Differences in Expression, Affinity, and Function of Soluble (s)IL-4Rα and sIL-13Rα2 Suggest Opposite Effects on Allergic Responses

Marat Khodoun, Christina Lewis, Jun-Qi Yang, Tatyana Orekov, Crystal Potter, Thomas Wynn, Margaret Mentink-Kane, Gurjit K. Khurana Hershey, Marsha Wills-Karp and Fred D. Finkelman
J Immunol November 15, 2007, 179 (10) 6429-6438; DOI: https://doi.org/10.4049/jimmunol.179.10.6429

Abstract

IL-4 and IL-13 are each bound by soluble receptors (sRs) that block their activity. Both of these sRs (sIL-4Rα and sIL-13Rα2) are present in low nanogram per milliliter concentrations in the serum from unstimulated mice, but differences in affinity and half-life suggest differences in function. Serum IL-4/sIL-4Rα complexes rapidly dissociate, releasing active IL-4, whereas sIL-13Rα2 and IL-13 form a stable complex that has a considerably longer half-life than uncomplexed IL-13, sIL-13Rα2, IL-4, or sIL-4Rα. Approximately 25% of sIL-13Rα2 in serum is complexed to IL-13; this percentage and the absolute quantity of sIL-13Rα2 in serum increase considerably during a Th2 response. sIL-13Rα2 gene expression is up-regulated by both IL-4 and IL-13; the effect of IL-4 is totally IL-4Rα-dependent while the effect of IL-13 is partially IL-4Rα-independent. Inhalation of an IL-13/sIL-13Rα2 complex does not affect the expression of IL-13-inducible genes but increases the expression of two genes, Vnn1 and Pira-1, whose products activate APCs and promote neutrophilic inflammation. These observations suggest that sIL-4Rα predominantly sustains, increases, and diffuses the effects of IL-4, whereas sIL-13Rα2 limits the direct effects of IL-13 to the site of IL-13 production and forms a stable complex with IL-13 that may modify the quality and intensity of an allergic inflammatory response.

Allergic immunopathology and host protection against helminth parasites are both mediated, in large part, by the type 2 cytokines IL-4, IL-5, IL-9, and IL-13 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14). Of these, a particularly prominent role is played by two related cytokines, IL-4 and IL-13. Both of these cytokines bind to cell membrane receptors that contain an IL-4Rα-chain and activate the transcription factor Stat6 (15, 16, 17), and both have prominent effects on multiple cell types that contribute to allergic inflammation (3, 5, 7, 12, 14, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27). There are, however, important differences between the biological effects of these two cytokines. Some of these differences stem from the failure of IL-13 to signal through the type 1 IL-4 receptor (IL-4R), which is composed of IL-4Rα and the cytokine receptor common γ-chain, γc (28, 29, 30). Because this is the only IL-4R expressed on T cells and mast cells, these cell types respond to IL-4 but not IL-13 (30). This probably accounts for the greater importance of IL-4 than IL-13 in the promotion of a Th2 response. In contrast, the type 2 IL-4R, which is composed of the IL-4Rα and IL-13Rα1 polypeptides (15, 29, 31, 32), is expressed by some bone marrow-derived cells, including macrophages, as well as most non-bone marrow-derived cells and is activated by both IL-4 and IL-13 (28, 29, 33, 34). Signaling through this receptor appears to be responsible for many proallergic effects of IL-4 and most proallergic effects of IL-13 (7, 10, 20, 28, 33, 34, 35), although IL-13 signaling through a cell membrane form of an additional IL-13-binding protein, cell membrane IL-13Rα2, may contribute to the profibrotic effects of this cytokine (36).

Additional differences between IL-4 and IL-13 function may reflect: 1) the ability of IL-4, but not IL-13, to stimulate NK and NK T cell production of IFN-γ (37), presumably through effects mediated by the type 1 IL-4R; 2) 1 IL-4R-dependent stimulation of other compounds that inhibit IL-13 effects (38), 3) the production of IL-13, but not IL-4, by some cell types (39, 40, 41), and 4) the production of more IL-13 than IL-4 during immune responses to allergens or worms (C. Perkins and F. D. Finkelman, unpublished data). The studies described in this article were performed to determine whether differences in the function of IL-4 and IL-13 might also be influenced by differences between the expression and function of soluble (s)3 forms of IL-4Rα and IL-13Rα2, proteins in blood and lymph that bind IL-4 and IL-13, respectively (42, 43, 44, 45). To address this possibility, we have evaluated the expression and functional characteristics of sIL-4Rα and IL-13Rα2 in mouse serum. We find that both of these soluble receptors (sRs) are present in low nanogram per milliliter quantities in the serum of naive BALB/c mice. However, the in vivo affinity of sIL-4Rα for IL-4 is considerably lower than that of sIL-13Rα2 for IL-13; IL-4/sIL-4Rα complexes rapidly dissociate while IL-13/sIL-13Rα2 complexes are very stable and have a considerably longer half-life than uncomplexed IL-13 or sIL-13Rα2. These differences suggest that sIL-4Rα may function more as an IL-4 carrier protein that prolongs and diffuses the effects of secreted IL-4 than as an IL-4 antagonist, whereas sIL-13Rα2 may have a pure antagonist function toward IL-13 that limits the effects of this cytokine to the site of its secretion. In addition, our studies suggest that IL-13/sIL-13Rα2 complexes themselves may have proinflammatory effects that differ from those of IL-13.

Materials and Methods

Mice

BALB/c wild-type and IL-4 receptor α-chain-deficient mice (BALB/cJ-Il4ratm1) (46) were purchased from Taconic Farms and IL-4-deficient mice (BALB/c-Il4tm2Nnt/J) (47) were purchased from The Jackson Laboratory. IL-13-deficient mice and IL-4/IL-13-double deficient mice (48) were a gift from Dr. A. McKenzie (Medical Research Council Laboratory of Molecular Biology, Cambridge, U.K.). IL-13Rα2-deficient mice (49) and Stat6-deficient mice (C.129S2-Stat6tm1Gru/J) (18) were a gift from M. Grusby (Harvard University, Boston, MA). IL-13/IL-13Rα2-double deficient mice were generated by breeding IL-13- and IL-13Rα2-deficient mice. Offspring were typed by PCR. Studies were reviewed and approved by the Institutional Animal Care and Use Committee at the University of Cincinnati and the Cincinnati Veterans Affairs Medical Center (Cincinnati, OH).

Reagents

The following antisera, Abs, and mAbs were prepared as described: goat antisera to mouse IgD (GaMD) and keyhole limpet hemocyanin (GaKLH) (50); EM-95 (rat IgG2a anti-mouse IgE (51); and 145-2C11 (hamster IgG anti-mouse CD3ε) (52). Affinity-purified goat anti-mouse IL-13Rα2 Ab was produced by immunizing a goat with mouse IL-13Rα2-human IgGFc fusion protein (a gift from D. Donaldson, then at Wyeth Research) in CFA (Difco), boosting with the same Ag in IFA (Difco), adsorbing with human IgG coupled to CNBr-activated Sepharose (Pharmacia), and affinity purifying by adsorption to and 3.5 M MgCl2 elution from mouse IL-13Rα2-mouse IgGFc fusion protein (D. Donaldson, then at Wyeth Research) coupled to cyanogen bromide-activated Sepharose. The purified Ab failed to stain mouse spleen cells more than a nonspecific control Ab (GaKLH) but captured sIL-13Rα2 in the serum of normal mice but not IL-13Rα2-deficient mice in a single Ab ELISA (data not shown). Affinity-purified goat anti-mouse IL-4Rα Ab was produced by immunizing a goat with mouse sIL-4Rα (a gift from C. Maliszewski, then at Immunex) in CFA, boosting with the same Ag in IFA, and affinity purifying by adsorption to and 3.5 M MgCl2 elution from mouse IL-4Rα coupled to cyanogen bromide-activated Sepharose. The purified Ab specifically stained spleen cells from wild-type mice, but not IL-4Rα-deficient mice, as compared with GaKLH (data not shown).

Affinity-purified goat anti-mouse IL-13 was purchased from R&D Systems. C531 (rat IgG anti-mouse IL-13) was a gift from S. Visvanathan (Centocor). IL-4 was purchased from PeproTech. IL-13 was a gift from M. Kasaian (Wyeth Research).

Administration of cytokines

IL-4 was administered by itself and as a complex with the neutralizing mAb, BVD4-1D11. IL-4/anti-IL-4 mAb complexes (IL-4C), which are prepared by mixing the cytokine and anti-cytokine mAb at a 2:1 molar ratio, slowly dissociate in vivo to release free IL-4. A single injection of IL-4C maintains the activity of the relevant cytokine for ∼3 days (53). These complexes are unable to activate C, bind more avidly than free IgG to FcγRs, or interact simultaneously with FcγRs and cytokine receptors because they contain a single IgG molecule and BVD4-1D11 blocks the IL-4 epitope that binds to IL-4Rα (54).

Measurement of free IL-4 and IL-13

Levels of free IL-4 and IL-13 in solution were measured by ELISA. IL-4 was captured by the non-neutralizing mAb BVD6-24G2.3 and detected with biotin-BVD4-1D11, followed serially by a HRP-streptavidin conjugate (Pierce) and a luminogenic substrate for HRP (SuperSignal ELISA Femto substrate; Pierce). IL-13 was captured by IL-13Rα2-IgGFc and detected with biotinylated anti-IL-13 mAb (C531, which binds IL-13 that is complexed to sIL-13Rα2; a gift from Centocor). Luminescence was measured with a Fluoroskan Ascent FL microtiter plate luminometer/fluorometer (Labsystems).

Measurement of free and cytokine-complexed sIL-13Rα2 and sIL-4Rα

Serum levels of the IL-13/sIL-13Rα2 complex were measured by ELISA using affinity-purified goat anti-IL-13Rα2 Ab to capture the complex onto microtiter plate wells followed by biotin-labeled anti-IL-13 mAb (C531), streptavidin-HRP, and luminogenic substrate. Serum levels of the IL-4/sIL-4Rα complex were detected by an ELISA in which microtiter plate wells were coated with goat anti-IL-4Rα mAb and the captured complex was detected with biotin-anti-IL-4 mAb (BVD6-24G2.3). Total levels of sIL-13Rα2 or sIL-4Rα were detected in the same way, except that rIL-13 (100 ng/ml) or IL-4 (20 ng/ml), respectively, was added to the serum before performing the assay. The percentage of saturation of the sR with the cytokine was determined by dividing the concentration of the cytokine/sR complex by the total soluble cytokine receptor concentration.

Preparation of IL-13-rich serum

IL-13Rα2-deficient mice were inoculated with 200 infective Nippostrongylus brasiliensis larvae on days 0 and 14 and injected i.p. with 10 μg of anti-CD3 mAb on day 21. Mice were bled on days 6, 7, 8, and 19 and 2 h after anti-CD3 mAb injection on day 21. Sera were pooled and concentrated 2-fold.

Worm inoculation

Mice were inoculated subcutaneously with 500 N. brasiliensis third-stage infectious larvae (L3) or inoculated percutaneously via the tail with 25–30 cercariae of a Puerto Rican strain of Schistosoma mansoni (Naval Medical Research Institute (NMRI) strain) that were obtained from infected Biomphalaria glabrata snails (Biomedical Research Institute).

Dissociation of IL-13/sIL-13Rα2 complexes

One hundred and fifty microliters of mouse IL-13Rα2-IgGFc-agarose was mixed with 4.5 ng of recombinant mouse IL-13. Agarose beads were washed first with PBS and then eluted sequentially with 20 mM phosphate buffer at pH 6.5, 6.0, 5.5, 5.0, 4.5, and 4.1, followed by 3.5 M MgCl2. Eluates were immediately brought to pH 7.0 by adding an equal volume of 100 mM phosphate buffer (pH 7.0) except for MgCl2 eluates, which were immediately desalted with Centricon ultrafiltration units (Amicon). IL-13 concentrations in eluates were measured by ELISA.

Microarray data analysis

Gene expression summary values for the Affymetrix GeneChip data in CEL files were computed using RMAExpress (55, 56, 57) (http://rmaexpress.bmbolstad.com/). Data analyses were conducted with GeneSpring version 7.3.1 (Agilent Technologies) software, including filtering, statistical analysis, and clustering. Hybridization signals were transformed from log base 2 to linear values and then the relative expression of each sequence on the array was normalized to its expression in mice treated with PBS or other appropriate controls in the same experiment.

Quantitative real-time RT-PCR

Total RNA was extracted from frozen lungs using TRIzol reagent (Invitrogen Life Technologies) per the manufacturer’s protocols, followed by purification using an RNeasy mini kit and DNase digestion (Qiagen). RNA purity was confirmed with a NanoDrop spectrophotometer (NanoDrop), and RNA integrity was confirmed using a bioanalyzer (model 2100; Agilent Technologies). Purified total lung RNA was reverse transcribed into single-stranded cDNA using random hexamers and SuperScript II (Invitrogen Life Technologies). Real-time RT-PCR was performed on the iCycler (Roche Diagnostics) using a total volume of 20 μl containing 100 μM iCycler-DNA Master SYBR Green (Roche Diagnostics), double-distilled H2O, and 4 μl of cDNA, which corresponds to ∼33 ng of total RNA. The cDNA was added as the template and 5 μl (3 mM) of the primer of interest was added to the PCR (β-actin: 5′-GTGACGTTGACATCCG-3′ (sense primer) and 5′-CAGTAACAGTCCGCCT-3′ (antisense primer); 2210421G13Rik/ApoA1: 5′-AGGTACCACTCTGGCAATGACCAA-3′ (sense primer) and 5′-TCTGCAGCAGTCTGTGACTTAGCA-3′ (antisense primer); Vnn1: 5′-AAGTGTTGCTGAGTGAGG-3′ (sense primer) and 5′-TGTGCTATGAAGTCTGAGG-3′ (antisense primer); Pira1: 5′-GAGGGTCGGGTGTATGTATTAGC-3′ (sense primer) and 5′-GCCACAAGGAACATCAACTAAGC-3′ (antisense primer); IL-13Rα2: 5′-GGACTCATCAGACTATAAAGA-3′ (sense primer) and 5′-GTGTGCTCCATTTCATTCTA-3′ (antisense primer); and 18S rRNA: 5′-GTAACCCGTTGAACCCCATT-3′ (sense primer) and 5′-CCATCCAATCGGTAGTAGCG-3′ (antisense primer)). The amount of mRNA transcripts encoding these genes was determined using the following formula: relative gene expression = (1.8(a − b)) × 100,000, where a is the crossing point of β-actin and b is crossing point of the gene of interest.

Statistics

Data were analyzed by ANOVA and Fisher’s protected least significant difference for statistical significance using StatView. Values of p < 0.05 were considered statistically significant. Figures show means ± SEM.

Results

Serum sIL-13Rα2, but not serum sIL-4Rα, is partially saturated with its ligand in naive mice

Serum levels of IL-4/sIL-4Rα complexes and IL-13/sIL-13Rα2 complexes were measured by ELISA using Ag-affinity purified goat Abs to the soluble sRs to capture the complexes in serum and the biotin-labeled mAbs to cytokine epitopes that were not blocked by the sRs to detect bound complexes. Serum levels of these complexes were then measured a second time after adding sufficient IL-4 or IL-13 to saturate serum sIL-4Rα or sIL-13Rα2, respectively. Results of several experiments with untreated 8- to16-wk-old BALB/c mice indicated that sIL-4Rα and sIL-13Rα2 are present in serum at similar concentrations (∼4–10 ng/ml; Fig. 1⇓). However, sIL-4Rα was never detectably complexed with IL-4 in naive mouse serum, whereas 15–40% of sIL-13Rα2 was complexed with IL-13.

FIGURE 1.
FIGURE 1.

Levels of sIL-13Rα2 and sIL-4Rα in normal mouse serum. Eight- to 16-wk-old untreated BALB/c mice (five per group in this and most subsequent studies) were bled and serum levels of sIL-13Rα2 and sIL-4Rα and their saturation with IL-13 and IL-4, respectively, were determined.

Worm infection and Ag immunization induce a greater increase in serum concentration of sIL-13Rα2 than that of sIL-4Rα

To determine whether sIL-4Rα and/or sIL-13Rα2 change in concentration and/or saturation during the course of a Th2 response, these parameters were followed in BALB/c mice infected with the nematode parasite N. brasiliensis or inoculated with GaMD. N. brasiliensis inoculation and GaMD immunization each stimulate a strong Th2 response (58). In both cases, sIL-4Rα concentrations increased 2–3-fold and sIL-13Rα2 concentrations increased 7–10-fold as the Th2 response peaked and then decreased back toward baseline levels (Fig. 2⇓). Saturation of sIL-13Rα2 with IL-13 increased to a peak of ∼90%, whereas sIL-4Rα complexed to IL-4 never became detectable. Infections of wild-type, IL-13-deficient, and IL-4/IL-13-double deficient mice with a second helminth parasite, S. mansoni, demonstrated that IL-4 and IL-13 secretion in infected mice both contributed to the increase in serum sIL-13Rα2 levels, with little increase seen in mice that are deficient in both IL-4 and IL-13 (Fig. 3⇓). Regardless of whether mice were stimulated with GaMD immunization or worm infection, the kinetics of the increases in the serum levels of sIL-4Rα and/or sIL-13Rα2 concentration followed the previously determined kinetics of increases in the serum levels of IL-4 (peak on days 5–6 for GaMD, days 6–8 for N. brasiliensis, and weeks 8–12 for S. mansoni (Refs. 43 and 46 and F. D. Finkelman, unpublished data).

FIGURE 2.
FIGURE 2.

sIL-13Rα2 and sIL-4Rα responses to Th2 stimulation. BALB/c mice were inoculated with N. brasiliensis (upper panels) or immunized with GaMD (lower panels) and bled at the time points shown. Serum concentrations of sIL-4Rα and sIL-13Rα2 and their saturation with their cytokine ligands were determined.

FIGURE 3.
FIGURE 3.

Cytokine dependence of the sIL-13Rα2 response to infection with Schistosoma mansoni. BALB/c wild-type, IL-13-deficient, and IL-4/IL-13-double deficient mice were infected with S. mansoni and bled at the time points shown. Serum levels of sIL-13Rα2 and their saturation with IL-13 were determined.

Only rapid production of large amounts of IL-4 induces detectable IL-4/sIL-4Rα complex formation in vivo

These results raised the possibility that sIL-4Rα does not bind IL-4 at all in vivo. To evaluate this possibility, experiments were performed that determined sIL-4Rα saturation with IL-4 during a rapid in vivo IL-4 response. Priming of mice with an anti-IgD mAb followed by challenge with an anti-IgE mAb, which induces massive basophil IL-4 and IL-13 secretion over a 4-h period (19), had little effect on serum levels of sIL-4Rα or sIL-13Rα2 but increased sIL-4Rα saturation with IL-4 from 0 to ∼8% and sIL-13Rα2 saturation with IL-13 from ∼20 to ∼60% (Fig. 4⇓). Treatment with anti-CD3 mAb, which causes even more massive cytokine secretion by NK T cells over a 2 h period (59, 60), caused an approximate doubling of sIL-4Rα and sIL-13Rα2 concentrations in serum and increased sIL-4Rα saturation with IL-4 to nearly 50% and sIL-13Rα2 saturation with IL-13 to nearly 100% (Fig. 4⇓). Thus, sIL-4Rα binds naturally produced IL-4 in vivo, but this is only detectable when massive amounts of IL-4 are secreted over a short period of time.

FIGURE 4.
FIGURE 4.

Stimuli that cause rapid production of IL-4 and IL-13 increase serum sIL-13Rα2 and sIL-4Rα concentration and saturation. BALB/c mice (five per group) were used in the experiment. BALB/c mice were injected with saline or GaMD and challenged 14 days later with saline or 100 μg of rat anti-IgE mAb and bled 4 h later (upper panels) or injected with saline or 10 μg of anti-CD3 mAb and bled 2 h later (lower panels). Serum concentrations of sIL-4Rα and sIL-13Rα2 and their saturation with their cytokine ligands were determined. Asterisks (*) in this figure and in subsequent figures indicate that the value for the indicated group is significantly increased (p < 0.05) as compared with the value for an untreated, saline-treated, or vehicle-treated group.

Both IL-4 and IL-13 induce IL-4Rα- and Stat6-dependent increases in serum levels of sIL-13Rα2, but only the IL-13-induced increase is partially IL-4Rα- and Stat6-independent

Previous studies have demonstrated that sIL-13Rα2 gene expression is up-regulated by IL-4 and IL-13 (61). To determine whether these cytokines have similar effects on serum sIL-13Rα2 protein levels and whether sIL-4Rα levels are also affected, mice were treated with IL-4 or IL-13 and serum concentrations of both sRs and their saturation with their ligands were determined (Fig. 5⇓). Injection of a single 1-μg dose of rIL-4, which has a very short in vivo half-life, caused a large increase in the percentage of sIL-4Rα that was saturated with IL-4 30 min postinjection; however, this percentage decreased by >50% during the next 1.5 h and was nearly back to baseline at 12 h (Fig. 5⇓A, right panel). Only a small increase in the serum concentration of sIL-4Rα was observed; this returned to baseline by 12 h after IL-4 injection (Fig. 5⇓A, left panel). IL-4 injection had a modest effect on the serum concentration of sIL-13Rα2 and no effect on its saturation with IL-13 (Fig. 5⇓B). Injection of a single 1-μg dose of rIL-13 had no effect on sIL-4Rα levels at 2 h or on sIL-4Rα saturation with IL-4 at any time point, but it increased sIL-4Rα levels by ∼80% at 12 h (Fig. 5⇓C). Injection of 1 μg of IL-13 also completely saturated sIL-13Rα2 with IL-13 for >12 h and caused a 2-fold increase in serum sIL-13Rα2 levels at 4 h and a 5-fold increase at 12 h (Figs. 5⇓, B and D). To compensate for a possible difference in IL-4 vs IL-13 half-life, we also evaluated the effect on serum sIL-13Rα2 concentration of treating mice with a long-acting form of IL-4 (IL-4C, produced by mixing IL-4 and a neutralizing anti-IL-4 mAb at concentrations that produce a complex that contains one molecule of anti-IL-4 mAb and two molecules of IL-4). IL-4C treatment substantially increased serum levels of sIL-13Rα2 (observed both 1 and 3 days later); this increase was totally IL-4Rα- and Stat6-dependent (Fig. 6⇓, A and B). In contrast, IL-13 induced considerable increases of serum sIL-13Rα2 levels in IL-4Rα- and Stat6-deficient mice, although it induced larger increases in wild-type mice (Fig. 6⇓C).

FIGURE 5.
FIGURE 5.

IL-4 and IL-13 each induce increases in serum levels of both sIL-4Rα (A and C) and sIL-13Rα2 (B and D). BALB/c mice were injected with 1 μg of IL-4 (A and B) or IL-13 (B, C, and D). Serum levels of sIL-4Rα and sIL-13Rα2 and their saturation of IL-4 or IL-13, respectively, were measured at the time points indicated.

FIGURE 6.
FIGURE 6.

IL-4Rα- and Stat6-dependence of IL-4 and IL-13 up-regulation of serum sIL-13Rα2 concentration. A, BALB/c wild-type mice were injected with saline or IL-4C that contained 2 μg of IL-4. Mice were bled 1 day later and serum sIL-13Rα2 levels were determined. B, Wild-type, IL-4Rα-, and Stat6-deficient mice were injected with IL-4C that contained 2 μm of IL-4. Mice were bled prior to and 3 days after IL-4C injection and serum levels of sIL-13Rα2 were determined. C, Wild-type, IL-4Rα-, Stat6-, and IL-13Rα2-deficient mice were left untreated or injected with 1 μg of IL-13. Mice were bled 1 day later and sIL-13Rα2 levels were determined.

IL-13/sIL-13Rα2 complexes have a longer in vivo half-life than free IL-13 or sIL-13Rα2 while IL-4/sIL-4Rα complexes have a shorter in vivo half-life than free sIL-4Rα

Follow-up studies were performed to more formally investigate the suggestion that the half-life of the IL-13/sIL-13Rα2 complex is considerably longer than the half-life of the IL-4/sIL-4Rα complex. Previous studies have shown that uncomplexed IL-4 has a very short half-life (<15 min), whereas complexes formed of rIL-4 and recombinant sIL-4Rα had a longer half-life (62, 63). Our current studies demonstrate that complexes formed of natural sIL-4Rα and rIL-4 dissociate rapidly when injected into IL-4Rα-deficient mice with a half-life well under 30 min (Fig. 7⇓A), while the half-life of the free sIL-4Rα formed by dissociation of IL-4/sIL-4Rα complexes is ∼2 h (consistent with a previous estimate of 2.3 h; Ref. 62) and was not affected by administering sIL-4Rα as a complex with IL-4. Two experiments that gave very similar results were performed to evaluate the half-lives of free sIL-13Rα2 and IL-13/sIL-13Rα2 complexes in mice deficient in both IL-13 and IL-13Rα2 (Fig. 7⇓B). Based on the last two points in the concentration curves that reflect the half-lives after injected proteins have fully distributed, uncomplexed sIL-13Rα2 has a half-life of ∼3.3 h and the IL-13/sIL-13Rα2 complex has a half-life of ∼18 h. Half-life calculations based on the average of all points in the concentrations curves, including points that may be affected by initial distribution, give injected uncomplexed sIL-13Rα2 a half-life of ∼1.3 h and IL-13/sIL-13Rα2 complex a half-life of ∼11.1 h (Fig. 7⇓C). A separate experiment demonstrated that the half-life of free, natural IL-13, when injected i.v. into IL-13/IL-13Rα2-double deficient mice, is ∼20 min and that this short half-life was associated with the loss of IL-13 in the urine (Fig. 7⇓D). Thus, whereas the IL-4/sIL-4Rα complex rapidly dissociates into free IL-4, which has an even shorter half-life, and free sIL-4Rα, which has a somewhat longer half-life, the IL-13/sIL-13Rα2 complex is remarkably stable and has a considerably longer half-life than either of its constituents.

FIGURE 7.
FIGURE 7.

Serum half-lives of free and cytokine-complexed sIL-13Rα2 and sIL-4Rα. A, IL-4Rα-deficient mice were injected with 0.5 ml of 5-fold concentrated normal mouse sera or normal mouse serum plus IL-4. Mice were bled at the times shown and serum levels of the IL-4/sIL-4Rα complex and total sIL-4Rα were determined. B, IL-13/IL-13Rα2-double deficient mice were injected with 0.5 ml of 10-fold concentrated serum from N. brasiliensis-infected, IL-13-deficient BALB/c mice (filled circles and open squares representing two independent experiments) or 10-fold concentrated serum from N. brasiliensis-infected wild-type BALB/c mice (gray triangles and open diamonds representing two independent experiments). Mice were bled at the times shown and the amounts of free sIL-13Rα2 or IL-13/sIL-13Rα2 complexes were determined. C, Mean values were calculated from the two experiments shown in B and half-life curves (dashed lines) were calculated for both sets of points using exponential equations. D, IL-13-rich serum (0.7 ml) from IL-13Rα2-deficient mice, prepared as described in Materials and Methods, was injected into nine IL-13/IL-13Rα2-double deficient mice. Three recipient mice were bled 20, 40, or 80 min later and serum IL-13 levels were determined by ELISA. IL-13 concentrations were also determined in pooled urine samples obtained from the same mice 30, 90, or 720 min after IL-13 injection.

Decreased renal excretion may account for the prolonged serum half-life of IL-13/sIL-13Rα2 complexes

Because sIL-13Rα2 is excreted intact in urine (44), it seemed possible that the increased serum half-life of the IL-13/sIL-13Rα2 complex might reflect reduced urinary secretion. To evaluate this possibility, we compared urine sIL-13Rα2 concentrations to serum concentrations of free and IL-13-complexed sIL-13Rα2 and evaluated how urine concentrations are modified by stimuli that increase the serum concentration of free or IL-13-bound sIL-13Rα2. In contrast to serum, which contains a mixture of free and IL-13-bound sIL-13Rα2, only free sIL-13Rα2 was detected in urine (Fig. 8⇓A). Injection of mice with 1 μg of rIL-13 increased the serum concentration of the IL-13/sIL-13Rα2 complex considerably but had little effect on either the serum or urine concentration of free sIL-13Rα2 (Fig. 8⇓A). In contrast, IL-4C injection of wild-type mice (Fig. 8⇓B) or IL-13-deficient mice (Fig. 8⇓C) induced considerable and proportionate increases in serum and urine concentrations of free sIL-13Rα2 (Fig. 8⇓, B and C). As expected, sIL-13Rα2 was undetectable in the serum and urine of IL-13Rα2-deficient mice (Fig. 8⇓B). Taken together, these observations suggest that complex formation with IL-13 drastically reduces renal excretion of sIL-13Rα2, which considerably increases its serum half-life.

FIGURE 8.
FIGURE 8.

Complex formation with IL-13 blocks the renal clearance of sIL-13Rα2. A, BALB/c mice were injected i.v. with saline or recombinant mouse IL-13. Blood and urine was collected 1 day later and serum and urine total sIL-13Rα2 concentration and IL-13/sIL-13Rα2 complex concentration were determined. Percentages shown in this and other panels indicate the urine concentration relative to serum concentration of total sIL-13Rα2. B, BALB/c wild-type and IL-13Rα2-deficient mice were injected with saline or IL-4C. Blood and urine was collected 1 day later and serum and urine total sIL-13Rα2 concentration and IL-13/sIL-13Rα2 complex concentration were determined. C, BALB/c IL-13-deficient mice were injected with saline or IL-4C. Blood and urine was collected 1 day later and serum and urine total sIL-13Rα2 concentration and IL-13/sIL-13Rα2 complex concentration were determined.

The increased half-life of IL-13/sIL-13Rα2 complexes only partially accounts for the IL-13-induced increase in serum concentration of sIL-13Rα2 in IL-4Rα-deficient mice

The increased in vivo half-life of IL-13/sIL-13Rα2 compared with free sIL-13Rα2 might explain the increase in serum levels of sIL-13Rα2 observed in IL-13-injected Stat6- and IL-4Rα-deficient mice; the longer half-life of the complex should cause serum levels to increase even if there is no increase in sIL-13Rα2 production and secretion. To test this possibility, we compared the increases in serum sIL-13Rα2 levels in IL-4Rα-deficient mice injected with IL-13 with the increases that would be expected if the rate of production and secretion of sIL-13Rα2 remained constant but its half-life increased from 3.3 to 18 h or from 1.3 to 11.1 h (Fig. 9⇓). In either case, the observed increase in sIL-13Rα2 levels was not entirely explained by the increased half-life, even though an assumption used in calculating the expected sIL-13Rα2 levels (that all secreted sIL-13Rα2 immediately becomes complexed with injected or secreted IL-13) maximizes the expected sIL-13Rα2 concentrations. Because differences between observed and expected sIL-13Rα2 concentrations should reflect sIL-13Rα2 production and secretion, these data suggest that IL-13 induces an IL-4Rα-independent increase in the rate of sIL-13Rα2 production and secretion. This increase is in addition to IL-4Rα-dependent, Stat6-dependent stimulation of sIL-13Rα2 production and secretion by IL-13 (based on observations that IL-13 induces a larger increase is sIL-13Rα2 in wild-type mice than in IL-4Rα-deficient mice and that the IL-4C-induced increase in IL-4Rα- and Stat6-dependent increase in sIL-13Rα2 levels induced by IL-4C is entirely IL-4Rα- and Stat6-dependent) (Fig. 6⇑).

FIGURE 9.
FIGURE 9.

Increased half-life does not entirely explain the IL-13-induced increase in sIL-13Rα2 concentration in IL-13-treated IL-4Rα-deficient mice. IL-4Rα-deficient BALB/c mice were injected with 1 μg of IL-13, and total sIL-13Rα2 levels were determined at the times indicated. These levels (filled circles) were compared with the expected levels based on no change in the rate of sIL-13Rα2 secretion and an increase in serum sIL-13Rα2 half-life from 3.3 h for free sIL-13Rα2 to 18 h for IL-13/sIL-13Rα2 complex (open squares, based on the last segment of each curve in Fig. 7⇑C) or an increase in serum sIL-13Rα2 half-life from 1.3 h for free sIL-13Rα2 to 11.1 h for the IL-13/sIL-13Rα2 complex (gray triangles, based on the entire curves (dashed lines) in Fig. 7⇑C).

The IL-13-induced increase in sIL-13Rα2 gene expression is partially IL-4Rα independent

Any increase in sIL-13Rα2 secretion might reflect an increase in gene transcription, mRNA stability, translation of mRNA, and/or secretion of translated proteins. To evaluate the effects of IL-4 and IL-13 stimulation on steady-state mRNA levels, which reflect both transcription and mRNA stability, we used real-time PCR to determine IL-4 effects on sIL-13Rα2 mRNA levels in wild-type and IL-13-deficient mice and IL-13 effects on sIL-13Rα2 mRNA levels in wild-type and IL-4Rα-deficient mice. IL-4-induced similar substantial increases in sIL-13Rα2 mRNA levels in wild-type and IL-13-deficient mice (Fig. 10⇓A), demonstrating that its stimulatory effect does not require the induction of IL-13 secretion or signaling through a receptor that is uniquely triggered by IL-13. IL-13 stimulated a large increase in sIL-13Rα2 mRNA levels in wild-type mice and a considerably smaller, but still statistically significant, increase in sIL-13Rα2 mRNA levels in IL-4Rα-deficient mice (Fig. 10⇓B). This result was reproduced in a second experiment and IL-13 enhancement of sIL-13Rα2 mRNA levels was not observed when injected IL-13 was mixed with an IL-13 antagonist (a sIL-13Rα2-IgGFc fusion protein), demonstrating that the IL-4Rα-independent effect of IL-13 is not due to a contaminant in our IL-13 preparation (Fig. 10⇓C). Thus, IL-13 can increase steady-state sIL-13Rα2 mRNA levels and rates of sIL-13Rα2 secretion by signaling through an IL-4Rα-containing receptor (presumably the type 2 IL-4R) and by signaling through a second distinct pathway.

FIGURE 10.
FIGURE 10.

IL-4Rα dependence of IL-4- and IL-13-induced increases in steady-state levels of IL-13Rα2 mRNA. A, Wild-type and IL-13-deficient BALB/c mice were injected i.v. with vehicle or IL-4C (2 μg IL-4/10 μg of BVD4-11D11 in 200 μl of saline) and sacrificed 3 days later. Levels of sIL-13Rα2 mRNA relative to mRNA levels for a housekeeping gene (18S RNA) were determined by real-time PCR. B, Wild-type and IL-4Rα-deficient BALB/c mice were injected with vehicle or 2 μg of IL-13 and sacrificed 1 day later. Levels of sIL-13Rα2 mRNA relative to 18S RNA levels were determined by real-time PCR. C, IL-4Rα-deficient BALB/c mice were injected i.v. with vehicle, 3 μg of IL-13, or 3 μg of IL-13 plus 9 μg of sIL-13Rα2-Fc and sacrificed 1 day later. Levels of sIL-13Rα2 mRNA relative to 18S RNA were determined by real-time PCR.

IL-13/sIL-13Rα2 complexes are acid stable and may suppress IL-13-induced gene expression

The considerably increased in vivo half-life of sIL-13Rα2 complexed with IL-13 as compared with free IL-13 suggests that IL-13/sIL-13Rα2 complexes may have a biological function. The most obvious possibility would be that these complexes act as a repository for IL-13 that can release this cytokine under the proper conditions. Against this possibility is the very high affinity of sIL-13Rα2 for IL-13 and the high stability of the complexes in vivo; both suggest that substantial dissociation of the complex with the release of bioactive IL-13 is unlikely. It seemed possible, however, that the complex might dissociate under conditions that vary from those seen in blood; for example, the acidic pH that can develop in the asthmatic lung (64). To test this possibility, we bound recombinant sIL-13Rα2-IgGFc fusion protein to agarose, saturated it with IL-13, and tested whether IL-13 could be eluted from the complex by decreasing pH. No elution of IL-13 was observed at a pH as low as 4.1, whereas the complex was dissociated by 3.5 M MgCl2 solution, a chaotropic agent (Fig. 11⇓).

FIGURE 11.
FIGURE 11.

IL-13/sIL-13Rα2 complexes are stable at acid pH. One hundred and fifty microliters of IL-13Rα2-IgGFc-agarose was saturated with 4.5 ng of IL-13, washed extensively with PBS, then stepwise eluted with buffers at the pHs shown before being eluted with 3.5 M MgCl2. The IL-13 concentration of each eluate was determined by ELISA.

As an alternative, we considered the possibility that intact IL-13/sIL-13Rα2 complexes might have a biological activity. To screen for this, we increased the serum levels of IL-13/sIL-13Rα2 complexes by infecting mice with N. brasiliensis, saturated serum sIL-13Rα2 with IL-13 by adding recombinant mouse IL-13 to the serum, and removed the excess, free IL-13 by adsorption with sIL-13Rα2-IgGFc fusion protein-agarose. Mice were anesthetized and inoculated intratracheally (i.t.) with this treated serum or, as a control, with serum from N. brasiliensis-infected IL-13/sIL-13Rα2-double deficient mice. Extracts from the lungs of both sets of mice were first tested for RNA specific for the Sprr1, Sprr2a, and Sprr2b genes, which are potently induced by IL-13 to a considerably greater extent than they are induced by IL-4 (38). No increase in the expression of these genes was observed in two experiments (data not shown), suggesting that IL-13/sIL-13Rα2 complexes lack IL-13 activity. To determine whether the expression of any lung genes was induced by IL-13/sIL-13Rα2 complexes, RNA purified from the lungs was tested for expression of >20,000 genes by hybridization to a gene chip. The expression of only two genes, Pira1 (paired Ig-like receptor A1) and Vnn1 (vanin-1), was found to be increased at least 2-fold, and the expression of only one gene, ApoA1 (apolipoprotein, A1), was found to be decreased by a factor <2 in the lungs of mice inoculated with IL-13/sIL-13Rα2 complex-enriched serum as compared with mice inoculated with serum that lacked IL-13 and sIL-13Rα2. No increased expression was observed for five genes that are strongly induced in the lungs by IL-13 (Kcnj15, Agr2, Itln2, Ccl11, and Retnlb; data not shown). Stimulation of Pira1 and Vnn1 and suppression of ApoA1 expression were confirmed by real-time PCR, using primers specific for Pira1, Vnn1, and ApoA1 (Fig. 12⇓A). An additional experiment was performed to determine whether these changes in gene expression are stimulated by IL-13/sIL-13Rα2 complexes as opposed to a different serum molecule that is dependent on IL-13 and/or IL-13Rα2. Serum from N. brasiliensis-infected wild-type mice was absorbed with anti-IL-13Rα2 Ab-agarose until it no longer had sIL-13Rα2 or IL-13 detectable by ELISA or was mock-absorbed and then used to inoculate IL-13/IL-13Rα2-double deficient mice i.t. Increases in Pira1 and Vnn1 gene expression were induced by the mock-absorbed as compared with the anti-IL-13Rα2-absorbed serum, whereas mice inoculated with either serum expressed similar levels of ApoA1 mRNA (Fig. 12⇓B). Thus, IL-13/sIL-13Rα2 complexes induce pulmonary Pira1 and Vnn1 gene expression, while a different constituent of serum that is induced directly or indirectly by IL-13 and/or IL-13Rα2 appears to suppress pulmonary ApoA1 expression.

FIGURE 12.
FIGURE 12.

Effect of IL-13/sIL-13Rα2 complexes on pulmonary gene expression. A, IL-13/sIL-13Rα2-double deficient mice were inoculated i.t. daily on three consecutive days with 50 μl of PBS, serum from N. brasiliensis-infected wild-type, or serum from N. brasiliensis-infected IL-13/sIL-13Rα2 double-deficient mice and sacrificed 16 h after the last dose of PBS or serum. Sera used for i.t. inoculation had been saturated with recombinant mouse IL-13 and then absorbed to remove any free IL-13. RNA was purified from the lungs of PBS- and serum-inoculated mice and used for a gene scan and, subsequently, for real time PCR (four individual mice per group in each of two separate experiments) to determine the levels of Pira1, Vnn1, and ApoA1 gene expression relative to levels of a housekeeping gene, β-actin. Results are pooled from the two identical experiments. Differences between values for lungs inoculated with serum that contained IL-13 and sIL-13Rα2 are all significantly different from values for lungs inoculated with IL-13/sIL-13Rα2 serum or PBS (p < 0.05). Numbers to the right of the diagonal bars show the ratio of expression in lungs from mice treated with IL-13/sIL-13Rα2-containing serum vs IL-13/sIL-13Rα2-deficient serum. B, A similar experiment was performed in which IL-13/sIL-13Rα2 double-deficient mice were inoculated i.t. with serum from N. brasiliensis-infected wild-type mice that had been adsorbed with anti-IL-13Rα2 Ab-agarose or control Ab-agarose (mock absorbed) before inoculation. Differences between values for Pira1 and Vnn1 but not ApoA1 gene expression were significantly greater (p < 0.05) for mice that had received the mock-absorbed serum.

Discussion

Our studies demonstrate similarities and differences between the sRs specific for IL-4 and IL-13. Both are present in low nanogram per milliliter amounts in the serum of immunologically naive mice and both increase in amount in response to IL-4 or IL-13 stimulation of IL-4Rα-dependent activation of Stat6. The increase in sIL-13Rα2 concentration is considerably greater than the increase in sIL-4Rα concentration during a Th2 response; however, much of this difference reflects a 4–5-fold increase in the serum half-life of the IL-13/sIL-13Rα2 complex as compared with that of free sIL-13Rα2. This increase in serum half-life is most likely caused by a marked decrease in the urinary excretion of sIL-13Rα2 when it is complexed by IL-13. This decrease in renal clearance may reflect a loss of the ability of sIL-13Rα2 to pass through the glomerular basement membrane as its molecular mass increases from 45 kDa (for the uncomplexed molecule) to 60 kDa (for the IL-13/sIL-13Rα2 complex); changes in shape and charge may also hinder glomerular filtration.

No similar increase in half-life is seen for IL-4/sIL-4Rα complexes; in fact, these complexes rapidly break down to release free sIL-4Rα and free IL-4 (which is rapidly used or eliminated). These distinctions reflect in part a difference in the in vivo affinity of sIL-4Rα and sIL-13Rα2 for their ligands; the former must bind IL-4 with relatively low affinity while the latter must bind IL-13 with high affinity. This difference is discordant with the similar initial in vitro determinations of sIL-4Rα affinity for IL-4 (Kd of ∼70 pM) and IL-13Rα2 for IL-13 (Kd of ∼50 pM) (65). Subsequent studies, however, revealed that the Kd for IL-4/sIL-4Rα complexes, which was initially determined at 4°C, increases substantially at 37°C with the t1/2 for complex dissociation decreasing from 112 to 2.3 min (66).

These physical differences suggest a difference in function. Previous studies indicate that complexes of IL-4 with recombinant sIL-4Rα have a stronger agonist effect than an equal quantity of free IL-4. The ease with which these complexes dissociate suggests that sIL-4Rα functions primarily as a carrier protein for IL-4 that increases its in vivo half-life sufficiently to increase the biological effect of secreted IL-4 and allow it to act at sites distant from its site of secretion. This function may become most apparent when large amounts of IL-4 are secreted over a short period of time, as happens physiologically when Ag activates basophils by crosslinking basophil-associated IgE (19). The relatively low in vivo affinity of sIL-4Rα for IL-4 also makes it unlikely that this molecule will inhibit IL-4 binding to cell membrane type 1 or type 2 IL-4R heterodimers, which have similar, very high affinities for IL-4.

Additional recent studies suggest that sIL-4Rα can also amplify responses to low doses of IL-13, possibly by stabilizing complexes produced between IL-13 and cell membrane IL-13Rα1 before these complexes can bind to cell membrane IL-4Rα to form a signaling complex (67). In contrast, the high stability of IL-13/sIL-13Rα2 complexes and our failure to find any evidence that saturated IL-13/sIL-13Rα2 complexes have IL-13 agonist activity in vivo suggest that sIL-13Rα2 has a purely antagonist effect toward IL-13 and that the large amount of sIL-13Rα2 secreted during the course of a Th2 response confines IL-13 activity to the site of IL-13 secretion. The higher affinity of sIL-13Rα2 than that of the cell membrane form of the same molecule for IL-13 (68) additionally suggests that sIL-13Rα2 may effectively limit the binding of IL-13 to cell membrane IL-13Rα2, which may signal directly (36) or act as a depot that can transfer IL-13 to the type 2 IL-4R.

One observation about IL-13/sIL-13Rα2 complexes, however, seems at odds with the hypothesis that sIL-13Rα2 functions as a simple antagonist for IL-13. There is no obvious selective advantage for an inert molecular complex to remain in circulation; hence, IL-13/sIL-13Rα2 complexes might be expected to have a short in vivo half-life. Instead, the in vivo half-life of IL-13/sIL-13Rα2 complexes is increased 4–5-fold as compared with the half-life of free sIL-13Rα2. This raised the possibility that these complexes are not inert. One possibility was that they have the capacity to dissociate with the release of active IL-13, but only under specific conditions. Because IL-13 is known to have effects on epithelial cells, smooth muscle cells, and fibroblasts that promote the physiological abnormalities associated with asthma (8) and lung pH can decline to 5 during an asthma attack (64), we determined whether IL-13/sIL-13Rα2 complexes dissociate at low pH. We found no evidence of dissociation at a pH as low as 4.1, although dissociation was induced by a chaotropic salt solution. Our negative results do not, however, preclude the possibility that other physiological or pathological conditions may increase IL-13/sIL-13Rα2 complex dissociation.

We also evaluated the possibility that IL-13/sIL-13Rα2 complexes have a biological effect of their own by comparing the ability of serum from N. brasiliensis-infected wild-type mice, which has a high concentration of IL-13/sIL-13Rα2 complexes, and serum from N. brasiliensis-infected mice deficient in both IL-13 and sIL-13Rα2 to stimulate pulmonary gene expression when inoculated i.t. into mice deficient in both IL-13 and sIL-13Rα2. To assure that serum from N. brasiliensis-infected wild-type mice contained IL-13/sIL-13Rα2 complexes but no free IL-13 or sIL-13Rα2, we added recombinant mouse IL-13 to this serum to fully saturate sIL-13Rα2 and then absorbed the serum repeatedly with sIL-13Rα2-agarose to remove free IL-13. Experiments in which RNA from the lungs of treated mice was evaluated for gene expression by real-time PCR and gene scan revealed no evidence of increased expression of IL-13-induced genes but demonstrated increased expression of two proinflammatory genes, Vnn1 and Pira1, and decreased expression of one anti-inflammatory gene, ApoA1, in the lungs of mice inoculated with serum that contained the IL-13/sIL-13Rα2 complex. Pulmonary expression of these genes was not changed in mice inoculated i.t. with serum from N. brasiliensis-infected mice that lacked both IL-13 and sIL-13Rα2. A subsequent experiment in which serum from N. brasiliensis-infected wild-type mice was absorbed with anti-IL-13Rα2 Ab-agarose or mock-absorbed before inoculating it i.t. into IL-13/IL-13Rα2-double deficient mice identified IL-13/sIL-13Rα2 complexes as the serum component responsible for Vnn1 and Pira1 up-regulation, but not ApoA1 down-regulation.

Vnn1 encodes a pantetheinase, Vanin-1, that releases cysteamine from pantetheine. Cysteamine promotes the production of chemokines that attract neutrophils and inhibits γ-glutamylcysteine synthetase, which is needed to synthesize the natural reducing agent glutathione (69, 70). This suggests that increased Vanin-1 is likely to exacerbate inflammation dependent on neutrophils and oxidation, which is consistent with the decreased inflammation observed in S. mansoni-infected, Vnn1-deficient mice (71).

In addition, macrophages in Vnn1-deficient mice infected with the intracellular bacterium Coxiella burnetii demonstrated decreased inducible NO synthase expression and increased arginase expression, suggesting that Vanin-1 promotes classical macrophage activation and inhibits alternative (allergic) macrophage activation (72).

The six mouse Pira genes are homologous to human leukocyte Ig-like receptors and encode cell membrane proteins that couple with a homodimer of the Fc receptor common γ-chain and bind MHC class I tetramers. As a result, the binding of Pira gene products by self MHC class I activates Fc receptor γ ITAMs and increases the basal activation state of the mast cells, macrophages, neutrophils, and dendritic cells that express these genes (73). The stimulatory effects of the Pira gene products are balanced by the single Pirb gene, which encodes an MHC class I binding protein with extracellular domains similar to those encoded by Pira genes and is expressed on the same cell types. The PIR-B protein, however, is associated with inhibitory ITIM motifs and down-regulates the basal level of mast cell, macrophage, neutrophil, and dendritic cell activation (73). Thus, an increase in Pira1 expression without a corresponding increase in Pirb expression is likely to promote inflammation by increasing the activation state of inflammatory and APCs.

In addition to demonstrating biologically important differences between the properties of sIL-4Rα and sIL-13Rα2 and a possible function for IL-13/sIL-13Rα2 complexes, our studies provide evidence for IL-4Rα-independent signaling by IL-13. Although all of the IL-4-induced increases in serum sIL-13Rα2 levels are IL-4Rα- and Stat6-dependent and most of the IL-13-induced increases in serum sIL-13Rα2 levels result from IL-4Rα/Stat6-dependent signaling and the increased half-life of IL-13/sIL-13Rα2 complexes, the increase in serum sIL-13Rα2 levels in IL-13-treated IL-4Rα-deficient mice is too rapid to be explained by the increase in half-life. Consistent with this, IL-13 but not IL-4 induces a small but significant increase in IL-13Rα2 mRNA levels in IL-4Rα-deficient mice. This increase is not due to LPS contamination; our rIL-13 preparation has very low levels of LPS, and LPS treatment does not increase serum levels of sIL-13Rα2 (data not shown). More importantly, the IL-13-induced increase in the IL-13Rα2 mRNA level in IL-4Rα-deficient mice is blocked by neutralizing IL-13 with recombinant sIL-13Rα2-Fc. We do not yet know the signaling pathway involved in IL-13-induced, IL-4Rα-independent up-regulation of sIL-13Rα2 expression. Possibilities include IL-13 signaling through cell membrane IL-13Rα1 in the absence of IL-4Rα or through cell membrane IL-13Rα2. Recent studies demonstrate that alternative splicing of mouse IL-13Rα2 mRNA allows the production of distinct transcripts for soluble and cell membrane forms of the IL-13Rα2 gene (68) and that IL-13 ligation of cell membrane IL-13Rα2 can induce a signaling pathway that activates an AP-1 variant (36). Additional studies are required to determine whether both this pathway and the Stat6 pathway can independently induce increased IL-13Rα2 gene transcription.

In sum, our observations: 1) demonstrate the presence of substantial quantities of both sIL-4Rα and sIL-13Rα2 in serum; 2) show a likely difference in the functions of sIL-4Rα and sIL-13Rα2; 3) indicate that the combination of IL-13 with sIL-13Rα2 simultaneously blocks IL-13 signaling and promotes a novel inflammatory response that modifies classical Th2 inflammation; and 4) provides a marker for an IL-4Rα-independent IL-13 signaling pathway that may allow IL-13 to have effects that are not reproduced by IL-4.

Acknowledgments

We are grateful to Amgen, Wyeth Research, and Centocor for their gifts of mice and reagents, Michael Grusby (Harvard University) for letting us use his IL-13Rα2-deficient mice, and Christa Nealeigh, D.V.M. (Arcanum Veterinary Service) for expert help with the preparation of polyclonal Abs.

Disclosures

Fred Finkelman has consulted for and received honoraria and research support from Amgen, Centocor, and Wyeth and honoraria from Abbott. None of the other authors report any disclosures.

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 a Merit Award (to F.D.F.) from the U.S. Department of Veterans Affairs, by National Institutes of Health grants to F.D.F. (R01 AI052099 and R01 AI55848) and G.K.H. (R01AI58157), and a National Institutes of Health P01 grant to M.W-K., G.K.K.H., and F.D.F. (HL076383).

  • ↵2 Address correspondence and reprint requests to Dr. Fred D. Finkelman, Cincinnati Veterans Authority, Medical Center, 3200 Vine Street, Cincinnati, OH 45220. E-mail address: ffinkelman{at}pol.net

  • ↵3 Abbreviations used in this paper: s, soluble (prefix); GaKLH, goat antiserum to keyhole limpet hemocyanin; GaMD, goat anti-mouse IgD antiserum; i.t., intratracheal(ly); sR, soluble receptor.

  • Received July 5, 2007.
  • Accepted August 28, 2007.

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