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. Author manuscript; available in PMC: 2010 Feb 16.
Published in final edited form as: J Immunol. 2009 Dec 15;183(12):7870. doi: 10.4049/jimmunol.0901028

IL-13 Receptor Alpha 2 Membrane and Soluble Isoforms Differ in Human and Mouse1

Weiguo Chen *, Umasundari Sivaprasad *, Yasuhiro Tabata *, Aaron M Gibson *, Matthew T Stier *, Fred D Finkelman †, Gurjit K Khurana Hershey *,‡,2
PMCID: PMC2822278  NIHMSID: NIHMS153663  PMID: 20007572

Abstract

Although mice have ng/ml serum levels of soluble (s) IL-13Rα2, humans lack sIL-13Rα2 in serum. Our data provide a mechanism for this biologic divergence. In mice, discrete transcripts encoding s and membrane (mem) forms of IL-13Rα2 are generated by alternative splicing. We utilized siRNA to specifically deplete the transcript encoding memIL-13Rα2 (full-length) or sIL-13Rα2 (ΔEx10) in murine cells. Depletion of the full-length transcript decreased memIL-13Rα2, but had no effect on the level of sIL-13Rα2 in cell supernatants at baseline or following cytokine stimulation. Depletion of the ΔEx10 transcript decreased sIL-13Rα2 in supernatants at baseline and following stimulation. In contrast to mice, we were unable to find a transcript encoding sIL-13Rα2 in humans and siRNA-mediated depletion of full-length IL-13Rα2 decreased both sIL-13Rα2 and memIL-13Rα2 in human cells. Inhibition of matrix metalloproteinases (MMPs)/MMP-8 abolished production of sIL-13Rα2 from human cells. Thus, sIL-13Rα2 is derived exclusively from the memIL-13Rα2 transcript in humans through MMPs/MMP-8 cleavage of memIL-13Rα2, supporting a limited role for sIL-13Rα2 in humans and highlighting the potential importance of memIL-13Rα2 in human immunity. These observations require consideration when results of murine IL-13 studies are applied to humans.

Keywords: IL-13 receptor, membrane form, soluble form, siRNA, IL-13, MMP-8

Introduction

IL-13 is a central mediator of allergic inflammation and asthma (1, 2). IL-13 mediates its effects through multiple receptors, including IL-4 receptor alpha (IL-4Rα), IL-13 receptor alpha 1 (IL-13Rα1) and IL-13 receptor alpha 2 (IL-13Rα2) (3–7). IL-13Rα1 binds IL-13 with low affinity by itself but it binds IL-13 with high affinity when paired with IL-4Rα, and is critical for allergen-induced airway hyperresponsiveness and mucus production (8, 9). In contrast, IL-13Rα2 has a high affinity for IL-13 but is insufficient to render cells responsive to IL-13 even in the presence of IL-4Rα (10). IL-13Rα2 has a short cytoplasmic tail that lacks box 1 and box 2 signaling motifs, suggesting that it has no direct signaling ability. Indeed, characterization of IL-13Rα2-deficient mice has revealed that IL-13Rα2 can downregulate IL-13 responses by sequestering IL-13 (11, 12), although there is evidence that IL-13Rα2 may contribute to IL-13-induced TGFβ1-dependent fibrosis (13, 14).

In mice, IL-13Rα2 exists in soluble (s) and membrane (mem) forms, which can both bind to IL-13 and modulate its activity (15–17). Alternative splicing in mice results in two discrete transcripts, one that encodes full-length receptor (mem form) and another that lacks exon 10 resulting in early termination (ΔEx10, s form). sIL-13Rα2 generated by alternatively splicing is functionally active and binds IL-13 with 2–3 fold greater affinity than memIL-13Rα2 (15). In mouse models, sIL-13Rα2 acts as an inhibitory protein that regulates IL-13 responses (7). A recombinant sIL-13Rα2-Fc fusion protein can bind and neutralize IL-13 (10) and attenuate airway hyperresponsiveness when administered to allergen-challenged mice (18). Notably, naïve mice have ng/ml quantities of sIL-13Rα2 in their serum, and this is upregulated under conditions of allergic inflammation. The serum concentration of sIL-13Rα2 is ~10-fold greater than that of IL-13 on a molar basis (19–21), which is sufficiently high to inhibit IL-13 signaling in serum and make sIL-13Rα2 a major in vivo modifier of IL-13 activity and allergic inflammation in mice.

In humans, the presence, source, and role of sIL-13Rα2 remain to be established. One recent study failed to detect serum sIL-13Rα2 in any human samples in contrast to previous observations (20). In this paper, we confirm that human serum, unlike mouse serum lacks sIL-13Rα2 and demonstrate that the mechanisms for the derivation of memIL-13Rα2 vs. sIL-13Rα2 are distinctly different in mouse and man.

Materials and Methods

Cell lines

HaCaT human keratinocytes were kindly provided by Dr. Tim Bowden (University of Arizona, Tucson, AZ). Primary human pulmonary artery smooth muscle cells (HPASMC) were kindly provided by Dr. Tim Le Cras (Cincinnati Children’s Hospital Medical Center, Cincinnati, OH). BC3H1 and U87 cell lines were purchased from American Type Culture Collection. The U937 human IL-13Rα2 transfectants was described previously (22).

siRNA transfection

Silencer® negative control #1 siRNA was purchased from Ambion. Human IL-13Rα2-specific siRNA targeting the 3′-untranslated region of the full-length transcript (5′-GGCCAAAUGUUCAAUAUGAdTdT-3′ and 5′-UCAUAUUGAACAUUUGGCCdTdT-3′), mouse IL-13Rα2-specific siRNA targeting the transmembrane domain (5′-GGGCCAGACUCAAAGAUUAdTdT-3′ and 5′-UAAUCUUUUGAGUCUGGCCCdTdT-3′) and mouse IL-13Rα2-specific siRNA targeting the junction of exon 9 and exon 11 of the soluble isoform (5′-UUGGGAAGAGCCUCCAUGUdTdT-3′ and 5′-ACAUGGAGGCUCUUCCCAAdTdT-3′) were synthesized by Invitrogen (23). 8×105 BC3H1, U87, HaCaT or HPASMC cells were transfected with 20 μM siRNA oligonucleotide (control or IL-13Rα2-specific) using Lipofectamine RNAiMAX reagent (Invitrogen). Three days later, media from the cells were collected and IL-13Rα2 was quantified by ELISA. Surface expression of IL-13Rα2 on the cells was assessed by flow cytometry or total cellular IL-13Rα2 was quantified by ELISA. A portion of the cells was used for RNA extraction for RT-PCR of IL-13Rα2 to verify mRNA depletion after siRNA transfection. For cytokine stimulation, IL-13 (50 ng/ml) (PeproTech), IL-4 (10 ng/ml) (PeproTech), or IL-13 (50 ng/ml) plus TNFα (10 ng/ml) (R&D Systems) were added to the media 48h after siRNA transfection and cells were harvested 24h later.

Flow cytometry

Flow cytometry was performed as previously described using a FACSCalibur (BD Biosciences) and analyzed with CellQuest Pro (BD Biosciences) or FlowJo (Tree Star) software (15, 22). HPASMC and BC3H1 smooth muscle cells were stained with goat anti-human IL-13Rα2 polyclonal antibody and goat anti-mouse IL-13Rα2 polyclonal antibody (R&D Systems) respectively. Normal goat IgG (Santa Cruz Biotechnology) was used as a control. In order to determine if human samples contained IL-13Rα2, which could compete for anti-IL-13Rα2, human samples were assayed for their ability to block surface staining of U937 cells stably transfected with human IL-13Rα2 (22). These cells were resuspended in buffer containing human plasma samples (1:5 dilution) before staining for flow cytometry.

ELISA

ELISA of soluble human IL-13Rα2 was performed as previously described (16, 22). Random de-identified human plasma samples (atopy status unknown) were provided by Dr. Paul Steele from the Clinical Laboratory at Cincinnati Children’s Hospital Medical Center. In order to test the ability of human plasma to compete for anti-human IL-13Rα2, 1:5 diluted human plasma samples were preincubated with 2 μg/ml of goat anti-human IL-13Rα2 polyclonal antibody or normal goat IgG at room temperature for 1h before being added to the plates. The ELISA for mouse IL-13Rα2 was performed as previously described (15, 16). To determine the effect of plasma on the detection of IL-13Rα2 by ELISA, human IL-13Rα2-Fc protein (R&D Systems) was diluted in 1:20 human plasma samples. To assess the effect of IL-13, human IL-13Rα2-Fc protein or medium from cultured cells was preincubated with human IL-13 (1, 10 or 50 ng/ml) at 37°C for 1h before added to plates.

RT-PCR

RNA was extracted from the cell lines using Trizol® (Invitrogen), treated with DNase and purified using RNeasy MinElute Kit (QIAGEN). Reverse transcription was done using Oligo-T First-Strand cDNA Synthesis Kit (GE Healthcare). Realtime PCR was done using SYBR Green Master Kit and LightCycler® 480 instrument (Roche Diagnostics). For the conventional PCR, cDNA for human memIL-13Rα2 was amplified using forward primer 5′-CAGAAGTTCAAAGTTCCTGGGCAG-3′ and reverse primer 5′-GCCATGACTGGAAACTGTTGAGTC-3′. cDNA for human GAPDH was amplified using forward primer (5′-ACCCCTTCATTGACCTCAACTACA-3′) and reverse primer (5′-AGTGATGGCATGGACTGTGGTCAT-3′). For realtime PCR, cDNA for mouse memIL-13Rα2 was specifically amplified using forward primer 5′-TGAAAGTGAAGACCTATGCTTT-3′ and reverse primer 5′-GACAAACTGGTACTATGAAAAT-3′. cDNA for mouse sIL-13Rα2 was specifically amplified using the same forward primer and reverse primer 5′-CAGATCCACATGGAGGCTCTT-3′. cDNA for mouse β-actin was amplified using forward primer 5′-TCATCACTATTGGCAACGA-3′ and reverse primer 5′-TGTGTTGGCATAGAGGT-3′.

Treatment with MMP inhibitor

Media from cultured U87 cells were collected for ELISA and the cells were washed with PBS. Fresh medium was added and the cells were incubated with the broad-spectrum MMP inhibitor GM6001, a GM6001 Negative Control, a specific MMP-8 Inhibitor or a MMP-8 Inhibitor Negative Control (EMD Biosciences) at concentrations of 10μM or 20μM in DMSO or DMSO alone. Twenty-four hours later, the culture medium and cells were collected for ELISA. This treatment did not affect the cell viability as determined by trypan blue staining.

Statistical analysis

All values are shown as mean±SD. The comparisons of means from independent experiments were analyzed with 2-tailed paired student’s t-test using Prism 5.0a for Mac OS X (GraphPad Software). A p value of <0.05 was considered statistically significant.

Results

sIL-13Rα2 is absent in human plasma

High (approximately 10 ng/ml) levels of sIL-13Rα2 are present in serum from BALB/c WT mice and absent in IL-13Rα2 KO mice (Fig 1A). We determined the level of sIL-13Rα2 in human plasma samples (N=120). When a rat anti-human IL-13Rα2 monoclonal antibody was used for capture, no sIL-13Rα2 was detected in any of the human plasma samples. Alternatively, when using a goat anti-human IL-13Rα2 polyclonal antibody for capture, binding was found in a few samples; however the same samples also showed some binding to normal goat IgG, suggesting that the binding was non-specific (Fig. 1B).

Figure 1. sIL-13Rα2 in mouse and human plasma samples.

Figure 1

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(A) ELISA of plasma samples from BALB/c WT and IL-13Rα2 KO mice (n=5). (B) ELISA of random human plasma samples using rat anti-human IL-13Rα2 monoclonal, goat anti-human IL-13Rα2 polyclonal or normal goat IgG for capture. (C) ELISA of human plasma samples preincubated with excess normal goat IgG or goat anti-human IL-13Rα2 polyclonal antibody at room temperature for 1h. (D) U937 human IL-13Rα2 transfectants were resuspended in buffer containing 1:5 diluted human plasma samples prior to flow cytometry using goat anti-human IL-13Rα2 antibody and FITC-rabbit anti-goat IgG antibody. Shaded area: unstained; Solid thin line: stained with normal goat IgG; Solid thick line: stained with anti-human IL-13Rα2; Dotted, dashed and long dashed lines: stained with anti-human IL-13Rα2 in the presence of three different human plasma samples. (E) ELISA of human IL-13Rα2-Fc fusion protein diluted in 1% BSA PBS or 1:20 human plasma samples. (F) ELISA of human IL-13Rα2-Fc fusion protein preincubated with IL-13 (1, 10 or 50 ng/ml).

In order to confirm the absence of sIL-13Rα2 in the human samples, human plasma samples were pre-incubated with excess normal goat IgG or goat anti-human IL-13Rα2 polyclonal antibody at room temperature for 1h. Then, an ELISA was performed to look for free sIL-13Rα2 in the samples. With only 1 exception, preincubation of human plasma samples with a goat anti-human IL-13Rα2 polyclonal antibody failed to suppress the non-specific binding observed with goat anti-human IL-13Rα2 polyclonal antibody (Fig. 1C). To further confirm the absence of sIL-13Rα2 in the human samples, we tested whether human plasma samples could compete for anti-IL-13Rα2 and block surface staining of IL-13Rα2 on U937 cells stably transfected with human IL-13Rα2 (22). The presence of human plasma samples had no effect on the surface staining of IL-13Rα2 on U937 human IL-13Rα2 transfectants even at a 1:5 dilution.

To eliminate the possibility that other components in the plasma were affecting the detection of IL-13Rα2, the human plasma samples were spiked with increasing concentrations of IL-13Rα2-Fc protein prior to the ELISA. The majority of the IL-13Rα2-Fc protein was detectable in the ELISA irrespective of the presence of plasma (Fig. 1E). We also determined if the presence of IL-13 interfered with the detection of IL-13Rα2 by ELISA. IL-13Rα2-Fc protein or medium from cultured U87 cells were preincubated with increasing concentrations of IL-13 before the ELISA was performed. The presence of IL-13 had no effect on the detection of IL-13Rα2 (Fig. 1F). These data collectively indicate that sIL-13Rα2 is absent in human plasma.

sIL-13R α2 at baseline and after stimulation with IL-4 or IL-13 depends on expression of the soluble ΔEx10 transcript in murine cells

We have previously reported sIL-13Rα2 and memIL-13Rα2 are encoded in mice by distinct transcripts (15). We designed siRNA to specifically deplete the transcript encoding mem (full-length) or s (ΔEx10) IL-13Rα2 in murine cells. BC3H1 cells were utilized because they do not express detectable memIL-13Rα2 at baseline, but can be induced to express memIL-13Rα2 by culture with IL-13 plus TNFα (Fig. 2A). memIL-13Rα2-specific siRNA transfection significantly suppressed memIL-13Rα2 without changing the level of sIL-13Rα2 in culture medium after IL-13 plus TNFα stimulation (Fig. 2B). sIL-13Rα2-specific siRNA transfection significantly decreased sIL-13Rα2 concentration in culture medium (Fig. 2B) but induced only a negligible decrease in memIL-13Rα2 expression after IL-13 plus TNFα stimulation (Fig. 2C). The depletion of memIL-13Rα2 or sIL-13Rα2 transcripts was confirmed by realtime PCR (Fig. 2C, 2D). These data indicate that most sIL-13Rα2 is generated in mice from the sIL-13Rα2 transcript.

Figure 2. Effect of memIL-13Rα2- or sIL-13Rα2-specific siRNA on the expression of IL-13Rα2 mRNA and protein in BC3H1 cells.

Figure 2

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(A) Surface expression of IL-13Rα2 determined by flow cytometry. Shaded area: unstained; Solid thin line: stained with normal goat IgG; Solid thick line: stained with goat anti-mouse IL-13Rα2 polyclonal antibody. (B) sIL-13Rα2 in culture medium. (C) Expression of memIL-13Rα2. (D) Expression of sIL-13Rα2. The data are shown as mean±SD of means from three independent experiments with relative concentration or expression normalized to control siRNA IL-13 plus TNFα group of each experiment. Mem: memIL-13Rα2-specific; Sol: sIL-13Rα2-specific.

sIL-13Rα2 detected in the culture medium of human cells is derived from the memIL-13Rα2 transcript

In mice, alternative splicing out of exon 10, which contains the transmembrane domain, generates a transcript for sIL-13Rα2. We searched for, but were unable to find a human IL-13Rα2 transcript that similarly lacked the transmembrane domain (data not shown). Thus, sIL-13Rα2 is likely absent in human serum and bronchoalveolar lavage fluid in humans because humans lack an alternatively spliced transcript that encodes sIL-13Rα2. Given the apparent lack of a transcript encoding human sIL-13Rα2, we sought to determine the source of sIL-13Rα2 in the culture medium of human cells. We transfected human U87 glioma cells (Fig 3A) and HaCaT keratinocytes (Fig 3B) with IL-13Rα2-specific siRNA that targets the full-length transcript. At baseline, U87 cells generate a high level of sIL-13Rα2 in the culture medium (Fig. 3A). HaCaT cells have a low level of sIL-13Rα2 in the culture medium, which is increased following stimulation with IL-4. Transfection with IL-13Rα2-specific siRNA depleted IL-13Rα2 mRNA in both U87 cells and HaCaT cells before and after IL-4 stimulation (Fig. 3C, 3D). The level of sIL-13Rα2 in the culture medium and cell-associated IL-13Rα2 in U87 cells and HaCaT cells were significantly decreased after transfection with IL-13Rα2-specific siRNA (Fig 3A, 3B).

Figure 3. Effect of memIL-13Rα2-specific siRNA on the expression of IL-13Rα2 mRNA and protein in U87 and HaCaT cells.

Figure 3

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(A) IL-13Rα2 in culture medium and cell lysate from U87 cells. (B) IL-13Rα2 in culture medium and cell lysate from HaCaT cells. (C) mRNA expression of IL-13Rα2 in U87 cells. (D) mRNA expression of IL-13Rα2 in HaCaT cells. The data are shown as mean±SD of means from three independent experiments with relative concentration normalized to control siRNA group (U87) or control siRNA IL-4 group (HaCaT) of each experiment.

We confirmed this observation in primary cells. Human pulmonary artery smooth muscle cells (HPASMC) express considerable IL-13Rα2 on their cell surface at baseline (Fig 4). We determined the effect of siRNA transfection on the surface expression of IL-13Rα2 in these cells. Transfection of IL-13Rα2-specific siRNA significantly decreased total IL-13Rα2 in the cell lysate (Fig 4A), memIL-13Rα2 (Fig 4B), and the level of sIL-13Rα2 (Fig 4A) in culture medium. The depletion of IL-13Rα2 mRNA was confirmed by RT-PCR (Fig 4C). These data support that the sIL-13Rα2 detected in culture medium of human cells is derived from memIL-13Rα2.

Figure 4. Effect of memIL-13Rα2-specific siRNA on the expression of IL-13Rα2 mRNA and protein in HPASMC cells.

Figure 4

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(A) Level of IL-13Rα2 in culture medium and cell lysate. (B) Surface expression of IL-13Rα2 by flow cytometry. Shaded area: unstained; Solid thin line: stained with normal goat IgG; Solid thick line: stained with goat anti-human IL-13Rα2 polyclonal antibody. (C) mRNA expression of IL-13Rα2. The data are shown as mean±SD of means from three independent experiments with relative mean channel fluorescence or concentration normalized to control siRNA group of each experiment.

Inhibition of MMPs/MMP-8 results in decreased production of sIL-13Rα2 from human cells

We have previously demonstrated the MMP-8 can cleave memIL-13Rα2 to generate sIL-13Rα2 in cells stably transfected with a memIL-13Rα2 plasmid (16). We tested whether endogenous sIL-13Rα2 could be generated by MMP-8 mediated cleavage in human cells. We treated U87 cells with GM6001, a broad-spectrum MMP inhibitor and then determined the levels of sIL-13Rα2 in the culture medium. GM6001 treatment resulted in significantly decreased sIL-13Rα2 in the culture medium with no effect on the total IL-13Rα2 in cell lysate, indicating that sIL-13Rα2 was generated following cleavage of memIL-13Rα2 by MMPs. In contrast, treatment with the GM6001 negative control did not affect the levels of sIL-13Rα2 in the culture medium (Fig. 5A, 5B). In all cases, the culture medium collected before treatment had comparable levels of sIL-13Rα2 (Fig. 5C). In order to further confirm these results, we utilized a specific MMP-8 inhibitor. As shown in Fig 5D, 5E and 5F, inhibition of MMP-8 resulted in a 69% decrease in the level of sIL-13Rα2 in the culture medium. Treatment with the MMP-8 inhibitor had no effect on the level of total IL-13Rα2 in cell lysates. These data strongly suggest that cleavage of memIL-13Rα2 by MMPs, especially MMP-8, contributes to the release of sIL-13Rα2 from human cells.

Figure 5. Effect of GM6001 or MMP-8 Inhibitor on production of sIL-13Rα2 from U87 cells.

Figure 5

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(A) sIL-13Rα2 in culture medium after treatment. (B) Total IL-13Rα2 in cell lysate after treatment. (C) sIL-13Rα2 in culture medium before treatment. (D) sIL-13Rα2 in culture medium after treatment. (E) Total IL-13Rα2 in cell lysate after treatment. (F) sIL-13Rα2 in culture medium before treatment. The cells were treated with GM6001, GM6001 Negative Control, MMP-8 Inhibitor or MMP-8 Inhibitor Negative Control (10μM or 20μM) for 24h. NC: Negative Control. The data are shown as mean±SD of means from three independent experiments with relative concentration normalized to DMSO group of each experiment.

Discussion

sIL-13Rα2 can be produced by both pre-mRNA alternative splicing and proteolytic cleavage of memIL-13Rα2 (15–17). Our data support that in the mouse, sIL-13Rα2 production at baseline and after stimulation with IL-4 or IL-13 depends on expression of the soluble ΔEx10 transcript. In contrast, in humans, s and memIL-13Rα2 are both derived from the mem form of the receptor. The absence of an alternatively spliced sIL-13Rα2 transcript in humans compared to mice is likely due to the low homology between human IL-13Rα2 intron 8, exon 9 (transmembrane domain), and intron 9 with the homologous mouse IL-13Rα2 intron 9, exon 10 (transmembrane domain), and exon 11 (Fig. 6) (24). Through evolution, the homology between human and mouse IL-13Rα2 was maintained from exon 2 to exon 8, but the downstream exons, that encode the respective transmembrane domains, are divergent with very low homology. In contrast, the exons and introns across the entire gene are well conserved between human and monkey. Interestingly, we did identify an EST clone derived from rhesus monkey placenta (GenBank accession number CN643593), which would encode a predicted soluble form of IL-13Rα2 lacking the transmembrane and intracellular domains. This transcript was generated by alternative polyadenylation in intron 7 resulting in an early termination. We identified an equivalent transcript in humans (GenBank accession number GQ494004), however we could not demonstrate expression of this transcript in normal or disease states and the protein expressed from this transcript was intracellularly localized (probably due to protein misfolding, data not shown).

Figure 6. Evolutionarily conserved regions (ECRs) in human, rhesus monkey and mouse IL-13Rα2 genes.

Figure 6

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Exon 9 of the human gene (top) contains the transmembrane domain. The level of nucleotide identity to human gene is shown by layer height from 50% to 100%. The ECRs in rhesus monkey and mouse genes are indicated by pink rectangles.

Our data confirm the recent failure by O’Toole et al to detect sIL-13Rα2 in human plasma or bronchoalveolar lavage fluid samples and calls into question whether sIL-13Rα2 exists at all in humans (25). The absence of sIL-13Rα2 in bronchoalveolar lavage fluid, even after allergen challenge, indicate that, in humans, (1) sIL-13Rα2 is not released from cells even under conditions of allergic inflammation; and (2) proteolytic cleavage of memIL-13Rα2 does not generate appreciable sIL-13Rα2 even under conditions of allergic inflammation. In contrast, high levels of sIL-13Rα2 are found in the serum of naïve mice and in the bronchoalveolar lavage fluid of mice following an allergen challenge (15). This striking mouse-human difference must be taken into consideration when applying observations about IL-13 function and regulation made in mouse models to humans.

Patients with asthma, chronic obstructive pulmonary disease or bronchiectasis have increased expression of MMPs including MMP-8 (26–28). MMPs are important mediators of airway inflammation and remodeling (29). Our data demonstrate that one mechanism by which memIL-13Rα2 is released into the soluble form involves MMP-8 activity. This is supported by our previous work demonstrating that MMP-8 can cleave human IL-13Rα2 in vitro (16). The relevance of soluble IL-13Rα2 in human systems, and specifically the role of MMP8-induced cleavage of memIL-13Rα2 in the diseased airways remains to be established. The absence of sIL-13Rα2 in the plasma suggests that the function of the soluble receptor might be localized to the tissues and the regulation of expression has evolved to be more tightly controlled in humans compared to mice. In the tissues, sIL-13Rα2 could sequester IL-13 away from its cognate signaling receptor IL-13Rα1, attenuating IL-13 signaling in the local environment. This would be consistent with the anti-inflammatory function for MMP-8 (30, 31). Alternatively, it is possible that the MMP-8 induced IL-13Rα2 cleavage is the first step in degradation of the memIL-13Rα2, once its role is completed.

In conclusion, sIL-13Rα2 is absent in human plasma and has a limited role in humans in contrast to mice, where the concentration of sIL-13Rα2 in serum is sufficiently high (ng/ml range) to inhibit IL-13 signaling. Indeed, sIL-13Rα2 suppresses IL-13 activity and allergic inflammation in vivo in mice, prolongs the serum half-life of IL-13 and regulates levels of circulating and tissue IL-13 (11, 12, 21). The lack of sIL-13Rα2 in humans is likely to limit the applicability of some murine studies of IL-13 to humans. Use of a humanized mouse model that expresses IL-13Rα2 in the same way that it is expressed in humans may improve the human applicability of studies of in vivo IL-13 and IL-13Rα2 function.

Acknowledgments

We thank Dr. Yuichi Machida for help with designing mouse soluble form IL-13Rα2 siRNA, and Cynthia Chappell for assistance with preparation of this manuscript.

Abbreviations

IL-13Rα2

IL-13 receptor alpha 2

IL-13Rα1

IL-13 receptor alpha 1

IL-4Rα

IL-4 receptor alpha

mem

membrane

s

soluble

HPASMC

human pulmonary artery smooth muscle cell

MMP

matrix metalloproteinase

ECR

evolutionarily conserved region

Footnotes

1

This work was supported by National Institutes of Health grants R01 AI58157 (GKKH), AR054490 (GKKH), P01 HL076383 (GKKH), PO1 HL076383 (FDF) and a Merit Award from the Department of Veterans Affairs (FDF).

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