Cardiovascular Research Institute and Departments of Medicine and Physiology, University of California, San Francisco, California 94143-0130
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ABSTRACT |
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Previous work
showed that the Th2 cytokine interleukin (IL)-13 induces goblet cell
metaplasia via an indirect mechanism involving the expression and
subsequent activation of epidermal growth factor receptor (EGFR).
Because Clara cell secretory protein (CCSP) expression has been
reported in cells that express mucins, we examined the effect of IL-13
on CCSP gene and protein expression in pathogen-free rat airways and in
pulmonary mucoepidermoid NCI-H292 cells. Intratracheal instillation of
IL-13 induced CCSP mRNA in epithelial cells without cilia within
8-16 h, maximal between 24 and 48 h; CCSP immunostaining increased in a time-dependent fashion, maximal at 48 h. The CCSP immunostaining was localized in nongranulated secretory cells and
goblet cells and in the lumen. Pretreatment with the selective EGFR
tyrosine kinase inhibitor BIBX1522, cyclophosphamide (an inhibitor of
bone marrow leukocyte mobilization), or a blocking antibody to IL-8
prevented CCSP staining. Treatment of NCI-H292 cells with the EGFR
ligand transforming growth factor-, but not with IL-13 alone,
induced CCSP gene and protein expression. Selective EGFR tyrosine
kinase inhibitors, BIBX1522 and AG1478, prevented CCSP expression
in NCI-H292 cells, but the platelet-derived growth factor receptor
tyrosine kinase inhibitor AG1295 had no effect. These findings
indicate that IL-13 induces CCSP expression via an EGFR- and
leukocyte-dependent pathway.
Clara cell 10-kDa protein; epidermal growth factor receptor; goblet cell; interleukin-13; secretoglobin; uteroglobin
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INTRODUCTION |
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CLARA CELL SECRETORY PROTEIN (CCSP), also known as uteroglobin, CC10, and CC16, is a 16-kDa homodimeric protein secreted by the mucosal epithelia of various organs including lung, uterus, prostate, and breast (28). Despite extensive biochemical characterization, a solved crystal structure (36), and the availability of CCSP null mice (16, 41), the in vivo physiological roles of CCSP remain obscure. Originally described as a protein expressed only in nonciliated bronchiolar epithelial cells (33), CCSP is now thought to be expressed in nongranulated secretory cells throughout the tracheobronchial tree and in some goblet cells (4, 5, 21). In human bronchial specimens, CCSP mRNA (14) and protein levels (2, 29) were reported to be decreased in areas of goblet cell hyperplasia compared with levels in normal-appearing mucosa. However, the regulation of CCSP expression in goblet cells remains unknown.
In pathogen-free rat airways, goblet cell metaplasia involves activation of epidermal growth factor receptor (EGFR) tyrosine kinase activity and subsequent phosphorylation of downstream target molecules including Ras, Raf, mitogen-activated protein (MAP) kinase kinase, and p44/42 MAP kinase (34). EGFR signaling results in the differentiation of basal cells into nongranulated secretory cells and then into the mucin-producing pregoblet and goblet cells (34). The total number of epithelial cells appears to remain constant during goblet cell production, suggesting that EGFR activation promotes selective cell differentiation and not proliferation. Many stimuli utilize EGFR signaling for MUC5AC production in pulmonary mucoepidermoid NCI-H292 cells, suggesting that the EGFR is a convergent pathway for mucin production by airway goblet cells (26).
The Th2 cytokine interleukin (IL)-13 induces goblet cell metaplasia in
rat airway epithelium via an indirect mechanism involving neutrophil
recruitment and the expression and subsequent activation of EGFR
(32). Because CCSP is expressed in some goblet cells, we
examined the effect of IL-13 on CCSP expression in pathogen-free rat
airways in vivo and on NCI-H292 airway epithelial cells in vitro. Here
we show that, in vivo, IL-13 induces CCSP gene and protein expression
in nongranulated secretory cells and in some goblet cells via an EGFR-
and leukocyte-dependent pathway. In vitro, ligand-dependent activation
of the EGFR with transforming growth factor (TGF)- also induced CCSP
gene and protein expression. However, IL-13 alone had no effect on CCSP
expression in vitro, suggesting that IL-13-induced CCSP expression in
the airway epithelium occurs by an indirect mechanism involving EGFR
signaling. These findings are the first to implicate the EGFR signaling
pathway in the regulation of CCSP expression.
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METHODS |
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Animals. Specific pathogen-free, male Fischer 344 rats, weighing 220-240 g (Simonsen Laboratories, Gilroy, CA) were housed in pathogen-free rooms with free access to standard laboratory chow and water. The experimental protocol was approved by the Committee on Animal Research, University of California, San Francisco, CA.
Intratracheal instillation of IL-13. Studies were first performed in rats to determine whether IL-13 influences CCSP expression in carinal epithelium. Groups of animals (n = 5 in each group) were anesthetized with pentobarbital sodium (Nembutal, 50 mg/kg ip; Abbott Laboratories, Abbott Park, IL) and allowed to breathe spontaneously. The laryngeal area and vocal cords were visualized using a high-intensity illuminator (Fiberlite; Dolan Jenner Industries, Lawrence, MA). Sterile phosphate-buffered saline (PBS, 200 µl, control) or IL-13 (recombinant murine IL-13, 500 ng/200 µl of PBS; R&D Systems, Minneapolis, MN) was instilled into the trachea of each animal via a 20-gauge Angiocath catheter (Becton Dickinson, Sandy, UT). This dose was chosen because it induces MUC5AC production maximally in pathogen-free rats (32). In all studies, carinal epithelium was examined to ensure consistent sampling. For the study of CCSP protein expression, carinal epithelium was examined 4, 16, 24, 48, and 72 h after IL-13 instillation or 48 h after instillation of PBS.
Tissue preparation. Animals were euthanized after IL-13 instillation with a lethal dose of pentobarbital sodium (200 mg/kg ip). The systemic circulation was perfused with 1% paraformaldehyde in diethylpyrocarbonate-treated PBS via the left ventricle. For frozen sections, carinal tissue was removed, placed in 4% paraformaldehyde overnight, and then placed in 30% sucrose for cryoprotection. The tissues were embedded in optimal cutting temperature compound (Sakura Finetek, Torrance, CA). For plastic or paraffin sections, tissues were placed in 4% paraformaldehyde overnight, dehydrated with ethanol, and embedded in JB-4 plus monomer solution A (Polysciences, Warrington, PA) or in paraffin. The embedded tissues were cut as 4-µm-thick cross sections and placed on glass slides.
Immunohistochemical staining for CCSP protein. Sections were treated with 0.3% H2O2 in methanol to block endogenous peroxidase activity and blocked with 2% bovine serum albumin (BSA) in PBS. A solution containing 0.05% Tween 20 and 1% BSA in PBS (assay buffer) was used to dilute antibodies. Next, sections were incubated with a rabbit polyclonal antibody to rat CCSP (generous gift from Gurmukh Singh, University of Pittsburgh, Pittsburgh, PA) at a 1:2,500 dilution for 2 h at room temperature. As a control for specific staining, sections were also incubated with assay buffer omitting the primary antibody. Sections were then incubated with biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA) at a 1:200 dilution for 1 h at room temperature. Bound antibody was visualized using the Vectastain ABC staining kit (Vector Laboratories) using 3,3'-diaminobenzidine (Sigma, St. Louis, MO) as a peroxidase substrate.
Quantification of CCSP protein expression in rat airways. Images were recorded as described (32). Starting from a point chosen at random, we measured the CCSP-stained area and total epithelial area in 10 consecutive high-power fields with a phase contrast lens at ×40. Results are expressed as the percentage of total epithelial area that stained for CCSP. The analysis was performed with the public domain NIH IMAGE program, developed at the National Institutes of Health and available by anonymous File Transfer Protocol from zippy.nimh.nih.gov or on floppy disk from the National Technical Information Service (part no. PB95-500195GEI, Springfield, VA).
In situ hybridization for CCSP mRNA in rat airway epithelium.
To assess CCSP gene expression in the airway epithelium of
IL-13-treated animals, we generated an in situ probe specific for rat
CCSP mRNA. Total cellular RNA from rat lung was obtained using TRIzol
reagent (Life Technologies, Grand Island, NY) per the manufacturer's instructions followed by treatment with DNase I. The rat CCSP cDNA has
been cloned and sequenced (27). On the basis of this sequence, we designed PCR primers containing PstI or
HindIII restriction sites at their 5'-ends (forward primer:
5'-TCT GCT GCA GCT CAG CCT CTT CGG A-3'; reverse primer: 5'-TCG AAG CTT
ATT GCA AAG AGG AAG GCG GGG TT-3') to amplify the entire coding region
for the mature protein. After reverse transcription of 1 µg of total
RNA and 35 cycles of PCR amplification with a 60°C annealing
temperature, the 359-bp amplified product was purified, digested
with PstI and HindIII, and directionally cloned
into pBluescript II-SK(-) (Stratagene, La Jolla, CA). The inserted cDNA
was sequenced to confirm fidelity and copy number. This recombinant
plasmid containing the rat CCSP cDNA was linearized and transcribed in
vitro with T7 or T3 polymerase to obtain the antisense or sense probe,
respectively. In situ hybridization with 2,500-3,000
counts · min1 · µl
1 of
antisense or sense probe was performed as previously described (23). After exposure for 7 days at 4°C, the slides were
developed, fixed, and counterstained with hematoxylin.
Pretreatment with inhibitors of EGFR activation and with
inhibitors of neutrophil recruitment in rat airways.
To determine whether IL-13-induced CCSP expression involves EGFR
signaling, we pretreated pathogen-free rats with a selective tyrosine
kinase inhibitor, BIBX1522 (30 mg · kg1 · day
1 ip;
Boehringer Ingelheim Pharma, Ingelheim, Germany), 1 day before IL-13 instillation and daily thereafter until the animals were euthanized. In previous work, this dose of BIBX1522 prevented IL-13-induced Alcian blue/periodic acid-Schiff (PAS) and MUC5AC staining (32). To evaluate the role of leukocytes in
IL-13-induced CCSP expression, we pretreated animals with
cyclophosphamide, an inhibitor of leukocyte mobilization from the bone
marrow (Sigma), or with a blocking antibody to IL-8 (rabbit anti-human
IL-8 antibody; Biosource, Camarillo, CA). Cyclophosphamide was
administered 5 days (100 mg/kg ip) and 1 day (50 mg/kg ip) before IL-13
instillation. Blocking antibody to IL-8 (10 µg) was administered into
the trachea together with IL-13; instillation of anti-IL-8 antibody was
repeated at 12-h intervals until the animals were euthanized. Both of
these treatments reduced neutrophil numbers in the carinal epithelium nearly to control levels (32). Because maximal
expression of CCSP protein was observed at 48 h after IL-13
instillation, carinal sections were examined at this time point in all
inhibition studies.
Cell culture.
Cells from the human pulmonary mucoepidermoid carcinoma cell line
NCI-H292 were grown in RPMI-1640 medium containing 10% fetal bovine
serum, penicillin (100 units/ml), streptomycin (100 µg/ml), and HEPES
(25 mM) at 37°C in a humidified 5% CO2 water-jacketed incubator. When confluent, cells were washed with PBS and incubated with fresh serum-free medium containing either TGF- (human
recombinant TGF-
, 20 ng/ml; Calbiochem, La Jolla, CA) or IL-13 (10 ng/ml; recombinant human IL-13; R&D Systems). Experiments were
terminated at preselected times (for mRNA, 4 and 8 h; for protein,
24 h). As controls, cells were incubated with serum-free medium
alone for the same time periods. In inhibition studies, cells were
pretreated with the selective EGFR tyrosine kinase inhibitors BIBX1522
(5 µg/ml) or tyrphostin AG1478 (10 µm; Calbiochem) 30 min
before the addition of growth factors. The effect of a selective
platelet-derived growth factor receptor (PDGFR) tyrosine kinase
inhibitor, tyrphostin AG1295 (10 µm; Calbiochem), was also examined.
Immunocytochemical staining for CCSP protein in NCI-H292 cells. Cells grown on eight-chamber slides were fixed with 4% paraformaldehyde for 30 min. A solution containing assay buffer was used to dilute antibodies. Cells were incubated with a rabbit polyclonal antibody to human CCSP (1:2,500; gift from Gurmukh Singh) for 1 h at room temperature. As a control for specific staining, cells were also incubated with assay buffer omitting the primary antibody. Incubation with the biotinylated goat anti-rabbit IgG secondary antibody and visualization of bound antibody using the Vectastain ABC staining kit were as described in Immunohistochemical staining for CCSP protein for the in vivo studies. CCSP immunostaining was graded as follows: 0 to 1+, negative to indeterminate staining; 2+, weak but clearly positive cellular staining; and 3 to 4+, strong staining. For statistical analysis, negative staining was considered 0 to 1+, and positive staining was considered 2 to 4+. Repeated cell counts and calculation of the percentage of CCSP-positive cells by the same observer and a blinded second observer yielded an intraobserver variability of 2.5 ± 1.2% and an interobserver variability of 4.4 ± 1.5%.
In situ hybridization for CCSP mRNA in NCI-H292 cells. To assess CCSP gene expression in pulmonary mucoepidermoid NCI-H292 cells, we generated an in situ probe specific for human CCSP mRNA. PstI digestion of the CCSP expression plasmid pGEL101 (Ref. 24; generous gift from Anil Mukherjee, National Institutes of Health, Bethesda, MD) yielded a 340-bp DNA fragment containing the entire coding region of mature human CCSP and 53 nucleotides of the pGEM 4Z polylinker. This fragment was purified by preparative low-melting agarose gel electrophoresis and subcloned into the PstI site of pBluescript II-SK(-). The orientation and copy number of the cDNA insert were determined by restriction digests using sites present in the pGEM 4Z polylinker and not present in the CCSP cDNA sequence. Sequence identity to the CCSP cDNA was confirmed by sequencing pGEL101. The preparation of RNA probes and in situ hybridization were performed as described above.
To obtain a quantitative estimate of relative levels of expressed CCSP mRNA, we recorded images at random using bright-field illumination at ×40 magnification. Each video image was stored as a 512 × 480-pixel digital image. Using the NIH IMAGE program described in Quantification of CCSP protein expression in rat airways, we found a threshold value by displaying each digital image in gray-scale and binary black-and-white formats, where the boundary between black and white was the threshold value. The threshold value of the image was adjusted until the threshold (black and white) image accurately represented the gray-scale image in terms of the area subtended by silver grains. The number of particles above the threshold value was then counted by the software program. The number of cells was determined by a manual count.Statistics. All data are expressed as means ± SE. One-way ANOVA was used to determine significant differences between groups. Scheffé's F-test was used for multiple comparisons when statistical significances were identified in the ANOVA. A probability of <0.05 for the null hypothesis was considered a statistically significant difference.
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RESULTS |
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IL-13 induces CCSP gene and protein expression in rat airway
epithelium.
We examined the effect of instilled IL-13 on CCSP expression in
pathogen-free rat airways. Control epithelium at 48 h expressed low levels of CCSP mRNA (Fig. 1) and
protein (Fig. 2). IL-13 induced CCSP
mRNA expression in the airway within 8-16 h; expression was maximal at 24-48 h and decreased by 72 h (Fig. 1). CCSP
protein was present at levels significantly above control at 24 h
and maximal at 48 h after instillation (Fig. 2). No specific
hybridization was observed with a sense probe for CCSP mRNA (Fig. 1).
These results show that IL-13 induces CCSP gene and protein expression in airway epithelium time dependently.
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Nongranulated secretory cells and goblet cells express CCSP gene
and protein.
Next, we characterized the cells that expressed CCSP gene and protein
in response to IL-13. CCSP gene expression in the airway epithelium was
heterogeneous (Fig. 1). Phase contrast microscopy revealed that CCSP
gene expression was restricted to nonciliated epithelial cells (data
not shown). Some epithelial cells containing CCSP protein were columnar
in shape, extended from the basal lamina to the lumen, and contained
PAS-positive granules, characteristics consistent with nongranulated
secretory cells and early pregoblet cells (Ref. 25; Fig.
3A). Other CCSP-positive cells
contained numerous PAS-positive granules (pregoblet and goblet cells).
Ciliated cells and basal cells contained no CCSP protein. No staining
for CCSP was observed when the primary antibody was omitted (Fig. 3B).
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Inhibition of EGFR tyrosine kinase activity and of neutrophil
recruitment prevents IL-13-induced CCSP expression.
IL-13-induced goblet cell metaplasia and mucin production in
pathogen-free rats involve EGFR signaling and neutrophil recruitment (32). We examined the effects of EGFR blockade and of
inhibition of neutrophil recruitment on IL-13-induced CCSP expression.
Control epithelium contained rare CCSP-positive nongranulated secretory cells, characterized by coarse vacuoles distributed throughout the
cytoplasm, without luminal staining. After IL-13 instillation, CCSP was
present in large secretory granules in the apexes of goblet cells,
nongranulated secretory cells, and on the luminal surface (Fig.
4A). Pretreatment with a
selective EGFR tyrosine kinase inhibitor, BIBX1522, cyclophosphamide
(an inhibitor of leukocyte mobilization from the bone marrow), or with
an anti-IL-8 antibody prevented IL-13-induced CCSP staining (Fig.
4B). Intraepithelial CCSP staining was almost completely
abolished by these treatments, but some CCSP remained on the luminal
surface after pretreatment with BIBX1522 or cyclophosphamide. Together,
these data implicate EGFR activation and leukocyte recruitment in
IL-13-induced CCSP expression.
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The EGFR ligand TGF-, but not IL-13 alone, induces CCSP
expression in NCI-H292 cells.
Next, we examined the effects of IL-13 and ligand-dependent EGFR
activation on CCSP expression in NCI-H292 cells. NCI-H292 cells express
EGFR on their surfaces and produce MUC5AC in response to EGFR
activation with TGF-
(34). NCI-H292 cells also express intercellular adhesion molecule (ICAM)-1 after IL-13 treatment (3), suggesting that these cells express receptors for
IL-13. Control NCI-H292 cells contained sparse staining for CCSP (Fig. 5A). IL-13 did not increase
the intensity of staining for CCSP (Fig. 5B) or the number
of CCSP-immunopositive cells (Fig. 5I) and had no effect on
CCSP gene expression (Fig. 6, B and
I). However, TGF-
markedly
increased the number of CCSP-positive cells (Fig. 5, C and
I) as well as CCSP mRNA levels (Fig. 6, C and
I). No staining for CCSP in NCI-H292 cells was observed when
the primary antibody was omitted (Fig. 5, G and
H). Pretreatment with selective EGFR tyrosine kinase
inhibitors (BIBX1522, AG1478) prevented the TGF-
-mediated
increase in CCSP-positive cells (Fig. 5, D, E, and I) and CCSP mRNA expression (Fig. 6, D,
E, and I). The sense probe for CCSP was negative
(Fig. 6, G and H). The selective PDGFR tyrosine
kinase inhibitor AG1295 was without effect. Together, these results
implicate EGFR activation in the induction of CCSP gene and protein
expression in NCI-H292 cells and show that IL-13 has no effect on CCSP
expression in vitro.
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DISCUSSION |
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The function and regulation of CCSP in the airway epithelium remain unknown. Because IL-13 has been shown to produce goblet cell metaplasia (7, 39) via an EGFR-dependent pathway (32) and because previous studies reported that goblet cell mucins and CCSP often colocalize in airway epithelial cells (4, 5, 21), we examined the effect of IL-13 on CCSP expression in pathogen-free rat airway epithelium in vivo. Here we show that, in vivo, instilled IL-13 results in CCSP gene and protein expression in nongranulated secretory cells, pregoblet cells, and some goblet cells.
To determine whether IL-13 causes CCSP expression by a direct effect on the airway epithelium, we studied CCSP expression in human airway epithelial NCI-H292 cells in vitro. NCI-H292 cells produce MUC5AC mucin via an EGFR-dependent pathway (34). They have not previously been reported to express CCSP. NCI-H292 cells express ICAM-1 (3) and release IL-1 receptor antagonist protein (20) in response to IL-13, suggesting that NCI-H292 cells express IL-13 receptors. IL-13 is reported to have no effect on MUC5AC mucin expression in NCI-H292 cells (22). Likewise, IL-4 is reported not to induce mucin expression in human bronchial epithelial cells in vitro (13). IL-13 also had no effect on CCSP secretion in human bronchial epithelial BEAS-2B cells (40). In the present study, IL-13 had no effect on CCSP gene or protein expression in vitro. This result suggests that, in vivo, IL-13 causes CCSP expression via an indirect effect on airway epithelial cells.
How does IL-13 cause CCSP expression in the airway epithelium? We know from recent work that instilled IL-13 causes time-dependent neutrophil and eosinophil recruitment into the airways (32) and that neutrophils induce mucin production via the activation of EGFR (35). To test the role of IL-13-induced leukocyte recruitment on CCSP expression, we examined the effects of cyclophosphamide and an IL-8 blocking antibody in vivo. Pretreatment with cyclophosphamide prevented IL-13-induced CCSP protein production, implicating leukocytes in the response. Furthermore, pretreatment with an IL-8-blocking antibody also blocked IL-13-induced CCSP production. IL-8 selectively activates neutrophils but not eosinophils, resulting in the adhesion of neutrophils to epithelium (11). These results implicate a cascade involving neutrophil recruitment and activation in IL-13-induced CCSP production.
Because IL-13-induced goblet cell metaplasia and MUC5AC mucin
production require EGFR activation (32), we examined the
effects of EGFR blockade on CCSP production in vivo and in vitro.
Selective inhibition of EGFR tyrosine kinase phosphorylation by
BIBX1522 prevented IL-13-induced CCSP protein production in vivo,
implicating EGFR activation in the response. In NCI-H292 cells in
vitro, the EGFR ligand TGF- induced CCSP gene and protein
expression. TGF-
-mediated induction of CCSP expression was prevented
by the selective EGFR tyrosine kinase inhibitors, BIBX1522 and
AG1478, but not by the PDGFR tyrosine kinase inhibitor AG1295,
implicating a pathway downstream of EGFR as being required for CCSP
expression. Together, the results of this study suggest that, in vivo,
IL-13 induces CCSP expression by an indirect mechanism involving
neutrophil recruitment and subsequent expression and activation of EGFR.
EGFR-mediated goblet cell metaplasia involves the differentiation of
nongranulated secretory cells that express EGFR into pregoblet and then
into goblet cells. Here we show that CCSP and MUC5AC mucin are induced
by the EGFR signaling pathway and that these two proteins are often
present in the same pregoblet and goblet cells. However, our results
suggest that CCSP is expressed early and MUC5AC expressed later, in
EGFR-mediated goblet cell differentiation. We observed the induction of
CCSP mRNA in nonciliated epithelial cells within 8 h and CCSP
protein in nongranulated secretory, pregoblet, and some goblet cells
within 24 h of IL-13 instillation. Others have also reported the
presence of CCSP mRNA in nonciliated columnar cells of human bronchial
epithelium (5, 9, 14, 21). However, in studies evaluating
CCSP expression in airway epithelium, CCSP mRNA and protein levels were
decreased in bronchi containing diffuse goblet cell metaplasia compared with bronchi containing normal-appearing epithelium (2, 14, 29). There were also fewer CCSP-immunopositive cells in the proximal airways of older IL-4 transgenic mice compared with their younger counterparts (12), suggesting that chronic
exposure to IL-4 results in downregulation of CCSP expression. In
contrast, several studies have shown that exposure to tobacco
smoke (15, 17), ozone (1), and insecticides
(6) results in increased CCSP content within
nongranulated secretory cells. We found that CCSP gene and protein
expression peaked at 48 h and decreased 72 h after a single
dose of IL-13. Nongranulated secretory cells expressed CCSP, but not
MUC5AC, in response to EGFR activation. Meanwhile, some goblet cells
expressed MUC5AC, but not CCSP, in response to the same signal.
Therefore, we suggest that differentiation of nongranulated secretory
cells into pregoblet cells and then into goblet cells is associated
with EGFR-mediated CCSP expression but that mature, terminally
differentiated goblet cells may no longer express CCSP in response to
EGFR signaling (Fig. 7). This model would
explain the differences in CCSP expression between acute and chronic
models of goblet cell metaplasia. It is also consistent with our
observations in pathogen-free rats: CCSP protein was increased in
nongranulated secretory cells, pregoblet cells, and some goblet cells
in the first few days after ovalbumin challenge or instillation with
tumor necrosis factor- and TGF-
. Both of these stimuli cause
goblet cell formation via an EGFR-mediated pathway (34).
However, in a model of chronic epithelial wounding (19),
no CCSP protein was apparent in goblet cells or in the lumen 2 wk after
agarose plug instillation (unpublished observations).
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The function of CCSP in the airway epithelium is unknown. However, a
role for CCSP is suggested by its expression in disease states. CCSP
levels in the airway epithelium are reported to be decreased in asthma
(31), chronic obstructive pulmonary disease (29), and bronchopulmonary dysplasia (30).
Common to these pathologies is neutrophilic inflammation of the
airways. Furthermore, exuberant neutrophil infiltration has also been
reported in CCSP-deficient mice after infection with Pseudomonas
aeruginosa (10) or adenovirus (8) and
after ovalbumin challenge (38). CCSP has been reported to
inhibit neutrophil chemotaxis in vitro (37) and to
function via a receptor-mediated pathway (18). In
addition, recent work has shown that peptides derived from CCSP
attenuate IL-8-induced upregulation of 2-integrins on
neutrophils and subsequent adhesion to endothelial cells
(42). In light of these findings and the results of our
study, we suggest that CCSP may act in a negative feedback fashion to
inhibit neutrophil recruitment and adhesion to the airway epithelium.
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ACKNOWLEDGEMENTS |
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We thank Dr. Gurmukh Singh for providing the polyclonal antibodies for rat and human CCSP, Dr. Anil Mukherjee for providing the cDNA for human CCSP, and Boehringer Ingelheim Pharma KG for providing BIBX1522. We also thank Dr. Yao-Wu Zheng for helpful suggestions and expert advice.
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FOOTNOTES |
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This work was supported in part by a Will Rogers Institute grant.
Address for reprint requests and other correspondence: J. A. Nadel, Cardiovascular Research Inst., Box 0130, Univ. of California, San Francisco, San Francisco, CA 94143-0130 (E-mail address: janadel{at}itsa.ucsf.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.
First published February 22, 2002;10.1152/ajplung.00404.2001
Received 17 October 2001; accepted in final form 14 February 2002.
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