IL-13-induced Clara cell secretory protein expression in airway epithelium: role of EGFR signaling pathway

Suil Kim, Jae Jeong Shim, Pierre-Regis Burgel, Iris F. Ueki, Trang Dao-Pick, Dominic Cheng-Wei Tam, and Jay A. Nadel

Cardiovascular Research Institute and Departments of Medicine and Physiology, University of California, San Francisco, California 94143-0130


    ABSTRACT
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INTRODUCTION
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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-alpha , 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


    INTRODUCTION
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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)-alpha 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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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 · min-1 · 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 · kg-1 · 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-alpha (human recombinant TGF-alpha , 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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ABSTRACT
INTRODUCTION
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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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Fig. 1.   Effect of interleukin (IL)-13 on Clara cell secretory protein (CCSP) gene expression in rat airway epithelium. In situ hybridization for CCSP mRNA was performed 48 h after instillation of vehicle alone (control) or at various times (4, 8, 16, 24, 48, and 72 h) after IL-13 administration. Sense probe for CCSP mRNA 48 h after IL-13 is shown as a control for specific hybridization (sense 48h). Results are representative of studies in 3 different animals for each time point. Photomicrographs are shown at ×40 magnification. Bar, 50 µm.



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Fig. 2.   Effect of IL-13 on CCSP protein expression in rat airway epithelium. Staining for CCSP was performed after instillation of vehicle alone (200 µl sterile PBS, control) or at various times (4, 16, 24, 48, and 72 h) after IL-13 administration. The area of epithelium that stained for CCSP in 10 consecutive high-power fields was measured with a phase contrast lens at ×40 magnification. Results are expressed as the epithelial area that stained for CCSP divided by the total epithelial area. Values are expressed as means ± SE (n = 5; *P < 0.05 compared with control; +P < 0.05 compared with IL-13 at 24 and 72 h).

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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Fig. 3.   Effect of IL-13 on CCSP and periodic acid-Schiff staining in rat airway epithelium. A: 48 h after instillation of IL-13, CCSP-positive staining was present in goblet (G), pregoblet (P-G), and nongranulated secretory cells (S), but not in basal (Ba) or ciliated cells (C). B: no staining for CCSP was evident when the primary antibody was omitted. Results are representative of studies in 5 different animals. Photomicrographs are shown at ×100 magnification. Bar, 20 µm.

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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Fig. 4.   Effect of inhibition of epidermal growth factor receptor (EGFR) tyrosine kinase activation and leukocyte recruitment on IL-13-induced CCSP protein expression in rat airway epithelium. A: sections from an animal treated with vehicle alone (control), IL-13 alone (IL-13/48H), or IL-13 in combination with BIBX1522 (IL-13 + BIBX), cyclophosphamide (IL-13 + cyclo), or anti-IL-8 antibody (IL-13 + anti-IL-B) were stained with a polyclonal antibody specific for rat CCSP. Results are representative of studies in 5 different animals for each condition. Photomicrographs are shown at ×100 magnification (bar, 20 µm). B: the area of epithelium represented in the sections in A that stained for CCSP in 10 consecutive high-power fields was measured with a phase contrast lens at ×40 magnification. Results are expressed as the epithelial area that stained for CCSP divided by the total epithelial area. Values are expressed as means ± SE (n = 5; *P < 0.05 compared with control; +P < 0.05 compared with IL-13 alone).

The EGFR ligand TGF-alpha , 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-alpha (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-alpha 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-alpha -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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Fig. 5.   Effects of IL-13 and of EGFR activation and inhibition on CCSP protein expression in NCI-H292 cells. Immunocytochemical analysis for CCSP protein in confluent NCI-H292 cells incubated for 24 h with: culture medium alone (control, A); IL-13 (B); transforming growth factor (TGF)-alpha (C); TGF-alpha  + BIBX1522 (D); TGF-alpha  + AG1478 (E); or TGF-alpha  + AG1295 (F). No staining for CCSP was evident when the primary antibody was omitted in cells incubated with culture medium alone (control, G) or with TGF-alpha (H). Results are representative of at least 3 independent experiments. Photomicrographs are shown at ×20 magnification. Bar, 50 µm. I: the number of cells represented in the photomicrographs in A-H that stained positive for CCSP was counted in 5 random high-power fields at ×20 magnification. Results are expressed as the number of CCSP-positive cells divided by the number of total cells. Values are expressed as means ± SE (n = 5; *P < 0.05 compared with control; +P < 0.05 compared with TGF-alpha alone).



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Fig. 6.   Effects of IL-13 and of EGFR activation and inhibition on CCSP gene expression in NCI-H292 cells. In situ hybridization for CCSP mRNA in confluent NCI-H292 cells incubated for 8 h with: culture medium alone (control, A); IL-13 (B); TGF-alpha (C); TGF-alpha  + BIBX1522 (D); TGF-alpha  + AG1478 (E); or TGF-alpha  + AG1295 (F). Results of in situ hybridization using a sense probe for CCSP mRNA in NCI-H292 cells incubated for 8 h with culture medium alone (control, G) or with TGF-alpha (H) are shown as a control for specific hybridization. Results are representative of at least 3 independent experiments. Photomicrographs are shown at ×40 magnification. Bar, 50 µm. I: the number of silver grains represented in the photomicrographs (A-H) was counted in 5 random high-power fields at ×40 magnification. Results are expressed as the number of silver grains divided by the number of total cells. Average background (no. of silver grains/cell from in situ using a sense probe, 1.3) was subtracted from each result before statistical analysis. Values are expressed as means ± SE (n = 5; *P < 0.05 compared with control; +P < 0.05 compared with TGF-alpha alone).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha induced CCSP gene and protein expression. TGF-alpha -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-alpha and TGF-alpha . 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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Fig. 7.   Proposed mechanism for IL-13-induced differentiation of nongranulated secretory cells into goblet cells. A: airway epithelium of pathogen-free animals contains few goblet cells and no EGFR. IL-13 administration results in the expression and release of IL-8 (). IL-8 recruits neutrophils (N) from the vascular space. Blockade of IL-8 activity with an anti-IL-8 antibody or inhibition of leukocyte recruitment by cyclophosphamide prevents IL-13-induced CCSP expression. B: neutrophils, upon recruitment and activation, induce EGFR expression and EGFR tyrosine kinase activity (P). Selective inhibition of EGFR tyrosine kinase activity by BIBX prevents CCSP and mucin expression. C: phosphorylation of downstream target molecules (including Ras, Raf, mitogen-activated protein (MAP) kinase kinase, and p44/p42 MAP kinase; not shown) results in a nuclear signal (arrow from P to nucleus, open oval at base of cell) leading to CCSP (paired open circles) and mucin (solid ovals) synthesis (small arrows from nucleus). CCSP is secreted into the extracellular space where it inhibits neutrophil recruitment. D: inhibition of neutrophil recruitment results in resolution of the inflammatory process and in downregulation of the EGFR. The goblet cell is now poised for degranulation.

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 beta 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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Barr, BC, Hyde DM, Plopper CG, and Dungworth DL. A comparison of terminal airway remodeling in chronic daily versus episodic ozone exposure. Toxicol Appl Pharmacol 106: 384-407, 1990[ISI][Medline].

2.   Barth, PJ, Koch S, Müller B, Unterstab F, Wichert P, and Moll R. Proliferation and number of Clara cell 10-kDa protein (CC10)-reactive epithelial cells and basal cells in normal, hyperplastic and metaplastic bronchial mucosa. Virchows Arch 437: 648-655, 2000[ISI][Medline].

3.   Bianco, A, Sethi SK, Allen JT, Knight RA, and Spiteri MA. Th2 cytokines exert a dominant influence on epithelial cell expression of the major human rhinovirus receptor, ICAM-1. Eur Respir J 12: 619-626, 1998[Abstract/Free Full Text].

4.   Boers, JE, Ambergen AW, and Thunnissen FBJM Number and proliferation of Clara cells in normal human airway epithelium. Am J Respir Crit Care Med 159: 1585-1591, 1999[Abstract/Free Full Text].

5.   Broers, JLV, Jensen SM, Travis WD, Pass H, Whitsett JA, Singh G, Katyal SL, Gazdar AF, Minna JD, and Linnoila RI. Expression of surfactant associated protein-A and Clara cell 10 kilodalton mRNA in neoplastic and non-neoplastic human lung tissue as detected by in situ hybridization. Lab Invest 66: 337-346, 1992[ISI][Medline].

6.   Elia, J, Aoki A, and Maldonado CA. Response of bronchiolar Clara cells induced by a domestic insecticide. Analysis of CC10 kDa protein content. Histochem Cell Biol 113: 125-133, 2000[ISI][Medline].

7.   Grünig, G, Warnock M, Wakil A, Venkayya R, Brombacher F, Rennick DM, Sheppard D, Mohrs M, Donaldson DD, Locksley RM, and Corry DB. Requirement for IL-13 independently of IL-4 in experimental asthma. Science 282: 2261-2263, 1998[Abstract/Free Full Text].

8.   Harrod, KS, Mounday AD, Stripp BR, and Whitsett JA. Clara cell secretory protein decreases lung inflammation after acute virus infection. Am J Physiol Lung Cell Mol Physiol 275: L924-L930, 1998[Abstract/Free Full Text].

9.   Hay, JG, Danel C, Chu C-S, and Crystal RG. Human CC10 gene expression in airway epithelium and subchromosomal locus suggest linkage to airway disease. Am J Physiol Lung Cell Mol Physiol 268: L565-L575, 1995[Abstract/Free Full Text].

10.   Hayashida, S, Harrod KS, and Whitsett JA. Regulation and function of CCSP during pulmonary Pseudomonas aeruginosa infection in vivo. Am J Physiol Lung Cell Mol Physiol 279: L452-L459, 2000[Abstract/Free Full Text].

11.   Jagels, MA, Daffern PJ, Zuraw BL, and Hugli TE. Mechanisms and regulation of polymorphonuclear leukocyte and eosinophil adherence to human airway epithelial cells. Am J Respir Cell Mol Biol 21: 418-427, 1999[Abstract/Free Full Text].

12.   Jain-Vora, S, Wert SE, Temann U-A, Rankin JA, and Whitsett JA. Interleukin-4 alters epithelial cell differentiation and surfactant homeostasis in the postnatal mouse lung. Am J Respir Cell Mol Biol 17: 541-551, 1997[Abstract/Free Full Text].

13.   Jayawickreme, SP, Gray T, Nettesheim P, and Eling T. Regulation of 15-lipoxygenase expression and mucus secretion by IL-4 in human bronchial epithelial cells. Am J Physiol Lung Cell Mol Physiol 276: L596-L603, 1999[Abstract/Free Full Text].

14.   Jensen, SM, Jones JE, Pass H, Steinberg SM, and Linnoila RI. Clara cell 10 kDa protein mRNA in normal and atypical regions of human respiratory epithelium. Int J Cancer 58: 629-637, 1994[ISI][Medline].

15.   Ji, C-M, Royce FH, Truong U, Plopper CG, Singh G, and Pinkerton KE. Maternal exposure to environmental tobacco smoke alters Clara cell secretory protein expression in fetal rat lung. Am J Physiol Lung Cell Mol Physiol 275: L870-L876, 1998[Abstract/Free Full Text].

16.   Johnston, CJ, Mango GW, Finkelstein JN, and Stripp BR. Altered pulmonary response to hyperoxia in clara cell secretory protein deficient mice. Am J Respir Cell Mol Biol 17: 147-155, 1997[Abstract/Free Full Text].

17.   Jones, R, and Reid L. Secretory cell hyperplasia and modification of intracellular glycoprotein in rat airways induced by short periods of exposure to tobacco smoke, and the effect of the antiinflammatory agent phenylmethoyloxadiazole. Lab Invest 39: 41-49, 1978[ISI][Medline].

18.   Kundu, GC, Mantile G, Miele L, Cordella-Miele E, and Mukherjee AB. Recombinant human uteroglobin suppresses cellular invasiveness via a novel class of high-affinity cell surface binding site. Proc Natl Acad Sci USA 93: 2915-2919, 1996[Abstract/Free Full Text].

19.   Lee, H-M, Takeyama K, Dabbagh K, Lausier JA, Ueki IF, and Nadel JA. Agarose plug instillation causes goblet cell metaplasia by activating EGF receptors in rat airways. Am J Physiol Lung Cell Mol Physiol 278: L185-L192, 2000[Abstract/Free Full Text].

20.   Levine, SJ, Wu T, and Shelhamer JH. Extracellular release of the type I intracellular IL-1 receptor antagonist from human airway epithelial cells: differential effects of IL-4, IL-13, IFN-gamma, and corticosteroids. J Immunol 158: 5949-5957, 1997[Abstract].

21.   Linnoila, RI, Jensen SM, Steinberg SM, Mulshine JL, Eggleston JC, and Gazdar AF. Peripheral airway cell marker expression in non-small cell lung carcinoma: association with distinct clinicopathologic features. Am J Clin Pathol 97: 233-243, 1992[ISI][Medline].

22.   Longphre, M, Li D, Gallup M, Drori E, Ordoñez CL, Redman T, Wenzel S, Bice DE, Fahy JV, and Basbaum C. Allergen-induced IL-9 directly stimulates mucin transcription in respiratory epithelial cells. J Clin Invest 104: 1375-1382, 1999[Abstract/Free Full Text].

23.   Lou, Y-P, Takeyama K, Grattan KM, Lausier JA, Ueki IF, Agustí C, and Nadel JA. Platelet-activating factor induces goblet cell hyperplasia and mucin gene expression in airways. Am J Respir Crit Care Med 157: 1927-1934, 1998[Abstract/Free Full Text].

24.   Mantile, G, Miele L, Cordella-Miele E, Singh G, Katyal SL, and Mukherjee AB. Human Clara cell 10-kDa protein is the counterpart of rabbit uteroglobin. J Biol Chem 268: 20343-20351, 1993[Abstract/Free Full Text].

25.   Mercer, RR, Russell ML, Roggli VL, and Crapo JD. Cell number and distribution in human and rat airways. Am J Respir Cell Mol Biol 10: 613-624, 1994[Abstract].

26.   Nadel, JA, and Burgel P-R. The role of epidermal growth factor in mucus production. Curr Opin Pharmacol 1: 254-258, 2001[Medline].

27.   Nordlund-Möller, L, Andersson O, Ahlgren R, Schilling J, Gillner M, Gustafsson J-Å, and Lund J. Cloning, structure, and expression of a rat binding protein for polychlorinated biphenyls. J Biol Chem 265: 12690-12693, 1990[Abstract/Free Full Text].

28.   Peri, A, Cordella-Miele E, Miele L, and Mukherjee AB. Tissue-specific expression of the gene coding for human Clara cell 10-kD protein, a phospholipase A2-inhibitory protein. J Clin Invest 92: 2099-2109, 1993[ISI][Medline].

29.   Pilette, C, Godding V, Kiss R, Delos M, Verbeken E, DeCaesteker C, de Paepe K, Vaerman J-P, DeCramer M, and Sibille Y. Reduced epithelial expression of secretory component in small airways correlates with airflow obstruction in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 163: 185-194, 2001[Abstract/Free Full Text].

30.   Ramsay, PL, DeMayo FJ, Hegemier SE, Wearden ME, Smith CV, and Welty SE. Clara cell secretory protein oxidation and expression in premature infants who develop bronchopulmonary dysplasia. Am J Respir Crit Care Med 164: 155-161, 2001[Abstract/Free Full Text].

31.   Shijubo, N, Itoh Y, Yamaguchi T, Imada A, Hirasawa M, Yamada T, Kawai T, and Abe S. Clara cell protein-positive epithelial cells are reduced in small airways of asthmatics. Am J Respir Crit Care Med 160: 930-933, 1999[Abstract/Free Full Text].

32.   Shim, JJ, Dabbagh K, Ueki IF, Dao-Pick T, Burgel P-R, Takeyama K, Tam D, and Nadel JA. IL-13 induces mucin production by stimulating epidermal growth factor receptors and by activating neutrophils. Am J Physiol Lung Cell Mol Physiol 280: L134-L140, 2001[Abstract/Free Full Text].

33.   Singh, G, Singh J, Katyal SL, Brown WE, Kramps JA, Paradis IL, Dauber JH, Macpherson TA, and Squeglia N. Identification, cellular localization, isolation, and characterization of human clara cell-specific 10 kD protein. J Histochem Cytochem 36: 73-80, 1988[Abstract].

34.   Takeyama, K, Dabbagh K, Lee HM, Agustí C, Lausier JA, Ueki IF, Grattan KM, and Nadel JA. Epidermal growth factor system regulates mucin production in airways. Proc Natl Acad Sci USA 96: 3081-3086, 1999[Abstract/Free Full Text].

35.   Takeyama, K, Dabbagh K, Shim JJ, Dao-Pick T, Ueki IF, and Nadel JA. Oxidative stress causes mucin synthesis via transactivation of epidermal growth factor receptor: role of neutrophils. J Immunol 164: 1546-1552, 2000[Abstract/Free Full Text].

36.   Umland, TC, Swaminathan S, Singh G, Warty V, Furey W, Pletcher J, and Sax M. Structure of a human Clara cell phospholipid-binding protein-ligand complex at 1.9 Å resolution. Nat Struct Biol 1: 538-545, 1994[ISI][Medline].

37.   Vasanthakumar, G, Manjunath R, Mukherjee AB, Warabi H, and Schiffmann E. Inhibition of phagocyte chemotaxis by uteroglobin, an inhibitor of blastocyst rejection. Biochem Pharmacol 37: 389-394, 1988[ISI][Medline].

38.   Wang, S-Z, Rosenberger CL, Espindola TM, Barrett EG, Tesfaigzi Y, Bice DE, and Harrod KS. CCSP modulates airway dysfunction and host responses in an Ova-challenged mouse model. Am J Physiol Lung Cell Mol Physiol 281: L1303-L1311, 2001[Abstract/Free Full Text].

39.   Wills-Karp, M, Luyimbazi J, Xu X, Schofield B, Neben TY, Karp CL, and Donaldson DD. Interleukin-13: central mediator of allergic asthma. Science 282: 2258-2260, 1999[Abstract/Free Full Text].

40.   Yao, XL, Levine SJ, Cowan MJ, Logun C, and Shelhamer JH. Tumor necrosis factor-alpha stimulates human Clara cell secretory protein production by human airway epithelial cells. Am J Respir Cell Mol Biol 19: 629-635, 1998[Abstract/Free Full Text].

41.   Zhang, Z, Kundu GC, Yuan CJ, Ward JM, Lee EJ, Demayo F, Westphal H, and Mukherjee AB. Severe fibronectin-deposit renal glomerular disease in mice lacking uteroglobin. Science 276: 1408-1412, 1997[Abstract/Free Full Text].

42.   Zouki, C, Ouellet S, and Filep JG. The anti-inflammatory peptides, antiflammins, regulate the expression of adhesion molecules on human leukocytes and prevent neutrophil adhesion to endothelial cells. FASEB J 14: 572-580, 2000[Abstract/Free Full Text].


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