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The Journal of Immunology, 2007, 178: 3244-3251.
Copyright © 2007 by The American Association of Immunologists, Inc.

IL-13 Mediates In Vivo IL-9 Activities on Lung Epithelial Cells but Not on Hematopoietic Cells1

Valérie Steenwinckel*, Jamila Louahed*, Ciriana Orabona*,¶, François Huaux{dagger}, Guy Warnier*, Andrew McKenzie{ddagger}, Dominique Lison{dagger}, Roy Levitt§ and Jean-Christophe Renauld2,*

* Ludwig Institute for Cancer Research and Experimental Medicine Unit, Université Catholique de Louvain, Brussels, Belgium; {dagger} Industrial Toxicology and Occupational Medicine Unit, Université Catholique de Louvain, Brussels, Belgium; {ddagger} Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, U.K.; § Genaera Corporation, Plymouth Meeting, Pennsylvania; and Department of Experimental Medicine, University of Perugia, Perugia, Italy


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Increased IL-9 expression, either systemically or under the control of lung-specific promoter, induces an asthma-like phenotype, including mucus overproduction, mastocytosis, lung eosinophilia, and airway hyperresponsiveness. These activities correlate with increased production of other Th2 cytokines such as IL-4, IL-5, and IL-13 in IL-9 Tg mice. To determine the exact role of IL-13 in this phenotype, mice overexpressing IL-9 were crossed with IL-13-deficient mice. In these animals, IL-9 could still induce mastocytosis and B lymphocyte infiltration of the lungs. Although IL-9-induced eosinophilia in the peritoneal cavity was not diminished in the absence of IL-13, IL-13 was required for IL-9 to increase eotaxin expression and lung eosinophilia. Mucus production and up-regulation of lung epithelial genes upon IL-9 overexpression were completely abolished in the absence of IL-13. Using hemopoietic cell transfer experiments with recipients that overexpressed IL-9 but were deficient in the IL-9 receptor (IL-9R), we could demonstrate that the effect of IL-9 on lung epithelial cells is indirect and could be fully restored by transfer of hemopoietic cells expressing IL-9R. Mucus production by lung epithelial cells was only up-regulated when hemopoietic cells simultaneously expressed functional IL-9R and IL-13 genes, indicating that IL-13 is not a cofactor but a direct mediator of the effect of IL-9 on lung epithelial cells. Taken together, these data indicate that IL-9 can promote asthma through IL-13-independent pathways via expansion of mast cells, eosinophils, and B cells, and through induction of IL-13 production by hemopoietic cells for mucus production and recruitment of eosinophils by lung epithelial cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Interleukin 9 (IL-9) is a pleiotropic Th2 cytokine that was originally described as a growth factor for T lymphocytes (1). Biological targets for IL-9 include mast cells, B lymphocytes, T lymphocytes, T cell clones, hemopoietic progenitors, and immature neuronal cell lines (2). IL-9 has also been proposed as a candidate gene for asthma susceptibility. This hypothesis is based on genetic studies that showed an association between the regions of the human and mouse genome containing the IL-9 gene, and asthma or bronchial hyperresponsiveness (3, 4, 5). Moreover, the expression of IL-9 and its receptor is increased in the airways of asthma patients (6). Further support for the role of IL-9 in asthma was provided by studies of transgenic (Tg)3 mice. IL-9 overexpression, either systemically or under the control of a lung-specific promoter, results in an asthma-like phenotype, with lung eosinophilia (7, 8, 9), goblet cells hyperplasia, increased mucus production by the pulmonary epithelial cells (9, 10), mastocytosis (11), high level of IgE and airway hyperreactivity (9, 12).

Conflicting results have been published regarding the efficacy of IL-9 inhibition in asthma. Two independent studies showed that administration of neutralizing IL-9 Abs could attenuate pulmonary eosinophilia, goblet cell hyperplasia, and airway hyperresponsiveness (AHR) in murine models of allergen-induced asthma (13, 14). In contrast, IL-9-deficient mice could still develop eosinophilia and AHR in similar models (15), although they showed a defective goblet cell hyperplasia and mastocytosis in a pulmonary granuloma model (16).

Accumulating observations point to a role for IL-13 in IL-9 activities. IL-13, like other Th2 cytokines, is overexpressed in IL-9 Tg mice (17). IL-13 overexpression in vivo stimulated mucus hypersecretion by lung epithelial cells, goblet cell hyperplasia and AHR (18, 19). Moreover, studies using mice lacking IL-13, STAT-6, or IL-4R{alpha} demonstrated that IL-13 is required for allergen-induced mucus overproduction and AHR (20, 21, 22). In line with these observations, mucus hypersecretion in IL-9 Tg mice was also suppressed in the absence of IL-13 signaling (23), indicating that IL-13 either directly mediates or is a required cofactor of IL-9 activity on lung epithelial cells.

The current study aimed at better determining the implication of IL-13 in IL-9 activities, not only on epithelial cells but also on eosinophils, mast cells, and B cells. By using IL-9 Tg/IL-13 knockout (KO) mice, we showed that the effects of IL-9 on lung epithelial cells, but not on hemopoietic cells, were dependent on IL-13. We next determined, with a model of bone marrow (BM) cell transfer, that IL-13 is not a cofactor but a direct mediator of IL-9 activity on epithelial cells and that IL-9 directly promotes IL-13 expression by hemopoietic cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

IL-13-deficient mice (129 x C57BL/6 genetic background) were obtained by insertion of a cassette containing a LacZ reporter gene and a neomycin resistance gene into exon 1 of the IL-13 locus, as described previously (24). Tg5 IL-9 Tg mice, obtained in the FVB/N background and expressing high levels of IL-9 in all organs, were described previously (25).

IL-9R{alpha} KO mice were generated in the C57BL/6 background by substitution of a 2826 nucleotide KpnI-SpeI fragment containing exons 2, 3, 4, 5, and 6, with a neomycin resistance gene. This construct was transfected into C57BL/6 embryonic stem (ES) cells by electroporation. ES cells containing the neomycin resistance gene were isolated in the presence of G418 and the prospective clones screened by PCR. The sequences of the primers were: IL-9R wild type (WT): 5'-AATGTCAGCTCTGGGCGTTG-3' (forward) and 5'-TCTGGGGAGAAGGAAAGGAC-3' (reverse); IL-9R KO: 5'-CTTCTATCGCCTTCTTGACG-3' (forward) and 5'-AGATGGGCACAACTACAAGG-3' (reverse). Homologous recombination was confirmed by Southern blot. ES cells containing the correct gene recombination were used to derive chimeric males that were crossed with FVB females to obtain heterozygous mice. For the current study, those heterozygous mice had been backcrossed for eight generations onto strain C57BL/6 before being interbred to generate homozygous IL-9R KO mice. These mice were born at mendelian proportions and showed normal viability and fertility.

To derive IL-9 Tg/IL-9R KO offspring, IL-9R KO mice were bred with Tg5 mice, to obtain F1 mice that were backcrossed with IL-9R KO mice. IL-9 Tg/IL-9R KO mice were identified using a TS1 bioassay on serum to detect circulating IL-9 in IL-9 Tg mice (1, 25), and using PCR on tail genomic DNA with the two primers sets described above and corresponding to IL-9R WT and IL-9R KO alleles.

IL-9 Tg mice were backcrossed with IL-13 KO mice to generate IL-9 Tg/IL-13 KO offspring. Mice were screened by PCR on genomic DNA for the IL-13 gene and with a TS1 bioassay for circulating IL-9. The sequences of the primers were: IL-13 WT: 5'-GGGTGACTGCAGTCCTGGCT-3' (forward) and 5'-GTTGCTCAGCTCCTCAATAAGC-3' (reverse); IL-13 KO: 5'-GGCGGATGAGCGGCATTTTCCGTG-3' (forward) and 5'-GCCGAAAGGCGCGGTGCCGCTGGC-3'(reverse).

Bleomycin (Aventis) was resuspended in sterile saline solution (NaCl 0.9%) and administered at a dose of 0.001 U/g by intratracheal instillation after anesthesia [mix of 10 mg of Ketalar (NV Warner-Lambert) and 2 mg of Rompun (Bayer AG)]. Bronchoalveolar lavages (BAL) were performed 3 or 7 days after bleomycin instillation.

Preparation and transfer of hemopoietic cells

BM was aseptically flushed from femora and tibias of 129, C57BL/6, IL-13 KO, or IL-9R KO 10- to 12-wk-old mice. Cells were resuspended and filtered through a 40-µm nylon cell strainer. For T cell depletion with anti-CD5 magnetic MicroBeads (MACS; Miltenyi Biotec), red cells were removed by osmotic shock, before hemopoietic cells were washed, counted, and resuspended in MiniMACS buffer (PBS supplemented with 2 mM EDTA, 2% decomplemented FCS, and 1.5 µg/ml gentamicin). Staining and depletion of cells were performed following the manufacturer’s protocol. In brief, cells were incubated 15 min, at 4°C, with anti-CD5 MicroBeads (10% v/v), at the concentration of 100 x 106 cells/ml, before washing and resuspension in 500 µl of MiniMACS buffer to pass through a MS separation column. The degree of T cell depletion was determined using FITC-labeled anti-mCD4 and PE-labeled anti-mCD3 Abs (BD Pharmingen). Analysis was performed on a FACScan apparatus (BD Biosciences). In a representative experiment, percentages of CD8+ and CD4+ T cells dropped from 1.86% and 2.19%, respectively, to 0.95% and 0.76%, after depletion.

For cell transfer experiments, IL-9 Tg/IL-9R KO recipient mice were exposed to two doses of 550 rad of ionizing radiation, given 3 h apart, followed by i.v. injection of 4 x 106 BM cells isolated from C57BL/6, 129, IL-13 KO, IL-9R KO mice. Cells were injected in a volume of 200 µl of sterile DMEM. Mice were killed 3 or 4 wk after the transfer.

BM-derived mast cell lines were obtained as previously described by culturing BALB/c BM in the presence of IL-3 or both IL-3 and IL-9 for 4 wk (11). CD4+ and CD4– cells were sorted from mesenteric lymph nodes using anti-CD4 magnetic MicroBeads (MACS) as described above.

Peritoneal and BAL

Cells from the peritoneal exudates were harvested by peritoneal lavage with 4 ml of PBS. BAL were performed with two subsequent 1-ml fractions of PBS. Cytospins were conducted with 50,000 cells and centrifuged for 5 min at 800 rpm (Cytospin 3; Shandon). Cellular composition of the lavages was determined based on morphology and staining with Diff-Quik (Dade Behring).

For FACS staining, cells obtained from peritoneal lavage or BAL were counted, washed and suspended in Hank’s medium supplemented with 3% decomplemented FCS and 0.01 M azide. Double staining was performed with FITC-conjugated anti-IgM (LOMM9; provided by H. Bazin, Université de Louvain, Brussels, Belgium) and PE-conjugated anti-Mac1 (M1/70; BD Pharmingen). After staining, cells were fixed with 1.25% paraformaldehyde. Analysis was performed on a FACScan apparatus (BD Biosciences) on 104 cells/sample.

RT-PCR

Total RNA was isolated from lung tissues using TriPure isolation reagent (Roche) according to the manufacturer’s instructions. Reverse transcription was performed on 1 to 5 µg of total RNA with an oligo(dT) primer (Roche) and Moloney murine leukemia virus reverse transcriptase (Invitrogen). PCR amplifications were performed from cDNA corresponding to 20 ng of total RNA at 94°C for 1 min, 58°C for 1 min, and 72°C for 2 min with a total number of 17 cycles for beta-actin and 35 cycles for IL-13. IL-13 PCR primers set was: 5'-TGGGTGACTGCAGTCCTGGCT-3' (forward) and 5'-GTTGCTTTGTGTAGCTGAGCA-3' (reverse). beta-actin PCR primers set was: 5'-ATGGATGACGATATCGGCTGC-3' (forward) and 5'-GCTGGAAGGTGGACAGTGAG-3' (reverse).

Quantitative PCR were performed using primers sets corresponding to murine MUC5AC, Igh-6, trefoil factor 2 (TFF2), resistin-like {alpha}, eotaxin, and beta-actin with qPCR Mastermix for SYBR Green I (Eurogentec). The sequences of the primers (final concentration: 300 nM) were: MUC5AC, 5'-GGACCAAGTGGTTTGACACTGAC-3' (forward) and 5'-CCTCATAGTTGAGGCACATCCCAG-3' (reverse); Igh-6, 5'-GTGACTCACAGGGATCTGCCTT-3' (forward) and 5'-GTATAGGTCTCTCCGGAGTTCC-3' (reverse); TFF2, 5'-GGGACTGCATGCTCTGGTAGA-3' (forward); 5'-GGGAAGAAACACCAGGGCACT-3' (reverse); resistin-like {alpha} (Retna), 5'-GCCCCAGGATGCCAACTTTGA-3' (forward), 5'-CTCCACTCTGGATCTCCCAAG-3' (reverse); eotaxin, 5'-GCGCTTCTATTCCTGCTGCTC-3' (forward), 5'-GCATCCTGGACCCACTTCTTC-3' (reverse); beta-actin, 5'-TCCTGAGCGCAAGTACTCTGT-3' (forward) and 5'-CTGATCCACATCTGCTGGAAG-3' (reverse). Samples were first heated 2 min at 50°C then 10 min at 95°C. cDNA was amplified as follows: 40 cycles of a two-step PCR program at 95°C for 15 s and 60°C for 1 min. Melting point analysis was conducted by heating the amplicon from 60°C to 95°C. For IL-13, similar cycling conditions were used with the addition of a internal TaqMan oligonucleotide probe. Primers for IL-13 were as follows: 5'-AGACCAGACTCCCCTGTGCA-3' (forward), 5'-TGGGTCCTGTAGATGGCATTG-3' (reverse) and 5'-CGGGTTCTGTGTAGCCCTGGATTCC-3' (Taqman probe).

Northern blots

Tissue expression of mCLCA3/gob5 was determined by Northern blot analysis of RNA extracted from lungs using the TriPure isolation reagent (Roche). A total of 10 µg of poly(A+) mRNA were fractionated by electrophoresis in 1.3% agarose gels containing 2.2 mol/L formaldehyde and blotted onto Hybon-C Extra membranes (Amersham Biosciences). Filters were hybridized to mCLCA3/gob5 probes, 32P-labeled by using Rediprime II labeling kit (Amersham Biosciences). beta-actin probes were used as controls for equal loading. Phosphor imager quantification was used to determine the level of signal.

ELISA mMCP-1

Serum mMCP-1 concentrations were measured in duplicate on 96-well microtiter plates using a commercial mMCP-1 kit (Morendun Scientific) according to the manufacturer’s instructions.

Statistical analysis

Statistical significance was analyzed using the InStat3 program. Mann-Whitney U test was run to determine the p value when comparing two groups. Differences with p < 0.05 were considered statistically significant. Values were presented as means ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
IL-13 is not required for systemic IL-9 activities on eosinophils, mast cells, and B lymphocytes

In line with previous observations made in mice that overexpressed IL-9 specifically in the lungs (17), Tg5 IL-9 Tg mice also show increased IL-13 expression in the lungs (Fig. 1A) and other organs (data not shown). To determine the precise role of IL-13 in these in vivo activities of IL-9, Tg5 mice were crossed with IL-13 KO mice to generate IL-9 Tg/IL-13 KO offspring. To investigate the contribution of IL-13 to IL-9-induced eosinophilia (7, 8), resident peritoneal cells were analyzed by cytospin. Both IL-9 Tg mice and IL-9 Tg/IL-13 KO mice showed a significant eosinophil expansion in the peritoneal cavity (Fig. 1B), indicating that IL-13 is not required for IL-9-induced eosinophilia.


Figure 1
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  		FIGURE 1. IL-13 expression is induced in IL-9 Tg mice, but is dispensable for IL-9-effects on eosinophils, mast cells, and B cells. A, Total RNA was extracted from lungs of IL-9 Tg or WT mice (Ctrl). RT-PCR amplification was performed using primers for IL-13 and the housekeeping gene beta-actin. PCR products were run on 1% agarose gel stained with ethidium bromide. Each lane corresponds to an individual mouse. B, Quantification of resident eosinophils from the peritoneal cavity based on morphology and histological staining. Results are expressed as mean and SEM (n = 5). C, mMCP-1 ELISA was performed on serum from control littermates, IL-9 Tg, IL-13 KO, and IL-9 Tg/IL-13 KO mice. Mean values and SEM were calculated from 5 mice of each genotype. D, Quantitative RT-PCR for Igh-6 expression was performed on lungs from control littermates, IL-9 Tg, IL-13 KO and IL-9 Tg/IL-13 KO mice. Results were normalized with beta-actin expression. Mean values and SEM were calculated from 5 mice of each genotype. E, Quantification of peritoneal B1 lymphocytes with a double FACS staining using anti-IgM and anti-Mac1 Abs. Results are expressed as mean and SEM (n = 5). For B–E, the effect of IL-9 was statistically significant (p < 0.03; Mann-Whitney U test) for both IL-13 +/– and –/– mice. Similar results were obtained in at least two independent experiments.

 
Systemic IL-9 overexpression also induces mast cell expansion, particularly in the lungs, gut, and kidneys, which results in increased serum concentration of mast cell protease mMCP-1 (11). Thus, we assessed the implication of IL-13 in these effects of IL-9 on mast cells by measuring mMCP-1 concentrations in IL-9 Tg/IL-13 KO mice. As shown in Fig. 1C, IL-9 Tg/IL-13 KO mice produced as much mMCP-1 as IL-9 Tg mice, indicating that IL-13 is not required for IL-9-induced mastocytosis.

We next determined whether IL-13 could play a role in the activity of IL-9 on B cells. IL-9 Tg mice are characterized by B lymphocyte expansion in the lungs or in the peritoneal cavity, and this expansion might be relevant for the activity of IL-9 in inflammatory processes such as those induced by silica particles in the lungs (26, 27). To quantify B cell infiltration in the lungs, we performed quantitative PCR for the Igh-6 gene (Ig H chain for IgM). As illustrated in Fig. 1D, IL-9 Tg lungs expressed higher levels of Igh-6 compared with controls, and this effect of IL-9 was not affected in IL-13 KO mice (Fig. 1D). Numbers of peritoneal B1 lymphocytes in IL-9 Tg mice were also assessed, and similarly, the expansion of B lymphocytes induced by IL-9 was not affected in the absence of IL-13 (Fig. 1E). Taken together, these observations indicate that the expansion of B cells induced by IL-9 are independent of IL-13.

IL-13 is required for IL-9 activities on lung epithelial cells

IL-9 overexpression induces mucus production by lung epithelial cells, and this effect of IL-9 was previously shown to be dependent on IL-13 (17). To address this question in our model, we analyzed the expression of three genes that are directly or indirectly related to mucus production: mCLCA3/gob5, MUC5AC, and TFF2. Although the function of mCLCA3/gob5 either as a Ca2+-activated chloride channel (28, 29) or as a secreted protein (30) remains elusive, its expression in lungs correlates with mucus production (31), pointing to this gene as a key factor in this process. As shown in Fig. 2A, mCLCA3/gob5 expression was strongly up-regulated in IL-9 Tg mice, but this effect was not observed in the absence of IL-13. The very same pattern of expression was observed for MUC5AC (Fig. 2B), a mucin gene that codes for a major component of lung mucus, and for TFF2, a mucus-associated peptide that promotes epithelial restitution and is responsible for mucus viscosity (32, 33).


Figure 2
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  		FIGURE 2. A functional IL-13 gene is required for IL-9 effects on pulmonary epithelial cells. A, Northern blot analysis of lungs from WT littermates, IL-9 Tg, IL-13 KO and IL-9 Tg/IL-13 KO mice with a mCLCA3/gob5 cDNA probe. The blot was reprobed with beta-actin as a control for equal loading. Each lane corresponds to an individual mouse. B, Quantitative RT-PCR analysis of lung RNA from WT littermates, IL-9 Tg, IL-13 KO, and IL-9 Tg/IL-13 KO mice for MUC5AC, TFF2, Retnla, and eotaxin. Results were normalized with beta-actin. Mean values and SEM were calculated from 4 to 5 mice of each genotype. The effect of IL-9 was statistically significant (p < 0.02; Mann-Whitney U test) only for IL-13 +/– mice. Similar results were obtained in three independent experiments for mCLCA3, TFF2, Retnla, and eotaxin.

 
We also assessed the expression of other epithelial genes such as Retna (also known as FIZZ1) and eotaxin. Retna is a cysteine-rich secreted protein produced by a subset of bronchial epithelial cells and by type II alveolar pneumocytes upon allergen-induced inflammation (34, 35, 36). Eotaxin/CCL11 is a chemokine implicated in eosinophil recruitment in the lungs (37) and was previously shown to be induced in IL-9-Tg lungs (7). Like mucus-related genes, both Retna and eotaxin/CCL11 were up-regulated in IL-9 Tg animals in an IL-13-dependent way (Fig. 2B).

As IL-9-induced lung eotaxin/CCL11 expression (Fig. 2B), but not systemic eosinophilia (Fig. 1B), was abolished in the absence of IL-13, we sought to determine whether IL-9 could still promote lung tissue eosinophilia in IL-13-deficient mice. When mice are bred in a specific pathogen-free (SPF) facility, IL-9 induces a systemic eosinophilia and lung eotaxin/CCL11 production but this is not reflected by a significant increase in eosinophil numbers in bronchoalveolar lavage. However, lung eosinophilia can be induced in such mice by triggering lung inflammation and injury with agents such as bleomycin (38). As illustrated in Fig. 3, intratracheal instillation of bleomycin in IL-9 Tg mice failed to induce lung eosinophilia in the absence of IL-13, as correlated with eotaxin expression. Taken together, these results indicate that IL-9 plays a double role in lung eosinophilia. On the one hand, it stimulates systemic eosinophil expansion, resulting in steady-state peritoneal eosinophilia, independently of IL-13. On the other hand, it promotes their recruitment to the lungs via IL-13-dependent up-regulation of eotaxin/CCL11 by epithelial cells.


Figure 3
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  		FIGURE 3. IL-9-induced lung eosinophilia is dependent of IL-13. BALs or lungs of control littermates, IL-9 Tg, IL-13 KO, and IL-9 Tg/IL-13 KO were harvested 3 days after intratracheal administration of bleomycin. Quantification of eosinophils in BAL was based on morphology and staining obtained with Diff-Quik (upper panel). Mean values and SEM were calculated from 4 mice of each genotype. Total RNA was extracted from the lungs of these mice and quantitative RT-PCR was performed on RNA for eotaxin (lower panel). Results were normalized with beta-actin. The effect of IL-9 was statistically significant (p < 0.03; Mann-Whitney U test) only for IL-13 +/– mice. Similar results were obtained in three independent experiments.

 
Characterization of IL-9Tg/IL-9R KO mice for an adoptive cell transfer model

The observations that IL-13 is up-regulated in IL-9 Tg mice, and that IL-9 activities on lung epithelial cells are abolished in IL-13-deficient mice suggest that IL-9 acts only indirectly on epithelial cells, and that IL-13 is the mediator of these effects. However, several groups reported that lung epithelial cells express the IL-9 receptor, or even directly respond to IL-9 (6, 10, 39, 40, 41). These observations raise the alternative hypothesis that IL-13 would act as an in vivo cofactor, synergizing with IL-9, rather than as a mediator of its activity. To address this question, we took advantage of IL-9R-deficient mice and from an adoptive cell transfer approach to generate animals chimeric for IL-9 responsiveness and IL-13 production. IL-9R-deficient mice were generated by deleting exons 2 to 6, which encode the entire extracellular domain of the receptor. We first generated IL-9 Tg/IL-9R KO mice, in which IL-9 overexpression did not have any effect, as illustrated in Fig. 4 for mCLCA3/gob5, MUC5AC expression in lungs, and for mMCP-1 serum concentration. Despite the presence of circulating IL-9, these mice also failed to show any expansion of B lymphocytes in the peritoneal cavity (data not shown) confirming that IL-9 Tg/IL-9R KO mice overexpressed IL-9 but were unable to respond to this cytokine.


Figure 4
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  		FIGURE 4. Mucus production and mMCP-1 expression in control littermates, IL-9 Tg, IL-9R KO, and IL-9 Tg/IL-9R KO mice. A, Structure of the IL-9R{alpha} gene. Targeting vector and the predicted homologous recombination events are indicated. The IL-9R{alpha} gene is shown as wide and narrow boxes corresponding respectively to exons and introns. B, Lung total RNA was extracted from mice with the indicated genotypes. Northern blot analysis was performed with a mCLCA3/gob5 probe, before reprobing with beta-actin as a control for equal loading. Each lane represents mRNA from individual mouse. C, Quantitative RT-PCR was performed for MUC5AC, and results were normalized with beta-actin. Mean values and SEM were calculated from 3 to 5 mice of each genotype. D, mMCP-1 ELISA was performed on sera from the same mice. For MUC5AC and mMCP-1, the effect of IL-9 was statistically significant (p < 0.04; Mann-Whitney U test) only for IL-9R+/– mice.

 
IL-9-responsive hemopoietic cells are sufficient to mediate IL-9-effects on epithelial cells

We next analyzed pulmonary mucus production after adoptive transfer of WT BM into irradiated IL-9 Tg/IL-9R KO recipient mice. In this model, only transferred cells were able to respond to IL-9 produced by the IL-9 Tg/IL-9R KO recipient mice. When we reconstituted recipient mice with C57BL/6 WT cells, but not with IL-9R KO cells, serum mMCP-1 concentrations increased, reflecting the effect of IL-9 on transferred mast cell progenitors (Fig. 5A). Interestingly, even the activities of IL-9 on lung epithelial cells, namely MUC5AC and mCLCA3/gob5 induction, were restored after BM transfer (Fig. 5, B and C), indicating that this effect can be indirectly mediated by hemopoietic cells. Similar results were obtained after transfer of spleen cells or of PBMC (data not shown).


Figure 5
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  		FIGURE 5. WT BM transfer restores IL-9 activities in IL-9 Tg/IL-9R KO recipient mice. IL-9 Tg/IL-9R KO mice were irradiated and reconstituted with 4 x 106 BM cells from C57BL/6 or IL-9R KO mice. Serum samples and lungs were harvested 3 wk after transfer. A, Serum mMCP-1 concentrations were determined by ELISA. B, For MUC5AC, quantitative RT-PCR analysis was performed, and results were normalized with beta-actin. C, mCLCA3/gob5 expression in lungs was quantified by phosphor imager analysis of Northern blots hybridized with a mCLCA3 probe. Mean and SEM were calculated from 3 mice and are representative of at least four independent experiments.

 
IL-13 produced by hemopoietic cells in response to IL-9, mediates IL-9 effects on lung epithelial cells

When IL-13 KO cells were transferred into IL-9 Tg/IL-9R KO mice, the effect of IL-9 on mast cell protease mMCP-1 was still restored but not the effect on lung epithelial cells (Fig. 6). As recipient mice had a normal capacity to produce IL-13, this observation suggests that IL-13 does not act as a cofactor for IL-9 but directly mediates its activity. To formally demonstrate this role of IL-13 on IL-9 activity, we transferred a mixed BM population containing IL-13 KO cells together with IL-9R KO cells in the IL-9 Tg/IL-9R KO mice. As shown in Fig. 7, whereas mMCP-1 expression demonstrated an efficient hemopoietic cell reconstitution, mice reconstituted with a mix of IL-13 KO and IL-9R KO cells failed to up-regulate mCLCA3/gob5 and MUC5AC expression. Taken together, these observations indicate that a functional IL-13 gene must be present in the hemopoietic cells that directly respond to IL-9, demonstrating that IL-13 is a direct mediator of IL-9 activity on lung epithelial cells.


Figure 6
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  		FIGURE 6. IL-9 Tg/IL-9R KO recipient mice reconstituted with IL-13 KO cells have a reduced expression of MUC5AC and mCLCA3 genes in lungs. IL-9 Tg/IL-9R KO mice were irradiated and reconstituted with 4 x 106 BM cells from 129, C57BL/6 (B6), or IL-13 KO mice. Serum samples and lungs were harvested 3 wk after transfer. Serum mMCP-1 concentrations were determined by ELISA (left panel). For MUC5AC, quantitative RT-PCR analysis was performed, and results were normalized with beta-actin (middle panel). mCLCA3/gob5 expression in lungs was quantified by phosphor imager analysis of Northern blots hybridized with a mCLCA3 probe (right panel). Mean and SEM were calculated from 3 to 5 mice per group and are representative of four independent experiments. The differences in MUC5AC and mCLCA3 expression between mice reconstituted with WT or IL-13 KO cells were statistically significant (p < 0.04; Mann-Whitney U test).

 

Figure 7
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  		FIGURE 7. IL-9-responsive cells must have a functional IL-13 gene to restore MUC5AC and mCLCA3 genes in IL-9 Tg lungs. IL-9 Tg/IL-9R KO recipient mice were irradiated and reconstituted with 4 x 106 BM cells from 129, C57BL/6 (B6), IL-9R KO mice, or with 2 x 106 BM cells from IL-13 KO mice together with 2 x 106 cells from IL-9R KO mice. Serum samples and lungs were harvested 3 wk after transfer. A, Serum mMCP-1 concentrations were determined by ELISA. B, mCLCA3/gob5 expression in lungs was analyzed by Northern blots with a mCLCA3 probe, before reprobing with beta actin as a control for equal loading. C, For MUC5AC, quantitative RT-PCR analysis was performed, and results were normalized with beta actin. Mean and SEM were calculated from 4 to 5 mice per group and are representative of 3 independent experiments. The differences in MUC5AC expression between mice reconstituted with WT (B6 or 129) or IL-9R KO cells (9RKO or 13 KO + 9R KO) were statistically significant (p < 0.03; Mann-Whitney U test).

 
To better characterize the hemopoietic cells that produce IL-13 in our model, cells from IL-9 Tg or WT mesenteric lymph nodes were isolated and sorted into CD4+ and CD4– populations, and IL-13 expression was analyzed by quantitative RT-PCR. As shown in Fig. 8A, IL-13 production was dramatically increased both in CD4+ and CD4– populations from IL-9 Tg mice, suggesting that both Th cells and at least another population are responsible for IL-13 production. Similar experiments performed in SCID mice overexpressing or not IL-9 also showed IL-9-dependent IL-13 induction in spleen cells (data not shown), confirming that nonlymphoid cells are involved in this process. Mast cells are obvious candidates as they respond to IL-9 and are known to produce a wide spectrum of cytokines including IL-13. To address this hypothesis BM-derived mast cell lines were obtained from WT mice in the presence of IL-3 alone or in the presence of IL-3 and IL-9. As shown in Fig. 8B, IL-9 induced a 3-fold increase in IL-13 production. A similar in vitro effect of IL-9 was found on a Th cell clone such as TS2 (1), which could be cultured in the presence of IL-2 or IL-9 (Fig. 8B).


Figure 8
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  		FIGURE 8. IL-13 gene expression in IL-9 stimulated T cells and mast cells. A, Mesenteric lymph nodes were isolated from WT and IL-9 Tg mice. CD4+ and CD4– cells were sorted by MACS and contained, respectively, >98% and <3% CD4+ cells. IL-13 quantitative RT-PCR analysis was performed, and results were normalized with beta-actin. Similar results were obtained in one independent experiment. B, RNA was extracted from TS2 Th cells cultured in the presence of IL-2 or IL-9, and from BM-derived mast cell lines (BMMC) cultured in the presence of either IL-3 alone or IL-3 and IL-9. IL-13 quantitative RT-PCR analysis was performed, and results were normalized with beta-actin.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The key role of TH2 cytokines in the pathogenesis of allergic asthma is supported by a multitude of studies in human and animal models, but the mechanism by which this TH2 cytokine network elicits the cardinal features of asthma remains partly unknown. In this article, we show that up-regulation of IL-13 expression in hemopoietic cells is responsible for the effects of IL-9 on lungs epithelial genes, whereas other activities of IL-9 on mast cells, eosinophils, and B cells are independent of IL-13.

The observation that IL-13 is dispensable for IL-9-induced mastocytosis is not surprising because mast cells express the IL-9 receptor (42) and as IL-9 directly promotes mast cell proliferation and differentiation in vitro, either alone or in synergy with other mast cell growth factors such as IL-3 and stem cell factor (11, 43). Interestingly, this mast cell-stimulating role of IL-9 appears to be the less redundant activity of this cytokine as IL-9-deficient mice show a defective mastocytosis in a pulmonary granuloma model (16), although IL-9 is not required for other mast cell-dependent processes such as systemic anaphylaxis (44).

The role of IL-9 on B lymphocyte responses remains poorly understood. In vivo, IL-9 promotes the expansion of the B1-b lymphocyte subset, and peritoneal B lymphocytes but not spleen B lymphocytes were found to bind IL-9 (27, 45). B1-b cells preferentially reside in the peritoneal cavity and are characterized by expression of the Mac1 cell surface Ag. Although they share these characteristics with CD5+ B1-a cells, IL-9-activated B1-b cells fail to recapitulate activities ascribed to B1-a cells, including natural IgM and autoantibody production (27, 46). Interestingly, in a silica-induced lung fibrosis model, the anti-fibrotic effect of IL-9 correlated with increased B cell numbers in BAL (26) and was lost in B cell-deficient mice (47). Here, by using quantitative RT-PCR for the IgM H chain gene, we showed that IL-9-induced B cell infiltration does not require IL-13. However, the relationship between this activity of IL-9 and the complex pathogenesis of asthma remains elusive.

Concerning eosinophils, the results presented here show that IL-9 acts at two distinct stages of eosinophilia. Increased eosinophil production in IL-9-Tg mice is illustrated by eosinophil expansion in the peritoneal cavity. This effect of IL-9 is not affected in IL-13-deficient mice. By contrast, the IL-9-dependent lung eosinophilia in response to bleomycin (38) is abrogated in these mice, in line with the effect of IL-9 on lung eotaxin expression. Interestingly, when bred under SPF conditions, IL-9 Tg mice showed increased eosinophil numbers in the peritoneal cavity and in the spleen, as well as increased eotaxin expression in lungs, but no BAL eosinophilia. This process requires an additional inflammatory stimulus such as bleomycin administration, or breeding in non-SPF animal facilities (data not shown). Taken together, these observations indicate that lung eosinophilia induced by IL-9 is at least a three-step process, involving increased production of eosinophils, an inflammatory stimulus that might promote expression of adherence molecules by lung endothelial cells, and an IL-13-dependent effect on eotaxin expression. Whether the effect of IL-9 on central eosinophilia is a direct effect of IL-9 on eosinophils remains to be established. Although a direct effect of IL-9 on eosinophils has been reported (8, 48), we failed so far to observe any STAT activation in mouse or human eosinophils incubated in vitro with IL-9 (unpublished data from our laboratory), and we cannot rule out the possibility that IL-9 promotes eosinophil differentiation indirectly by up-regulating IL-5 expression by other cells such as mast cells or T lymphocytes. The observation that IL-5 is required for IL-9-induced eosinophilia (38) demonstrates that there is no redundancy between IL-9 and IL-5 activities on these cells, but does not allow determination about whether IL-5 is the direct mediator or an essential cofactor for IL-9 effect on eosinophils.

Our data clearly show that the effects of IL-9 overexpression on lung epithelial cells, including up-regulation of mucus-related genes (MUC5AC, mCLCA3/gob5) and of eotaxin, are dependent on IL-13. The key role of IL-13 on lung epithelial cells and, more specifically on the regulation of MUC5AC, mCLCA3/gob5, and TFF2 genes in allergic models has been elegantly described in mice that overexpress IL-13 but express STAT6 only in non-ciliated airway epithelial cells (49, 50). Our observations support the hypothesis that the regulation of these genes in lung epithelial cells is a unique feature of IL-13, and that IL-9 has no direct effect on this cell type. However, this contrasts with several reports that suggested that lung epithelial cells directly respond to IL-9 (6, 10, 39, 40). Interestingly, it has been reported that the effect of IL-9 on such cells might depend on their state of differentiation. IL-9 could indeed stimulate goblet cells proliferation in differentiating primary cultures, but not in differentiated human airway epithelial cells (41). In addition, IL-9 has so far never been shown to activate STAT6 in any cell type, and the mucus-related genes expressed in lung airways appear to be specifically regulated by STAT6 (49), raising the possibility that IL-9 could still act directly on epithelial cells but without being able by itself to promote the mucus production pathway, which seems to be a unique IL-13 target. Further in vitro studies on highly purified cell populations will probably be needed to clarify this issue.

The fact that IL-9-induced goblet cell hyperplasia and mucus production was abolished in mice deficient for IL-13 or the IL-4R{alpha}-chain, a component of IL-13R, has been previously demonstrated (23). However, as mentioned before for IL-5 and eosinophils, this observation did not allow determination about whether IL-13 is the direct mediator or a required cofactor for IL-9 in this model. Here, by using mice Tg for IL-9 but deficient in its receptor in a cell transfer experimental model, we demonstrate that the effect of IL-9 on mucus-related genes specifically required a functional IL-13 gene in IL-9-responsive cells. Reconstitution of mice with BM cells that had a functional IL-13 gene but no IL-9 receptor, together with cells that had a functional IL-9 receptor but were deficient in IL-13, did not restore the up-regulation of lung epithelial genes obtained with WT BM. These data formally demonstrate that this activity of IL-9 is the result of IL-13 up-regulation by hemopoietic-derived cells. BM transfer experiments similar to those reported here have been performed in IL-4R{alpha}-deficient mice and showed that, in sharp contrast with IL-9R{alpha}, IL-4R{alpha} is required on epithelial cells but not on hemopoietic cells for allergic airway inflammation (51). Taken together, these data indicate that IL-9 promotes allergic inflammation by acting upstream IL-13, which is the main TH2 effector cytokine for lung epithelial cells.

Interestingly, recent data in the leishmania model indicate that blocking IL-9 down-regulates the production of TH2 cytokines by T lymphocytes, pointing to IL-9 as an upstream regulator of TH2 responses in vivo (52). In the Tg mice used in our model, IL-13 was found to be up-regulated by IL-9 in lymph node and spleen cells, and cell sorting experiments indicated that both CD4+ and CD4– cells were involved in this process. In vitro, IL-9 stimulation of T lymphoma cell lines, Th cell clones, and mast cell lines resulted in increased production of IL-13 (Fig. 8 and Ref. 53), suggesting that at least T lymphocytes and mast cells are responsible for IL-13 up-regulation by IL-9. Further in vitro studies will be required to unravel the molecular mechanisms that are responsible for this regulation of IL-13 expression.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by the Belgian Federal Service for Scientific, Technical, and Cultural Affairs, by the Actions de Recherche Concertées of the Communauté Française de Belgique and the opération Télévie. F.H. is a research associate with the Fonds National de la Recherche Scientifique, Belgium. Back

2 Address correspondence and reprint requests to Dr. Jean-Christophe Renauld, Ludwig Institute for Cancer Research and Experimental Medicine Unit, Université Catholique de Louvain, Avenue Hippocrate 74, Brussels, Belgium. E-mail address: Jean-Christophe.Renauld{at}bru.licr.org Back

3 Abbreviations used in this paper: Tg, transgenic; AHR, airway hyperresponsiveness; KO, knockout; mMCP-1, mouse mast cell protease 1; mCLCA3, mouse calcium-activated chloride channel 3; TFF2, trefoil factor 2; Retnla, resistin-like {alpha}; WT, wild type; SPF, specific pathogen free. Back

Received for publication August 23, 2006. Accepted for publication November 30, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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J. Xiang, J. Rir-Sim-Ah, and Y. Tesfaigzi
IL-9 and IL-13 Induce Mucous Cell Metaplasia That Is Reduced by IFN-{gamma} in a Bax-Mediated Pathway
Am. J. Respir. Cell Mol. Biol., March 1, 2008; 38(3): 310 - 317.
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