© 2003 American Thoracic Society DOI: 10.1165/rcmb.2002-0207OC Interleukin-9 Induces Mucous Cell Metaplasia Independent of InflammationCenter for Comparative Respiratory Biology and Medicine, University of California-Davis, Davis, California; Genentech Inc., South San Francisco, California; and Genaera Corporation, Plymouth Meeting, Pennsylvania Address correspondence to: Dr. J. Rachel Reader, Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, 1 Shields Avenue, Davis, CA 95616-8734. E-mail: db4{at}pacbell.net
Interleukin-9 (IL-9) has been strongly implicated in the pathogenesis of asthma, including the overproduction of mucus, in humans and in animal models. We evaluated the inflammatory changes associated with the upregulation of mucus production by examining the time course of inflammation after daily intratracheal IL-9 administration to naive C57Bl6 mice for 9 d. IL-9 induced an asthmatic phenotype, which in general took several days to develop, as assessed by the measurement of airway hyperresponsiveness, pulmonary inflammation, and serum immunoglobulin E. However, within 24 h of a single dose of IL-9, muc5ac mRNA upregulation occurred, and increased numbers of periodic acid Schiff/Alcian blue-positive mucous cells appeared. This response occurred before the development of an inflammatory cell influx and was the result of epithelial metaplasia. It seemed that IL-9 evoked mucous cell metaplasia independent of IL-13 because mRNA tissue evaluation indicated that muc5ac upregulation preceded any increase in IL-13 mRNA expression or detectable levels of IL-13 in the brochoalveolar lavage fluid. Therefore, the upregulation of IL-13 by IL-9 may be responsible for the amplification of mucus production but is not required for its initiation. IL-9 seems to directly stimulate mucous cell metaplasia without the requirement of inflammatory cell influx.
Abbreviations: airway hyperresponsiveness, AHR • analysis of variance, ANOVA • airway pressure-time index, APTI • bronchoalveolar lavage, BAL • bronchoalveolar lavage fluid, BALF • bovine serum albumin, BSA • 5-chloro-2-deoxyuridine, Cldu • Dulbecco's phosphate-buffered saline, DPBS • 6-carboxyfluorescein, FAM • hematoxylin and eosin, H&E • 5-hydroxytryptamine, 5-HT • immunoglobulin E, IgE • interleukin, IL • intratrachael, IT • knock out, KO • lavage fluid protein, LFP • periodic acid Schiff/Alcian blue, PAS/AB • phosphate-buffered saline, PBS • reverse transcriptase/polymerase chain reaction, RT-PCR • 6-carboxy-N,N,N',N'-tetramethylrhodamine, TAMRA • white blood cells, WBC
Allergic asthma is a chronic TH2-mediated inflammatory disease characterized physiologically by airway hyperresponsiveness (AHR) and intermittent airflow obstruction. Recent data suggest that the underlying etiology is orchestrated by TH2 lymphocytes, which result in airway remodeling. One aspect of airway remodeling, excessive epithelial mucus production, is an important cause of airflow obstruction, especially in patients who die in status asthmaticus. It is widely accepted that specific genetic and environmental factors play an important role in the etiology of asthma, and this has led to an intensive search for candidate genes that may predispose individuals to asthma. Several groups have reported a linkage between asthma or its risk factors with human chromosome 5q31-q33, which shares synteny with segments of mouse chromosomes 11, 13, and 18. Chromosome 5q31-q33 contains several candidate genes that may be associated with atopic asthma, including cytokines, growth factors, and growth-factor receptors. In particular, interleukin (IL)-9 has been suggested as a candidate based on linkage disequilibrium between log serum total immunoglobulin E (IgE) and a marker within this gene (1). In mice, these data are supported by evidence that differences in bronchial responsiveness between hyporesponsive C57Bl/6J and hyper-responsive DBA/2J mice is determined in part by a qualitative trait locus that maps to the syntenic region of chromosome 13 (2). IL-9 transgenic and knock out (KO) mice and the intratracheal (IT) installation of recombinant murine IL-9 provide further evidence for an important role for IL-9 in asthma. These models illustrate that IL-9 plays a fundamental role in the development of AHR (3), elevated serum IgE (4), eosinophilic airway inflammation (5–7), and marked upregulation of mucous genes (8, 9). The relevance of these results in mice to humans with asthma is illustrated by the observation that airways of people with asthma show elevated immunohistochemical staining for IL-9 protein and mRNA levels that correlate with AHR and airflow obstruction (10, 11) and increased IL-9 receptor expression as compared with healthy control subjects (12). Expanding knowledge of the wide-ranging effects of IL-9 upon a multitude of key cells that play a role in asthma, including eosinophils, mast cells, lymphocytes, and lung epithelial cells, suggests that IL-9 can induce an array of mediators that may be responsible for the down-stream effects of IL-9 (13). One potentially important cytokine is the TH2 cytokine IL-13, which alone can induce an asthmatic phenotype, including the upregulation of mucous genes and increased numbers of mucous cells in the epithelium (14–16). IL-9 (8) and IL-13 (16, 17) can increase mucous gene expression in a variety of animal models. Although other studies have shown that the IT instillation of IL-9 can induce an asthmatic phenotype (8, 11), the present study enhances our understanding of the pathogenesis by examining key events at different times over a 10-d time course. In addition, we hypothesized that IL-13 is a key downstream mediator associated with increased IL-9 and plays an important role in the induction of the early events seen in the model, specifically the mucous cell metaplasia. These data suggest that IL-9 can initiate mucous cell metaplasia independently of IL-13. The early upregulation of muc5ac expression implies and is consistent with in vitro data suggesting that IL-9 directly contributes to and influences mucous cell differentiation in the mouse airway.
Protocol Male C57Bl6 mice (6 to 8 wk old upon arrival) were purchased from Jackson Laboratories (West Grove, PA). The animals were housed in an Association for the Assessment of Laboratory Animal Care-accredited facility under specific pathogen free conditions. The animal housing facilities were maintained at 72° ± 2°F with a light cycle of 12:12 h (light/dark). Autoclaved rodent chow (Purina, Richmond, IN) and water were provided ad libitum. Sentinel animals were found to be free from disease and parasites. All studies were conducted in accordance with an Institutional Animal Care and Use Committee-approved protocol. On Day 0, mice were ear tagged and anesthetized with isofluorane. Osmotic pumps (Alzet pump model 2002; Durect Co., Cupertino, CA) loaded with 5-chloro-2-deoxyuridine (Cldu) (Sigma, St. Louis, MO) diluted in phosphate-buffered saline (PBS) (75 mg/ml) were implanted subcutaneously between the scapulae. The pumps delivered 9.9 mg Cldu at 37.5 µg/h for 11 d. On Days 1 to 9, mice were anaesthetized, placed on a vertical platform, and dosed daily with 5 µg recombinant murine IL-9 (R&D Systems, Minneapolis, MN). Each dose of IL-9 was delivered intratracheally in 20 µl of 0.1% bovine serum albumin (BSA) (Sigma) in sterile Dulbecco's phosphate-buffered saline (DPBS), administered with a 100-µl, gas-tight glass syringe (Hamilton, Reno, NV) using a 26-gauge, blunt, low-deadspace needle. Sham control animals were also anaesthetized and given 20 µl IT 0.1% BSA in DPBS daily. Mice were killed at 2, 6, 12, and 24 h after a single dose of IL-9 (five animals per group) and at 4, 7, and 10 d after multiple doses of IL-9 (4-d and 7-d time points: five animals per group; 10-d time point: seven animals per group). At the later time points, animals were killed 24 h after their last IT instillation. Sham control mice were killed on Day 10 after daily doses of BSA in DPBS (seven animals). AHR and airway epithelial proliferation were assessed only on Day 10, but bronchoalveolar lavage (BAL) white blood cell (WBC) differential counts, BAL total lavage fluid protein (LFP), and total serum IgE were measured on Days 4, 7, and 10. Additional time points at 2, 6, 12, and 24 h after a single dose of IL-9 were examined for histopathology scores, BAL IL-13 protein, and muc5ac and IL-13 mRNA.
Airway Hyperresponsiveness
Sample Collection
BAL WBC and Cell Differentials
Serum Total IgE and BAL Protein and IL-13 Analysis
Histology and Immunohistochemistry Five-micron sections stained with H&E and PAS/AB were evaluated by light microscopy, and the severity of the inflammatory response and mucous cell metaplasia were each scored on a scale of 0 to 4. The inflammatory response was graded according to the severity of the perivascular and peribronchiolar inflammation, which was largely eosinophilic and lymphocytic with a few neutrophils. A score of 0 was given for no inflammation, 1 (minimal) for rare focal accumulations of inflammatory cells, 2 (mild) for scattered focal accumulations of inflammatory cells, 3 (moderate) for numerous focal accumulations of inflammatory cells and some areas of confluent infiltrate, and 4 (severe) for extensive areas of confluent inflammation. The amount of PAS/AB-positive staining material in the airways was scored 0 for no mucous cells in the airway epithelium, 1 for a few scattered mucous cells, 2 for multiple foci of mucous cells, 3 for confluent epithelial mucous cells, and 4 for confluent mucous cells extending into more distal airways.
Stereology
RNA Isolation
TaqMan Reverse Transcriptase/Polymerase Chain Reaction
Oligonucleotide sequences and target-specific fluorescence-labeled DNA probes were chosen using an internally developed software program. To detect muc5ac, the primer probe set used was forward primer 5'-CCCTTGGATCCATCATCTACA-3' (position 1,295), reverse primer 5'-CCTGGCTACACATCGCATAG-3' (position 1,363), and probe 5'-CCAGACAGACCTTGATGGCCACTG-3' (position 1,317) (total amplicon length 68 bp). To detect IL-13, the primer probe set used was forward primer 5'-CCTACAGAAAACTGCAGCAAGA-3' (position 672), reverse primer 5'-GCCTCAGTTGCCCTGTGT-3' (position 738), and probe 5'-ACCAGGCCACAGGCTGGACTC-3' (position 718) (total amplicon length 66 bp). The probes were equipped with a 5'-reporter dye (6-carboxyfluorescein [FAM]) and a 3'-quencher dye (6-carboxy-N,N,N',N'-tetramethylrhodamine [TAMRA]). The exact concentration of the RNA placed in the reaction plate was determined using the RiboGreen RNA Quantitation Kit (Molecular Probes, Eugene, OR). The Ct values were corrected for RNA concentration before control animal data were subtracted from the experimental group (
Statistical Analysis
Airway Hyperresponsiveness Airway responsiveness was determined on Day 10 after nine doses of IL-9. Mice treated with IL-9 had a significant increase in APTI (Figure 1) when compared with sham control mice (P = 0.003). The response of the IL-9–treated mice was approximately twofold that of the control mice. However, there was no difference in the baseline resistance between the IL-9–treated and sham control groups (baseline pulmonary resistance: IL-9–treated mice 2.14 ± 0.11 sham control mice 2.09 ± 0.11; P = 0.78). The increase in APTI indicates that the mice were hyper-responsive to the 5-HT challenge, a cardinal feature of human asthma.
Pulmonary Inflammatory Response Analysis of BALF from IL-9–treated mice revealed a significant increase in eosinophil numbers at Days 4, 7 (P = 0.02), and 10 (P = 0.005) compared with sham control animals. The total number of BAL inflammatory cells/lung (i.e., WBCs) was significantly increased only at Day 10 (P = 0.02), as was the lymphocyte count (P = 0.02) (Figure 2). Histologic evaluation of tissue sections by light microscopy after staining with H&E demonstrated perivascular and peribronchiolar eosinophilic and lymphocytic inflammation, which increased in severity with increasing numbers of IL-9 doses. At Day 4, no to minimal inflammation was evident; by Day 7, the majority of the animals had mild inflammation; and by Day 10, the severity had increased to moderate in most of the animals (Figure 3). The increase in severity of the inflammatory response in the tissues over time corresponded with the increasing inflammatory infiltrate observed in the BALF. Although all the lung tissues were also stained with toluidine blue, only rare individual mast cells were visualized at any of the time points examined. These mast cells were observed as frequently in sham-exposed mice as they were in IL-9–treated mice.
Lavage Fluid Protein To assess the integrity of the airway epithelium, total LFP levels were measured in the BALF. When compared with sham control animals, LFP increased over time, and the levels were significantly elevated by Day 10 (P = 0.001) (Figure 4). Increases in LFP may signify loss of epithelial integrity, allowing serum proteins to cross the epithelial barrier. Alternatively, an increase in protein may reflect increased protein transport or production of secretory products, including mucins, within the lung.
Serum IgE To investigate the ability of IL-9 to nonspecifically induce IgE production from B cells, serum IgE levels were measured in the sham control animals and at Days 4, 7, and 10 after daily IL-9 installation. No significant increase in serum IgE was seen until Day 10 (P = 0.02) (Figure 4).
Mucus Cell Metaplasia and Induction of muc5ac
IL-13 Protein and mRNA Expression Using Taqman RT-PCR, IL-13 mRNA was examined at the same time points as those used for the evaluation of muc5ac mRNA. The first time point at which a significant increase in IL-13 mRNA could be detected was at Day 4, when mRNA levels in IL-9 treated animals increased to fourfold above that of sham control animals (Figure 6) (P = 0.016 for IL-9 Ct 26.01 ± 0.46 versus sham control Ct 27.64 ± 0.15). To confirm the presence of IL-13 protein, levels were measured in the BALF using a commercially available ELISA. The earliest that IL-13 protein could be detected (detection limit 1.5 pg/ml) in the BALF was at Day 7 (P = 0.002). With continued administration of IL-9, IL-13 levels continued to increase in the BALF (Figure 6).
Epithelial Cell Proliferation
This study extends and clarifies the potential role for IL-9 in the lung. In vivo administration of IL-9 to the airways of mice induced an asthmatic phenotype. The key finding suggests that IL-9 plays a direct role in the initiation of mucus production in airway epithelial cells, an effect that manifests within 24 h of a single IL-9 dose. Furthermore, data derived from the Cldu proliferation assay indicate that IL-9 increases the number of mucous cells in the epithelium by inducing metaplasia rather than hyperplasia. This mucous cell metaplasia appeared before the development of an inflammatory response. We have demonstrated that IL-9 can upregulate IL-13, probably as a result of an indirect mechanism. However, the immediate initiation of mucus production seemed to be independent of IL-13 or required levels of IL-13 below our detection threshold.
Many of the features of an asthmatic phenotype, including AHR, elevated BAL protein, and serum IgE and a predominantly lymphocytic and eosinophilic infiltrate in the BALF and the lung tissue, were observed after nine daily IT doses of IL-9. Although the installation of IL-9 resulted in AHR, AHR may have been dependent on the infiltration of inflammatory cells and the mediators they subsequently produce. Alternatively, a critical threshold dose or repeated exposures of IL-9 may be required to directly affect resident cells (smooth muscle, nervous system, epithelium) before AHR develops. Similarly, significant increases in serum IgE and LFP did not occur until Day 10. The later increase in serum IgE was not unexpected because the induction of B cells to produce IgE in a naive animal takes at least 7 d. Significant inflammation appeared in the BALF and tissue after Day 7, suggesting that the upregulation of inflammatory mediators, including the C-C chemokines eotaxin, MIP-1 The time course of these different responses discussed above varied greatly, suggesting that IL-9 may act primarily as a proinflammatory stimulus by enhancing the production and activity of other cytokines and growth factors. One possibility is that IL-9 causes the growth, differentiation, and recruitment of mast cells. IL-9 was originally described as a mast cell growth factor, and it can also prime mast cells to respond to allergen (18). Similar arguments can be made for T lymphocytes, B cells, and eosinophils because the IL-9 receptor is found on many of the other cell types thought to be crucial in the asthmatic response. IL-9 can potentiate TH2 responses in vivo and in vitro (19) and can increase the release of IgE from IL-4–primed B cells (20). By promoting the upregulation of the IL-5R, IL-9 stimulation results in enhanced eosinophil maturation and increased longevity (21). Airway epithelial cells can be activated by IL-9, resulting in the stimulation of a number of chemokines. In addition, IL-9 causes the upregulation of various T-cell chemoattractants, proteases, ion channels, and selective mucus genes (6, 8, 22, 23), leaving the epithelial cell poised to play a pivotal role in the development and maintenance of asthma. Other animal models involving the manipulation of IL-9 largely support this data. Two independent IL-9 transgenic models had an asthmatic phenotype characterized by AHR, elevated IgE levels, pulmonary eosinophilia, increased numbers of intra-epithelial mast cells, basement membrane thickening, and mucus overproduction (4, 5, 7). One transgenic model using the Clara cell CC10 promoter that resulted in lung selective expression of IL-9 demonstrated an asthmatic phenotype in naive mice (7). When the same group generated a transgenic mouse with inducible lung specific expression of IL-9, transgene induction during adulthood resulted in a phenotype similar to that seen in mice constitutively expressing IL-9 from birth (5). In contrast, the transgenic mouse used by McLane and colleagues, which resulted in ubiquitous expression of IL-9, required antigen stimulation to produce a similar lesion (4). IL-9 over-expression in this model dramatically enhanced the effect of antigenic stimulation compared with similarly treated congenic controls. Limited data using an IT installation of IL-9 produced a similar lesion (3). Recent data suggests that IL-9 may not be obligatory for the development of an asthmatic phenotype. An IL-9 KO mouse sensitized and challenged with ovalbumin developed AHR, pulmonary eosinophilia, IgE production, and mucous cell hyperplasia and maintained increased levels of IL-4, IL-5, and IL-13 in the BALF, suggesting that other TH2 cytokines can act in a compensatory fashion (24). However, this was a constitutive KO mouse, so these compensatory mechanisms may have been promoted during development. Although these KO data suggest that IL-9 may not play a crucial role in driving all of the asthmatic phenotype, KO mice demonstrated an important role for mastocytosis. After a primary lung challenge with a parasitic nematode, wild-type mice displayed a prominent mast cell influx, a feature not observed in IL-9 KO mice. However, with secondary antigen challenge, mastocytosis was restored to a magnitude equivalent to wild-type control mice, indicating that IL-9 is not the sole factor involved in pulmonary recruitment of mast cells (9). Furthermore, IL-9 transgenic mice are characterized by the abnormal appearance of mast cells in the lung parenchyma (7). However, unlike the transgenic IL-9 models, in our IL-9 model, toluidine blue stains on the lungs for the identification of mast cells only demonstrated rare, individual mast cells in the lung tissue at any time point. This suggests that the asthmatic phenotype we observed was probably not caused by the influx of mast cells and that a more chronic exposure or higher local concentrations may be required to increase mast cell numbers within the lung.
In contrast to the effects described above, mucous cells appeared as early as 24 h after the first IL-9 installation. This was accompanied by a marked increase in PAS/AB-positive staining in the airway epithelium largely confined to the proximal airways, although by Day 10, small numbers of mucous cells were appearing in more distal airways. In control mice, only occasional mucous cells were seen in the epithelium. At these earlier time points, when mucous cells were clearly visible in the airway epithelium, BAL and tissue inflammation were minimal, and there was no significant increase in LFP or serum IgE. The rapid appearance of mucous cells in the epithelium after antigen challenge has been previously reported, suggesting a possible role for IL-9 (25, 26). However, contrary to our IL-9 IT model, antigen challenge also resulted in a neutrophil influx that may have released neutrophil elastase or tumor necrosis factor Data from IL-9 transgenic mice further support a role for IL-9 in the upregulation of mucus. Selective over-expression of IL-9 in the lung results in the accumulation of mucus in the airway epithelium (7), and, in a similar IL-9–inducible transgenic mouse, strong PAS/AB staining appeared in the epithelium 7 d after gene induction. However, in that model, only one earlier time point (1 d) was examined (5) The appearance of mucous cells in the epithelium could be a result of epithelial hyperplasia or metaplasia. Immunohistochemistry for Cldu indicated that epithelial proliferation in the proximal airways of IL-9–treated animals was not significantly different from that in the control animals. Because the Cldu-positive staining is indicative of the cumulative incorporation of Cldu from the time of its delivery, these data demonstrate that neither IL-9 nor any of the inflammatory mediators induced by the installation of IL-9 cause epithelial proliferation. This strongly supports a metaplastic rather than hyperplastic mechanism and is likely to be a direct effect of IL-9. Metaplasia rather than hyperplasia is not unprecedented and concurs with experiments in other species and model systems where transdifferentiation rather than proliferation was responsible for increases in mucous cells (29). It has been suggested that this occurs as a result of the differentiation of nongranulated secretory cells to mucous cells (30). Furthermore, in vitro data support the hypothesis that IL-9 can directly upregulate mucin genes (8, 11). Mucin overproduction in asthma contributes to airflow obstruction. In mouse asthma models, the mucous gene responsible for the majority of the increase in mucus is muc5ac, and goblet cell metaplasia in the airways is associated with pulmonary expression of muc5ac mRNA (17). Similarly, in humans with asthma, muc5ac most likely accounts for the increased mucin stores (31). The TH2-derived cytokines are considered important in asthma and have been shown to upregulate the mucous genes in vivo (IL-9, IL-13, and IL-4). Whereas IL-4 is presumed to be necessary for the generation of IL-9– and IL-13–producing TH2 cells, IL-9 and IL-13 are believed to directly regulate goblet cell production and function (8, 14, 15, 32, 33). The inter-related roles of IL-9 and IL-13 seem to be complex, with IL-13 over-expression and IT installation demonstrating many of the same effects produced by IL-9. The exaggerated production of IL-13 is well documented in asthma (34), and a central role for IL-13 in mouse allergic asthma has been demonstrated in several models (14–16). In particular, IL-13 is a potent inducer of mucous cell hyperplasia in the lung (16, 17), and mucous cell metaplasia in an ovalbumin mouse model can be significantly ameliorated by the administration of a soluble IL-13 receptor Fc fusion protein (14, 15). Because IL-13 is a potent mucus inducer and we (unpublished data) and others (5) have shown that IL-9 can induce IL-13, we postulated that IL-9 may cause mucous cell metaplasia indirectly through IL-13 upregulation. To test that hypothesis, we examined the time course of these events in our model. Increased IL-13 mRNA levels were seen at 4 d, but IL-13 protein could not be detected in the BALF until Day 7. In contrast, increases in muc5ac mRNA were detected as early as 24 h after a single IL-9 installation. These data suggest that IL-13 was not necessary for the induction of muc5ac by IL-9 or that the levels of IL-13 required are so low that they are below the threshold of detection. Although quantitative polymerase chain reaction is extremely sensitive and can detect very small increases in RNA levels, it is possible that a very localized release of small amount of IL-13 may go undetected. In our model, the time course of the appearance of IL-13 correlated with BAL WBC counts. Because IL-13 is largely produced by activated TH2 lymphocytes and significant numbers of lymphocytes were not seen in the BALF until Day 10 of the model, this might help dissociate the different roles of IL-9 and IL-13. However, this distinction may be unimportant in asthmatics and possibly in antigen-derived mouse asthma models because lymphocytes that have already undergone maturation and activation are already present within the lung tissue and are probably the source of IL-9 and IL-13. In our experiments, the appearance of PAS/AB-positive mucous cells in the epithelium at 24 h after the installation of a single dose of IL-9 was accompanied by increased levels of muc5ac mRNA at this time point. The magnitude of the muc5ac increase over subsequent time points reflected the severity of the pathology tissue score for PAS/AB-positive cells in the epithelium. In agreement with our data, IL-9 KO mice used in a pulmonary nematode model showed that IL-9 was necessary for the initiation of mucus production (9). This seemed to be independent of IL-13 because IL-13 expression was not impaired and did not compensate for the lack of IL-9 in the primary antigen response. On the contrary, in the same model with repeated exposure or in mice sensitized and multiply challenged with ovalbumin, mucus production occurred despite the absence of IL-9 (24). It is presumed that other TH2 cytokines, notably IL-13, may be acting in a compensatory manner. Two other studies demonstrated a partial reduction in mucus production when antibodies against IL-9 or its receptor were used (11, 35). Interestingly, the BAL from an antigen challenged dog caused in vitro muc5ac upregulation in H292 lung epithelial cells. This upregulation was blocked with an IL-9R antagonist, but an anti-IL-13 antibody had no effect (11). On the other hand, an IL-9–inducible transgenic model has shown that IL-13 is necessary for the increased production of mucus by IL-9 (5). The presence of increased levels of IL-13 mRNA and, later, IL-13 protein, in our study confirms that IL-9 can upregulate IL-13. Because there was a time lag before the IL-13 message appeared, it is likely that this increase is via an indirect effect rather than a direct effect and is potentially caused by the recruitment of IL-13–producing cells into the lung. In conclusion, the data presented here demonstrate that the IT installation of IL-9 produces an asthmatic phenotype that includes AHR, eosinophils and lymphocytes, IgE, LFP, and an increased number of mucous cells. We speculate that many of the features of the IL-9–induced asthmatic response require the upregulation of other inflammatory mediators. On the other hand, IL-9 seemed to initiate a direct increase in mucus-containing epithelial cells as an early event that seemed to be the result of epithelial metaplasia. IL-9 is capable of upregulating IL-13, which may be responsible for the amplification of the response but may not be required for the initiation of mucus production.
The authors thank Lei Putney for the immunohistochemistry, Frank Ventimiglia for the photography, Nancy Tyler for assistance in preparing the manuscript, and the pathology department at Genentech Inc. for processing tissue and lavage samples. Financial support was provided by Genentech, Inc., South San Francisco, California. Received in original form October 8, 2002 Received in final form December 4, 2002
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Abstract
Introduction
60% lung capacity (0.017 ml x body weight in grams). Lung tissue was stained with hematoxylin and eosin (H&E), periodic acid-Schiff/Alcian blue (PAS/AB) (pH 2.5), and toluidine blue. The duodenum was collected as a control for the Cldu immunohistochemistry. 
Ct parameter). Data were expressed as fold change from the control animal RNA. Four mice in each group were evaluated and compared with baseline values from three control animals. 
, MCP-1, MCP -3 and MCP 5 (
