Introduction

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The normal respiratory epithelium is coated with mucus, secreted by both goblet cells and submucosal gland mucous cells, which provides a variety of protective functions (1). Respiratory mucus protects the lower airways and alveoli from dehydration and damage from inhaled particles, pathogens, or chemical irritants (2). However, excessive secretion from hyperplastic goblet cells contributes to the morbidity and mortality associated with a variety of diseases, including asthma, chronic bronchitis, and cystic fibrosis (3). Moreover, histochemical studies have described abnormalities in mucin glycosylation in airway diseases where an increased amount of intracellular acidic (sulfated) mucins are present (2, 4). Similarly, abnormalities of mucin-type glycoproteins have been reported in lung carcinoma (5), although the regulatory mechanisms of mucus hypersecretion remain poorly understood.

Interleukin (IL)-9 is a T helper (Th) 2 cytokine that was discovered based on its growth-promoting effect on T helper cells (6). Subsequently, several activities were attributed to this protein, including immunoglobulin (Ig) E production by B cells, differentiation of hematopoietic and neuronal progenitor cells, as well as differentiation of mast cells (7). Recent studies have demonstrated that IL-9 and its receptor are strongly implicated in the pathogenesis of allergic asthma in humans and in murine models (10). Moreover, IL-9 transgenic (Tg5) mice develop an exaggerated Th2-type response after allergic sensitization, including an abnormally enhanced tissue eosinophilia (14, 15).

The present study was undertaken to assess the effect of IL-9 on the regulation of mucin hyperexpression from airway epithelial cells in vitro and in vivo. Here, we report that IL-9 upregulates mucin gene expression and production of mucous glycoconjugate in lung epithelial cells in vivo and in vitro. We show that upon IL-9 exposure, patterns of epithelial mucin expression correlated more closely with hypersecretory respiratory disorders than with the normal respiratory mucosae. These observations demonstrate a direct biologic role for IL-9 on airway epithelial cell function(s), in addition to our findings that human airway epithelial cells express the IL-9 receptor (IL-9R). Together, these data add additional support for IL-9 as a mediator in regulating biologic and physiologic responses associated with asthma.

	Materials and Methods

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Animals

Tg5 mice were generated in an FVB/N background as described previously (16). Tg5, FVB/NJ (control mice), and C57BL/6J mice 6 to 8 wk of age were used in this study according to protocol approved by the Institutional Animal Care and Use Committee of Magainin Pharmaceuticals. Control (FVB/NJ) and C57BL/6 mice were purchased from Jackson Laboratory (Bar Harbor, ME). Tg5 mice were generated using a fusion gene consisting of an IL-9 genomic fragment linked to the promoter of the murine pim-1 gene, including the TATA box and the cap site, followed by two copies of the Eµ enhancer and one copy of the Moloney murine leukemia virus long terminal repeat. The circulating IL-9 level is > 1 µg/ml in Tg5 mice, whereas IL-9 is undetectable in the serum of control FVB.

Intratracheal Instillation

C57BL/6 mice were anesthetized daily by inhalation of 1.5% halothane and placed on a vertical platform. Endotoxin-free (as specified by the manufacturer's analysis) recombinant cytokines (R&D Systems, Minneapolis, MN) were diluted in 0.1% bovine serum albumin (BSA) fraction V in sterile phosphate-buffered saline (PBS) solution (GIBCO BRL, Grand Island, NY). The relative biologic activity of each cytokine was adjusted proportional to the mean effective dose of each preparation as determined by the manufacturer. Twenty microliters of recombinant murine (rm) IL-9 (5 µg), recombinant IL-13 (2.5 µg), or recombinant interferon  (IFN-) (5 µg) were administered once each day for 10 d with a 50-µl glass syringe (Hamilton, Reno, NV) containing a 2-in, 26-gauge blunt-end needle inserted directly above the trachea. Control mice were injected with 20 µl of BSA and saline solution.

Tissue Preparation

Lung tissue was derived from various mice and dissected free of hilar lymph nodes and snap-frozen in liquid nitrogen. The tissues were embedded in cryomatrix embedding resin (Shandon Lipshaw, Pittsburgh, PA). Frozen sections (8 µm) were made on an IEC Minotone plus (Shandon Lipshaw) at 20°C. Sections were transferred onto silane-treated glass slides and allowed to dry at room temperature for 1 h. Sections were fixed in formalin (5% formaldehyde in ethanol) for 1 min and stained with alcian blue/periodic acid-Schiff (AB/PAS) and counterstained with hematoxylin and eosin (H&E) (Sigma, St. Louis, MO).

Cells

Lung tissues were obtained from patients undergoing thoracic surgery. After surgery, lung tissues were immediately placed in medium containing 10% heat-inactivated fetal bovine serum (FBS) plus antibiotic. Specimens were rinsed with Hanks' balanced salt solution and then incubated in 0.1% protease (Sigma Type XIV) overnight at 4°C. After digestion, the tissue was sheared through a 20-gauge needle and filtered successively through 45-µm and 15-µm nylon mesh (Tetko Inc., Kansas City, MO). Cells were washed, counted, and then plated at a density of 1 × 10⁶ cells in collagen-coated six-well plates (Falcon Biocoat; Beckton Dickinson, MA) in Dulbecco's modified Eagle's medium/F12 medium supplemented with 2% heat-inactivated FBS, 1% penicillin/streptomycin, 1% fungizon, 0.1 mg/ml L-glutamine, 5 µg/ml transferrin, 5 µg/ml insulin, and 10⁷ M retinoic acid. Cells were cultured in the presence or absence of 50 ng/ml of recombinant human IL-9 (rhIL-9) (R&D Systems). Using these conditions, greater than 95% of the cells were of epithelial origin, as recognized by an antipan cytokeratin antibody.

NCI-H292, a human pulmonary muccoepidermoid carcinoma cell line, was purchased from the American Type Culture Collection (Manassas, VA) and cultured in RPMI 1640 medium supplemented with 10% FBS, 1% penicillin/streptomycin (GIBCO BRL) in a humidified, 5% CO₂ supplemented air-containing incubator at 37°C. Confluent cells were stimulated with or without 50 ng/ml of rhIL-9 for 24 h.

Determination of Mucous Glycoconjugate Production in NCI-H292 Cells

NCI-H292 cells were cultured in eight-well chamber slides and were treated with control or IL-9-supplemented medium for 24 h. Cells were then fixed with formalin, and mucous glycoconjugates were visualized by AB/PAS staining.

Reverse Transcription and Polymerase Chain Reaction

Total RNA was isolated from cells and lung tissues using the Trizol method (GIBCO BRL) following the manufacturer's protocol. Reverse transcriptase/polymerase chain reaction (RT-PCR) was performed on 2 µg of total RNA with an oligo(dT) primer. Complementary DNA (cDNA) corresponding to 20 ng of total RNA was amplified with specific primers (Table 1) by PCR as described (17). Amplification conditions for all reactions were done at 94°C for 30 s, 54° to 62°C for 1.5 min, 72°C for 1.5 min, for 35 cycles. Products of the predicted molecular weight were sequenced on each terminus to confirm their authenticity.

Dot Blot

Tissues were lysed in sample buffer (60 mM Tris, pH 6.8, 1% sodium dodecyl sulfate, 10% glycerol, 5 mM dithiothreitol) and boiled for 5 min. Equal amounts of protein lysate (50 µg) were blotted onto nitrocellulose membrane (Schleider & Schuell, CA) and let dry at room temperature. Membranes were blocked in 5% condensed milk and incubated with mouse monoclonal antibody to MUC2 (#sc-7314, 1:500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or to MUC5AC (clone 45 M1, 1:500; Research Diagnostic, Inc., NJ) overnight at 4°C and with a horseradish peroxidase-conjugated, goat antimouse secondary antibody, using chemiluminescence for detection (Pierce).

Immunocytochemistry

Biopsies were taken from the segmental divisions of the main bronchi of the right lung using alligator forceps (Olympus Corp., Tokyo, Japan) and were fixed immediately in 4% paraformaldehyde/PBS solution for 2 h, washed three times in 15% PBS/sucrose, embedded in optimal cutting temperature compound (Tissue-Tek; Miles Inc., Elkhart, IN), and snap-frozen in isopentane cooled in liquid nitrogen. Cryostat sections 10-µm thick were cut on 0.1% polylysine-coated slides, baked in an oven at 37°C, and stored at 80°C. In order to detect IL-9R, immunostaining was performed using the alkaline phosphatase-antialkaline phosphatase technique as previously described (18). Briefly, sections were incubated with the primary antibody, rabbit antihuman IL-9 receptor polyclonal antibody (#sc-698; Santa Cruz Biotechnology, Inc.) overnight at 4°C. Sections were then incubated with the swine antirabbit immunoglobulin as secondary antibody, followed by the streptavidin complex. The reaction was visualized with fast red alkaline phosphatase substrate. For negative control preparation, the primary antibody was replaced by either nonspecific antibody immunoglobulin or Tris-buffered saline.

	Results

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Induction of Mucous Glycoprotein in the Lung of IL-9 Transgenic Mice

To assess the effect of IL-9 on mucus hypersecretion in the respiratory tract, we studied the effect of IL-9 on mucous glycoconjugate production in transgenic mice that constitutively overexpress murine IL-9 in all tissues. Histologic examination of the airways from Tg5 mice revealed that the epithelium from most conducting airways were stained positive for mucin using AB/PAS (Figure 1C). Higher magnification of stained airway epithelium found the accumulation of mucous glycoconjugate material in the cytoplasm of epithelial cells (Figure 1D). By contrast, airway epithelia from control FVB mice were AB/PAS negative (Figures 1A and 1B).

View larger version (128K): [in this window] [in a new window]   Figure 1.   Histologic staining for mucous glucoconjugate in lung sections from IL-9 transgenic (Tg5) and control (FVB) mice. Frozen sections of lung tissue from FVB mice (A and B) and Tg5 mice (C and D) were stained with AB/PAS. The airway epithelium from Tg5 mice showed a robust AB/ PAS staining in contrast to cells from FVB mice. Higher magnification reveals that mostly nonciliated epithelial cells were hypertrophic and stained positive for mucin in the Tg5 mice (D) in contrast to the airway epithelial cells from the FVB mice (B). (A and C) Original magnification: ×100; (B and D) original magnification: ×200.

IL-9 and IL-13, but Not IFN-, Induce Mucin Hypersecretion from Mouse Airway Goblet Cells

We next determined if the elevated expression of mucin is an inherent feature of lung cells expressing an IL-9 transgene or if it is due to the biologic effects of IL-9. Correlation between strain-specific IL-9 messenger RNA (mRNA) and protein level with airway hyperresponsiveness (AHR) has been previously demonstrated (11, 19). C57BL/6 mice that have very low amounts of steady-state IL-9 protein level in the lung were used to evaluate the ability of rmIL-9 to induce mucin production in vivo. rmIL-9 and control cytokines were administered directly to the lung by intratracheal instillation. The Th2-associated murine IL-13 cytokine has previously been shown to induce mucin expression in epithelial cells and was therefore used as a positive control (20). Both BSA plus saline alone and the Th1-associated IFN- cytokine were used as negative controls. Local administration of IL-9 (Figure 2C) resulted in enhanced AB/PAS staining of airway epithelia, thus confirming the results observed in the Tg5 mice. In contrast, the airway epithelia from mice treated with saline/BSA solution or IFN- were not hypertrophic and were PAS negative (Figures 2A and 2B, respectively). In both IL-9- and IL-13-treated mice, epithelial cells were enlarged by accumulation of glycoconjugate material, shown as purple by the PAS reaction. These results indicate that Th2 cytokines such as IL-9 and/or IL-13 stimulate the production of mucus and induce goblet cell hyperplasia in these animals.

View larger version (141K): [in this window] [in a new window]   Figure 2.   Intratracheal instillation of rmIL-9 induces mucin production. Light micrographs of frozen sections from lung tissue of C57BL/6 mice treated by intratracheal administration with BSA/saline (A), rmIFN- (B), rmIL-9 (C), or rmIL-13 (D). Sections were fixed and stained with AB/PAS. Instillation of IL-9 increased PAS staining in epithelial cells similar to mice treated with IL-13, which was used as a positive control. In contrast, the airway epithelium from mice treated with saline/BSA or IFN- was negative for mucin staining, demonstrating a direct effect of Th2-type cytokines on the regulation of mucus production. Original magnification: ×200.

IL-9 Induces MUC2 and MUC5AC Expression In Vivo

To determine what glycoprotein(s) is induced by IL-9 in airway epithelial cells, we examined the steady-state expression of mucin genes that are normally expressed by epithelial cells. We evaluated mRNA expression of MUC2, MUC4, MUC5AC, and surfactant D (Surf D) in the lung of Tg5 and control (FVB) mice. Surf D is a collagenous glycoprotein produced by lung type II cells (21), whereas MUC2 and MUC5AC are mainly produced by goblet cells (22). Representative RT-PCR analysis is shown in Figure 3. Expression of MUC2 and MUC5AC were found to be upregulated in the Tg5 mice but not in control mice (Figure 3A). In contrast, similar levels of MUC4 and Surf D were found in both mouse strains. IL-9 induction of MUC2 and MUC5AC was confirmed at the protein level by dot blot analysis of whole lung protein using specific monoclonal antibodies. Proteins were extracted from the lungs of FVB and Tg5 mice, and serial dilutions of proteins were blotted onto nitrocellulose membrane. Spleen lysate was used as a negative control because mucin production does not occur in the cell types of this organ. Protein extracts from small intestine and stomach were used as positive controls for MUC2 and MUC5AC, respectively, where the steady-state protein levels for these subtypes have been previously reported for these tissues (32). An increase of MUC2 protein in the lungs of Tg5 mice as compared with FVB mice is shown clearly in Figure 3B. Although MUC5AC was found in the FVB mice, the abundance of this protein was much higher in Tg5 mice (Figure 3B). We confirmed the effect of IL-9 on the induction of MUC2 and MUC5AC in IL-9-treated C57BL/6 mice (Figure 4), thus corroborating the results observed in the Tg5 mice. Interestingly, we found that IL-13 also upregulated the expression of MUC2 and MUC5AC in the lung. This induction was not seen in the absence of reverse transcriptase, therefore ruling out the amplification of this product from genomic DNA by PCR amplification. Intratracheal instillation of IFN- did not affect the pattern of mucin gene expression (Figure 4). MUC4 and Surf D were both found to be constitutively expressed in all samples analyzed (Figure 4).

View larger version (27K): [in this window] [in a new window]   Figure 3.   IL-9 induces MUC2 and MUC5AC expression. (A) Total RNA was extracted from lung tissues of control mice (FVB) and IL-9 transgenic mice (Tg5). RNAs were reverse transcribed and amplified by PCR. RT-PCR amplification was performed as described in MATERIALS AND METHODS using specific oligonucleotides listed in Table 1. Amplifications were performed at 94°C for 30 s, 58°C for 1 min, and 72°C for 1.5 min with a total number of 22 cycles for  actin, 35 cycles for MUC2, MUC3, and MUC5AC, and 25 cycles for MUC4 and Surf D. Samples where the reverse transcriptase was omitted ( lanes) were used as negative control for each condition. PCR products were analyzed on 1.5% agarose gels stained with ethidium bromide. The identity of each PCR product was verified by sequencing the amplification products. (B) Threefold dilutions of equal amounts of lung protein extract from FVB and Tg5 mice were blotted onto nitrocellulose and hybridized with anti-MUC2 or MUC5AC antibodies as described in MATERIALS AND METHODS. Spleen protein lysate was used as a negative control. Proteins extracted from small intestine and stomach were used as positive control for MUC2 and MUC5AC, respectively. The first dilution of all lanes corresponds to 50 µg of protein.

View larger version (57K): [in this window] [in a new window]   Figure 4.   Instillation of IL-9 induces MUC2 and MUC5AC expression. Total RNA was extracted from lung tissue of C57BL/6 mice treated by intratracheal administration of endotoxin-free BSA/saline, recombinant murine IFN-, recombinant murine IL-9, or recombinant murine IL-13. RT-PCR amplification was performed as described in Figure 3.

Induction of Mucous Glycoprotein and Mucin Gene Expression by IL-9 in Human Epithelial Cells

To determine whether IL-9-induced mucin production by epithelial cells was a direct effect of IL-9, we assessed the ability of IL-9 to induce mucous glycoconjugate production in the human pulmonary muccoepidermoid cell line NCI-H292 in vitro. Cells were cultured with or without rhIL-9 and assayed for mucin production by AB/PAS (Figure 5). Although NCI-H292 cells displayed a basal PAS staining, IL-9 significantly stimulated the number and intensity of PAS-positive stained cells (Figures 5B and 5D). No effect on cell density was observed after IL-9 stimulation, suggesting a role of IL-9 in the differentiation of these cells into mucous glycoconjugate-containing goblet cells (data not shown).

View larger version (168K): [in this window] [in a new window]   Figure 5.   IL-9 induces mucous glycoconjugate staining in the human muccoepidermoid NCI- H292 cell line. HE/PAS staining was performed on confluent NCI-H292 cells stimulated with control medium (A and C) or with medium supplemented with 50 ng/ml of rhIL-9 (B and D) for 24 h (original magnification: ×100). IL-9 stimulation increased the number and intensity of cells staining positive for PAS (arrows). (C and D) A higher magnification (×200) of each culture is shown.

Next, we analyzed mucin gene expression in these cells. The housekeeping PMS2 gene was used as an internal standard to correct for variation in the starting concentrations of template cDNA (17). Analysis of steady-state mRNA expression levels for MUC1, MUC6, MUC7, and MUC8 found them to be constitutive in the NCI-H292 cells (Figure 6). We could not detect the expression of MUC3, which is predominantly expressed in goblet cells of the small intestine and duodenum (23). In contrast, MUC2 and MUC5AC expression was induced in the presence of IL-9. To investigate whether tumor necrosis factor (TNF) , IL-4, or IL-13 (all of which have been previously reported to induce mucin gene expression) (20, 24, 25) were involved in mediating the stimulatory effects of IL-9 on mucous glycoconjugate production in these cells, we analyzed the expression of these cytokines by RT-PCR in the various culture treatments. Neither IL-4, IL-13, nor TNF- was expressed upon IL-9 stimulation indicating that IL-9 induction of mucin was independent of these cytokines (data not shown).

View larger version (46K): [in this window] [in a new window]   Figure 6.   IL-9-mediated MUC2 and MUC5AC expression from human airway epithelial NCI-H292 cells. Confluent NCI-H292 cells were stimulated for 24 h with or without 50 ng/ml of rhIL-9. Total RNA was extracted and RT-PCR amplification was performed for mucin gene expression using specific primers listed in Table 1. Amplification of the hPMS2 housekeeping gene was used as a control for sample loading and integrity. Samples where reverse transcriptase was omitted ( lanes) were used as negative control for each condition. PCR products were analyzed on 1.5% agarose gels stained with ethidium bromide. The identity of each RT-PCR amplification product was verified by sequencing.

IL-9-Mediated Mucin Production from Human Primary Lung

The effect of IL-9 on mediating mucin production was also assessed on primary cultures of human airway epithelia. These cultures consisted primarily of lung epithelial cells as determined by morphology and histologic staining (data not shown). Cells were grown in the presence or absence of 50 ng/ml of rhIL-9 for 4 d, total RNA was extracted, and gene expression was determined by RT-PCR. Interestingly, the same pattern of mucin induction was observed in the human primary lung culture, therefore confirming the results observed in vivo in IL-9-expressing mice. Figure 7 shows the increase of MUC2 and MUC5AC cDNA levels after IL-9 stimulation, whereas the other mucin genes are constitutively expressed. MUC4 and MUC5B were also found to be constitutively expressed in both conditions (data not shown).

View larger version (43K): [in this window] [in a new window]   Figure 7.   IL-9-mediated MUC2 and MUC5AC expression from human primary airway epithelial cell cultures. Human primary airway epithelial cells were cultured with either 50 ng/ml of rhIL-9 or control medium (Ctr) for 4 d. RT-PCR amplifications were performed as described in Figure 6.

IL-9 Expression on Human Airway Epithelial Cells

To demonstrate that IL-9 has a direct effect on human primary airway epithelial cells in vivo, we performed immunostaining analysis for the IL-9R chain, which is the only receptor known to transduce an intracellular signal by IL-9 (7) in human bronchial biopsy samples. As shown in Figure 8, human airway epithelial cells constitutively express IL-9R, thus supporting the view that IL-9 directly induces mucin gene expression in airway epithelial cells by binding to its receptor.

View larger version (116K): [in this window] [in a new window]   Figure 8.   IL-9R expression in human airway epithelium. Immunostaining of endoscopic biopsy sections from patients incubated with an anti-IL-9R antibody (A) or an isotype control antibody (B). These data show that positive immunoreactivity to IL-9R occurs mainly in the epithelium (stained red), whereas no positive stain appeared in the control antibody section. Magnifications of sections are ×400.

	Discussion

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Genetic mapping studies have linked bronchial hyperresponsiveness, atopy, and asthma to human chromosome 5q31 (13, 26), which contains several genes involved in allergic disorders. Subsequent analysis of the genes within this region identified the IL-9 gene as a candidate for asthma susceptibility (9, 19). These data were further supported by two independent studies using IL-9 transgenic mice, both of which found that mice overexpressing IL-9 exhibited increased AHR, elevated airway eosinophilia, and elevated serum IgE levels in contrast to control mice (12, 15). In addition to the physiologic data, several studies have shown a wide range of biologic activities for IL-9 on many of the cell types involved in the allergic inflammatory response (7). Most recently, IL-9 has been shown to induce C-C chemokine expression in lung epithelial cells, which is associated with airway eosinophilia in Tg5 mice (14).

In this report, we investigated the role of IL-9 on mucin production by airway epithelial cells in vitro and in vivo. Here, we show that IL-9 expression in the lungs induces the differentiation of airway epithelial cells into mucous glycoconjugate-containing cells and induces the expression of a subset of mucin genes. These results are in accord with the recently published data by Temann and coworkers (12) that find an increase in goblet cells and mucus hypersecretion in transgenic mice that locally overexpress IL-9 in the lung. Expression studies showed that the increase in mucin production by IL-9 in whole lungs correlated with elevated steady-state expression of the MUC2 and MUC5AC genes in the lung of Tg5 mice, whereas no expression was observed in the control mice. In contrast, Surf D and MUC4 genes were expressed at similar levels in both the Tg5 and control mice, suggesting that IL-9 regulates a specific subset of mucin genes. IL-9-mediated mucus hypersecretion was further corroborated in studies using C57BL/6 mice where recombinant IL-9 protein was directly administered into the airway. These studies showed that the presence of IL-9 in the airway results in an increased number of PAS-positive epithelial cells and induced MUC2 and MUC5AC gene expression in the lung. Similar results were observed in IL-13-treated mice but not in mice treated with the Th1-associated IFN- cytokine. These data suggest that IL-9 might contribute to the airway obstruction, mucus hypersecretion, and goblet cell hyperplasia that are commonly associated with Th2-polarized inflammatory responses in the airway (27). Importantly, IL-9 induction of mucin was also confirmed in human primary lung cultures and in the pulmonary epithelial NCI-H292 cell line, further supporting a direct role of IL-9 on mucin production in airway epithelial cells. These data also demonstrate that IL-9-induced mucin production in the airway is a conserved process in rodents and humans and suggests an important role for this biologic response in allergic inflammation. The expression of all other mucin genes tested were constitutively expressed, which is in accordance with previous reports (22, 28, 29). The MUC2, MUC5AC, and MUC-6 genes are clustered at chromosomal location 11p5.5 (30), but only MUC2 and MUC5AC were upregulated by IL-9, suggesting a gene-specific regulation of these genes exists instead of a locus-activating mechanism since MUC6 expression was unaffected by IL-9 levels. Whereas MUC5AC has been previously shown to be expressed in epithelial goblet cells (22), and correlates well with cytokine-induced mucin hypersecretion (31), a role for MUC2 appears to be equivocal. Previous reports suggest that MUC2 is not a prominent mucin subtype in respiratory secretion (32), whereas others have suggested that it is involved in the pathogenesis of inflammatory airway disorders (33, 34). The latter view is supported by the finding that its expression is elevated in the respiratory tract after inflammation and in the bronchial cells of smokers and patients with chronic bronchitis (35).

Airway epithelial hypertrophy, mucus hypersecretion, and goblet cell hyperplasia are also characteristics of IL-4 and IL-13 transgenic mice (20, 24). This suggests a mechanism by which Th2-polarized inflammatory responses can result in airway obstruction, mucus hypersecretion, and goblet cell hyperplasia, which are commonly seen in the airways of rodent asthma models. To gain further insights into the mechanism(s) by which IL-9 mediates its effect(s) in the lung, studies were undertaken to determine whether IL-9 induces the production of cytokines that have been previously implicated in mucus hypersecretion. Analysis of IL-4, IL-13, and TNF- (20, 24, 25) expression showed that none of these cytokines was upregulated in the lung of IL-9-treated mice (data not shown), suggesting that IL-9- induced mucin gene expression is independent of at least these other cytokines. Vice versa, no expression of IL-9 or IL-4 was found in IL-13-treated mice, indicating that mucin expression by IL-13 is also independent of these two cytokines (data not shown). It is unclear as to why the various Th2 cytokines are able to independently induce mucin production in epithelial cells. The immunomodulatory functions of Th2 cytokines have identified IL-4, IL-13, and IL-9 as key targets in mucosal inflammation and exacerbation of disease in models of AHR. These cytokines share a variety of effects that are relevant to asthma and other Th2-dominated inflammatory disorders, including the ability to induce IgE production, chemokine expression, and increased number of eosinophils (7, 14, 20, 38, 39). However, there is evidence as to the comparative significance of each cytokine in the mechanisms underlying the induction of inflammation and airway hypereactivity. For example, IL-4 transgenic mice did not present hyperreactivity after challenge with methacholine (40), whereas expression of IL-9 or IL-13 induced hyperreactivity on methacholine challenge (12, 20). One possibility for the need of epithelial cells to be able to respond independently to various Th2 cytokines is to overcome antigen-specific induction of cytokines that may occur during Th2-associated host defense, where a particular antigen induces only one Th2 cytokine. Additional studies will be required to address this hypothesis and to get a better understanding of these mechanisms.

Mucins are localized in the intracellular granules of goblet cells and are discharged in response to a wide variety of stimuli, including irritants, inflammatory mediators, and nerve activation. Under normal conditions, ciliated goblet cells proliferate and differentiate into mucin-producing cells in order to maintain airway homeostasis and the epithelial cell population. In addition to participating in airway defense, the number of mucin-producing cells increases upon chronic airway insults, resulting in an increased output of mucus. Hypersecretion of mucin in the lung is a common clinical feature of asthma (41, 42), although the physiologic consequence of mucus hypersecretion on the pathogenesis of the disease is not clear. The upregulation by IL-9 of mucous glycoconjugate-containing cells and mucin gene expression might therefore be one of the mechanisms mediating the asthmalike phenotype in IL-9-expressing mice. The finding that human association studies performed on normal and asthmatic patients showed a significant increase in IL-9-expressing cells in the lung of asthmatics compared with controls, suggests that a similar mechanism exists in humans (Dr. Q. Hamid, personal observation). Moreover, our data demonstrate that human epithelial cells express the IL-9R and IL-9 responsive. In particular, the data shown in Figure 7 demonstrate the ability of IL-9 to induce mucin gene expression in human primary lung cultures. Additional studies will be needed to determine the relationship of how IL-9 and the other Th2-associated cytokines may work together to coordinate their effects on airway epithelial cells to produce mucin. The data presented here further support the growing evidence that the IL-9 pathway is critical in the pathogenesis of the asthmatic response and that therapeutic strategies to target IL-9 and/or its signaling pathway may prove to be an effective approach for the treatment of respiratory disorders such as asthma.