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Am. J. Respir. Cell Mol. Biol., Volume 17, Number 5, November 1997 541-551

Interleukin-4 Alters Epithelial Cell Differentiation and Surfactant Homeostasis in the Postnatal Mouse Lung

Shilpa Jain-Vora, Susan E. Wert, U.-Angela Temann, John A. Rankin, and Jeffrey A. Whitsett

Division of Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, Ohio; and Pulmonary Disease Section, VA Medical Center, West Haven, Connecticut


    Abstract
Top
Abstract
Introduction
Results
Discussion
References

Interleukin-4 (IL-4) is a pleotrophic cytokine which is increased during lung injury and inflammation. Epithelial cell morphology and surfactant homeostasis were assessed in 4-52-wk-old transgenic mice in which IL-4 was expressed in the bronchial and bronchiolar epithelial cells under the control of the Clara cell secretory protein promoter (CCSP-IL-4 mice). IL-4 caused progressive pulmonary infiltration with macrophages, lymphocytes, neutrophils, and eosinophils. Epithelial cell hypertrophy and mucus cell metaplasia were observed in the lungs of CCSP-IL-4 mice at all ages. Airway epithelial cells contained increased neutral glycoproteins and expressed gastric mucin, normally absent in the bronchiolar epithelium of the mouse. Immunohistochemical and biochemical studies demonstrated increased surfactant proteins A and B in lung sections and lung homogenates of CCSP-IL-4 transgenic mice. Increased immunostaining for surfactant proprotein C was also detected in type II epithelial cells of the transgenic mice. In contrast, surfactant protein B and CCSP expression was decreased or was absent in hypertrophic epithelial cells lining the conducting airways of transgenic mice. Lung-specific increase in T-cell proliferative responses to mitogenic stimulation and antibody secretion were detected in CCSP-IL-4 mice. Differentiated characteristics of respiratory epithelial cells were dramatically influenced by the chronic production of IL-4 in the conducting airways. Alterations in lung morphology in the CCSP-IL-4 mice are similar to some of those induced by antigenic stimulation or associated with chronic airway inflammation.


    Introduction
Top
Abstract
Introduction
Results
Discussion
References

Asthma is a common lung disease associated with chronic airway hyperreactivity and inflammation. Pathologic changes seen in the lungs of individuals with asthma include bronchiolar obstruction, mucosal edema, goblet cell hyperplasia, excessive mucus production, collagen deposition, and injury of airway epithelial cells (1). Typically, pulmonary inflammatory changes seen in asthma consist of infiltrates with both eosinophils and Th2-lymphocytes (4). While the precise mechanisms causing asthma are unclear, there is evidence that interleukin (IL)-4 and IL-5 are increased in the lungs of asthmatic individuals, and both cytokines have been proposed to play a role in the pathogenesis of asthma (5, 6).

IL-4 is a cytokine produced primarily by the second subset of T helper lymphocytes (Th2 cells) and by mast cells. Th2 cells also produce a variety of other cytokines, including IL-3, IL-4, IL-5, IL-6, IL-10, IL-13, and granulocyte macrophage colony-stimulating factor (GM-CSF) (7). IL-4 exhibits antitumor activity and is known to increase graft survival after transplantation (7). Antigen presentation in an IL-4-dominated microenvironment leads to the formation of IgE antibodies and enhances the migration of eosinophils and mast cells into regions of inflammation (8). IL-4 induces the differentiation of uncommitted precursor T cells toward the Th2-type cells and inhibits the differentiation of Th1-type cells, thereby encouraging the continued enhancement of Th2-type responses during asthma (8). In animal models of asthma, antigenic challenge results in pulmonary infiltration and changes in respiratory epithelial cells that are similar to those seen in human asthma. IL-4 expression is increased in the lungs of asthmatic individuals in humans and in animal models of asthma and is associated with Th2-type cellular responses (5, 6, 9). The underlying basis of excessive Th2 cell activity in the airways of individuals with asthma is presently unknown.

To assess the role of IL-4 in airway inflammation, a transgenic mouse was created in which murine IL-4 cDNA was selectively expressed in the conducting airway epithelium under the regulation of the Clara cell secretory protein (CCSP) promoter (16). Initial pulmonary studies of the CCSP-IL-4 mice showed increased lymphocytic infiltration without detectable changes in airway resistance after methacholine challenge. However, baseline airway resistance was increased, consistent with narrowing of the airways. Although a correlation between airway inflammation and airway hyperresponsiveness in asthmatic lungs has been described (17, 18), inflammatory responses were dissociated from airway hyperreactivity in the CCSP-IL-4 transgenic mice (16).

In the present study, lung inflammation, respiratory epithelial cell morphology, and surfactant protein expression were assessed during the postnatal period. IL-4 caused progressive pulmonary infiltration with immune cells, as well as mucus cell metaplasia and bronchial and bronchiolar cell hypertrophy as shown previously (16, 19), similar to some of the features associated with human asthma or mouse models of asthma (9, 20). Additionally, an age-dependent increase in surfactant protein concentration was observed in the lung homogenates of CCSP-IL-4 mice. These data indicate that IL-4 produced by airway epithelial cells induced migration of immune cells into the lung and influenced respiratory epithelial cell differentiation and surfactant homeostasis.

    Material and Methods
Top
Abstract
Introduction
Results
Discussion
References

Animals

Mice were housed and studied under IACUC-approved protocols and viral-free conditions in the animal facility at The Children's Hospital Research Foundation, Cincinnati, Ohio. The generation of IL-4 overexpressing mice has been described previously (16). Transgenic mice contain a construct with 2.4 kb of the rat CCSP promoter, 0.5 kb of the murine cDNA, the SV40 small t intron, and the polyadenylation sequence. Two founder CCSP-IL-4 transgenic mice (founder line #29) were crossed into FVBN mice (Charles River Laboratories, Wilmington, MA) and bred to produce homozygous transgenic mice. Fourth generation, 4-52-wk-old, homozygote positive (CCSP-IL-4) and negative (wild-type) mice were used in all experiments (n  >= 3 per experimental group). Since none of the measured parameters changed with age in the wild-type mice, only data generated in the 4-wk-old wild-type mice is represented in the figures. Transgene-positive animals were identified using polymerase chain reaction (PCR) analysis of tail DNA. PCR was performed using the following primers to identify the CCSP-IL-4 construct: 5'-CCC CAG CTA GTT GTC ATC C-3' (sense); 5'-TGA TGC TCT TTA GGC TTT CC-3' (antisense). A Perkin-Elmer thermocycler was used to amplify the inserted IL-4 cDNA: 30 cycles at 94°C, 54.1°C and 72°C for 30 s each, to yield a CCSP-IL-4 PCR product of 391 bp that was detected after ethidium bromide staining of agarose gels (Figure 1).


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  		Figure 1.   PCR of tail DNA. PCR was performed on tail DNA for CCSP-IL-4 transgenic mice produced by breeding heterozygotic CCSP-IL-4 mice. Transgenic mice were identified by a 391 bp CCSP-IL-4 PCR product on a 1% agarose gel, visualized by ethidium bromide. Lane 1: markers; lane 2: nontransgenic mouse; lane 3: homozygous transgenic mouse.

Histologic Methods

Lungs were inflation-fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), pH 7.4, and immersed in fixative for 24 h, as described previously (23). Tissues were then washed with PBS, dehydrated through a series of graded alcohols, and processed into paraffin blocks using an automated tissue processor. Paraffin-embedded tissues were serially sectioned to 5 µm thickness and stained with hematoxylin and eosin for histopathology, McManus's periodic acid-Schiff (PAS) reaction to detect neutral glycoproteins (24, 25), or Masson's trichrome stain for collagen detection (Poly Scientific R & D Corp., Bay Shore, NY) (26).

Immunohistochemical Studies

Antibodies to mature SP-B protein (27, 28), SP-B proprotein (pro SP-B) (29, 30), and SP-C proprotein (pro SP-C) (29) were generated in this laboratory and have been well characterized previously. Antisera to SP-A (31), CCSP (32), and mouse gastric mucin (MGM) (33) were gifts from Dr. Francis McCormack (University of Cincinnati College of Medicine, Cincinnati, OH) (SP-A), Dr. Gurmukh Singh (VA Medical Center, Pittsburgh, PA) (CCSP), and Dr. Samuel Ho (VA Medical Center, Minneapolis, MN) (MGM).

Serial sections of 5 µm were loaded onto polysine-coated slides (Fisher, Atlanta, GA), deparaffinized, and rehydrated. Immunostaining for SP-B, pro SP-B, pro SP-C, CCSP, and MGM was performed as previously described (29, 30, 34). Endogenous peroxidase was quenched for 15 min with 3% H2O2 in methanol. Sections were blocked with 2% normal goat serum in PBS with 0.2% Triton for 2 h at room temperature before incubating overnight at 4°C with the primary antibody at the following dilutions: anti SP-A at 1:5,000 and 1:7,000, anti SP-B at 1:2,000 and 1:4,000 (29, 30, 35), anti pro SP-B at 1:2,000 and 1:4,000 (29, 30, Wert and Profitt, unpublished data), anti pro SP-C at 1:2,000 and 1:4,000 (29, 30, 34), anti CCSP at 1:5,000 and 1:10,000 (30, 34), and anti-mouse gastric mucin (MGM-human MUC5A analog) at 1:15,000 and 1:20,000. The sections were washed in PBS with 0.2% Triton and then incubated for 30 min at room temperature with biotinylated goat anti-rabbit antibody (for surfactant and CCSP proteins) or anti-chicken antibody (for MGM) diluted 1:200 in the blocking solution. Staining was detected using an avidin/ biotin/peroxidase detection system (a standard Vectastain Elite ABC horseradish peroxidase kit; Vector Laboratories, Inc., Burlingame, CA). Sections were incubated with the avidin/biotin/peroxidase complex diluted in blocking solution for 30 min. The enzymatic reaction product was enhanced with nickel cobalt, wherein sections were incubated with nickel-containing diaminobenzidene in 0.1 M acetate buffer for 4 min, then Tris-cobalt for 4 min, to give a black precipitate. The sections were then counterstained with nuclear fast red for 2 min.

Optimal immunostaining for SP-A required pretreatment with 6 N guanidine hydrochloride (36) and predigestion of the sections with 0.02% trypsin in a 0.1 M Tris-saline buffer. Controls included formalin-fixed, paraffin-embedded lung and stomach from wild-type mice of the same age. Omission of the primary antibody with substitution of the blocking buffer was used as a control to check for endogenous biotin and peroxidase activity, as well as nonspecific binding of the secondary antibody. In order to determine relative changes in staining intensity among the various experimental time points, representative sections of transgenic and wild-type mice at each postnatal age were immunostained together by immersion in coplin jars containing the appropriate dilution of each antibody.

In order to assess the number of proliferating inflammatory cells in the lungs of the transgenic mice, 4- and 8-wk-old wild-type and CCSP-IL-4 transgenic mice were injected with 0.3-0.5 ml (1 ml/100 g of body weight) of 5-bromo-2'-deoxyuridine (BrdU)-labeling reagent (Zymed Laboratories, Inc., South San Francisco, CA) 2 h prior to killing the animals. Paraffin-embedded sections were stained for incorporated BrdU in proliferating cells using a biotinylated mouse monoclonal antibody to BrdU and a streptavidin-peroxidase detection system (Zymed). Controls included intestine from each of the BrdU-injected animals. Quantitative analysis of BrdU-positive, proliferating inflammatory cells was performed by counting the number of labeled cells found in 20 random fields from 2 lung sections from each of 3 animals at each time point.

In Situ Hybridization

Transcription vectors, containing mouse SP-B cDNA (pBluescript; Stratagene, La Jolla, CA), and rat CCSP cDNA (pGEM-4Z; Promega, Inc., Madison, WI; a gift from Dr. Sikandar L. Katyal, University of Pittsburgh, Pittsburgh, PA) were used to generate riboprobes labeled with [35S]UTP (specific activity: 1412 Ci/mmole; New England Nuclear Inc., Boston, MA). Plasmids were linearized and RNA synthesis was performed using either the SP6 and T7 (CCSP) or T3 and T7 (SP-B) polymerase and reagents contained in the Riboprobe Gemini Core System II transcription kit (Promega). In situ hybridization was performed as described previously (37). Briefly, fixed, cryoprotected, frozen tissue sections were pretreated with 10 µg/µl proteinase K for 5 min. Hydrolyzed riboprobe fragments of 0.2 Kb (106 cpm/µl) were applied to 5-µm paraffin sections and hybridized at 55°C for 13-16 h. Tissue sections were then washed in 50% formamide, 2× SSC, and 20 mM DTT at 60°C for 60 min; rinsed in 500 mM NaCl, 10 mM Tris-HCl (pH 7.5), and 5 mM EDTA; and treated with 20 µg/ml RNase A for 30 min at 37°C; followed by a series of washes at room temperature with decreasing concentrations of 2× to 0.1× SSC and 1 mM DTT. Slides were autoradiographed with Ilford K5 nuclear track emulsion (Polysciences, Inc., Warrington, PA) for 3-7 d at 4°C and developed with Kodak D19 developer (Eastman Kodak Company, Rochester, NY). Selected examples of hybridization results were photographed with darkfield illumination and phase optics.

Bronchoalveolar Lavage Studies

Animals were killed with a lethal i.p. injection of sodium pentobarbital. The abdomen was opened by a midline incision and the animal was exsanguinated by transection of the inferior vena cava to reduce pulmonary hemorrhage. Lungs were lavaged three times with 1-ml aliquots of sterile PBS, which were pooled and the volume was measured. The total number of viable cells was assessed by trypan blue exclusion using a hemocytometer. The bronchoalveolar lavage (BAL) fluids were then centrifuged at 2,000 rpm for 10 min, and resuspended in 1 ml PBS. The samples were then cytospun, and differential cell counts were performed on cytospin preparations stained with Diff-Quik (Scientific Products, McGaw Park, IN).

ELISA

Lungs from 4- to 24-wk-old wild-type and CCSP-IL-4 transgenic mice were harvested and lung tissue was homogenized in 1 ml of 50 µM phosphate buffer. Additionally, mouse lungs from 8-wk-old wild-type and transgenic mice were lavaged 3 times with 1-ml aliquots of sterile PBS, which were pooled and the volume was measured. SP-A and SP-B protein concentrations were measured in lung homogenates and BAL fluid by ELISA as described previously (38).

T-cell Proliferation Assay

Single-cell suspensions of lung and spleen homogenates were treated with Tris-ammonium chloride to lyse red blood cells (39). T-lymphocytes from lungs and spleens were enriched using discontinuous, 2-layer (40 and 80%) Percoll gradient centrifugation (Pharmacia Biotech, Piscataway, NJ). T cells collected from the interface were > 70% enriched, as confirmed by flow cytometry, using fluorescein isothiocyanate-conjugated antimouse Thy 1.2 antibody (Pharmingen, San Diego, CA). T cells (2 × 104) were cultured in triplicate in 96-well sterile plates (Falcon, Lincoln Park, NJ) along with 1 × 104 mitomycin-treated, allogeneic naive splenocytes as antigen-presenting cells (40) and 2.5 µg/ml Concanavalin A (Con A; Sigma Chemical Co., St. Louis, MO) in RPMI media supplemented with 10% heat-inactivated fetal calf serum (FCS). Control cells were stimulated with media. (3-[4,5-dimethylthiazol-2-yl]- 5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium) (MTS) and phenazine methosulfate were added 48 h later (Promega). The quantity of MTS bioreduced compound-formazan produced was measured by the amount of 490 nm absorbance, which is directly proportional to the number of living cells in culture.

Antibody-secreting Cell Assay

Nitrocellulose-based 96-well sterile plates (Millipore Co., Bedford, MA) were coated with 10 µg/ml antimouse IgA  + IgG + IgM (Zymed) in sterile PBS. Lung and spleen cells were cultured in triplicate at varying cell numbers in cRPMI with 1% heat-inactivated FCS. Antibody-secreting cells were detected 16 h later with alkaline phosphate conjugated anti-mouse IgA, IgG, IgG1, and IgG2a antibodies (Zymed). Plates were developed 1-3 h later using 5-bromo-4-chloro-3-indoyl phosphate and nitroblue tetrazolium. Individual purple plaques representing single antibody-secreting cells were counted.

Statistics

Statistical analyses were performed using Student's t-test or one-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls test to detect differences among individual group means (SigmaStat statistical software for Windows; Jandel Scientific Software, San Rafael, CA). P values < 0.05 were considered significant. Values are reported as mean ± SD.

    Results
Top
Abstract
Introduction
Results
Discussion
References

Chronic Production of IL-4 Causes Progressive Airway Inflammation

As early as 4 wk of age, pulmonary infiltrates consisting of macrophages, neutrophils, and lymphocytes were observed in lungs of CCSP-IL-4 transgenic mice. Histologic analysis of lung sections showed the infiltrating cells to be primarily in the lung parenchyma (16). Total numbers of white blood cells in BAL collected from CCSP-IL-4 transgenic mice increased in an age-dependent manner (P < 0.05; Figure 2A). While the total numbers of infiltrating cells increased with age, differential counts of BAL from transgenic mice showed a decrease in the percentage of macrophages, with a concomitant increase in the relative abundance of other infiltrating cells, especially neutrophils (Figure 2B). However, the numbers of macrophages, neutrophils, lymphocytes, and eosinophils obtained by BAL from CCSP-IL-4 mice were similar at 4 and 8 wk of age (Figure 2C). Increased numbers of macrophages, neutrophils, and lymphocytes were seen in older transgenic mice, when compared with those from 4-wk-old CCSP-IL-4 transgenic mice (P < 0.05). No pathologic changes were observed in lungs of nontransgenic mice under identical vivarium conditions. BrdU labeling and immunohistochemistry were used to assess whether the cellular infiltration was related to increased proliferation of resident inflammatory cells. There was no detectable increase (P > 0.05) in the BrdU labeling in lung sections from 4- and 8-wk-old transgenic mice (4-wk: 11.5 ± 1.5 BrdU positive cells/0.04 mm2, and 8-wk: 12 ± 3 BrdU positive cells/0.04 mm2) when compared with wild-type controls (4 wk: 10 ± 1 BrdU positive cells/0.04 mm2, and 8-wk: 11 ± 1 BrdU positive cells/0.04 mm2).


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  		Figure 2.   Analyses of inflammatory cells in BAL of CCSP-IL-4 transgenic mice. Four-week-old wild-type and 4- to 24-wk-old CCSP-IL-4 mice were lavaged. Total number of viable cells was assessed by trypan blue exclusion (A). Differential percentage of infiltrating cells was assessed for each BAL sample to determine the relative abundance of leukocytes (B). Total number of each infiltrating cell type was assessed for each BAL sample (C). All values are mean ± SD of data collected from 3 mice per group. Significantly different from wild-type mice: *P < 0.05; significantly different from 4-wk-old CCSP-IL-4 transgenic mice: §P < 0.05; and significantly different from 8-wk-old mice: P < 0.05. 

Histologic Changes in the Airway Epithelial Cells

As shown previously, pulmonary epithelial cells in the bronchial and bronchiolar airways were enlarged and hypertrophic (16, 19). Airway epithelial cells stained with PAS, indicating the presence of neutral glycoproteins (Figure 3D). When serial sections were immunostained for the presence of MGM (33), the staining of MGM-protein coincided with that of PAS (Figure 3E). Both PAS-reactive and mucin-positive proteins persisted in the airway epithelia of older CCSP-IL-4 transgenic mice (8-52 wk; data not shown) (19). In contrast, neither PAS-reactivity nor MGM-staining were observed in lungs of nontransgenic mice (Figures 3A and 3B). CCSP protein was also detected in the PAS- and mucin-positive airway epithelial cells of CCSP-IL-4 transgenic mice (Figure 3F). CCSP antiserum uniformly stained the epithelial cells of the conducting airways of nontransgenic mice (Figure 3C). However, the number of CCSP immunopositive cells decreased in the hypertrophic pulmonary epithelial cells of the more proximal airways of older transgenic mice (8-52 wk; data not shown). Modestly increased trichrome staining for collagen was observed surrounding the airways of CCSP-IL-4 transgenic mice at 4 wk of age (Figures 4A and 4B). By visual inspection, this abnormal collagen deposition increased with advancing age in transgenic mice (8-52 wk; data not shown).


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  		Figure 3.   Lung histology. Lungs from 4-wk-old wild-type (A-C) and CCSP-IL-4 transgenic mice (D-F) were inflation-fixed and serial paraffin sections were stained. MGM protein was present in the airway epithelial cells of CCSP-IL-4 transgenic mice (D). PAS- reactive material was also noted in CCSP-IL-4 mouse lungs (B). Bronchial and bronchiolar epithelial cells showed staining for CCSP in CCSP-IL-4 mice (F). Altered immunostaining of epithelial cells in CCSP-IL-4 transgenic mouse lungs is shown by arrows. Lungs from 3 mice were analyzed for each group. b = bronchioles; scale bar is 124 µm.


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  		Figure 4.   Trichrome staining of lung sections. Paraffin sections from 4-wk-old wild-type (A) and CCSP-IL-4 transgenic mice (B) were stained with Masson's trichrome. Collagen is stained in blue and indicated by arrows. Lungs from 3 mice were analyzed for each group. b = bronchioles; scale bar is 124 µm.

Surfactant Proteins in CCSP-IL-4 Mice

At 4 wk of age, SP-A and SP-B immunoreactive material was seen in the alveolar lumen and bronchiolar air spaces of the CCSP-IL-4 transgenic mice (Figures 5E and 5F). Surfactant-positive proteinaceous material persisted in bronchial and bronchiolar airways of 8-52-wk-old transgenic mice (data not shown). In control mice, SP-A and SP-B proteins were detected in bronchiolar and alveolar type II cells, but staining was not detected in the air spaces (Figures 5A and 5B). The number of pro SP-B and mature SP-B immunopositive bronchial and bronchiolar epithelial cells was decreased in the conducting airways of the transgenic mice, but staining in the alveolar type II cells was unchanged (Figures 5C, 5G, 5B, and 5F). Immunostaining for pro SP-C was consistently more intense in the type II cells within the lung parenchyma of CCSP-IL-4 transgenic mice, when compared with wild-type mice (Figures 5H and 5D). In situ hybridization analyses of older founder and offspring CCSP-IL-4 transgenic mice confirmed the loss of SP-B and CCSP mRNA in proximal bronchiolar epithelial cells that normally express high levels of these mRNAs (Figure 6).


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  		Figure 5.   Surfactant protein expression in CCSP-IL-4 transgenic mouse lungs. Lungs from 4-wk-old wild-type (A-D) and CCSP-IL-4 (E-H) transgenic mice were inflation-fixed and embedded in paraffin. Immunopositive SP-A staining was observed in the alveolar spaces of CCSP-IL-4 transgenic mice, shown by arrowheads, when compared with wild-type mice (E, A). Immunoreactive staining for SP-B was also detected in the alveolar spaces of transgenic mice, indicated by arrowheads; loss of SP-B staining in airway epithelial cells is shown with arrow (F, B). Fewer conducting airway epithelial cells from transgenic mice stained positive with anti-pro SP-B antibody when compared with wild-type mice, indicated with arrow (G, C). Pro SP-C staining of type II epithelial cells was more intense in transgenic mouse lungs as shown by arrows (H, D). Lungs from 3 mice were analyzed for each group. b = bronchioles; scale bar is 62 µm.


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  		Figure 6.   In situ hybridization analysis for CCSP and SP-B mRNA expression in 8-16-wk-old wild-type (A-D) and CCSP-IL-4 transgenic founder mice (E-H). Paraffin sections were hybridized with [35S]UTP-labeled riboprobes synthesized from full-length cDNA templates for rat CCSP or mouse SP-B gene. The sections were then photographed using either darkfield illumination to detect the hybridization signals (A, C, E, G) or phase optics to show the underlying morphology (B, D, F, H). CCSP mRNA was detected at high levels in the bronchiolar epithelium (arrows) of wild-type mice (A, B). In the CCSP-IL-4 transgenic mouse lung, CCSP mRNA was detected in the terminal bronchioles (arrows), but not in the larger segmental bronchioles (asterisk) (E, F). SP-B mRNA was detected in alveolar type II cells scattered throughout the lung parenchyma and in the bronchiolar epithelium (arrows) of wild-type mice (C, D). In the CCSP-IL-4 transgenic mouse lung, SP-B mRNA was detected in alveolar type II cells and in the terminal bronchioles (arrows), but not in the larger segmental bronchioles (asterisk) (G, H). In the 8-16-wk-old founder mouse, the terminal bronchioles are hyperplastic and tortuous, and the larger bronchioles contain hypertrophic, PAS /mucin-positive cells (data not shown). The lung parenchyma appears to be consolidated with inflammatory cells. Scale bar is 250 µm.

SP-A and SP-B protein concentrations were increased in lung homogenates from 4-24-wk-old CCSP-IL-4 transgenic mice, when compared with wild-type controls as assessed by ELISA (P < 0.05; Table 1). Similarly, SP-A and SP-B protein concentrations were also markedly increased in BAL fluid of 8-wk-old CCSP-IL-4 transgenic mice, consistent with increased lumenal staining detected by immunohistochemistry (P < 0.05; Table 1).

                              
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TABLE 1
SP-A and SP-B protein concentration in lung homogenate and BAL

Lymphocytic Proliferative Response and Antibody Production in CCSP-IL-4 Transgenic Mice

Since increased numbers of lymphocytes and other inflammatory cells were observed in the CCSP-IL-4 mouse lungs, we proceeded to study some of the immunologic changes associated with chronic IL-4 production. Proliferative responses to stimulation with Con A were increased in T cells isolated from lung homogenates of CCSP-IL-4 transgenic mice when compared with wild-type controls (P < 0.05; Figure 7A). In contrast, response to Con A stimulation by splenic T cells from the same mice, used as an internal control, was not different. Analyses of antibody-secreting cells from lungs of CCSP-IL-4 transgenic mice demonstrated an increase in the total number of IgA, IgG, and IgG1 isotype antibody-secreting cells compared with those from nontransgenic mice (P < 0.05; Figure 7B). There was no difference in the numbers of antibody-secreting cells isolated from spleens of the same mice.


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  		Figure 7.   Immunologic changes in CCSP-IL-4 transgenic mouse lungs. Lung cells and splenocytes were isolated from wild-type (shaded bars) and CCSP-IL-4 transgenic (open bars) mice. (A) Enriched populations of T cells were cultured with mitomycin C- treated, naive allogeneic spleen cells (antigen-presenting cells) and stimulated with 2.5 µg/ml Con A. Increase in cell number was assessed 48 h later. Results are expressed as the percentage of increase in cell number compared with nonstimulated cells (number of cells after 48 h in the absence of Con A). Data are represented as mean ± SD of triplicate cultures; significant difference from wild-type mice: *P < 0.05. Lung cells (B) and splenocytes (C) were isolated from wild-type (shaded bars) and CCSP-IL-4 transgenic (open bars) mice. Cells were cultured on antimouse Ig-coated plates and antimouse IgA, IgG, IgG1, and IgG2a isotype antibody-secreting cells were counted 24 h later. Data are represented as mean ± SD of triplicate cultures; significant difference from wild-type mice: *P < 0.05.

    Discussion
Top
Abstract
Introduction
Results
Discussion
References

Expression of IL-4 in the conducting airway cells of transgenic mice (CCSP-IL-4) caused marked infiltration of immune cells into the lung, epithelial cell hypertrophy, mucus cell metaplasia, increased collagen staining, and increased concentration of surfactant proteins in lung homogenates. Together, these findings indicate that chronic production of IL-4 in the conducting airways of transgenic mice alters both respiratory epithelial cell differentiation and surfactant homeostasis.

IL-4 enhances differentiation, proliferation, and activation of a variety of inflammatory cells, including eosinophils, B cells, T cells, fibroblasts, endothelial cells, neutrophils, basophils, macrophages, and mast cells (7). The intense pulmonary infiltration with macrophages, lymphocytes, neutrophils, and eosinophils in the CCSP-IL-4 transgenic mouse lungs is consistent with the previously described functions of IL-4. IL-4-dependent pulmonary inflammation occurred in the absence of any further antigenic stimulation, and both macrophage and neutrophil numbers changed with advancing age of the transgenic mice. These age-dependent changes in differential cell counts may be related to continued effects of IL-4 or to the local production of other proinflammatory mediators and chemotactic cytokines that may alter migration, proliferation, or differentiation of both pulmonary parenchymal cells and inflammatory cells themselves. Since there was no significant increase in the number of proliferating inflammatory cells in lungs of transgenic mice, it suggests that the age-dependent increases in leukocytes represent an infiltrative response to the local, chronic production of IL-4.

The sites of pulmonary infiltration are consistent with the expression of IL-4 in bronchial and bronchiolar epithelial cells under the regulation of the CCSP promoter (16). In mice, CCSP is expressed in nonciliated tracheal, bronchial, and bronchiolar epithelial cells. Expression of CCSP begins in late gestation and increases postnatally (37). The lack of systemic toxicity or splenic activation supports the concept that the IL-4 transgene and its effects are confined to the lung.

Bronchial and bronchiolar epithelial cells of CCSP-IL-4 transgenic mice were hypertrophic and stained for neutral glycoproteins and gastric mucin. CCSP and SP-B protein and mRNA expression in nonciliated epithelial cells lining the proximal conducting airways of CCSP-IL-4 transgenic mice were decreased, especially in older mice. The distribution of changes in epithelial cell morphology correlated with the gene expression of the IL-4 transgene in the bronchial and bronchiolar epithelium, under the control of CCSP promoter. Abnormalities in epithelial cell morphology were noted as early as 4 wk of age, suggesting that respiratory epithelial cell differentiation is influenced by IL-4. Since numerous complex exocrine, autocrine, or paracrine signals influence airway epithelial cell differentiation, it remains unclear whether changes in the surfactant protein gene expression and mucous cell metaplasia are directly or indirectly modulated by IL-4.

Preliminary visual analysis revealed mildly increased collagen deposition surrounding the bronchial and bronchiolar airways, consistent with transgene expression in the conducting airway. IL-4 is known to cause cell proliferation, as well as cytokine and extracellular matrix production in both fibroblasts and endothelial cells in vitro (41- 44). It is possible that increased leukocytic infiltration and collagen staining in the lungs of CCSP-IL-4 mice may be influenced by paracrine interactions among epithelial, stromal, and infiltrating cells.

Increased concentration of surfactant proteins in lung homogenates and BAL were consistently observed in the lungs of CCSP-IL-4 mice. Pulmonary surfactant is a complex mixture of phospholipids and proteins including SP-A, SP-B, SP-C, and SP-D. Synthesis and secretion of surfactant is carried out primarily by type II airway epithelial cells (45). Pulmonary surfactant is recycled and catabolized by type II epithelial cells and alveolar macrophages to regulate the surfactant pool sizes (46). The concentrations of SP-A and SP-B were increased approximately 3- to 12-fold in the CCSP-IL-4 transgenic mouse lung homogenates and lipid-laden alveolar macrophages were readily detected in lung sections (data not shown). The mechanisms underlying disrupted surfactant homeostasis in the CCSP-IL-4 mice are unknown at present. Pulmonary alveolar proteinosis, defined as an increase in concentration of surfactant proteins and phospholipids in the alveolar and bronchial spaces, has been observed in GM-CSF null mutant mice (38, 47). Recent evidence suggests a role for GM-CSF and its receptors in surfactant clearance (48, 49). Gene-targeted disruption of the beta  subunit of the GM-CSF receptor caused alveolar proteinosis and was corrected by bone marrow transplantation (49). Together, these findings implicate GM-CSF signaling in the clearance of surfactant by alveolar macrophages. IL-4 is known to inhibit GM-CSF-regulated signaling in vitro (50, 51); however, in our study, GM-CSF mRNA was detected in the lungs of CCSP-IL-4 mice by reverse transcription-polymerase chain reaction (RT-PCR) analyses (data not shown). Whether the increased concentration of surfactant proteins seen in the CCSP-IL-4 mice is caused by changes in macrophage function remains to be determined.

Numbers of eosinophils and lymphocytes were also increased in the lungs of CCSP-IL-4 mice. It is known that IL-4 stimulates expression of vascular cell adhesion molecule-1 on endothelial cells, which binds to very late activation antigen-4 expressed on eosinophils and lymphocytes (52). Thus, IL-4 may recruit eosinophils and lymphocytes into the lungs of CCSP-IL-4 transgenic mice. Eosinophils also secrete proinflammatory mediators that may contribute to cellular changes in the respiratory epithelium and bronchial hyperactivity (53, 54). In human asthma, activated CD4 T-cells are found in the respiratory tract (55- 57). The enhanced proliferative responses of T-lymphocytes to mitogenic stimulation with Con A indicate the presence of activated T-cells in transgenic mouse lungs that are probably of the Th2 subset of T-lymphocytes.

Activation of allergen-specific Th2 cells has been linked to the pathology of atopic allergy (58). IL-4 induces B cells to produce IgE, IgA, and IgG1 isotype antibodies which provide protection for the mucosal surfaces in the body (7). In CCSP-IL-4 transgenic mouse lungs, an increase in the total number of IgA and IgG1 isotype antibody- secreting cells was noted. These results indicate that the Th2-associated antibody isotypes are enhanced, as seen in asthma and other mucosal disorders.

Pathologic features including lung inflammation, increased mucin, and surfactant protein secretion seen in CCSP-IL-4 transgenic mice are also seen in asthma and other lung disorders. The present findings indicate that chronic IL-4 production by airway epithelial cells is sufficient to cause pathologic changes similar to those associated with asthma, chronic obstructive pulmonary disease, cystic fibrosis, and other forms of chronic lung inflammation. CCSP-IL-4 mice may be useful in further defining the potential role of IL-4 in the pathogenesis of chronic lung disorders.

    Footnotes

Address correspondence to: Jeffrey A. Whitsett, M.D., Children's Hospital Research Foundation, Division of Neonatology and Pulmonary Biology; 3333 Burnet Ave., Cincinnati, OH 45229-3039. E-mail: JEFF.WHITSETT{at}CHMCC.ORG

(Received in original form December 30, 1996 and in revised form February 24, 1997).

Acknowledgments: The authors thank William Hull for estimating the surfactant protein concentrations using ELISA; Sherri Profitt for advice with histochemical stains and immunohistochemistry; and Dr. Robert Mason (National Jewish Center for Immunology and Respiratory Medicine, Denver, CO) for advice on immunostaining for SP-A. This work was supported by the National Institute of Health grants HL 51832 (J.A.W., S.E.W.) and HL 28623 (J.A.W.).

Abbreviations BAL, bronchoalveolar lavage; BrdU, 5-bromo-2'-deoxyuridine; CCSP, Clara cell secretory protein; Con-A, Concanavalin-A; GM-CSF, granulocyte macrophage colony-stimulating factor; IL, interleukin; MGM, mouse gastric mucin; PAS, periodic acid-Schiff base reaction; PBS, phosphate-buffered saline; RT-PCR, reverse transcription-polymerase chain reaction; -B, surfactant protein (-A; SP (-A, -C); -C), -B; Th2 cells, second subset of T helper lymphocytes.

    References
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Abstract
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References

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