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The Journal of Immunology, 2004, 172: 1092-1098.
Copyright © 2004 by The American Association of Immunologists

Polymorphisms in IL-4R{alpha} Correlate with Airways Hyperreactivity, Eosinophilia, and Ym Protein Expression in Allergic IL-13-/- Mice 1

Dianne C. Webb2,*, Klaus I. Matthaei*, Yeping Cai*, Andrew N. J. McKenzie{dagger} and Paul S. Foster*

* Division of Molecular Bioscience, The John Curtin School of Medical Research, Australian National University, Australian Capital Territory, Australia; and {dagger} Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The development of airways hyperreactivity in allergic IL-13-/- mice is controversial and appears to correlate with the number of times that the original 129 x C57BL/6 founder strain has been crossed to the BALB/c background. In this investigation, we compared allergic responses in founder IL-13-/- mice crossed for either 5 (N5) or 10 (N10) generations to BALB/c mice. Whereas allergic N5 IL-13-/- mice developed airways hyperreactivity, tissue eosinophilia, elevated IgE, and pulmonary expression of Ym proteins, these processes were attenuated in N5 IL-13-/- mice treated with an IL-4-neutralizing Ab, and in N10 IL-13-/- mice. These data showed that IL-4 was more effective in regulating allergic responses in N5 IL-13-/- mice than in N10 IL-13-/- mice. To elucidate the mechanism associated with these observations, we show by restriction and sequence analysis that N5 IL-13-/- mice express the C57BL/6 form of IL-4R{alpha} and N10 IL-13-/- mice express the BALB/c form. Despite the near identical predicted molecular mass of these isoforms, IL-4R{alpha} from N5 IL-13-/- mice migrates with a slower electrophoretic mobility than IL-4R{alpha} from N10 IL-13-/- mice, suggesting more extensive posttranslational modification of the N5 form. The Thre49Ile polymorphism in the extracellular domain of BALB/c IL-4R{alpha} has been demonstrated to disrupt N-linked glycosylation of Asn47 and increase the dissociation rate of the IL-4R{alpha}/IL-4 interaction. Collectively, these data show that polymorphisms in IL-4R{alpha}, which have been shown to affect the interaction with IL-4, correlate with the ability of IL-4 to regulate allergic responses in IL-13-/- mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Asthma arises from the dysregulation of pulmonary immune responses to normally innocuous environmental allergens. CD4+ Th2 lymphocytes are a predominant cellular infiltrate in the asthmatic lung (1, 2), and the cytokines produced by these cells underlie the development of key phenotypic features of asthma that include eosinophilia, elevated IgE, enhanced mucus production, airway remodeling, and changes in the contractility of the smooth muscle layer surrounding the airways. These processes ultimately result in thickening of the airway wall, occlusion, and acute airway constriction. Research into the mechanisms by which Th2 cytokines regulate asthma has recently focused on IL-4 (3, 4, 5, 6, 7, 8) and IL-13 (9, 10), cytokines whose functions are linked by their dependence on the IL-4R{alpha} subunit (IL-4R{alpha}) for signal transduction.

The type I receptor comprising IL-4R{alpha} and the common {gamma}-chain is expressed by hemopoietic cells and is exclusively responsive to IL-4. This receptor plays an important role in the proliferation, differentiation, and phenotype stabilization of Th2 lymphocytes (11, 12) and in isotype switching of B cell Ab production to IgE (13). In contrast, the type II receptor comprising IL-4R{alpha} and IL-13R{alpha}1 is responsive to both IL-4 and IL-13 (14) and is expressed by a diverse range of cells, including those of nonhemopoietic origin (15, 16). However, despite IL-4 and IL-13 potentially targeting common cells expressing IL-4R{alpha}/IL-13R{alpha}1, the transient early peak in levels of IL-4 compared with the delayed, but more sustained expression of IL-13 following T cell stimulation (17, 18) suggests that the latter cytokine plays a more dominant role in downstream effector responses. In mouse models, blockade of IL-13 with sIL-13R{alpha}2Fc, a soluble high affinity IL-13 antagonist, ablates allergen-induced accumulation of eosinophils in the airway lumen, the hypersecretion of mucus, and airways hyperreactivity (AHR)3 to cholinergic challenge (9, 10, 19). In addition, overexpression of IL-13 in naive transgenic mice induces features of asthma including subepithelial fibrosis (20, 21). Collectively, these data suggest that IL-13 is pivotal in modulating allergic inflammation, mucus secretion, airway remodeling, and AHR.

Although a number of studies have investigated the development of allergic responses in mice in which IL-13 was neutralized with the sIL-13R{alpha}2Fc antagonist, or in transgenic mice that overexpress IL-13 specifically in the lungs, our laboratory was the first to report the somewhat controversial finding that AHR, Th2 responses, eosinophilia, and enhanced expression of the novel allergy-associated Ym2 protein persist in the lungs of allergic IL-13-/- mice (8, 22). In addition, treatment of these allergic IL-13-/- mice with an IL-4-neutralizing Ab ablated AHR, Ym2 expression, and tissue eosinophilia, suggesting that IL-4 was able to substitute for IL-13 to induce aspects of allergic disease. In direct contrast to our work, a subsequent report by Walter et al. (23) showed that IL-13-/- mice failed to develop AHR. The IL-13-/- mice used in the two studies originated from the same laboratory (24), and similar allergy models had been used. However, a distinct difference was the number of generations that the IL-13-/- mice had been crossed with the BALB/c strain from the founder 129 x C57BL/6 background. Our study had used 129 x C57BL/6 mice crossed for five generations onto the BALB/c strain (N5), whereas in the Walter et al. study, the mice had been crossed for a further two generations.

Therefore, the aim of the current study was to address the conflicting observations relating to the development of allergic disease in IL-13-/- mice. In this study, we report characterization of N10 IL-13-/- mice and show that polymorphisms in IL-4R{alpha} from N5 and N10 IL-13-/- mice correlate with the development of AHR, eosinophilia, and expression of the Ym proteins.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Induction of allergic airways inflammation

Two breeding pairs of N5 (129/C57BL/6 x BALB/c) IL-13-/- mice were obtained from the Animal Facilities, Medical Research Council Laboratories (Cambridge, U.K.) (24), to generate a breeding colony at the John Curtin School of Medical Research. These mice were also crossed for a further five generations onto the BALB/c background to generate an N10 IL-13-/- colony. Pulmonary allergy was induced in equal numbers of 6- to 8-wk-old male and female mice by i.p. sensitization with 50 µg of OVA mixed with 1 mg of Rehydrogel (Reheis, Berkeley Heights, NJ) in 0.9% sterile saline (Sal). Sal-sensitized control mice received 1 mg of alhydrogel in 0.9% Sal. On days 12, 14, 16, and 18, all mice were aeroallergen challenged with an aerosol of 10 mg/ml OVA in 0.9% Sal for 3 x 30 min per day with 30-min breaks between aerosols, as previously described (7, 8, 25). In some experiments 24 h before the first priming with OVA, IL-4 was neutralized by i.p. injection with 1 mg of 11B11 mAb and then mice were treated weekly throughout the experimental period. Twenty-four hours after the last challenge, AHR was measured. Mice were then sacrificed by cervical dislocation, and bronchoalveolar lavage fluid (BALF) was obtained by cannulating the trachea and gently flushing the airways with two 1-ml vol of HBSS. Mice were treated according to Australian National University Animal Welfare guidelines (Protocol 59/01) and were housed in a specific pathogen-free facility.

Measurement of AHR

Responsiveness to {beta}-methacholine was assessed in conscious, unrestrained mice by barometric plethysmography, using apparatus and software supplied by Buxco (Troy, NY). Measurement was performed essentially as previously described (8, 26). Briefly, mice were placed in the plethysmograph chamber and exposed to an aerosol of water (baseline readings) and then cumulative concentrations of {beta}-methacholine ranging from 3.125 to 25 mg/ml. The aerosol was generated with an ultrasonic nebulizer and drawn through the chamber for 2 min. The inlet was then closed, and enhanced pause (Penh) readings were taken for 3 min and averaged. We have previously shown a direct correlation between Penh and airway resistance in response to {beta}-methacholine challenge in BALB/c mice (27). Values were reported as the mean percentage increase over baseline ± SEM (n = 6–8 mice per group).

Characterization of eosinophils in pulmonary tissue and BALF

Lungs were fixed in 10% phosphate-buffered Formalin, sectioned, and stained with Carbol’s chromotrope-hematoxylin, and the number of eosinophils in the bronchi-bronchiole region of 20 high power fields (x1000 magnification) was counted per mouse. Leukocytes in the BALF were identified by morphological criteria, as previously described (7, 25), using cytospins stained with May-Grünwald Giemsa.

Determination of OVA-specific IgE by ELISA

OVA-specific IgE was measured with an ELISA capture assay. Total serum IgE was bound by solid-phase rat anti-mouse IgE (Zymed Laboratories, San Francisco, CA). OVA-specific IgE was then detected with biotinylated OVA and streptavidin-APase (Amersham Pharmacia Biotech, Sydney, Australia). Data were expressed as ELISA units, which were the product of the dilution and the OD. All comparative assays were conducted concurrently.

Proliferation and cytokine production by IL-13-/- Th2 CD4+ cells cultured in vitro

Equal numbers (4 and 5) of male and female mice were sensitized at 6–8 wk of age by i.p. injection with 50 µg of OVA mixed with 1 mg of Rehydrogel in 0.9% sterile Sal. Splenocytes were recovered after 7 days, erythrocytes were lysed, and the washed splenocytes were stimulated with 200 µg/ml of OVA and polarized toward a Th2 phenotype with 40 ng/ml of rIL-4 (kind gift from S. Ford and I. Young, John Curtin School of Medical Research) and anti-IFN-{gamma} Ab (clone R46A2, 70 µg/ml) in MLC medium for 7 days at 37°C and 5% CO2. CD4+ cells were then purified using the Minimacs magnetic bead system following the manufacturer’s recommendations (Miltenyi Biotec, Nordheim West Falen, Germany). CD4+ cells (5 x 106/ml) were incubated in MLC medium containing 200 µg/ml OVA, and with 1 x 106 mitomycin C-treated (25 µg/ml for 60 min at 37°C, followed by extensive washing in PBS), naive splenocytes to serve as APCs. Mitomycin C-treated splenocytes cultured in MLC medium containing 200 µg/ml OVA without T cells were used as a control. After cells had been cultured for 2 days, proliferation was determined with the CellTitre 96 reagent (Promega, Madison, WI) and following the manufacturer’s recommendations. Supernatants were also collected and assayed for IL-4, IL-5, and IFN-{gamma}. Cytokines were measured by sandwich ELISA, as described elsewhere (8). The sensitivity of detection was 12 pg/ml for IL-4, 24 pg/ml for IL-5, and 50 pg/ml for IFN-{gamma}. The paired IL-4, IL-5, and IFN-{gamma} Abs and standards were obtained from BD PharMingen (San Diego, CA).

SDS-PAGE and Western blotting

BALF supernatants (10 µl/well from a pool derived from 4–5 mice per group) were heated to 90°C in SDS-PAGE sample buffer (Invitrogen, Carlsbad, CA) containing 5% 2-ME and electrophoresed on 4–12% SDS-PAGE gels (Invitrogen). Western immunoblots were then performed on BALF proteins that were electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane using a Multiphor Novablot semidry transfer system (Pharmacia Biotech, Uppsala, Sweden). The membrane was blocked with 2% BSA in TBST (TBS, 0.05% Tween 20), probed with a 1/1000 dilution of rabbit anti-Ym Ab, which detects both Ym1 and Ym2 proteins (22), and then with alkaline phosphatase-conjugated anti-rabbit Ab (Sigma-Aldrich, St. Louis, MO). The alkaline phosphatase was detected with stabilized Western Blue substrate (Promega). The relative intensities of bands, after correcting for individual lane background, were determined using a Sygene BioImaging System (Sygene International, Frederick, MD).

RT-PCR, restriction analysis, DNA sequence analysis, and genomic DNA preparation

Total RNA was purified from lung tissue using TRIzol reagent (Life Technologies, Rockville, MD) and reverse transcribed to cDNA using oligo(dT) and Thermoscript enzyme (Life Technologies, Rockville, MD). DNA specific for the gene encoding IL-4R{alpha} was amplified by PCR using the forward primer, DW93, 5'-GCAGGCACCTTTTGTGTCCCC, which hybridizes at 22 bp 5' of the methionine start codon, and the reverse primer, DW95, 5'-CCCTGGCCTCAGCACAGACTC, which hybridizes at 24 bp 3' of the stop codon (28). PCR was performed for 35 cycles with high fidelity Pfx polymerase (Life Technologies) at an annealing temperature of 59°C to generate a product of 2614 bp encoding the complete IL-4R{alpha} reading frame. PCR products were purified with the QIAquick purification kit (Qiagen, Victoria, Australia) and then either digested with Eco47III restriction endonuclease (Promega) and analyzed by gel electrophoresis or A-tailed and cloned into the plasmid vector pGEM-T (Promega). The resultant clones were sequenced using PRISM Ready Reaction Dye-Deoxy Terminator Cycle Sequencing reagents and an Applied Biosystems (Foster City, CA) automated sequencing system model 373A (PerkinElmer Roche, Branchburg, NJ) with the SP6 and T7 promoter primers, which hybridize to vector sequences, and internal primers based on the sequence of IL-4R{alpha} GenBank entry NM_010557. At least two independent clones per mouse strain were sequenced. Genomic DNA was prepared from mouse tails that were incubated overnight at 56°C in lysis buffer (0.2% SDS, 5 mM EDTA, 200 mM NaCl, and 100 µg/ml proteinase K). PCR for exon 4, which contains the Eco47III restriction site, was performed using the forward primer, DW106, 5'-GGGAGCATCAAGGTCCTGGG and the reverse primer, DW107, 5'-TCAGAGAACTCGAAGAACATCAG to generate a product of 142 bp. PCR products were purified and then digested with Eco47III. Genomic DNA and Eco47III screening was conducted on at least eight male and female mice from different litters per IL-13-/- strain and from two wild-type (WT) BALB/c and C57BL/6 mice.

Electrophoretic mobility of IL-4R{alpha} from IL-13-/- CD4+ T cells

Th2-biased CD4+ cells were purified, as described above, and freeze thawed, treated with 1 mg/ml DNase 1 (Roche Diagnostics, Sydney, New South Wales, Australia) for 20 min at 37°C, and then heated to 90°C for 5 min in SDS-PAGE sample buffer. The cell extract equivalent to 4 x 105 CD4+ cells was loaded per lane of a 4–12% SDS-PAGE gel. Western transfer and blotting were as described above, using 1/400 dilution of anti-murine IL-4R{alpha} Ab (S-20; Santa Cruz Biotechnology, Santa Cruz, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
AHR, eosinophilia, and IgE in N5 and N10 IL-13-/- mice

We previously reported that OVA sensitization induced AHR and a vigorous tissue and BALF eosinophilia in N5 IL-13-/- mice (8) (Fig. 1). In contrast, OVA sensitization failed to induce AHR in N10 IL-13-/- mice (Fig. 1B) and this was accompanied by a less robust tissue eosinophilia with negligible numbers of eosinophils in the airway lumen (Fig. 1, C and D). The AHR and spatial distribution of eosinophils in OVA-sensitized N10 IL-13-/- mice were similar to that observed in OVA-sensitized N5 IL-13-/- mice treated with IL-4-neutralizing Ab (Fig. 1, A–D). OVA-sensitized N5 IL-13-/- mice developed elevated levels of Ag-specific IgE by an IL-4-dependent mechanism (Fig. 1E). In contrast, no significant increase in IgE was observed in OVA-sensitized N10 IL-13-/- mice. Collectively, these data suggested that IL-4 is able to function in N5 IL-13-/- mice to regulate AHR, eosinophilia, and IgE, but is less efficient at regulating these processes in N10 IL-13-/- mice.



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  		FIGURE 1. AHR, eosinophilia, and IgE in OVA-sensitized N5 and N10 IL-13-/- mice. Airway reactivity (Penh: mean of six to eight mice per group ± SEM) to cumulative concentrations of {beta}-methacholine is represented as the percent increase over a baseline of 100% in response to water (A and B: *, p < 0.05). The mean number of peribronchial eosinophils (C) in 20 similar high power microscopic fields per mouse was determined. Total airway eosinophils (D) was determined by correlating total cell numbers in BALF with differential counts on May-Grünwald Giemsa-stained cytospins. Serum IgE was measured by an ELISA capture assay (E). (#, p < 0.05 for N10 IL-13-/- compared with N5 IL-13-/- OVA-sensitized mice).

 
Ym expression in N5 and N10 IL-13-/- mice

Expression of the Ym proteins, which are homologous to a family of proteins associated with vascular smooth muscle differentiation and tissue remodeling (29, 30), is enhanced in allergic mice by a process that is dependent on IL-4R{alpha} (22). OVA sensitization also induces secretion of Ym proteins into the airways of N5 IL-13-/- mice, but not in N5 IL-13-/- mice that were also deficient in IL-4 (22) (Fig. 2). In contrast, expression of Ym was reduced in OVA-sensitized N10 IL-13-/- mice to 45% of that seen in N5 IL-13-/- mice (Fig. 2). Therefore, while it seems that IL-4 can partially substitute for IL-13 in regulating Ym expression in N5 IL-13-/- mice, it does so less effectively in N10 IL-13-/- mice.



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  		FIGURE 2. Western blot for Ym protein expression in cytokine-deficient mice. Proteins in the supernatants from BALF were separated by SDS-PAGE and then electrophoretically transferred to a PVDF membrane. Ym proteins were detected on Western blots with rabbit anti-Ym Ab and alkaline phosphatase-conjugated anti-rabbit Ab (A). Densitometry of bands (B) was determined with a Sygene BioImaging System. Lane 1, N5 IL-13-/- Sal; lane 2, N5 IL-13-/- OVA; lane 3, N5 IL-13-/- OVA/anti-IL-4; lane 4, N10 IL-13-/- Sal; lane 5, N10 IL-13-/- OVA. The blot is representative of two independent experiments.

 
Differential proliferation and cytokine production by N5 and N10 IL-13-/- CD4+ T cells

The proliferative and cytokine responses of N5 IL-13-/- and N10 IL-13-/- CD4+ T cells biased under Th2-polarizing conditions were determined. Splenocytes from OVA-sensitized mice were cultured in the presence of OVA, anti-IFN-{gamma}, and IL-4. Th2-biased CD4+ cells were then purified and recultured in the absence of either exogenous IL-4 or anti-IFN-{gamma} Ab. After 2 days, cellular proliferation and the secretion of IL-4 and IL-5 were significantly lower in N10 IL-13-/- compared with N5 IL-13-/- CD4+ T cell cultures (Fig. 3). Notably, the most significant disparity was the production of IL-4 by N10 IL-13-/- cells, which was 42% lower than that produced by N5 IL-13-/- cells. No proliferation, or IL-4 and IL-5 production, by the control mitomycin C-treated splenocytes cultured in the absence of CD4+ T cells, or IFN-{gamma} in any culture supernatants, was detected (data not shown). These observations show that N5 IL-13-/- Th2-biased CD4+ cells have a more robust proliferative response, and higher IL-4 and IL-5 production than N10 IL-13-/- CD4+ cells.



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  		FIGURE 3. Proliferation and cytokine production by Th2-biased CD4+ cells from N5 IL-13-/- and N10 IL-13-/- mice. Splenocytes from OVA-sensitized mice were polarized toward a Th2 phenotype, and then purified CD4+ cells were incubated with mitomycin C-treated naive splenocytes and OVA. Proliferation was determined after 2 days with CellTitre reagent (A). IL-4 and IL-5 levels in culture supernatants on day 2 were determined by ELISA (B and C). The data are presented as the mean ± SEM of three independent cytokine/proliferation assays and representative of two independent cell culture assays.

 
Polymorphisms in the genes encoding the N5 and N10 IL-4R{alpha} subunits

Difference in sequences of the genes encoding the C57BL/6 and BALB/c IL-4R{alpha} has been identified and results in amino acid substitutions that alter the avidity of the receptor for IL-4 (28). As IL-4 was more effective in regulating allergic responses in N5 than in N10 IL-13-/- mice, and as these mice were derived from a 129 x C57BL/6 background, the genes encoding IL-4R{alpha} in both N5 and N10 IL-13-/- mice were characterized to determine whether polymorphisms in IL-4R{alpha} were associated with altered IL-4 production and function. The cDNA encoding IL-4R{alpha} was amplified by RT-PCR from the lungs of OVA-sensitized N5 IL-13-/- mice. Because the T to C substitution at nucleotide position 78 (numbering is counted from either the first coding nucleotide or the first amino acid in the mature IL-4R{alpha}) results in the loss of an Eco47III restriction endonuclease site in the BALB/c, but not the C57BL/6 IL-4R{alpha} (28), the IL-4R{alpha} PCR products were incubated with this enzyme. The IL-4R{alpha} genes from N5 IL-13-/- mice were digested with Eco47III, suggesting that the IL-4R{alpha} in these mice was of C57BL/6 origin (Fig. 4A). In comparison, the IL-4R{alpha} genes from N10 IL-13-/- mice and from WT BALB/c mice were resistant to digestion, suggesting that the IL-4R{alpha} in N10 IL-13-/- mice was of BALB/c origin. Moreover, complete digestion of the PCR products from exon 4 of IL-4R{alpha} genomic DNA from N5 IL-13-/- mice demonstrated that these mice were homozygous for the C57BL/6 form of IL-4R{alpha}. In contrast, digestion resistance of DNA from N10 IL-13-/- mice demonstrated that these mice were homozygous for the BALB/c form of IL-4R{alpha} (Fig. 4B). Notably, the T78C nucleotide substitution in the Eco47111 site (Fig. 4C) is silent and does not result in amino acid substitution of the encoded Ala26. The IL-4R{alpha} cDNA from N5 and N10 IL-13-/- mice was then sequenced. As suggested by the restriction digests, the sequence of the IL-4R{alpha} cDNA from N5 IL-13-/- mice was identical with the published C57BL/6 sequence (GenBank NM 010557). In comparison, apart from one nucleotide change, the sequence of the IL-4R{alpha} cDNA from N10 IL-13-/- mice was identical with the published BALB/c IL-4R{alpha} sequence (GenBank AF000304). An A to G nucleotide substitution identified at 1514 in N10 IL-13-/- mice is novel and results in a Glu505Gly mutation in the intracytoplasmic domain of the receptor (Fig. 5). The IL-4R{alpha} gene from WT BALB/c mice was also sequenced and a Glu505Gly substitution was observed (data not shown), suggesting that this substitution is inherent in our colony of WT BALB/c mice. Overall, the IL-4R{alpha} expressed in N5 IL-13-/- mice differed from the IL-4R{alpha} in N10 IL-13-/- mice by 9 aa (Fig. 5)



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  		FIGURE 4. Eco47III restriction endonuclease digestion of cDNA and genomic DNA from N5 and N10 IL-13-/- mice. A, The mRNA encoding IL-4R{alpha} was isolated from the allergic lungs of N5 IL-13-/-, N10 IL-13-/-, and WT BALB/c mice and reverse transcribed into cDNA. The corresponding IL-4R{alpha}-specific PCR products were then digested with Eco47III. B, Genomic DNA was prepared from tail samples from WT C57BL/6, WT BALB/c, N5, and N10 IL-13-/- mice, and then exon 4 of IL-4R{alpha} was amplified by PCR. The PCR products were digested with Eco47III, which cuts the 142-bp exon 4 to give 61- and 81-bp fragments. These two closely migrating restriction fragments are seen as one broad band on the 2% agarose gel (* indicates Eco47III-digested samples, and unmarked samples are untreated controls). The Eco47III restriction digestion pattern of exon 4 shown in B is representative of at least eight male and female mice selected from different litters of the N5 and N10 IL-13-/- mice and two WT BALB/c and C57BL/6 mice. C, Schematic representation of exon 4 of IL-4R{alpha}. The underlined T nucleotide in the Eco47III recognition sequence in N5 IL-13-/- IL-4R{alpha} is substituted with C in N10 IL-13-/- IL-4R{alpha}.

 


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  		FIGURE 5. The translated protein sequence of the IL-4R{alpha} genes derived from N5 and N10 OVA-sensitized IL-13-/- mice. IL-4R{alpha}-specific PCR of cDNAs derived from the lungs of OVA-sensitized mice were cloned into the pGEM-T vector, and representative clones were sequenced. The IL-4R{alpha} gene from N5 IL-13-/- mice (C57BL/6 derived: upper sequence) and from N10 IL-13-/- mice (BALB/c derived: lower sequence) differs overall by 9 aa. The leader sequence is boxed, and numbering is from the first amino acid of the mature protein. Asn47 (*) is glycosylated in C57BL/6 IL-4R{alpha}, but not BALB/c IL-4R{alpha} (28 ). The Glu(E)505Gly(G) substitution (bold) in N10 IL-13 IL-4R{alpha} has not been previously reported in the BALB/c IL-4R{alpha} sequence.

 
Differential electrophoretic mobility of the IL-4R{alpha} in N5 and N10 IL-13-/- mice

Comparison of the BALB/c and C57BL/6 IL-4R{alpha} genes expressed heterologously in the human TF-1 erythroleukemic cell line suggested that the C57BL/6 receptor was differentially glycosylated and as a consequence bound IL-4 with greater avidity compared with BALB/c IL-4R{alpha} (28). The predicted molecular mass of the C57BL/6 and BALB/c IL-4R{alpha} proteins is 87.62 and 87.63 kDa, respectively. However, IL-4R{alpha} proteins expressed by Th2-biased CD4+ T cells from N5 and N10 IL-13-/- mice migrated with a molecular mass of ~98 kDa, suggesting posttranslational modification (Fig. 6). Notably, the slower mobility of the N5 IL-13-/- IL-4R{alpha} protein suggests a higher molecular mass and is indicative of more extensive modification compared with N10 IL-13-/- IL-4R{alpha}. This observation is consistent with the previously observed absence of N-linked glycosylation of Asn47 in BALB/c IL-4R{alpha} due to the Thre49Ile mutation and disruption of the Asn-Xaa-Ser/Thr recognition sequence (Fig. 5) (28, 31).



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  		FIGURE 6. Western blot for IL-4R{alpha} in Th2-biased CD4+ cells from N5 IL-13-/-, N10 IL-13-/-, and IL-4R{alpha}-/- (negative/Ctrl) mice. Proteins from lysates (equivalent to 4 x 105 cells) were resolved by 4–12% SDS-PAGE and then electrophoretically transferred to a PVDF membrane. IL-4R{alpha} was detected with anti-murine IL-4R{alpha} Ab. The position of phosphorylase B in the far right lane (Invitrogen; SeeBlue prestained protein standards), which migrates with a molecular mass corresponding to 98 kDa, is indicated.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we provide evidence that IL-4 functions more efficiently in N5 IL-13-/- mice, than in N10 IL-13-/- mice, to regulate AHR, eosinophilia, IgE, expression of Ym proteins, and Th2-biased CD4+ cell function. We have identified polymorphisms in the IL-4R{alpha} expressed by these IL-13-/- mice, and together with supportive data from a previous study (28), propose that differential glycosylation of the extracellular domain of these receptor isoforms underlies the efficacy with which IL-4 can substitute for IL-13.

Our previous observation that the AHR that developed in allergic N5 IL-13-/- mice could be inhibited with anti-IL-4 Ab (8) suggested that IL-4 could substitute for IL-13, either directly by targeting effector cells or indirectly through regulation of other Th2 cytokines. AHR and subepithelial remodeling have also been shown to develop in N6 IL-13-/- mice in a chronic model of asthma (32). These observations are in contrast to those reported by Walter et al. (23), who showed that IL-13 was critical for the development of allergen-induced AHR in N7 IL-13-/- mice. To address whether the disparity between N5 and N7 IL-13-/- mice was associated with the number of generations that the IL-13-/- mice had been crossed from the original 129 x C57BL/6 background, we crossed the N5 IL-13-/- mice onto the BALB/c background for a further five generations. Interestingly, similar to N7 IL-13-/- mice, N10 mice failed to develop AHR in response to an allergy-promoting regime. Further investigation revealed that although N10 IL-13-/- mice developed tissue eosinophilia (albeit reduced compared with allergic N5 IL-13-/- mice), the negligible numbers of eosinophils in the airway lumen resembled the spatial distribution of eosinophils in N5 IL-13-/- mice deficient in IL-4. This suggested that some aspect of eosinophil recruitment that was regulated by IL-4 was deficient in N10 IL-13-/- mice. Notably, this may be due to the observation that IL-4 can modulate allergy-induced inflammation by regulating expression of the adhesion molecule VCAM-1, which binds to the VLA-4 integrin expressed on eosinophils and lymphocytes (3, 33, 34, 35).

The differential function of IL-4 in allergic IL-13-/- mice is further demonstrated by the levels of OVA-specific IgE Ab. Whereas IgE is elevated in allergic N5 IL-13-/- mice, no allergy-induced IgE was seen in N5 IL-13-/- mice treated with anti-IL-4 Ab or in allergic N10 IL-13-/- mice. Although isotype switching to IgE has been shown to occur independently of IL-4 in response to chronic expression of IL-13 (36) or prolonged Ag exposure (37), IL-4 is important in generating IgE in similar models as used in the current study (6, 38). Thus, the absence of IgE in allergic N10 IL-13-/- mice suggests that IL-4 is less efficient at regulating IgE in N10 IL-13-/- mice than in N5 IL-13-/- mice.

We recently identified the Ym2 lectin as a novel allergy-related protein (22). Although homologous proteins have been associated with tissue remodeling, the role of Ym proteins in allergy-related processes to date is unclear. However, we have shown that enhanced expression of Ym is lost in WT allergic mice depleted of CD4+ T cells and in allergic IL-4R{alpha}-/- mice (22). In comparison, Ym expression is enhanced in allergic N5 IL-13-/- mice, but not in the same strain treated with anti-IL-4 Ab. Similarly, expression of Ym is reduced (but not ablated) in allergic N10 IL-13-/- mice, suggesting IL-4 is less efficient at substituting for IL-13 in N10 IL-13-/- mice than in N5 IL-13-/- mice to regulate Ym expression.

To investigate why IL-4 plays a differential role in allergic N5 and N10 IL-13-/- mice, we examined CD4+ T cell responses. The manner by which lymphocytes proliferate and bias toward a Th2 phenotype and thus expression of IL-4 is a co-coordinated process of Ag-driven signals from the TCR combined with signals elicited by ligation of IL-4R{alpha} by IL-4 (39). Signals through IL-4R{alpha} recruit Janus family tyrosine kinases that phosphorylate the receptor to activate STAT6 and enhance expression of GATA-3 (reviewed in Ref. 12). Although the combination of transcription factors that regulate the expression of IL-4 and IL-5 is complex, GATA-3 has been suggested as an important Th2-specific factor in chromatin remodeling of the flanking regions of the closely linked IL-4 and IL-13 genes and the intergenic regulatory region controlling expression of the IL-4/IL-13/IL-5 gene cluster (40). Several studies have shown that GATA-3 also directly activates the IL-5 promoter (41, 42). In addition to the Janus kinase-STAT6 pathway, IL-4 has been shown to stimulate cell proliferation by IL-4R{alpha} activation of the insulin receptor substrate-1/2 pathway (reviewed in Refs. 12 and 43). In the current study, proliferation and IL-4 and IL-5 production by N10 IL-13-/- CD4+ T cells were reduced compared with N5 IL-13-/- CD4+ T cells. As IL-4R{alpha} plays a central role in both proliferation and Th2 cytokine production, it seems plausible that IL-4R{alpha} from N10 IL-13-/- mice is less efficient in regulating these processes than IL-4R{alpha} from N5 IL-13-/- mice. Notably, we have observed that similarly biased CD4+ T cells from IL-4R{alpha}-/- mice show a pronounced defect in proliferative and Th2 cytokine responses compared with CD4+ T cells from WT BALB/c mice (manuscript in preparation).

In an attempt to understand why the IL-4R{alpha}/IL-4 pathway is less efficacious in N10 IL-13-/- mice than in N5 IL-13-/- mice, we drew on a previous study that examined polymorphisms in IL-4R{alpha} from C57BL/6 and BALB/c mice (28). Importantly, this study demonstrated that the Thre49Ile substitution in BALB/c mice disrupted the Asn-Xaa-Ser/Thre N-linked glycosylation recognition sequence (31) and glycosylation of Asn47. When expressed heterologously in the human TF-1 cell line, IL-4R{alpha} with N-glycosylated Asn47 (C57BL/6 IL-4R{alpha}) bound IL-4 with greater avidity than when Asn47 is unmodified. In the current study, we show, by restriction and sequence analysis, that the IL-4R{alpha} expressed by N5 IL-13-/- mice is the C57BL/6 form of IL-4R{alpha} and the N10 IL-13-/- mice express the BALB/c form. Furthermore, the IL-4R{alpha} from N5 IL-13-/- mice migrates with a slower electrophoretic mobility than IL-4R{alpha} from N10 IL-13-/- mice, suggesting, consistent with Schulte et al. (28), that the N5 form (C57BL/6), but not the N10 form (BALB/c) of IL-4R{alpha} is glycosylated at Asn47. Schulte et al. showed a marked increase in the dissociation rate of IL-4 for BALB/c IL-4R{alpha} (20 x 10-3 min-1) compared with the C57BL/6 receptor (3 x 10-3 min-1) (28). The faster dissociation rate of IL-4 probably limits the intensity of signaling by IL-4R{alpha}, either through direct contact or by modulating the interaction with the heterologous receptor partner. We extend these kinetic analyses to suggest that the more rapid dissociation rate of the IL-4R{alpha} expressed in N10 IL-13-/- mice is associated with the efficacy with which IL-4 can substitute for IL-13.

The concept that C57BL/6 IL-4R{alpha} binds IL-4 with greater avidity than BALB/c IL-4R{alpha} and enables IL-4 to substitute for IL-13 is seemingly in conflict with the paradigm developed in parasite models that C57BL/6 mice are low Th2 responders. However, recent studies have demonstrated that while allergic C57BL/6 mice ultimately develop both a robust pulmonary eosinophilia and Th2 cytokine bias, these responses were delayed by 24 h compared with similarly treated BALB/c mice (44, 45). Nonetheless, C57BL/6 mice do not develop AHR to the same degree as BALB/c mice, and this correlates with the failure of eosinophils to localize to the peribronchial region (45). Therefore, the capacity to mount a Th2 response is delayed, but not impaired per se in allergic C57BL/6 mice, and our observations suggest that differences in the development of allergy-related processes in these mice may be due to factors other than those associated with the IL-4/IL-4R{alpha} signaling process.

Although controversial, several studies have implicated the development of atopy with gain of function mutations in human IL-4R{alpha} (46, 47, 48, 49). Of relevance to the current study is the observation that the Ile50Val variant, which is the only known extracellular amino acid polymorphism in human IL-4R{alpha}, enhances activation of STAT6, cellular proliferation, and IgE synthesis (47, 50). Although both the mouse and human extracellular polymorphisms, which occur in a loop between two {beta} strands of IL-4R{alpha}, are adjacent to a highly conserved cysteine (51), the human variant is not thought to influence glycosylation and the kinetics of IL-4 binding (47). Despite the disparity in the effect that the human and mouse polymorphisms have on the avidity for IL-4, this region is clearly a hot spot with respect to regulating intracellular signals affecting the functionality of IL-4R{alpha}.

The presence of a homozygous C57BL/6 IL-4R{alpha} in the genome of IL-13-/- mice backcrossed to BALB/c mice for five generations is perplexing. We can only assume that the original two breeding pairs we obtained had some degree of heterogeneity of IL-4R{alpha}, which resulted in homozygous offspring that may have been randomly selected as a basis for our breeding colony. IL-4R{alpha} maps to mouse chromosome 7, indicating that selection for the IL-13 mutation (chromosome 11) would not influence selection for the C57BL/6 IL-4R{alpha}. In addition, as the IL-4/IL-5/IL-13 gene cluster is only 160 kb in length and the IL-4/IL-13 genes only 13 kb apart, it is highly likely that these genes are of 129/C57BL/6 origin, considering that the IL-13 mutation is selected for in the BALB/c crosses. Thus, heterogeneity in the genomic DNA encoding IL-4R{alpha}, rather than in IL-4 coding or regulatory regions, is a likely explanation for variability in IL-4 expression by CD4+ T cells from N5 and N10 IL-13-/- mice.

The Tapr locus, which maps to 5–10 cM from the IL-4 gene cluster, has been implicated as playing a role in the disparity in the development of AHR in BALB/c and HBA strains (52). We have considered that this locus may influence the disparity that we see in the N5 and N10 IL-13-/- mice. However, a study examining piggy-backed genes in 129/BALB/c crosses has shown that after 11 crosses, at least 21 cM of residual 129 DNA flanked the selected p53 allele (A. C. Blackburn, unpublished observations). Therefore, as this region of chromosome 11 is specifically selected in the IL-13-/- offspring, we consider it likely that the Tapr locus would be from the founder 129 x C57BL/6 strain and consistent in both the N5 and N10 IL-13-/- strains.

In summary, the current study suggests that the N5 IL-13-/- IL-4R{alpha} binds IL-4 with greater avidity than N10 IL-13-/- IL-4R{alpha} to regulate AHR, spatial aspects of eosinophilia, Ym expression, and Th2-biased CD4+ T cell responses. This study has not addressed whether IL-4 indirectly regulates these processes by inducing expression of other Th2 cytokines or by directly targeting effector cells that are also modulated by IL-13. These issues and the impact of polymorphisms in IL-4R{alpha} on intracellular signaling and the interaction with the heterologous receptor partners are the focus of our ongoing research interest.


   Acknowledgments
 
We thank Vane Damcevski for breeding the N5 IL-13-/- mice to the N10 BALB/c cross.


   Footnotes
 
1 This work was supported by National Health and Medical Research Peter Doherty Training Fellowship 179841 (to D.C.W.), National Health and Medical Research Council Program Grant 224207 (to P.S.F. and K.I.M.), and a Human Frontiers grant (to P.S.F.). Back

2 Address correspondence and reprint requests to Dr. Dianne C. Webb, Division of Molecular Bioscience, The John Curtin School of Medical Research, Australian National University, Canberra, Australian Capital Territory, Australia 2601. E-mail address: dianne.webb{at}anu.edu.au Back

3 Abbreviations used in this paper: AHR, airways hyperreactivity; BALF, bronchoalveolar lavage fluid; Penh, enhanced pause; PVDF, polyvinylidene difluoride; Sal, saline; WT, wild type. Back

Received for publication August 1, 2003. Accepted for publication November 6, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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