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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 593-602, 2002
© 2002 American Thoracic Society
DOI: 10.1165/rcmb.4838

Interleukin-13 Mediates a Fundamental Pathway for Airway Epithelial Mucus Induced by CD4 T Cells and Interleukin-9

Laurie Whittaker, Naiqian Niu, U.-Angela Temann, Amy Stoddard, Richard A. Flavell, Anuradha Ray, Robert J. Homer and Lauren Cohn

Sections of Pulmonary and Critical Care Medicine and Immunobiology, Department of Pathology, Yale University School of Medicine, New Haven, Connecticut; Howard Hughes Medical Institute, New Haven, Connecticut; Department of Medicine, Pulmonary Division, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; and Pathology and Laboratory Medicine Service, V. A. Connecticut Health Care System, West Haven, Connecticut

Address correspondence to: Lauren Cohn, M.D., Section of Pulmonary and Critical Care Medicine, Yale University School of Medicine, 333 Cedar Street, P.O. Box 208057, New Haven, CT 06520-8057. E-mail: lauren.cohn{at}yale.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mucus hyperproduction in asthma results from Th2-induced airway inflammation. Controversy exists about the precise mechanism of this Th2 effect. Although we showed that mucus can be induced by Th2 cells in the absence of interleukin (IL)-4, IL-5, eosinophils, and mast cells, but not without IL-4R{alpha} signaling, others demonstrated that IL-4 and IL-9 can directly stimulate airway epithelial mucus. Using a system in which in vitro-generated T cell receptor transgenic Th2 cells are transferred into recipient mice and activated in the respiratory tract with inhaled antigen, we now show that CD4 Th cells can stimulate mucus only through a common, IL-13–mediated pathway. All Th cytokines depend on IL-13 for this effect and IL-13 acts, not through intermediate inflammatory cells, but on structural cells within the lung, likely the airway epithelium itself. The potency of IL-13 is shown, requiring its complete blockade for a significant reduction in mucus production. We show that mucus induction by Th2 cells does not require nuclear factor-{kappa}B, unlike mucins induced by gram-negative infection. These studies define in vivo pathways that lead to mucus induction and indicate that, whereas IL-13 mediates a dominant pathway for CD4 Th induced inflammation, other inflammatory stimuli activate the epithelium to produce mucus by different pathways.

Abbreviations: antigen-presenting cells, APC • bronchoalveolar lavage, BAL • enzyme-linked immunosorbent assay, ELISA • fluorescence-activated cell sorter, FACS • fluorescence in situ hybridization, FISH • histologic mucus index, HMI • interferon, IFN • interleukin, IL • nuclear factor-{kappa}B, NF-{kappa}B • ovalbumin, OVA • periodic acid Schiff, PAS • phosphate-buffered saline, PBS • peripheral blood mononuclear cells, PBMC • OVA peptide 323–339, pOVA323–339 • reverse transcription–polymerase chain reaction, RT-PCR • T cell receptor, TCR • transgenic, Tg


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In chronic inflammatory diseases of the airways like asthma, excess mucus production causes significant morbidity, as it obstructs airways and contributes to symptoms, including coughing and wheezing (1, 2). In autopsy specimens from patients who died in status asthmaticus, obstructing plugs of mucus and cellular debris have been identified in the small airways. Activated CD4 Th2 cells infiltrate the airways in patients with asthma, and in animal models of asthma Th2 cells have been shown to stimulate mucus hyperproduction (3, 4).

Identifying the specific factors which stimulate mucus has been complex, because Th2 cells, through release of cytokines and chemokines, lead to the activation and recruitment of various inflammatory cells and the release of mediators. We previously showed that mucus production could be induced by Th2 cells that lacked interleukin (IL)-4 or IL-5, and in inflammatory responses that were devoid of eosinophils or mast cells (4, 5). Mucus could not be induced by Th2 cells in mice lacking IL-4R{alpha}, which signals by receptor engagement of IL-4 and IL-13. From these and other studies it was clear that IL-13 played a role in Th2-induced mucus, yet many studies suggested a reciprocal role for IL-4 in mucus production (6–9). In addition, the Th2 cytokine IL-9 appeared to play a key role in the induction of airway epithelial mucus (10–13).

The current studies were undertaken to determine the pathways for mucus induction by Th2 cells, and to find out which cytokines can stimulate mucus, which cells interact to produce mucus, and whether mucus is induced by a common signaling mechanism. Our goal was also to gain insight into how universal are these pathways in chronic inflammatory airway diseases. We now define a single IL-13–mediated pathway by which CD4 T cells stimulate mucus and we map in vivo how IL-13 leads to this highly potent effect on the airway epithelium.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice
IL-13-/- BALB/c mice were graciously provided by Dr. Andrew McKenzie. DO11.10 mice, which are transgenic (Tg) for the T cell receptor (TCR) recognizing ovalbumin (OVA) peptide 323–339 (pOVA323–339) were kindly provided to us on BALB/c background by Ken Murphy (Washington University, St. Louis, MO) and were bred in our facilities. DO11.10 were crossed to IL-13-/- BALB/c mice. Transfer recipients were 6- to 12–wk-old BALB/c, IL-13-/-, p50-/-, or syngeneic, wild-type mice (B6129PF2) (all from Jackson Laboratories, Bar Harbor, ME) and interferon (IFN)-{gamma}R-/- (kindly provided by J. Aguet, Molecular Biology Institute, Zurich, Switzerland and backcrossed 6 generations onto BALB/c). CC10 IL-9 Tg mice were bred at least six generations to BALB/c and then crossed to IL-4R{alpha}-/- mice (generously provided by F. Brombacher, University of Capetown, Capetown, South Africa) and killed at 6–8 wk of age. To generate bone marrow chimeras, female BALB/c (IL-4Ra+/+) and IL-4R{alpha}-/- mice were treated with 1,000 cGy of ionizing radiation followed by intravenous transfer of 5 x 106 bone marrow cells isolated from BALB/c IL-4R{alpha}+/+ or IL-4R{alpha}-/- male mice. Donor cell engraftment was confirmed by fluorescence in situ hybridization (FISH) for the Y chromosome on peripheral blood mononuclear cells (PBMC). Male cells made up >= 95% of the transplanted PBMC, and this was similar to FISH results with nontransplanted male PBMC.

Generation of Th1 and Th2 Cells
To generate Th2 cells from IL-13+/+ or IL-13-/- DO11.10 mice, CD4 T cells were isolated by negative selection as previously described (4) using monoclonal antibodies to CD8 and Class II MHC I-A and anti-Ig–coated magnetic beads (Collaborative Research, Inc., Bedford, MA). In some cases naive CD4 T cells were further isolated using anti-CD62L (Mel14; BD Pharmingen, San Diego, CA) and MACS (Milltenyi, Auburn, CA). To generate Th2 cells from non-Tg mice, C57BL/6 mice were injected intraperitoneally with 100 µg of OVA (Sigma Chemical Co., St. Louis, MO) in 4.5 mg of alum. Seven days after immunization, spleens and local draining lymph nodes were harvested and CD4 T cells isolated. Syngeneic T-depleted splenocytes were used as antigen-presenting cells (APC) and prepared by negative selection using anti-CD4, -CD8, and anti–Thy-1 antibodies and treatment with rabbit complement. APCs were mitomycin-C treated. To generate Th2 cells, CD4 T cells were stimulated with pOVA323–339 (5 µg/ml), IL-4 (1–10 ng/ml; Peprotech, Rocky Hill, NJ) and anti–IFN-{gamma} at inhibitory concentration. To generate Th1 cells, CD4 T cells were stimulated with pOVA323–339 (5 µg/ml), IL-12 (7.5 ng/ml; Genetics Institute, Cambridge, MA) and anti–IL-4 at inhibitory concentration. All cultures were set up in flasks containing a 1:2 ratio of CD4 T cells and APCs at a concentration of 2.5 x 105 CD4 T cells/ml for TCR Tg and 1 x 106 for non-Tg cells, and were maintained for 4 d.

Transfer of Cells and Aerosol Administration of OVA
Cultured Th2-like cells were harvested after 4 d, washed with phosphate-buffered saline (PBS), and 2.5–6 x 106 cells were injected intravenously into syngeneic recipient mice. One day after transfer of cells, mice were challenged with inhaled 1% OVA in PBS as previously described (4), for 20 min/d for a total of 7 d over a period of 9 d (four consecutive days exposed, 2 d rested, three consecutive days exposed). Control mice received inhaled OVA only. For experiments using IL-13 inhibitor, 400 µg sIL-13R{alpha}2-Fc or control Hu Ig (generously provided by Dr. Debra Donaldson, Genetics Institute) was administered intranasally to mice each day before OVA exposure. Mice were killed 24 h after the final OVA exposure.

Cytokine Assays
At the time of transfer, an aliquot of Th2-like cells was retained for restimulation. 2.5 x 105 CD4 T cells/ml, 2.5 x 105/ml freshly isolated APCs and pOVA (5 µg/ml) were cultured, and supernatants were collected at 24 h. IFN-{gamma}, IL-4, IL-5 (Endogen, Cambridge, MA), and IL-13 (BD Pharmingen) levels from cell supernatants were determined by ELISA. The lower limit of sensitivity for each of the ELISAs was 0.5 ng/ml for IFN-{gamma}, 0.010 ng/ml for IL-4, 0.010 ng/ml for IL-5, and 0.025 ng/ml for IL-13. IFN-{gamma}, IL-4, IL-13 (R&D Systems, Minneapolis, MN), and IL-9 levels from bronchoalveolar lavage (BAL) fluid were determined by enzyme-linked immunosorbent assay (ELISA). The lower limit of sensitivity for each of the ELISAs was 8 pg/ml for IFN-{gamma}, 4 pg/ml for IL-4, 8 pg/ml for IL-13, and 0.140 ng/ml for IL-9.

Fluorescence-Activated Cell Sorter Analysis
At the time of transfer, fluorescence-activated cell sorter (FACS; Becton Dickinson, San Jose, CA) analysis was performed on Th2 cell preparations to determine the purity of transferred cell populations. Cells were stained with anti-CD4 (Quantum Red-L3T4; Sigma) and in DO11.10 Tg populations, the biotinylated anticlonotypic antibody, KJ1–26, and fluorescein isothiocyanate-avidin D (Vector Laboratories, Burlingame, CA). KJ1–26 is specific for the Tg TCR in the DO11.10 mice. Transferred cells were uniformly more than 96% CD4 positive. After a period of inhalational exposure, BAL and lung cells were analyzed by FACS using these antibodies. Isolation of lung lymphocytes was performed after BAL and perfusion of blood from lungs. Lung tissue was passed through a wire mesh, digested with collagenase Type IV (150 U/ml; Worthington Biochemical, Freehold, NJ) and DNase 10 units/ml (Sigma) for 1 h at 37°C and passed again through a wire mesh to dissociate cells.

Lung Histology
Lungs were prepared for histology by perfusing the animal via the right ventricle with 20 ml of PBS. Lungs were then inflated with 1.0 ml of fixative instilled through a tracheostomy tube. Samples for paraffin sectioning were formalin fixed, sectioned in the coronal plane at 5 µm, and periodic acid Schiff (PAS) and Alcian blue stains were performed. Histologic mucus index (HMI) was performed on PAS-stained sections that included both central and peripheral airways on which marker dots in a grid with 2-mm spacing were placed over the entire lung section. The slide was examined at x100 final magnification on an Olympus BH-2 microscope (Olympus, Tokyo, Japan) with a rectangular 10-mm square reticule grid (American Optical Corp., Buffalo, NY) inserted in one eyepiece. Each marker dot was placed in the lower left corner of the field, and all intersections of airway epithelium with the reticle grid were counted in that field, distinguishing mucus-containing or normal epithelium. Approximately 25% of the total lung section was scored. The ratio of total number of mucus positive intersections and the total of all intersections, which we call the HMI, is equivalent to the linear percent of epithelium positive for mucus. This index was calculated for each mouse lung and then the mean of the HMI was calculated for each experimental group. HMI results were previously validated on Alcian blue– and mucicarmine-stained sections.

Reverse Transcription-Polymerase Chain Reaction
Total RNA was isolated from the left lung of each mouse after being homogenized in TrizolReagent (Gibco BRL, Gaithersburg, MD) and extracted according to the manufacturer's instructions. The RNA pellet was suspended in nuclease-free H2O. To synthesize cDNA, random primers were annealed to 4 µg of total lung RNA by incubation at 65°C for 5 min. Reverse transcription (RT) was performed by adding dNTP, an RNAse inhibitor (RNAse OUT; Gibco BRL), 0.1 M DTT, 50 U SuperScriptII (Gibco BRL), and buffer, and incubating at 42°C for 50 min and finally at 70°C for 15 min to terminate the reaction. The cDNAs synthesized were used in polymerase chain reaction (PCR). PCR was performed using primer sets corresponding to murine muc5ac (14) and HPRT. The sequences of the primers were: muc5ac: 5'-GGA CCA AGT GGT TTG ACA CTG AC-3' (forward) and 5'-CCT CAT AGT TGA GGC ACA TCC CAG-3' (reverse); HPRT: 5'-GTT GGA TAC AGG CCA GAC TTT GTT G-3' (forward) and 5'-GAG GGT AGG CTG GCC TAT AGG CT-3' (reverse). The reactions were assembled with 10 µM dNTP, 1x reaction buffer (Promega, Madison, WI), 25 mM MgCl2, primers (100 pM), cDNA (1–2 µl) and 2.5 U of DNA polymerase (Promega). The samples were first heated for 2 min at 94°C and the PCR conditions were: 94°C for 45 s, 67°C for 45 s (muc5ac), or 64°C for 45 s (HPRT), 72°C for 1 min for a total of 30 cycles. The reactions were terminated by incubation at 72°C for 7 min to complete chain extension. A single PCR product of the appropriate size was visualized by electrophoresis on a 1% agarose gel stained with ethidium bromide.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IL-4R{alpha} Deficiency Totally Blocks, and an IL-13 Inhibitor Partially Blocks, Th2-Induced Mucus Production
Using an adoptive transfer system in which in vitro–generated TCR Tg DO11.10 CD4 Th1 or Th2 cells were transferred into recipient mice, we previously showed that Th2, but not Th1, cells stimulate airway epithelial mucus (4). IL-4-/- Th2 cells were able to stimulate mucus production, but mucus was not induced when IL-4-/- Th2 cells were transferred into IL-4R{alpha}-/- mice (5). We now show that mucus staining is also blocked when 2.5 x 106 DO11.10 IL-4+/+ Th2 cells are transferred into IL-4R{alpha}-/- mice (Figure 1). The lack of induction of mucus is independent of recruitment of DO11.10 cells to and/or proliferation of these cells in the respiratory tract, because 1.5 x 104 CD4, KJ1.26-expressing cells were present in the BAL of IL-4R{alpha}+/+ mice and 4.4 x 104 CD4+KJ1.26+ cells were present in the BAL of IL-4R{alpha}-/- mice that received Th2 cells and inhaled OVA. Furthermore, both IL-4 and IL-13 were measured in the BAL fluid from both groups of mice, whereas IFN-{gamma} was not detectable (Table 1). The levels of IL-4 and IL-13 were higher in IL-4R{alpha}-/- mice, consistent with the increase in CD4, KJ1.26-expressing cells in the BAL. We further confirmed that the lack of mucus staining in IL-4R{alpha}-/- mice was not a result of IFN-{gamma}, an inhibitor of mucus (16), because mucus was not induced when Th2 cells were transferred into IL-4R{alpha}-/- x IFN-{gamma}R-/- mice (data not shown). Thus, it appears that the absence of mucus induction in IL-4R{alpha}-/- mice is not due to a block in Th2 cell recruitment, proliferation, activity, or a shift in the cytokines produced in vivo.



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  		Figure 1. IL-4R{alpha} deficiency totally blocks, and inhibition of IL-13 partially blocks, airway mucus staining in mice after transfer of Th2 cells. DO11.10 CD4 Th2 cells (2.5 x 106) were transferred into IL-4R{alpha}+/+ or IL-4R{alpha}-/- recipient mice and mice were exposed to inhaled OVA. Intranasal sIL-13R{alpha}2-Fc, control (HuIg), or neither (-) was administered during inhaled OVA exposure. A histologic mucus index (HMI) was performed on lung sections stained with PAS. Mean HMI (± SEM) is shown (n = 3–4 mice per group). One experiment is shown and is representative of three experiments. Statistical significance was determined by unpaired Student's t test. The difference between IL-4R{alpha}+/+ mice that received sIL-13Ra2-Fc or HuIg was not significant. *P = 0.0012.

 

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  		TABLE 1 Cytokine levels in bronchoalveolar lavage fluid of IL-4R{alpha}+/+ and IL-4R-/- mice

 
Because IL-4R{alpha} is the common receptor chain for both IL-4 and IL-13, these data indicated a role for IL-13 in the stimulation of mucus. However, it was not clear if both IL-13 and IL-4 or only IL-13 regulated Th2-induced mucus production. We administered an inhibitor of IL-13 (sIL-13R{alpha}2-Fc) or a control inhibitor (HuIg) to recipient mice after transfer of 2.5 x 106 DO11.10 Th2 cells, and mice were exposed to inhaled antigen for 7 d. Mice that received Th2 cells and the control inhibitor exhibit mucus staining in 80% of the bronchial epithelial cells examined, whereas mice that received treatment with the inhibitor of IL-13 show mucus in 40% of airway epithelial cells (Figure 1). These results are similar to studies showing that sIL-13R{alpha}2 led to a partial reduction in goblet cell hypertrophy in mice immunized and aerosol challenged with OVA (8, 17). It was still unclear if the persistence of mucus was due to incomplete blockade of IL-13 activity or an ability of IL-4 to stimulate mucus.

IL-13 is Essential for Th2-Induced Mucus
To determine the precise role of IL-13 in airway mucus production, we generated Th2 cells in vitro from DO11.10 TCR Tg IL-13+/+ and IL-13-/- mice. When restimulated with pOVA323–339 at the time of cell transfer, IL-13+/+ and IL-13-/- Th2 cells produced high levels of IL-5 and IL-4, low levels of IFN-{gamma}, but only the IL-13+/+ Th2 cells produced IL-13 (Figure 2A). Although IL-4 levels were high in both populations of cells, IL-13-/- Th2 cells produced less IL-4 compared with IL-13+/+ Th2 cells, as has been previously shown (18). In four different experiments, between 2.5 and 6 x 106 IL-13+/+ or IL-13-/- Th2 cells were transferred into IL-13+/+ or IL-13-/- recipient mice, and mice were exposed to inhaled OVA for 7 d. IL-13+/+ mice that received IL-13+/+ or IL-13-/- Th2 cells and IL-13-/- mice that received IL-13+/+ Th2 cells and inhaled OVA had airway inflammation and a marked increase in airway mucus (Figure 2B). However, in the complete absence of IL-13, when IL-13-/- mice received IL-13-/- Th2 cells and inhaled antigen, there was no mucus staining in airway epithelial cells, despite marked airway inflammation. There were more CD4, KJ1.26-expressing cells in the lungs (Figure 2C) and BAL (data not shown) of IL-13-/- mice that received IL-13-/- Th2 cells compared with IL-13+/+ mice that received IL-13+/+ Th2 cells. Thus, despite a 2-fold reduction in IL-4 production by IL-13-/- Th2 cells, there was a 10-fold increase in transgenic cells within the lung, indicating that IL-4–producing cells were present in ample quantity in the respiratory tract. These data show that IL-13 is required for Th2-induced mucus, even when high levels of IL-4 are present. Together with the data in Figure 1, these experiments show that only complete blockade of IL-13 will eliminate airway epithelial mucus staining.



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  		Figure 2. Airway mucus staining is not induced by Th2 cells in the complete absence of IL-13. (A) Cytokine production by IL-13+/+ (filled bars) and IL-13-/- (open bars) Th2 cells. At the time of transfer into recipient mice, in vitro generated IL-13+/+ and IL-13-/- DO11.10 CD4 Th2 cell populations were cultured with antigen-presenting cells in the presence of pOVA323–339. Supernatants were collected after 24 h and cytokine ELISAs were performed. (B) IL-13+/+ or IL-13-/- DO11.10 CD4 Th2 cells were transferred into IL-13+/+ or IL-13-/- recipient mice and mice were exposed to inhaled OVA. An HMI was performed on lung sections stained with PAS. Mean histologic mucus index (± SEM) is shown (n = 3–4 mice per group). One experiment is shown and is representative of four experiments. *P < 0.0002 all groups compared with IL-13-/- mice that received IL-13-/- Th2 cells. (C) CD4, KJ1.26-expressing cells were isolated from the lungs of IL-13+/+ mice that received IL-13+/+ Th2 cells or IL-13-/- mice that received IL-13-/- Th2 cells and inhaled OVA. One experiment is shown and is representative of three experiments. Statistical significance was determined by unpaired Student's t test. **P = 0.05.

 
Despite a lack of mucus staining, lung histopathology of IL-13-/- mice that received IL-13-/- Th2 cells showed airway epithelial cell hypertrophy (Figure 3). This finding shows that in the absence of IL-13, Th2 cells can activate epithelial hypertrophic changes typically associated with mucus hyperproduction, but the inflammatory response is not sufficient to induce mucus metaplasia.



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  		Figure 3. Airway epithelial changes after transfer of IL-13+/+ or IL-13-/- Th2 cells. (A) IL-13+/+ DO11.10 Th2 cells were transferred into an IL-13+/+ recipient mouse. (B) IL-13-/- DO11.10 Th2 cells were transferred into an IL-13-/- recipient mouse. (C) No cells were transferred into an IL-13+/+ recipient mouse. All mice were exposed to inhaled OVA for 7 d. Epithelium from a large airway is shown (PAS stain, x100 magnification). Only epithelial cells in A contain intracellular, black staining material, indicating the presence of mucins.

 
IL-13 Stimulates Increased Muc5ac Gene Expression
Increased mucin gene expression in airway epithelial cells has been shown to correlate with histologic mucus staining (19). In mice and humans, the mucin glycoprotein genes MUC2 and MUC5ac can be upregulated in airway epithelial cells in response to various stimuli (20). RT-PCR on RNA samples from whole lung showed that muc5ac gene expression was increased in wild-type (IL-4R{alpha}+/+ or IL-13+/+) mice that received transfer of wild-type (IL-13+/+) Th2 cells and inhaled OVA (Figure 4). These were mice that exhibited increased mucus staining by PAS and Alcian blue staining. Muc2 expression was not detectable (data not shown). Muc 5ac expression was markedly reduced in lungs from IL-13-/- mice that received IL-13-/- Th2 cells and IL-4R{alpha}-/- mice that received Th2 cells compared with wild-type recipient mice (IL-13+/+ and IL-4R{alpha}+/+) that received Th2 cells. There was a low level of muc5ac gene expression in IL-4R{alpha}+/+ or IL-4R{alpha}-/- mice that received no transfer of cells and inhaled OVA, and these mice had no appreciable histologic mucus staining. In these experiments, mucus staining correlates with a striking increase in muc5ac mRNA. Thus, Th2 cells producing IL-4 alone, in the absence of IL-13, do not stimulate a robust increase in muc5ac mRNA, supporting our findings that IL-4 cannot stimulate mucus production in vivo.



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  		Figure 4. Muc5ac gene expression is reduced after Th2 cell transfer in the absence of IL-13 or IL-4R{alpha} signaling. IL-4R{alpha}+/+ or IL-4R{alpha}-/-, IL-13+/+, or IL-13-/- recipient mice received transfer of wild-type DO11.10 Th2 cells (Th2 or IL-13+/+ Th2) or IL-13-/- Th2 cells. Control mice did not receive cells. All mice were exposed to inhaled OVA. Total RNA was extracted from one lung of each animal in an experimental group and RT-PCR was performed using primers for muc5ac and the housekeeping gene, HPRT. PCR products were run on an agarose gel stained with ethidium bromide. Each lane represents RNA from an individual mouse; lanes 1–3, 4–6, 7–8, or 9–10 show RNA from mice in the same experimental group.

 
IL-9–Induced Mucus Production Is Dependent on IL-4R{alpha}
IL-9 is a Th2 cytokine that is believed to play an important role in the pathogenesis of asthma (21). Airway epithelial mucus production was a striking feature of CC10 IL-9 Tg mice that overexpressed IL-9 in a lung-specific fashion (10). To investigate the mechanism of IL-9–induced mucus production, we crossed BALB/c CC10IL-9 Tg mice to IL-4R{alpha}-/- mice. Airway epithelial mucus staining was striking in CC10 IL-9 Tg x IL-4R{alpha}+/- or +/+ mice, but in CC10 IL-9 Tg x IL-4R{alpha}-/- mice mucus staining was absent (Figure 5A). Muc 5ac gene expression was increased in the lungs of CC10 IL-9 Tg x IL-4R{alpha}+/- mice, but undetectable in lungs from CC10 IL-9 Tg x IL-4R{alpha}-/- and in Tg-negative IL-4R{alpha}-/- or +/+ littermates (Figure 5A). CC10 IL-9 Tg x IL-4R{alpha}+/- or +/+ and CC10 IL-9 Tg x IL-4R{alpha}-/- mice had comparable, detectable levels of IL-9 in BAL fluid, whereas Tg-negative IL-4R{alpha}-/- or +/+ littermates had no detectable IL-9 in the BAL fluid. IL-13 was detectable by ELISA at low levels in the BAL of CC10 IL-9 Tg mice (data not shown). To determine if this low level of IL-13 was responsible for IL-4R{alpha}–dependent increased mucus staining in IL-9Tg mice, sIL-13R{alpha}2-Fc or Hu Ig control were administered intranasally to CC10 IL-9 Tg mice for 8 d. Mucus staining was blocked in mice that received sIL-13R{alpha}2-Fc, whereas mice that received the control Ig exhibited extensive PAS staining of the airways (Figure 5B). These studies show that IL-9–induced airway mucus is mediated by IL-13.




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  		Figure 5. Role of IL4R{alpha} and IL-13 in IL-9–induced mucus. (A) Mucus staining and mucin gene expression in CC10 IL-9 Tg bred to IL-4R{alpha}-/- mice. CC10 IL-9 Tg mice were bred to IL-4R{alpha}-/- mice and four groups of animals were analyzed as shown. A histologic mucus index (HMI) was performed on lung sections stained with PAS. Mean HMI (± SEM) is shown (n = 2–6 mice per group). Total RNA was extracted from one lung of each animal and RT-PCR was performed using primers for muc5ac and the housekeeping gene, HPRT. PCR products were run on an agarose gel stained with ethidium bromide. Each lane represents RNA from an individual mouse; lanes 1 and 2 are from IL-9Tg+ IL-4R{alpha}+/- mice, lanes 3 and 4 are from IL-9Tg+ IL-4R{alpha}-/- mice, and a single mouse is shown for the groups in lanes 5 and 6. These data are representative of five mice tested in each group. *P < 0.003 compared with IL-9 Tg+ IL-4R{alpha}+/- mice. (B) Mucus staining is inhibited in CC10 IL-9 Tg mice given sIL-13R{alpha}2-Fc. CC10 IL-9 Tg mice were treated intranasally with sIL-13Ra2-Fc or control (HuIg) for 8 d. An HMI was performed on lung sections stained with PAS. Mean HMI (± SEM) is shown (n = 3 mice per group). *P = 0.0002. Statistical significance was determined by unpaired Student's t test.

 
Th2-Induced Mucus Requires IL-4R{alpha} on Structural Cells in the Lung
We hypothesized two possible mechanisms by which IL-13 might mediate mucus induction: IL-13 might recruit and/or activate inflammatory cells to produce mediators that stimulate mucus production, or IL-13 might directly stimulate airway epithelial cells. To determine if Th2-induced mucus production is mediated by hematopoietic cells or nonhematopoietic cells, we generated bone marrow chimeras with IL-4R{alpha}+/+ and IL-4R{alpha}-/- mice. IL-4R{alpha}+/+ and IL-4R{alpha}-/- mice were irradiated and bone marrow was transplanted from either IL-4R{alpha}+/+ or IL-4R{alpha}-/- donor mice into IL-4R{alpha}+/+ or IL-4R{alpha}-/- host mice (IL-4R{alpha}+/+ -> IL-4R{alpha}+/+, IL-4R{alpha}-/- -> IL-4R{alpha}+/+, IL-4R{alpha}+/+ -> IL-4R{alpha}-/-, IL-4R{alpha}-/- -> IL-4R{alpha}-/-). Two months after bone marrow reconstitution, DO11.10 Th2 cells were transferred into each of the chimeras and mice were exposed to inhaled OVA. As expected, mucus was induced in IL-4R{alpha}+/+ -> IL-4R{alpha}+/+ mice (Figure 6). In IL-4R{alpha}-/- -> IL-4R{alpha}+/+ mice, mucus staining was still induced. Th2 cells did not stimulate mucus production in IL-4R{alpha}+/+ -> IL-4R{alpha}-/- and IL-4R{alpha}-/- -> IL-4R{alpha}-/- chimeras, both situations where host mice were IL-4R{alpha}-/-. Bone marrow chimeras that received inhaled OVA only had no significant mucus staining (data not shown). In all groups of mice that received Th2 cells and inhaled OVA, there was airway inflammation with comparable numbers of cells recovered after BAL. The number of CD4, KJ1.26-expressing cells in the BAL was increased in IL-4R{alpha}-/- -> IL-4R{alpha}-/- compared with IL-4R{alpha}+/+ -> IL-4R{alpha}+/+ chimeras (0.56 [± 0.1] x 105 versus 2.29 [± 0.47] x 105, P = 0.005). These data indicate that mucus induction by Th2 cells requires IL-4R{alpha} on nonhematopoietic cells. The presence of IL-4R{alpha}–expressing inflammatory hematopoietic cells in the lung cannot promote mucus production when IL-4R{alpha} is absent on structural cells. IL-13 may bind to its receptor on nonhematopoietic cells within the lung, possibly the bronchial epithelial cells, and stimulate mucus production.



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  		Figure 6. Airway mucus staining in IL-4R{alpha}-/- bone marrow chimeras after transfer of Th2 cells. Bone marrow chimeras were generated as described in MATERIALS AND METHODS using IL-4R{alpha}+/+ or IL-4R{alpha}-/- donor cells transplanted into IL-4R{alpha}+/+ or IL-4R{alpha}-/- host mice. DO11.10 Th2 cells were transferred into chimeric mice and mice were exposed to inhaled OVA. A histologic mucus index (HMI) was performed on lung sections stained with PAS. Mean HMI (± SEM) is shown (n = 3 mice per group). One experiment is shown and is representative of two experiments. *P <= 0.002 compared with chimeras whose host cells were IL-4R{alpha}+/+. Statistical significance was determined by unpaired Student's t test.

 
Th2-Induced Mucus Is Independent of Nuclear Factor-{kappa}B
The signaling mechanism through IL-4R{alpha} that leads to mucus induction is not known. In vitro studies have attempted to define some of the pathways by which mucins are induced. Muc2 gene expression induced by bacterial cell components (LPS) was shown to depend on the transcription factor, nuclear factor (NF)-{kappa}B (22). The classic NF-{kappa}B complex is a heterodimer of two polypeptide subunits, p50 and RelA (p65), that binds to a consensus sequence in the promoter of multiple genes associated with immune responses. Because different inflammatory stimuli lead to activation of NF-{kappa}B and there is structural homology among some of the mucin genes (23), we investigated if NF-{kappa}B was required for mucus induction by a Th2 inflammatory stimulus. We transferred OVA-specific Th2 cells (5 x 106) into wild-type or p50-/- mice, which do not display DNA binding activity for NF-{kappa}B (24). After exposure to inhaled OVA, both p50+/+ and p50-/- recipient mice exhibited comparable mucus staining of the airway epithelium, HMI 54% (± 13) in p50+/+ mice versus 77% (± 6) in p50-/- mice (n = 4 mice per group, P = NS). These studies show that the induction of mucus by Th2 cells does not require NF-{kappa}B.

Th1-Induced Mucus, in the Absence of IFN-{gamma}, Is IL-13–Dependent
Th1 cells typically do not stimulate mucus production due to the inhibitory action of IFN-{gamma} (4, 16). We showed that Th1 cells transferred into IFN-{gamma}R-/- mice could readily induce mucus, associated with a strong neutrophilic inflammatory response. BAL cells recovered from IFN-{gamma}R-/- mice that received Th1 cells were restimulated with pOVA323–339 in vitro, and the production of IL-13 was ~ 100 times less when compared with BAL cells from mice that received Th2 cells (0.6 [± 0.3] ng/ml versus 52 [± 29] ng/ml). To determine if this low level of IL-13 was responsible for the induction of mucus by Th1 cells, in the absence of IFN-{gamma}, we administered sIL-13R{alpha}2-Fc or HuIg control intranasally to IFN-{gamma}R-/- mice after transfer of Th1 cells and during exposure to inhaled OVA. In IFN-{gamma}R-/- mice that received HuIg, mucus was detected in 50% of airway epithelial cells and was completely blocked in these mice when sIL-13R{alpha}2-Fc was administered (Figure 7). CD4, KJ1.26-expressing cells were present in the BAL in both groups of mice (Th1 + HuIg, 5 x 104 ± 1.2; Th1+ sIL-13R{alpha}2-Fc, 9 x 104 ± 1.1). Wild-type mice that received HuIg and Th1 cells did not exhibit mucus staining. Th1 cells, therefore, are capable of mucus induction, not by an alternate pathway mediated by neutrophils, but via the same IL-13–mediated pathway of Th2 cells. In addition, the low level of IL-13 produced by Th1 cells is sufficient to stimulate mucus.



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  		Figure 7. Mucus induced by Th1 cells is blocked by an IL-13 inhibitor. DO11.10 Th1 cells (2.5 x 106) were transferred into IFN-{gamma}R+/+ (w.t.) or IFN-{gamma}R-/- mice and mice received intranasal sIL-13Ra2-Fc (+) or control HuIg (-) during exposure to inhaled OVA. A histologic mucus index (HMI) was performed on lung sections stained with PAS. Mean HMI (± SEM) is shown (n = 3–4 mice per group). One experiment is shown and is representative of two experiments. *P <= 0.03 compared with IFN-{gamma}R-/- mice that received Th1 cells and HuIg. Statistical significance was determined by unpaired Student's t test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These studies define a unique function for IL-13 in the induction of airway epithelial mucus in vivo. Neither IL-4, nor IL-9 produced by Th2 cells, nor any factor produced by Th1 cells upon activation, can stimulate mucus in the absence of IL-13. IL-13 is highly potent in its ability to stimulate mucus, as shown by Th1 cells, which produce 100-fold less IL-13 than Th2 cells, yet still can stimulate mucus staining in 60–70% of the bronchial epithelial cells. Therefore, inhibiting IL-13 must be absolute to eliminate mucus production, because partial blockade only leads to a modest reduction in mucus staining of airway epithelial cells.

Multiple different inflammatory mediators have been associated with airway epithelial mucus production in asthma. Cytokines, leukotrienes, histamine, platelet-activating factor, eosinophil cationic protein, and reactive oxygen species are elevated in the airways of individuals with asthma and have been shown to stimulate mucus secretion (25–29). In murine models of antigen-induced airway inflammation, the induction of mucus requires the presence of activated CD4 Th2 cells (4). Because CD4 T cells activate cascades of inflammation, dissecting out the critical factors that influence mucus production and secretion has been complex. In experiments employing blockade of leukotrienes or PAF, there was a reduction in airway mucus, but these data suggested that Th2 lymphocytes in the respiratory tract were also decreased (30, 31). Thus, these investigations did not identify whether a specific mediator could enhance mucus indirectly by increasing Th2 inflammation or directly by stimulating mucus production. In earlier studies we showed that Th2 cells could stimulate mucus in the absence of IL-5, eosinophils, and mast cells. Furthermore, mucus was induced by Th2 cells in the absence of IL-4, but not when both IL-4 and IL-13 signaling were blocked in IL-4R{alpha}-/- mice (5). IL-4R{alpha} appeared to mediate Th2-induced mucus production. Wills-Karp and Grunig showed that an IL-13 inhibitor caused a modest reduction in mucus production in an antigen-driven model of asthma (8, 17). Yet these studies did not clarify whether IL-4 could stimulate mucus in the absence of IL-13 or if mucus resulted from insufficient blockade of IL-13. Furthermore, in IL-13-/- mice, mucus was inhibited in one antigen-driven model of lung inflammation (32), but only partially reduced using another antigenic stimulus (13). Thus, it was unclear if factors other than IL-13 could stimulate mucus in a Th2-driven model of lung inflammation.

Some in vivo models suggested that IL-4 could stimulate mucus. In many cases, though, IL-13–producing cells were not completely absent from the experimental systems. In mice that overexpressed IL-4 in the respiratory tract, mucus production was markedly increased (6, 7). Yet these mice had marked airway inflammation with lymphocytes and eosinophils, both with the potential to produce IL-13. IL-4 instilled in the airways of lymphocyte-deficient mice also led to increased mucus staining and eosinophilia (8). IL-13 is produced predominantly by Th2 cells, but other inflammatory cells like mast cells, basophils, and NK cells have been shown to produce IL-13 (33, 34), and our recent studies in RAG-/- mice indicate that sufficient IL-13 is produced by nonlymphoid cells to stimulate mucus production (L. Cohn, unpublished data). High levels of recombinant IL-4 were also shown to increase mucin gene expression in cultured human airway epithelial cell lines (9). Although this system appears to be free of contaminating IL-13, it is unknown whether the bronchial epithelium can produce very low levels of IL-13. An alternate explanation is that high and sustained levels of IL-4 stimulate mucus. If this is the case, then the threshold for mucus induction was not achieved in our transfer system despite the magnitude of IL-4 produced by and the increased number of activated, antigen-specific Th2 cells in the respiratory tract of IL-13-/- recipient mice. Under physiologic conditions, it is unlikely that IL-4 will manifest an effect on mucus in the absence of IL-13.

IL-4 and IL-13 have many overlapping functions due to signaling through the common receptor chain, IL-4R{alpha}, and activation of the transcription factor, Stat6. Both IL-4 and IL-13 stimulate Th2 cell generation, IgE production, and most aspects of host protective immunity to parasites (35). IL-13 has a unique in vivo function in gastrointestinal expulsion of the parasitic nematode, Nippostrongylus braziliensis (36, 37). Our current studies support the conclusions of others that IL-13 has another unique function in the induction of airway epithelial mucus production (32, 38, 39). These IL-13 effects on the epithelium could be due to differences in either signal transduction of IL-4 and IL-13 through their receptors (40), the magnitude and kinetics of IL-13 and IL-4 production, or cellular expression of the receptors (35). Although these appear to be direct effects of IL-13 on the epithelium, there is also enhanced accumulation of Th2 cells in the respiratory tract in the absence of IL-13; therefore, these studies cannot rule out an undefined, indirect effect of IL-13 on mucus.

Th2 cytokines, aside from IL-4 and IL-13, have been shown to stimulate mucus. Most recently, IL-9 was proposed to be a key cytokine determining asthma susceptibility, given its chromosomal localization in humans and its association with bronchial hyperresponsiveness and mucus hypersecretion in mice (10, 21). IL-9 is produced by Th2 cells and mast cells and has been shown to promote T cell growth and IgE production, and to stimulate growth and differentiation of mast cells (41). Tg mice that constitutively overexpress IL-9 in the respiratory tract had marked mucus staining in the airway epithelium and airway inflammation with increased numbers of Th2 cells, eosinophils, and mast cells (10). IL-9 applied in vitro to cultured airway epithelial cells led to mucus production and muc5ac gene expression (11, 12). Although these studies support a direct role for IL-9 in mucus induction, other studies suggest that IL-9 activates an inflammatory cascade that leads to mucus induction. In IL-9–deficient mice, mucus production was reduced in the lung after a primary infection with Schistosoma, but it was not impaired in a secondary response (13). These experiments, together with other recent studies (42), show clearly that IL-9 stimulates mucus production by increasing production of IL-13, because mucus staining was negligible in IL-9 Tg mice that were deficient in IL-4R{alpha} or that received an IL-13 inhibitor, despite continued local production of IL-9. IL-9 may enhance IL-13 levels through proinflammatory effects on mast cells, which are activated early in pulmonary immune responses and can produce IL-13 (33).

IL-10 overexpressed in the respiratory tract of mice was recently shown to cause increased airway epithelial mucus production and airway inflammation. Mucus was inhibited when these animals were backcrossed to IL-4R{alpha}-/- or IL-13-/- mice (43). Together with the data presented herein, it appears that Th2 cytokines can promote mucus production by enhancing inflammation, Th2 cell generation, and ultimately IL-13 production, because Th2 cells stimulate mucus exclusively by an IL-13–mediated pathway.

We previously showed that Th1 cells could stimulate mucus production if IFN-{gamma} effects were blocked (16). Th1 cells stimulate a vigorous neutrophilic airway inflammatory response and we initially theorized that Th1 cells might be stimulating mucus by a neutrophilic pathway. Neutrophil elastase, epithelial growth factor, and TNF-{alpha} have been shown to stimulate airway epithelial mucus (44, 45). Yet, our studies showed that despite the presence of neutrophilia, an IL-13 inhibitor blocked mucus production after transfer of Th1 cells into IFN-{gamma}R-/- mice. These findings argue against a neutrophil-stimulated pathway of mucus induction, such as the model proposed by Takeyama and coworkers in which IL-13–induced mucus is mediated by neutrophils through the elaboration of TNF-{alpha}, epithelial growth factor, and oxidants causing EGFR activation (44, 46, 47). Additionally, our studies using bone marrow chimeras indicate that IL-13 cannot stimulate mucus solely through effects on inflammatory cells, like neutrophils. When IL-4R{alpha} was present on bone marrow derived inflammatory cells, but not on the structural cells in the lung, mucus was not induced by Th2 cells producing IL-13. IL-13–induced mucus may involve complex interactions between IL-4R{alpha} and other receptors, like EGFR or IL-9R, yet these interactions appear to depend on IL-4R{alpha} for the induction of mucus. Our data suggests that IL-9 stimulates IL-13 production, placing IL-4R{alpha} signaling downstream of IL-9 in the inflammatory cascade that leads to mucus production. The detailed interactions of IL-13/IL-4R{alpha} and EGFR or other inflammatory signals that have been shown to correlate with mucus gene activation have yet to be elucidated.

The pathways from IL-4R{alpha} engagement to mucin gene expression have not been characterized. Yet studies have begun to map some of the signaling pathways that activate mucin genes. Pseudomonas/LPS activates MUC2 gene transcription through a signaling pathway that is dependent on activation of NF-{kappa}B and binding to the promoter of the MUC2 gene (22). The transcription factor NF-{kappa}B controls the expression of multiple genes involved in immune responses that affect lung inflammation, including proinflammatory cytokines, adhesion molecules, chemokines, and mucins (48, 49). The classic NF-{kappa}B heterodimer is a complex of two polypeptide subunits, p50 and Rel A (p65). In p50-/- mice immunized and challenged with OVA, inflammation and mucus production were reduced, suggesting an important role for NF-{kappa}B in Th2-induced inflammatory responses in vivo (24). Recent studies showed that inhibition of NF-{kappa}B prevented GATA-3 expression and Th2 cytokine expression, including IL-4, IL-5, and IL-13, in developing Th2 cells (50). We now show that committed Th2 cells that produce cytokines can stimulate mucus production in the absence of NF-{kappa}B. Therefore, the original defect in mucus production we observed in immunized p50-/- mice resulted from defective Th2 cell generation and not from an effect of NF-{kappa}B on mucus. These data suggest that NF-{kappa}B p50 is not essential for muc5ac gene expression. Although in vivo studies characterizing Muc2 regulation have not yet been performed, it appears that the mucin genes, Muc2 and Muc5AC, are regulated differently by different inflammatory stimuli. In keeping with these findings, when LPS was instilled intranasally into IL-4R{alpha}-/- and IL-13-/- mice, mucus was induced (L. Cohn, unpublished observation), further supporting the theory that different host responses have evolved to promote mucus production.

Mucus production is one of many mechanisms by which the airway epithelium responds to pathogens and injury. The epithelial layer also secretes other substances important in host defense, functions as a physical barrier to entry of foreign matter, and interacts with cells in close apposition through local signaling mechanisms and at distant sites through release of chemical mediators. Th2 inflammation in the airway stimulates epithelial hypertrophy and mucus metaplasia. In the absence of IL-13, Th2 cells still activate the epithelium to undergo hypertrophy, yet none of the cytokines produced by these cells can provide the appropriate stimulus to activate mucus production. These are the first studies to show a dissociation of epithelial hypertrophy and mucus metaplasia. Although it is well known that hypertrophy is an adaptive response of different cell types to increased metabolic demands (51), the functions affected by airway epithelial hypertrophy, aside from mucus induction, have not been previously studied. Future studies using this model system will allow us to study the epithelial responses in asthma that are distinct from mucus metaplasia, and may provide new insights into epithelial effects in the airway remodeling response.

Many Th2 cytokines have been shown to stimulate mucus. We now established a single, common pathway by which mucus is induced in a CD4 Th inflammatory response. Cytokines, including IL-4, IL-5, IL-9, IL-10, and inflammatory cells including eosinophils and mast cells, activate mucus and muc5ac expression through increased secretion of IL-13. These effects are dependent on IL-4R{alpha} expression on structural cells within the lung, presumably the airway epithelium itself, which has been shown to express both IL-4R{alpha} and IL-13R{alpha}1 (52). Blocking mucus production and reducing airway obstruction in asthma and chronic bronchitis, both of which have been shown to have elevated levels of IL-13 (53; J. Elias, personal communication), is now a realistic goal. These studies show that total inhibition of IL-13 may be required for a significant effect, given the potency of this cytokine in mucus induction. Furthermore, this work suggests that IL-13 is not the primary stimulus of mucus in gram-negative infectious etiologies of mucus hypersecretion, such as cystic fibrosis. These concepts advance our understanding of the pathways that stimulate mucus and will aid in guiding studies of new immunotherapeutic agents to regulate mucus production.


   Acknowledgments
 
The authors wish to thank Dr. Jeffrey Tepper for technical assistance and for helpful comments, Dr. Debra Donaldson for providing sIL-13R{alpha}, and Dr. Andrew McKenzie for providing IL-13-/- mice. This work was supported by the National Institutes of Health grants R01-HL64040 (LC), P50-HL56389 (LC, RJH), T32-HL07778 (LW).

Received in original form February 11, 2002

Received in final form June 17, 2002


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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