kodak NTB2
emulsion, exposed for 16 h, and developed with Kodak D19 developer
following the manufacturer's protocol. Images from WT and CCSP(/
)
tissue were captured under identical conditions, and pseudocolored
silver grains (red) were superimposed over the bright-field image in
Adobe Photoshop. Sense probes were used as negative controls and
demonstrated no nonspecific hybridization under the conditions used.
Dual in situ hybridization and immunohistochemistry. WT tissue was hybridized as described in In situ hybridization with the exception that 3H-labeled riboprobes were used. After RNase treatment, the sections were blocked and incubated with rabbit anti-rat CCSP (1:16,000) overnight at 4°C. Antigen-antibody complexes were detected as previously described (32) with biotinylated goat anti-rabbit Ig secondary antibody, streptavidin-horseradish peroxidase, and diaminobenzidine substrate. The slides were then washed sequentially at 65°C for 30 min in 50% formamide-2× SSC-10 mM dithiothreitol (DTT), at room temperature for 15 min in 2× SSC-10 mM DTT, and at room temperature for 15 min in 0.1× SSC-10 mM DTT. Autoradiography and imaging were done as described in In situ hybridization.
IgA ELISA.
Six WT and six CCSP(/
) mice were lavaged twice with 1 ml of saline.
Cells were pelleted at 300 g, and the supernatants were concentrated two- to fourfold with Centricon 10 microconcentrators (Amicon, Beverly, MA). Serum was obtained from a similar number of WT
and CCSP(
/
) mice. Protein concentrations for lavage fluid and serum
were determined by bicinchoninic acid assay (Pierce, Rockford, IL).
Lavage fluid and serum IgA concentrations were determined with an IgA
ELISA kit as described by the manufacturer (Bethyl Laboratories,
Montgomery, TX). All samples were assayed in triplicate, and IgA levels
were normalized to protein concentration. Significance was determined
by Student's t-test.
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RESULTS |
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Altered lung gene expression associated with CCSP deficiency.
Our laboratory and others have previously demonstrated that CCSP
deficiency among CCSP(/
) mice is associated with ultrastructural changes to Clara cells (39, 41) and susceptibility to
inhaled oxidant gases (23, 48). Experiments performed in
the present study were designed to test the hypothesis that CCSP
deficiency is associated with changes in lung gene expression and that
the identity of differentially expressed genes would provide insight into functions for Clara cells and/or CCSP in lung homeostasis. Differences in steady-state lung expression of 8,700 genes were determined for pools of six adult male strain 129 CCSP(
/
) mice and
six age-, sex-, and strain-matched WT control mice with Incyte mouse
GEM1 cDNA microarrays (Fig. 1).
Differentially expressed genes were those whose estimated mRNA
abundance was increased by at least 190% or decreased by at least 53%
in total lung RNA from CCSP(
/
) mice relative to that in WT control
mice as suggested by Incyte Pharmaceuticals (24). To
validate these findings, five downregulated genes and five upregulated
genes were selected for Northern blot analysis based on their abundance
in total lung RNA of CCSP(
/
) and WT mice. Fluorescence intensity
(Table 1) was used as an index of
abundance for the corresponding mRNA. Northern hybridization indicated
that only 2 of the 10 genes identified as differentially expressed
through microarray analysis could be confirmed with a standard
quantitative methodology (Table 1). No further analysis of quantitative
gene expression data from microarray screening was undertaken without
prior validation with alternative approaches to confirm differential
gene expression.
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|
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CCSP-dependent changes in airway epithelial and peribronchial
lymphoid tissues.
To precisely localize sites of altered gene expression in lungs of
CCSP(/
) mice, the distribution of IgA and EST mRNAs were compared in lung tissue sections from WT and CCSP(
/
) mice (Fig. 3). Consistent with a previous study by
Stripp et al. (39), hybridization of an antisense
riboprobe for the Clara cell-specific CYP2F2 mRNA revealed no apparent
differences between strains (Fig. 3, A and
B). Antisense riboprobes generated toward EST W82219 hybridized to transcripts localized exclusively in conducting airways
of both WT and CCSP(
/
) mice, with an intensity that decreased in a
proximal to distal gradient (Fig. 3, C, D,
G, and H). Longer (1 mo) exposures to
photographic emulsion failed to detect EST W82219 transcript in
non-airway epithelial cells. Despite similarities in the distribution
of EST W82219 and Clara cell-specific CYP2F2 mRNAs, the extent of
EST W82219 mRNA hybridization was noticeably higher in airways of
CCSP(
/
) mice relative to that in WT control mice. Cellular
colocalization of EST W82219 mRNA with CCSP by dual in situ
hybridization with a 3H-labeled EST W82219 antisense
riboprobe coupled with immunohistochemical detection of CCSP
revealed a Clara cell-specific pattern of EST W82219 mRNA expression
(Fig. 3J) in the bronchiolar epithelium. These data, coupled
with previous observations by Stripp et al. (39, 41) of
ultrastructural changes to Clara cells of CCSP(
/
) mice, demonstrate
that CCSP deficiency results in phenotypic changes to Clara cells.
|
CCSP-dependent alterations in airway and systemic IgA production.
ELISA was used to determine whether changes in IgA mRNA expression in
cells of the BALT resulted in either local or systemic alterations in
the abundance of IgA protein (Fig. 4).
Bronchoalveolar lavage fluid and plasma were recovered from healthy
adult male WT and CCSP(/
) mice with no history of infection or
other environmental challenges. Levels of IgA were elevated ~2.5-fold
in the bronchoalveolar lavage fluid and 2.7-fold in the plasma of
CCSP(
/
) mice relative to those in WT control mice. Collectively,
changes in IgA expression in the lungs of CCSP(
/
) mice indicate a
potential role for Clara cells and/or CCSP in the modulation of local
lymphoid populations.
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DISCUSSION |
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We demonstrate that CCSP deficiency results in changes in lung
gene expression, indicating perturbations in lung homeostasis. A novel
gene was identified whose expression was confined to Clara cells and
upregulated in airways of CCSP(/
) mice. However, changes in lung
gene expression in CCSP(
/
) mice were not confined to Clara cells.
Local production of IgA was significantly elevated at both the mRNA and
protein levels in addition to a significant elevation in serum IgA
levels. Increased production of lung IgA involved a dramatic expansion
of peribronchial IgA-expressing lymphocytes, consistent with expansion
of BALT.
CCSP mRNA and protein abundance have been used as biomarkers to follow
changes in airways associated with either acute or chronic lung disease
(11, 19, 39, 44). Even though these studies have revealed
relationships between the abundance of CCSP and the type or magnitude
of lung pathology, functional consequences of these changes are likely
to be complex, particularly as they relate to chronic alterations in
airway homeostasis and perpetuation of airway disease. CCSP(/
)
mice have the potential to reveal functional roles for Clara cells and
their secretions in airway homeostasis. A number of related phenotypes
have been described for CCSP(
/
) mice, including increased
susceptibility to inhaled oxidant gases (13, 23) and
altered responses to pathogens (8, 9). A common finding of
these studies is the induction of a more profound inflammatory reaction
among CCSP(
/
) mice. However, a previous demonstration by Johnston
et al. (13) that CCSP(
/
) and WT mice show identical
inflammatory responses when challenged with low doses of inhaled
endotoxin argues against a direct role for CCSP in mediating these
differential responses. Moreover, findings of the present study
reinforce the notion that CCSP(
/
) mice have more complex changes to
the lung than simple CCSP deficiency.
Altered expression of a novel lung and Clara cell-specific genes in
total lung RNA of CCSP(/
) mice clearly indicates the potential for
functional changes to Clara cells. The many possible explanations for
altered Clara cell gene expression include compensatory upregulation of
related genes, nonspecific alterations in Clara cell gene expression
that result from the dramatic reduction in secretory product
expression, or differences in the accumulation and/or sequestration of
endogenous ligands. A study by McDowell et al. (24)
highlights the possibility of similar CCSP and EST W82219 regulatory
mechanisms by demonstrating a similar decrease in their mRNA levels
after murine lung injury induced by nickel exposure (24).
Preliminary analysis of the predicted protein product of EST W82219
indicates that it shares distant homology with the secretoglobin family
of proteins for which CCSP is the founding member (16, 30; Reynolds, P,
Reynolds S, and Stripp B, unpublished observations). This
raises the possibility that elevated expression of EST W82219 in Clara
cells of CCSP(
/
) mice represents a mechanism to compensate for CCSP
deficiency. Importantly, functional changes to Clara cells indicated by
altered expression of EST W82219 are consistent with earlier
observations by Stripp et al. (39, 41) of ultrastructural
perturbations observed in Clara cells of CCSP(
/
) mice. Prominent
ultrastructural changes to Clara cells of CCSP(
/
) mice include the
absence of secretory granules and the frequent appearance of
multilamellar inclusions surrounding cytoplasmic organelle components.
The basis for these ultrastructural changes in Clara cells of
CCSP(
/
) mice is not known. However, similar ultrastructural
alterations have been observed in Clara cells in association with
airway pathology such as equine COPD (14). Similarities in
Clara cell alterations occurring between clinical chronic airway
disease and CCSP(
/
) mice are further supported by studies (4,
20, 35) demonstrating that CCSP levels are significantly
decreased in the airways and serum of asthmatic patients, cigarette
smokers, and individuals with COPD. As such, functional changes to
Clara cells of CCSP(
/
) mice and the associated changes in airway
homeostasis represent a model of a primary Clara cell defect that may
yield important new insights into the pathobiology of chronic airway disease.
Alterations in IgA expression highlight a potentially important role
for Clara cells and/or CCSP in local immunoregulation. Increased
IgA mRNA abundance observed in peribronchial lymphoid tissue of
CCSP(/
) mice is associated with a significant elevation in
IgA protein levels in both the airway lining fluid and serum. IgA
serves important functions in innate mucosal defense against pathogens
through its ability to nonspecifically opsonize bacteria and contribute
to their clearance (33). Elevated steady-state levels of
IgA observed in the airway lining fluid of CCSP(
/
) mice may
therefore contribute to an increased clearance rate and resistance to
Pseudomonas infection (9). However, mechanisms contributing to elevated IgA production in the lung are unknown. CCSP
has been proposed to act as an anticytokine as a result of its ability
to modulate inflammation and the activity of interferon (IFN)-
(7, 29). Moreover, in vitro studies (22, 45, 46) indicated that CCSP may be directly regulated at the
transcriptional level by IFN-
and tumor necrosis factor (TNF)-
.
Even though a study (12) in CCSP(
/
) mice showed no
evidence for altered inflammatory responses to stimuli that do not
cause concomitant cellular injury, it is possible that altered local
production or activity of IFN-
or other cytokines such as TNF-
could contribute to altered immunoregulation. Both IFN-
and members
of the TNF family have been shown to function as potent regulators of
the immune response (15, 37, 47).
Interestingly, even though CCSP(/
) mice show no signs of autoimmune
disease (32), an independently generated line of mice harboring a different knockout of the same gene show systemic disease
including IgA glomerulonephropathy and focal pancreatic necrosis
(48, 49). Based on observations presented herein and those
of Zheng et al. (49), we propose that immunoregulatory changes and increases in local production of IgA that are associated with CCSP deficiency may contribute to systemic organ dysfunction. The
potential contribution of altered Clara cell function to systemic disease is suggested from epidemiological studies demonstrating that
smoking, which is associated with a significant decline in CCSP
abundance (2, 4), represents a significant risk factor for
progression to renal failure (31). Additional studies are necessary to determine the mechanisms whereby CCSP deficiency leads to
altered IgA production and to determine whether these changes are
associated with increased risk of systemic disease.
In summary, our results demonstrate that CCSP deficiency is associated
with changes in gene expression in both Clara cells and other cells in
the peribronchial region. Changes in the expression of IgA mRNA are
associated with local increases in IgA protein, suggesting that CCSP
deficiency leads to altered regulation of the immune response in the
lung. Additional studies are necessary to determine whether
immunoregulatory alterations observed among CCSP(/
) mice are a
direct function of CCSP deficiency or whether other changes to Clara
cells contribute to this phenotype.
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ACKNOWLEDGEMENTS |
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This study was supported by National Institute of Environmental Health Sciences Grants ES-08964 and ES-01247.
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FOOTNOTES |
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T. M. Watson was supported by National Institute of Environmental Health Sciences Toxicology Training Grant T32-ES-07026.
Address for reprint requests and other correspondence: B. R. Stripp, Univ. of Pittsburgh, Dept. of Environmental and Occupational Health, 3343 Forbes Ave, Rm. 314, Pittsburgh, PA 15260 (E-mail: bstripp{at}server.ceoh.pitt.edu).
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 26 May 2001; accepted in final form 10 September 2001.
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