The JI PBL Intereron Source
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tabata, Y.
Right arrow Articles by Hershey, G. K. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tabata, Y.
Right arrow Articles by Hershey, G. K. K.
The Journal of Immunology, 2006, 177: 7905-7912.
Copyright © 2006 by The American Association of Immunologists, Inc.

Allergy-Driven Alternative Splicing of IL-13 Receptor {alpha}2 Yields Distinct Membrane and Soluble Forms1

Yasuhiro Tabata, Weiguo Chen, Manoj R. Warrier, Aaron M. Gibson, Michael O. Daines and Gurjit K. Khurana Hershey2

Division of Allergy and Immunology, Cincinnati Children’s Hospital Medical Center and Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 45229


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
IL-13 is a key mediator of allergic inflammation. Its diverse functions are mediated by a complex receptor system including IL-4R{alpha}, IL-13R{alpha}1, and IL-13R{alpha}2. IL-4R{alpha} and IL-13R{alpha}1 form a high-affinity signaling heterodimer. IL-13R{alpha}2 binds IL-13 with high affinity and has been found to exist in membrane and soluble forms. Soluble IL-13R{alpha}2 has been postulated as a critical endogenous modulator of IL-13 responses. However, the mechanism of generation for the soluble form remains unclear. We present the initial study that a mechanism for generation of the soluble form is alternative splicing and that alternative splicing yields a distinct form of soluble IL-13R{alpha}2. We found that several mouse organs expressed two IL-13R{alpha}2 transcripts, the 1152-bp transcript encoding the full-length protein and the 1020-bp transcript lacking exon10, which encodes the transmembrane region. Deletion of exon 10 ({Delta}Ex10) caused a frameshift resulting in a different amino acid sequence from position 327 to position 339 and early termination. Constructs encoding both splice variants were transfected into WEHI-274.1 cells. Transfectants expressing the full-length transcript had IL-13R{alpha}2 on the cell surface but produced minimal soluble IL-13R{alpha}2 in the supernatants. In contrast, transfectants expressing the {Delta}Ex10 transcript displayed no membrane IL-13R{alpha}2 but secreted high levels of soluble IL-13R{alpha}2 capable of inhibiting IL-13 signaling. Both variants bound IL-13, but the {Delta}Ex10 variant displayed ~2-fold increase in IL-13 binding activity. Expression of the two IL-13R{alpha}2 transcripts was differentially regulated in vivo in an experimental allergic asthma model. Thus, alternatively spliced variants of IL-13R{alpha}2 may have a distinct biologic function in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Clinical and experimental investigations have identified IL-13 as crucial in developing allergic inflammatory responses (1, 2, 3). Its diverse functions are mediated by a complex receptor system including IL-4R{alpha}, IL-13R{alpha}1, and IL-13R{alpha}2. IL-4R{alpha} and IL-13R{alpha}1 form a high affinity IL-13R that signals. IL-13R{alpha}2 has been postulated to be a decoy receptor, a notion supported by the characterization of the IL-13R{alpha}2 knockout mice (4, 5). IL-13R{alpha}2 may also contribute to IL-13 responses as suggested by a recent report demonstrating that IL-13-induced TGF-beta-mediated fibrosis is dependent on IL-13R{alpha}2 (6). Interestingly, IL-13R{alpha}2–/– mice have greatly reduced levels of IL-13 in the serum but significantly greater tissue levels of IL-13 when compared with wild-type mice. Thus, IL-13R{alpha}2 regulates serum and tissue levels of IL-13. This finding was further supported by a report that treatment of IL-13R{alpha}2–/– mice with a soluble IL-13R{alpha}2-Fc recombinant protein increases circulating IL-13 (5). IL-13 has been shown to induce IL-13R{alpha}2 expression, demonstrating a complex feedback loop between IL-13 and IL-13R{alpha}2 whereby each one modulates the level of the other. Regulation of the level of expression of IL-13R{alpha}2, as well as its relative distribution among the membrane and soluble compartments, likely impacts IL-13 responses.

Unlike IL-4, IL-13 does not appear to be important in the initial differentiation of CD4 T cells into Th2 type cells (7) but rather appears to be important in the effector phase of allergic inflammation. This effector role is further supported by many observations in vivo, including findings that the administration of IL-13 results in allergic inflammation (2, 8), the tissue-specific overexpression of IL-13 in the lungs of transgenic mice brings on airway inflammation and mucus hypersecretion (9), and IL-13 appears to be more important than IL-4 in mucus hypersecretion (10). Further evidence that IL-13 is a critical effector molecule was provided by a study in which IL-13 was inducibly expressed in the lungs of mice (11). Thus, the modulatory role of IL-13R{alpha}2 may be more evident in the late phase of allergic inflammation.

In patients with allergic asthma, allergen challenge leads to an early-phase response occurring within 15–30 min following allergen challenge. Approximately 60% of patients also develop a late-phase response occurring ~3–5 h after allergen challenge and characterized by airway obstruction and increased airway inflammation (12). Similarly, in mice with already established airway disease, allergen challenge can evoke both phase responses (13). In a mouse model of allergic asthma, inhibition of the late-phase response was achieved following systemic administration of an IL-13R{alpha}2-Fc recombinant protein (14). The IL-13R{alpha}2-Fc recombinant protein was capable of binding and neutralizing IL-13 (15) and also of attenuating airway hyperresponsiveness when administrated in allergen-challenged mice (16). Thus, soluble IL-13R{alpha}2 has been postulated as being a critical endogenous modulator of IL-13 responses. However, the mechanism of generation for soluble IL-13R{alpha}2 has not yet been reported. Soluble cytokine receptors can be generated by several mechanisms, which include alternative splicing of mRNA transcripts and proteolytic cleavage of receptor ectodomains (17). In this study we demonstrate, for the first time, that alternative splicing yields a distinct form of soluble IL-13R{alpha}2.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animal protocols

Mice were purchased from The Jackson Laboratory, maintained in a pathogen-free vivarium, and handled under Institutional Animal Care and Use Committee-approved procedures and the guidance of the National Research Council. For the experimental allergic asthma mouse model, FVB/N strain mice were sensitized i.p. with 10 µg of standardized house dust mite (HDM)3 extracts (Greer Laboratories) in PBS or an equivalent amount of PBS alone on days 0 and 7. On days 14 and 21, mice were anesthetized and challenged intratracheally with 100 µg of HDM or PBS. On day 25, the lungs were lavaged, bronchoalveolar fluid (BALF) was collected as previously reported (18), and the organs were harvested.

3'-RACE

RNA was extracted from BALB/c mouse spleens. Alternatively, spliced transcripts of IL-13R{alpha}2 were examined by 3'-RACE analysis with the following primers: exon8-F (5'-GAT CCG AGA AGA CGA TAT TTC CTG-3') and a RACE adaptor primer (Invitrogen Life Technologies) followed by nested PCR with primers complementary to exon 9 (5'-GAA TGG AGT GAA GAG GAA TGT TGG-3') and the same adaptor primer. Amplified products were subcloned into a pCR2.1 cloning vector (Invitrogen Life Technologies) and sequenced.

Plasmid construction and transfection

IL-13R{alpha}2 transcripts following the signal peptide (15) were amplified with the forward primer 5'-CGA CGC GTG ACT ATA AGG ATG ACG ATG ACA AGT TGG AGA TAA AAG TTA ATC CT-3' to add a FLAG tag at the N terminus and with the reverse primer 5'-CGA GAT CTG GTA CCT TAA CAG AGG GTA TCT TCA TAA GC-3' for the full-length transcript or 5'-GCG GTA CCT CAT AAG CAC ACA CTT CTT TGT TCA GAT CCA CAT GGA GGC TCT TCC CAA CAT TCC TCT TCA-3' for the {Delta}Ex10 transcript. Amplified products, confirmed by sequence analysis, were cloned into a pEF-Neo-PPL-SP vector that was kindly provided by Dr. Y. Tagaya (National Cancer Institute, Bethesda, MD) (19). This vector contains the bovine preprolactin signal peptide. WEHI-274.1 mouse monocytic cells (American Type Culture Collection) were washed, resuspended in RPMI 1640 containing 20 µg of uncut plasmid and pulsed with a Gene Pulser II electroporation device (Bio-Rad) set at 960 µF and 200 V. After electroporation, the cells were grown for 24 h and then selected for resistance to G-418 at 1.2 mg/ml for 3 wk. Cell populations were screened by flow cytometry or ELISA as described below. Positive transfectant pools were cloned by limiting dilution.

Flow cytometry

Surface IL-13R{alpha}2 expression was assessed by flow cytometry. Briefly, cells were incubated with goat anti-mouse IL-13R{alpha}2 Ab (R&D Systems) or irrelevant goat IgG for 30 min on ice. IL-13 binding was determined by incubating cells with or without 200 ng/ml mouse IL-13 (PeproTech) for 30 min on ice, followed by incubation with goat anti-mouse IL-13 Ab (R&D Systems). In both procedures, bound Abs were detected with Alexa Fluor 488 chicken anti-goat Ab (Invitrogen Life Technologies). Samples were analyzed using a flow cytometer. Positive regions were set using isotype-matched negative controls, and the percentage positive for each region was reported.

ELISA

Soluble IL-13R{alpha}2 was quantified by ELISA with standard techniques. Briefly, 96-well plates were coated with goat polyclonal anti-mouse IL-13R{alpha}2 Ab (1 µg/ml; R&D Systems) for capture overnight at 4°C. Samples were applied to the plate and incubated at room temperature for 2 h. Wells were washed and then incubated with biotinylated anti-mouse IL-13R{alpha}2 Ab (0.5 µg/ml; R&D Systems) for another 2 h. This was followed by streptavidin-HRP conjugate and substrate solution (R&D Systems) at room temperature for 20 min each. Absorbance was read at 450 nm, and OD readings were converted to nanograms per milliliter using a standard curve generated from serial dilutions of a mouse IL-13R{alpha}2-Fc recombinant protein that was provided by Dr. F. Finkelman (Cincinnati Children’s Hospital Medical Center, Cincinnati, OH). This fusion protein has been described previously and consists of the extracellular domain of mouse IL-13R{alpha}2 coupled to Fc (15). The Abs used for ELISA are specific to the extracellular domain of the mouse IL-13R{alpha}2 protein.

EMSA

Conditioned medium was taken from untransfected WEHI cells, full-length transfectants, or {Delta}Ex10 transfectants (5 x 105 cells/ml) after incubation for 72 h. The conditioned medium or control medium was incubated with murine IL-4 or IL-13 (5 ng/ml; PeproTech) on ice for 10 min. Then, 5 x 106 WEHI cells were incubated with the conditioned medium at 37°C for 20 min. Nuclear protein extraction and EMSA for STAT6 were performed as described previously (20, 21). Briefly, cells were lysed in 20 µl of EMSA lysis buffer (10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 1.5 mM MgCl2, 0.2% Nonidet P-40, 1.0 mM DTT, and 0.5 mM PMSF) for 5 min on ice and centrifuged for 5 min at 10,000 x g. Pellets were resuspended in 20 µl of EMSA extraction buffer (20 mM HEPES (pH 7.9), 420 mM NaCl, 0.1 mM EDTA, 1.5 mM MgCl2, 25% glycerol, 1.0 mM DTT, and 0.5 mM PMSF) for 30 min on ice and centrifuged for 20 min at 20,000 x g. The resulting nuclear extracts were incubated with 32P-labeled STAT6 probe (Santa Cruz Biotechnology), resolved by gel electrophoresis, and visualized by autoradiography.

IL-13 binding assay

Cell lysates were prepared from untransfected WEHI cells, full-length transfectants, or {Delta}Ex10 transfectants. Cells were lysed in the EMSA lysis buffer (1.25 x 105 cells/µl) for 5 min on ice and centrifuged for 5 min at 10,000 x g. The supernatants were dialyzed in PBS overnight and then cleared by centrifugation. The concentrations of IL-13R{alpha}2 protein in each lysate were determined by ELISA as described above. IL-13 binding activity of the membrane and soluble forms of IL-13R{alpha}2 was quantified by ELISA. Briefly, 96-well plates were coated with goat polyclonal anti-mouse IL-13 Ab (2.0 µg/ml; R&D Systems) for capture overnight at 4°C. The lysates from full-length or {Delta}Ex10 transfectants were diluted to 10 ng of IL-13R{alpha}2 protein in 50 µl of PBS with 1% BSA. Lysates from the same cell count of WEHI cells were used as negative control. Serial dilutions of mouse IL-13R{alpha}2-Fc recombinant protein were used as standards. The samples were incubated with mouse IL-13 (100 ng/ml; PeproTech) for 30 min at 37°C and then applied to the plate and incubated overnight at 4°C. Wells were washed and then incubated with biotinylated anti-mouse IL-13R{alpha}2 Ab (0.2 µg/ml; R&D Systems) at room temperature for 2 h. This was followed by streptavidin-HRP conjugate and substrate solution (R&D Systems) at room temperature for 30 min each. Absorbance was read at 450 nm and OD readings were converted to relative IL-13 binding ratio using a standard curve generated from serial dilutions of mouse IL-13R{alpha}2-Fc recombinant protein.

RT-PCR for IL-13R{alpha}2 in mouse organs

Conventional RT-PCR analysis was performed using primers flanking exon 8 (5'-GAA ATG GAG CAC ACC TGG AGG A-3') and exon 11 (5'-CAC TGA TTT TCA AGA AGA TGT ATT C-3'). Quantitative RT-PCR analysis was performed using primers for full-length forward (5'-TGG AGA AGG AAG AAC CTG AAC CC-3'), reverse 5'-TGC TGG CTG GCT CTA TGT CAA G-3', and {Delta}Ex10 forward (5'-GAA GAG GAA TGT TGG GAA GAG CC-3') with the same reverse primer. The PCR products were quantified using LightCycler 480 system with SYBR Green I Master Mix according to the manufacturer’s instructions (Roche Diagnostics). Values were normalized to 18S rRNA (22). Quantitative PCR assays used for the full-length and {Delta}Ex10 transcripts were specific. The specificity of each primer set was confirmed using cDNA encoding the full-length or {Delta}Ex10 transcript as a template.

Statistical analysis

Reported values are expressed as mean and SD. Microsoft Excel was used to determine the levels of difference between all groups. Comparisons for all pairs were performed by unpaired two-tailed Student’s t test. Significance was set at a p = 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Characterization of murine IL-13R{alpha}2 transcripts

Soluble IL-13R{alpha}2 is an important modulator of IL-13 responses, but the mechanisms underlying its generation are unknown. We hypothesized that alternative splicing may yield a secreted form. To address this possibility, we investigated whether there were multiple transcripts for IL-13R{alpha}2 by using 3'-RACE. Fig. 1A shows the result of a 3'-RACE assay of the murine IL-13R{alpha}2 gene. Mouse spleens showed two amplified fragments (Fig. 1Aa, 387 bp; Fig. 1Ab, 261 bp). Sequence analysis revealed that the longer fragment was identical with the murine IL-13R{alpha}2 mRNA sequence in GenBank (accession no. NM_008356). The shorter fragment lacked exon 10, which encodes the transmembrane motif (Fig. 1, B and C). Fig. 1C shows the comparison of the predicted amino acid sequences from the two transcripts. The shorter transcript conserves IL-13 binding motifs but lacks the transmembrane motif. The deletion of exon 10 causes a frameshift resulting in a different amino acid sequence after the end of exon 9 from amino acid position 327 to position 339 (EPPCGSEQRSVCL) and an early termination at position 340. The longer transcript was designated as full length, and the shorter transcript was designated as {Delta}Ex10. The two transcripts were confirmed as identical except for the exon 10 deletion by sequencing of the entire coding regions (data not shown).


Figure 1
View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 1. Detection and characterization of two IL-13R{alpha}2 transcripts. A, 3'-RACE for IL13-R{alpha}2 reveals two transcripts (a, 387 bp; b, 261 bp); M, molecular marker; lane 1, spleen from a BALB/c mouse; lane 2, no reverse transcription control of subject in lane 1; lane 3, spleen from a FVB/N mouse; lane 4, no reverse transcription control of subject in lane 3; B: no cDNA control. B, Sequence analysis of 261-bp transcript. C, Schema and sequences of the two transcripts. TM, Transmembrane motif. Nucleotide sequence and predicted amino acid sequence from mid-exon 9 to the stop codon of both transcripts are shown. Highlighted nucleotides represent exon 10. Underlined nucleotides are the coding sequence of exon 11. *, A stop codon. The amino acid sequence in italics represents the transmembrane motif.

 
Expression of IL-13R{alpha}2 full-length and {Delta}Ex10 transcripts results in expression of the membrane and soluble forms of IL-13R{alpha}2, respectively

The two IL-13R{alpha}2 transcripts were amplified, subcloned into an expression vector, and transfected into WEHI-274.1 cells, which respond readily to IL-13 and express negligible endogenous IL-13R{alpha}2, and then the transfectants were assessed for membrane IL-13R{alpha}2 (Fig. 2A and B). Cells transfected with the full-length IL-13R{alpha}2 transcript demonstrated positive staining for surface IL-13R{alpha}2 by flow cytometry (64 ± 17%; p = 0.0044), whereas untransfected WEHI cells and the {Delta}Ex10 transfectant had negligible surface IL-13R{alpha}2 expression (1.2 ± 0.2 and 1.8 ± 0.4%, respectively). Similar results were obtained when surface IL-13 binding activity was assessed in untransfected and transfected cells (Fig. 2, C and D). Only full-length transfectants showed positive staining (69 ± 19%; p = 0.032), whereas untransfected WEHI cells and {Delta}Ex10 transfectants demonstrated minimal surface IL-13 binding (1.0 ± 0.1 and 1.1 ± 0.1%, respectively).


Figure 2
View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 2. Membrane-bound IL-13R{alpha}2 is produced by the full-length transcript. A, Surface IL-13R{alpha}2 expression was measured by flow cytometry. Cells were stained with goat anti-IL-13R{alpha}2 (solid line) or irrelevant goat IgG (dotted line). B, Percentage of cells positively defined by the M1 gate: untransfected WEHI cells, 1.2 ± 0.2%; full-length transfectants, 64 ± 17%; {Delta}Ex10 transfectants, 1.8 ± 0.4%. C, IL-13 binding ability. Cells were incubated with (solid lines) or without (dotted lines) mouse IL-13 (200 ng/ml) followed by goat anti-mouse IL-13 Ab. D, Percentage of cells positively defined by the M1 gate: untransfected WEHI cells, 1.0 ± 0.1%; full-length transfectants, 69 ± 19%; {Delta}Ex10 transfectants, 1.1 ± 0.1%. A representative picture from three separate experiments examining at least three separate stably transfected clones is shown.

 
We next characterized the transfectants for expression of soluble IL-13R{alpha}2. IL-13R{alpha}2 protein in the conditioned medium was measured by ELISA (Fig. 3A). Conditioned medium from {Delta}Ex10 transfectants contained high levels of IL-13R{alpha}2 (201.6 ± 33.8 ng/ml) compared with the full-length transfectants (p = 0.0013). Full-length transfectants and untransfected WEHI cells had minimal soluble IL-13R{alpha}2 protein in the supernatants (0.85 ± 0.15 and 0.05 ± 0.003 ng/ml, respectively). Conditioned medium from the full-length transfectant did contain detectable soluble IL-13R{alpha}2 when compared with untransfected WEHI cells (Fig. 3B; p = 0.011), supporting the theory that soluble IL-13R{alpha}2 can be generated from membrane IL-13R{alpha}2 as well.


Figure 3
View larger version (43K):
[in this window]
[in a new window]
  		FIGURE 3. Soluble IL-13R{alpha}2 is produced by the {Delta}Ex10 transcript. A, Conditioned medium was taken from untransfected WEHI cells, full-length transfectants, or {Delta}Ex10 transfectants (5 x 105 cells/ml) after incubation for 72 h. IL-13R{alpha}2-protein concentrations in the conditioned medium were measured by ELISA: {Delta}Ex10 transfectant medium, 201.6 ± 33.8 ng/ml soluble IL-13R{alpha}2; full-length transfectant medium, 0.85 ± 0.15 ng/ml; and untransfected cell medium, 0.05 ± 0.003 ng/ml. B, Data from A graphed with a different y-axis to highlight difference between untransfected and full-length transfected cells. C, The conditioned medium or control medium (DMEM with 10% FCS) were incubated with IL-4 or IL-13 (5 ng/ml) for 10 min. WEHI cells (5 x 106 cells) were incubated with the medium for 20 min. Then, nuclear protein extraction and EMSA for STAT6 activation were performed. C, Control medium; UT, conditioned medium from untransfected cells; DE, conditioned medium from {Delta}Ex10 transfectants. Serial dilutions represent IL-13R{alpha}2 vs IL-13 in number of moles from 10/1 (x1), 1/1 (x10) and 1/10 (x100). A representative picture from three separate experiments examining at least three separate stably transfected clones is shown.

 
Soluble IL-13R{alpha}2 derived from {Delta}Ex10 IL-13R{alpha}2 transcript is biologically active

To determine whether soluble IL-13R{alpha}2 generated by the alternatively spliced {Delta}Ex10 transcript was functionally active, conditioned medium was transferred to untransfected WEHI cells and IL-13 signaling was assessed (Fig. 3C). IL-13-dependent, but IL-4-independent, STAT6 activation was completely abolished by medium from transfectants expressing {Delta}Ex10 IL-13R{alpha}2 in a concentration-dependent manner. Equimolar amounts of soluble IL-13R{alpha}2 inhibited IL-13 activity almost completely. In contrast, medium from untransfected WEHI cells had no effect. IL-4-dependent STAT6 activation was slightly decreased by the {Delta}Ex10-conditioned medium. However, this effect was not dose dependent and most likely represents nonspecific inhibition.

We next compared the IL-13 binding activities of the full-length and {Delta}Ex10 IL-13R{alpha}2 variants by ELISA (Fig. 4). The soluble form ({Delta}Ex10) of IL-13R{alpha}2 has 2.3-fold higher IL-13 binding ability compared with the membrane form (full length; p = 0.0023).


Figure 4
View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 4. Membrane and soluble forms of IL-13R{alpha}2 are functionally different. Cell lysates from untransfected WEHI cells, full-length, or {Delta}Ex10 transfectants were incubated with mouse IL-13. IL-13R{alpha}2/IL-13 complexes were detected by ELISA using anti-IL-13 Ab for capture and anti-IL-13R{alpha}2 Ab for detection. OD readings were converted to relative IL-13 binding ratio by using a standard curve generated from serial dilutions of mouse IL-13R{alpha}2-Fc recombinant protein. At least three separate stably transfected clones were examined in each group.

 
Distribution and regulation of the two IL-13R{alpha}2 transcripts by IL-4 and IL-13

We next examined whether alternative splicing of IL-13R{alpha}2 was regulated by allergic inflammation. Expression of the two transcripts in several mouse organs was assessed by conventional RT-PCR (Fig. 5A). The full-length and {Delta}Ex10 transfectants expressed the expected transcripts of 496 bp (Fig. 5Aa) and 380 bp (Fig. 5Bb), respectively. Bone marrow, brain, spleen, liver, and kidney expressed both the full-length and {Delta}Ex10 transcripts at baseline. Heart and lung showed minimal expression of IL-13R{alpha}2. Expression of both IL-13R{alpha}2 transcripts was up-regulated when lungs were treated with IL-4 or IL-13 ex vivo, whereas expression was minimal without cytokines (Fig. 5B).


Figure 5
View larger version (37K):
[in this window]
[in a new window]
  		FIGURE 5. Expression of the two IL-13R{alpha}2 transcripts in mouse organs (data not shown). A, Conventional RT-PCR for IL-13R{alpha}2 transcripts in mouse organs using primers flanking exon 8 and 11. beta-Actin mRNA was amplified as a control. a, Full-length transcript (496 bp); b, {Delta}Ex10 transcript (380 bp); M, molecular marker; lane 1, bone marrow; lane 2, brain; lane 3, spleen; lane 4, liver; lane 5, kidney; lane 6, heart; lane 7, lung; B, blank (no cDNA control). B, RT-PCR for IL-13R{alpha}2 in cultured mouse lungs. Lungs were harvested from mice and cultured for 16 h in DMEM and 10% FCS without (lane 1), or with 10 ng/ml IL-4 (lane 2), or IL-13 (lane 3). a, Full-length transcript (496 bp); b, {Delta}Ex10 transcript (380 bp). M, molecular marker.

 
Regulation of IL-13R{alpha}2 in an experimental model of asthma

We next examined the impact of allergic inflammation on IL-13R{alpha}2 expression in a mouse model of allergic asthma. Mice were sensitized twice with HDM i.p. and then challenged with HDM twice intratracheally. Total eosinophils were increased in the BALF in HDM- vs PBS-treated mice (Fig. 6A).


Figure 6
View larger version (9K):
[in this window]
[in a new window]
  		FIGURE 6. Experimental mouse model of asthma. A, Bronchoalveolar eosinophil counts from HDM- or PBS-treated mice. B, IL-13R{alpha}2 concentration in bronchoalveolar fluid from HDM- or PBS-treated mice. C, IL-13R{alpha}2 concentration in serum from HDM- or PBS-treated mice (means ± SD; n = 4 mice per group).

 
We next compared the IL-13R{alpha}2 protein concentration in the BALF and serum obtained from PBS- and HDM-treated mice. BALF from HDM-treated mice contained significantly higher levels of soluble IL-13R{alpha}2 compared with PBS-treated mice (0.26 ± 0.095 ng/ml and 0.048 ± 0.031 ng/ml, respectively; p = 0.0021) (Fig. 6B). Similarly, HDM-treated mice contained higher levels of serum IL-13R{alpha}2 (28.44 ± 3.54 ng/ml), compared with PBS-treated mice (14.43 ± 4.19 ng/ml) (Fig. 6C).

Thus, soluble IL-13R{alpha}2 was induced following allergen challenge. We next examined the expression of the full-length and {Delta}Ex10 IL-13R{alpha}2 transcripts in this model. HDM-sensitized and -challenged mice demonstrated significantly higher expression of both IL-13R{alpha}2 transcripts in their lungs as compared with PBS-treated mice 1 day after challenge. PBS-treated mice had undetectable IL-13R{alpha}2 expression. Expression of both IL-13R{alpha}2 transcripts was significantly decreased in lungs at 5 days after challenge as compared with 1 day after challenge (Fig. 7A; p = 0.0047 in full-length transcript, p = 0.017 in {Delta}Ex10 transcript). In contrast, in the spleen the {Delta}Ex10 transcript, but not the full-length transcript, was significantly decreased in HDM-treated mice compared with spleens of PBS-treated mice (Fig. 7B; p = 0.014). In the bone marrow, expressions of both transcripts did not differ between the HDM and PBS-treated groups.


Figure 7
View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 7. Quantitative expression of the two IL-13R{alpha}2 transcripts in mouse organs from the experimental model of asthma. A, Quantitative RT-PCR for full-length or {Delta}Ex10 IL-13R{alpha}2 transcripts in lungs harvested from mice after PBS or HDM challenge using a mouse model of allergic asthma. Both full-length and {Delta}Ex10 transcripts were analyzed using specific primers 1 day and 5 days after the second HDM challenge (n = 5 mice in each group). B, Quantitative RT-PCR for IL-13R{alpha}2 transcripts in the spleen and bone marrow harvested from mice 1 day after the second challenge (n = 5 mice in each group).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Our data demonstrate alternative splicing of IL-13R{alpha}2 and define two distinct transcripts for IL-13R{alpha}2 in most mouse tissues. The smaller transcript lacks exon 10 and results in a frameshift and early termination. Deletion of exon 10 ({Delta}Ex10) results in removal of the transmembrane motif. Transfection experiments of the two transcripts revealed that the larger full-length transcript and the smaller {Delta}Ex10 transcript specifically encode membrane and soluble forms of IL-13R{alpha}2, respectively. Interestingly, conditioned medium from transfectants expressing only the full-length transcript did contain detectable soluble IL-13R{alpha}2, albeit at a much lower level than the transfectants expressing the {Delta}Ex10 transcript. Thus, generation of soluble IL-13R{alpha}2 can occur from an alternative transcript encoding a soluble form and by cleavage of membrane IL-13R{alpha}2. However, our data suggest that IL-13R{alpha}2 generated from the {Delta}Ex10 transcript accounts for >99.5% of the total soluble IL-13R{alpha}2. Soluble IL-13R{alpha}2 encoded by the {Delta}Ex10 transcript is biologically active; an equimolar amount of soluble IL-13R{alpha}2 secreted by {Delta}Ex10 transfectants inhibited IL-13 signaling.

Soluble cytokine receptors are important regulators of inflammation and immunity and can either down-regulate or contribute to cytokine signaling and responses (17). Soluble IL-13R{alpha}2 in the serum has been reported to act as an inhibitory protein regulating IL-13 responses (23). A rIL-13R{alpha}2-Fc fusion protein was capable of binding and neutralizing IL-13 (15) and attenuated airway hyperresponsiveness when administrated in allergen-challenged mice (16). Soluble IL-13R{alpha}2 has been measured at high levels in mouse serum, with up to 10 times higher molar concentrations than IL-13 (4, 24). Thus, the amount of soluble IL-13R{alpha}2 is high enough to inhibit IL-13 signaling in serum and supports the belief that soluble IL-13R{alpha}2 is a major modifier of IL-13 activity and allergic inflammation in vivo. Recently, Fichtner-Feigl et al. (6) reported that membrane-bound IL-13R{alpha}2 may contribute to IL-13-dependent lung fibrosis. Thus, membrane and soluble IL-13R{alpha}2 may have divergent roles. Our data demonstrate that membrane and soluble forms of IL-13R{alpha}2 are generated from distinct transcripts. The predicted amino acid sequence of the soluble form has a different C-terminal region from that of the membrane-bound form. This C-terminal alteration may result in a distinct effect of alternatively spliced IL-13R{alpha}2 after the binding of IL-13. Indeed, we observed that the {Delta}Ex10 IL-13R{alpha}2 protein product displayed 2-fold increased binding activity as compared with full-length membrane IL-13R{alpha}2.

The soluble and membrane IL-13R{alpha}2 transcripts have not been reported or observed previously. Donaldson et al. (15) observed two IL-13R{alpha}2 transcripts of 1.5 and 2.1 kb in mouse organs by Northern blotting. The sizes of these two transcripts are compatible with two cDNA sequences reported in GenBank (accession no. NM_008356 (1567 bp) and AK089687 (2206 bp)). These sequences differ in their 5'-untranslated regions, but both share the same coding region including the exon 10, which encodes the membrane form of IL-13R{alpha}2. The {Delta}Ex10 IL-13R{alpha}2 transcript differs by only 132 bp from the membrane form. Thus, it may be difficult to distinguish the two transcripts by Northern blotting. IL-13R{alpha}2 was initially cloned from a kidney cell line (25) and then found to be expressed in brain, spleen, liver, and thymus (15). We also found expression in the bone marrow. These organs are hemopoietic or hypervascular tissues. These results suggest that IL-13R{alpha}2 is expressed predominantly in blood cells. To distinguish the expression in lung tissue from circulating blood cells, we investigated the expression of IL-13R{alpha}2 in lung tissue ex vivo after washing blood cells away. Mouse lung tissue had minimal expression of IL-13R{alpha}2 at the baseline. Lung IL-13R{alpha}2 expression was up-regulated after cytokine activation similar to what we observed in vivo. IL-13R{alpha}2 has also been reported to be expressed in human lungs (26). The negligible expression in the mouse lungs at baseline may be in part because the mice in this study were maintained in a pathogen-free environment. These results are compatible with previous studies using human bronchial epithelial cells or mouse lung tissues (27, 28).

IL-13R{alpha}2 knockout mice, which have no soluble IL-13R{alpha}2 in the serum, have greatly reduced levels of IL-13 in the serum but significantly greater tissue levels of IL-13 compared with wild-type mice (5). This finding demonstrates a complex feedback loop between IL-13 and IL-13R{alpha}2 whereby they each modulate the level of the other, because IL-13 has been shown to induce IL-13R{alpha}2 expression (29). Thus, regulation of the levels of expression of IL-13R{alpha}2, as well as its relative distribution among the membrane and soluble compartments, likely impacts IL-13 responses. In our mouse model of allergic asthma, HDM-treated mice demonstrated significantly higher IL-13R{alpha}2 expression in their lungs as compared with PBS-treated mice 1 day after challenge, but the IL-13R{alpha}2 expression was decreased at 5 days after challenge. The relative decrease of the {Delta}Ex10 transcript was greater than the full-length transcript at day 5, suggesting that expressions of the two transcripts were differently regulated. This was further supported when we examined splenocytes.

The amount of soluble IL-13R{alpha}2 protein in the serum is substantial even at baseline (4). The source of this IL-13R{alpha}2 is unknown. It is possible that the soluble IL-13R{alpha}2 is generated in significant levels in the spleen or bone marrow. When we investigated the expression of the two transcripts in these tissues, we indeed observed a relatively high expression of the {Delta}Ex10 transcript in both the spleen and bone marrow. Interestingly, the {Delta}Ex10 transcript was significantly decreased in spleens of HDM-treated mice compared with spleens of PBS-treated mice, whereas no significant changes were observed in the full-length transcript. These results demonstrate that expression of the two transcripts is differentially regulated. The observed decrease of the {Delta}Ex10 transcript in spleens from HDM-treated mice may represent negative feedback due to high levels of serum-soluble IL-13R{alpha}2, which may preferentially suppress transcription of the soluble form.

In conclusion, we demonstrate that an alternatively spliced transcript of the mouse IL-13R{alpha}2 gene generates biologically relevant soluble IL-13R{alpha}2 protein in vitro, which can effectively block IL-13-dependent STAT6 activation. The truncated {Delta}Ex10 transcript, which encodes soluble IL-13R{alpha}2, was found in multiple mouse organs and was up-regulated in lungs following allergen challenge. Allergic inflammation differentially modulated expression of the membrane and soluble IL-13R{alpha}2 transcripts. Genetic factors may also impact the regulation of expression of the two IL-13R{alpha}2 transcripts. Further studies are warranted to further elucidate the mechanisms that regulate and control alternative splicing of the IL-13R{alpha}2 gene.


   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 by National Institutes of Health Grants R01AI58157 and P01HL076383 (both to G.K.K.H.). Back

2 Address correspondence and reprint requests to Dr. Gurjit K. Khurana Hershey, Division of Allergy and Immunology, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229. E-mail address: Gurjit.Hershey{at}cchmc.org Back

3 Abbreviations used in this paper: HDM, house dust mite; {Delta}Ex10, deletion of exon 10. Back

Received for publication April 5, 2006. Accepted for publication August 16, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Wills-Karp, M.. 2004. Interleukin-13 in asthma pathogenesis. Immunol. Rev. 202: 175-190. [Medline]
  2. Wills-Karp, M., J. Luyimbazi, X. Xu, B. Schofield, T. Y. Neben, C. L. Karp, D. D. Donaldson. 1998. Interleukin-13: central mediator of allergic asthma. Science 282: 2258-2261. [Abstract/Free Full Text]
  3. Hershey, G. K.. 2003. IL-13 receptors and signaling pathways: an evolving web. J. Allergy Clin. Immunol. 111: 677-691. [Medline]
  4. Wood, N., M. J. Whitters, B. A. Jacobson, J. Witek, J. P. Sypek, M. Kasaian, M. J. Eppihimer, M. Unger, T. Tanaka, S. J. Goldman, et al 2003. Enhanced interleukin (IL)-13 responses in mice lacking IL-13 receptor {alpha} 2. J. Exp. Med. 197: 703-709. [Abstract/Free Full Text]
  5. Chiaramonte, M. G., M. Mentink-Kane, B. A. Jacobson, A. W. Cheever, M. J. Whitters, M. E. Goad, A. Wong, M. Collins, D. D. Donaldson, M. J. Grusby, T. A. Wynn. 2003. Regulation and function of the interleukin 13 receptor {alpha} 2 during a T helper cell type 2-dominant immune response. J. Exp. Med. 197: 687-701. [Abstract/Free Full Text]
  6. Fichtner-Feigl, S., W. Strober, K. Kawakami, R. K. Puri, A. Kitani. 2006. IL-13 signaling through the IL-13{alpha}(2) receptor is involved in induction of TGF-beta(1) production and fibrosis. Nat. Med. 12: 99-106. [Medline]
  7. Zurawski, G., J. E. de Vries. 1994. Interleukin 13, an interleukin 4-like cytokine that acts on monocytes and B cells, but not on T cells. Immunol. Today 15: 19-26. [Medline]
  8. Grunig, G., M. Warnock, A. E. Wakil, R. Venkayya, F. Brombacher, D. M. Rennick, D. Sheppard, M. Mohrs, D. D. Donaldson, R. M. Locksley, D. B. Corry. 1998. Requirement for IL-13 independently of IL-4 in experimental asthma. Science 282: 2261-2263. [Abstract/Free Full Text]
  9. Zhu, Z., R. J. Homer, Z. Wang, Q. Chen, G. P. Geba, J. Wang, Y. Zhang, J. A. Elias. 1999. Pulmonary expression of interleukin-13 causes inflammation, mucus hypersecretion, subepithelial fibrosis, physiologic abnormalities, and eotaxin production. J. Clin. Invest. 103: 779-788. [Medline]
  10. Webb, D. C., A. N. McKenzie, A. M. Koskinen, M. Yang, J. Mattes, P. S. Foster. 2000. Integrated signals between IL-13, IL-4, and IL-5 regulate airways hyperreactivity. J. Immunol. 165: 108-113. [Abstract/Free Full Text]
  11. Zheng, T., Z. Zhu, Z. Wang, R. J. Homer, B. Ma, R. J. Riese, Jr, H. A. Chapman, Jr, S. D. Shapiro, J. A. Elias. 2000. Inducible targeting of IL-13 to the adult lung causes matrix metalloproteinase- and cathepsin-dependent emphysema. J. Clin. Invest. 106: 1081-1093. [Medline]
  12. Durham, S. R.. 1991. The significance of late responses in asthma. Clin Exp. Allergy 21: 3-7. [Medline]
  13. Cieslewicz, G., A. Tomkinson, A. Adler, C. Duez, J. Schwarze, K. Takeda, K. A. Larson, J. J. Lee, C. G. Irvin, E. W. Gelfand. 1999. The late, but not early, asthmatic response is dependent on IL-5 and correlates with eosinophil infiltration. J. Clin. Invest. 104: 301-308. [Medline]
  14. Taube, C., C. Duez, Z. H. Cui, K. Takeda, Y. H. Rha, J. W. Park, A. Balhorn, D. D. Donaldson, A. Dakhama, E. W. Gelfand. 2002. The role of IL-13 in established allergic airway disease. J. Immunol. 169: 6482-6489. [Abstract/Free Full Text]
  15. Donaldson, D. D., M. J. Whitters, L. J. Fitz, T. Y. Neben, H. Finnerty, S. L. Henderson, R. M. O’Hara, Jr, D. R. Beier, K. J. Turner, C. R. Wood, M. Collins. 1998. The murine IL-13 receptor {alpha} 2: molecular cloning, characterization, and comparison with murine IL-13 receptor {alpha} 1. J. Immunol. 161: 2317-2324. [Abstract/Free Full Text]
  16. Leigh, R., R. Ellis, J. Wattie, D. D. Donaldson, M. D. Inman. 2004. Is interleukin-13 critical in maintaining airway hyperresponsiveness in allergen-challenged mice?. Am. J. Respir. Crit. Care Med. 170: 851-856. [Abstract/Free Full Text]
  17. Levine, S. J.. 2004. Mechanisms of soluble cytokine receptor generation. J. Immunol. 173: 5343-5348. [Abstract/Free Full Text]
  18. Chen, W., M. B. Ericksen, L. S. Levin, G. K. Khurana Hershey. 2004. Functional effect of the R110Q IL13 genetic variant alone and in combination with IL4RA genetic variants. J. Allergy Clin. Immunol. 114: 553-560. [Medline]
  19. Marks-Konczalik, J., S. Dubois, J. M. Losi, H. Sabzevari, N. Yamada, L. Feigenbaum, T. A. Waldmann, Y. Tagaya. 2000. IL-2-induced activation-induced cell death is inhibited in IL-15 transgenic mice. Proc. Natl. Acad. Sci. USA 97: 11445-11450. [Abstract/Free Full Text]
  20. Daines, M. O., Y. Tabata, B. A. Walker, W. Chen, M. R. Warrier, S. Basu, G. K. Hershey. 2006. Level of expression of IL-13R{alpha}2 impacts receptor distribution and IL-13 signaling. J. Immunol. 176: 7495-7501. [Abstract/Free Full Text]
  21. Chen, W., M. O. Daines, G. K. Khurana Hershey. 2004. Methylation of STAT6 modulates STAT phosphorylation, nuclear translocation, and DNA-binding activity. J. Immunol. 172: 6744-6750. [Abstract/Free Full Text]
  22. Schmittgen, T. D., B. A. Zakrajsek. 2000. Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR. J. Biochem. Biophys Methods 46: 69-81. [Medline]
  23. Zhang, J. G., D. J. Hilton, T. A. Willson, C. McFarlane, B. A. Roberts, R. L. Moritz, R. J. Simpson, W. S. Alexander, D. Metcalf, N. A. Nicola. 1997. Identification, purification, and characterization of a soluble interleukin (IL)-13-binding protein. Evidence that it is distinct from the cloned Il-13 receptor and Il-4 receptor {alpha}-chains. J. Biol. Chem. 272: 9474-9480. [Abstract/Free Full Text]
  24. Morimoto, M., A. Zhao, K. B. Madden, H. Dawson, F. D. Finkelman, M. Mentink-Kane, J. F. Urban, Jr, T. A. Wynn, T. Shea-Donohue. 2006. Functional importance of regional differences in localized gene expression of receptors for IL-13 in murine gut. J. Immunol. 176: 491-495. [Abstract/Free Full Text]
  25. Caput, D., P. Laurent, M. Kaghad, J. M. Lelias, S. Lefort, N. Vita, P. Ferrara. 1996. Cloning and characterization of a specific interleukin (IL)-13 binding protein structurally related to the IL-5 receptor {alpha} chain. J. Biol. Chem. 271: 16921-16926. [Abstract/Free Full Text]
  26. Jakubzick, C., E. S. Choi, S. L. Kunkel, H. Evanoff, F. J. Martinez, R. K. Puri, K. R. Flaherty, G. B. Toews, T. V. Colby, E. A. Kazerooni, et al 2004. Augmented pulmonary IL-4 and IL-13 receptor subunit expression in idiopathic interstitial pneumonia. J. Clin. Pathol. 57: 477-486. [Abstract/Free Full Text]
  27. Zheng, T., Z. Zhu, W. Liu, C. G. Lee, Q. Chen, R. J. Homer, J. A. Elias. 2003. Cytokine regulation of IL-13R{alpha}2 and IL-13R{alpha}1 in vivo and in vitro. J. Allergy Clin. Immunol. 111: 720-728. [Medline]
  28. Yasunaga, S., N. Yuyama, K. Arima, H. Tanaka, S. Toda, M. Maeda, K. Matsui, C. Goda, Q. Yang, Y. Sugita, et al 2003. The negative-feedback regulation of the IL-13 signal by the IL-13 receptor {alpha}2 chain in bronchial epithelial cells. Cytokine. 24: 293-303. [Medline]
  29. David, M. D., J. Bertoglio, J. Pierre. 2003. Functional characterization of IL-13 receptor {alpha}2 gene promoter: a critical role of the transcription factor STAT6 for regulated expression. Oncogene 22: 3386-3394. [Medline]



This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
A. Munitz, E. B. Brandt, M. Mingler, F. D. Finkelman, and M. E. Rothenberg
Distinct roles for IL-13 and IL-4 via IL-13 receptor {alpha}1 and the type II IL-4 receptor in asthma pathogenesis
PNAS, May 20, 2008; 105(20): 7240 - 7245.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
S. Fichtner-Feigl, M. Terabe, A. Kitani, C. A. Young, I. Fuss, E. K. Geissler, H.-J. Schlitt, J. A. Berzofsky, and W. Strober
Restoration of Tumor Immunosurveillance via Targeting of Interleukin-13 Receptor-{alpha}2
Cancer Res., May 1, 2008; 68(9): 3467 - 3475.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. Khodoun, C. Lewis, J.-Q. Yang, T. Orekov, C. Potter, T. Wynn, M. Mentink-Kane, G. K. Khurana Hershey, M. Wills-Karp, and F. D. Finkelman
Differences in Expression, Affinity, and Function of Soluble (s)IL-4R{alpha} and sIL-13R{alpha}2 Suggest Opposite Effects on Allergic Responses
J. Immunol., November 15, 2007; 179(10): 6429 - 6438.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tabata, Y.
Right arrow Articles by Hershey, G. K. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tabata, Y.
Right arrow Articles by Hershey, G. K. K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS