This information is current as
of June 18, 2017.
Differences in Expression, Affinity, and
Function of Soluble (s)IL-4R α and sIL-13Rα
2 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
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2007 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2007; 179:6429-6438; ;
doi: 10.4049/jimmunol.179.10.6429
http://www.jimmunol.org/content/179/10/6429
The Journal of Immunology
Differences in Expression, Affinity, and Function of Soluble
(s)IL-4R␣ and sIL-13R␣2 Suggest Opposite Effects on
Allergic Responses1
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. Finkelman2*†‡
A
llergic 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 (1–14). 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-4R␣-chain and activate the transcription factor
Stat6 (15–17), and both have prominent effects on multiple cell types
that contribute to allergic inflammation (3, 5, 7, 12, 14, 18 –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-4R␣ and the cytokine receptor common ␥-chain, ␥c
(28 –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-4R␣ and IL-13R␣1 polypeptides (15, 29, 31, 32),
is expressed by some bone marrow-derived cells, including macro-
*Cincinnati Veterans Affairs Medical Center, Cincinnati, OH 45220; †Department of
Medicine, University of Cincinnati College of Medicine, Cincinnati, OH 45267; ‡Division of Immunobiology and §Division of Allergy and Immunology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229; and ¶National Institute of
Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
Received for publication July 5, 2007. Accepted for publication August 28, 2007.
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: [email protected]
www.jimmunol.org
phages, 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, 33–35), although
IL-13 signaling through a cell membrane form of an additional IL13-binding protein, cell membrane IL-13R␣2, 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-␥ (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 – 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-4R␣ and IL-13R␣2, proteins
in blood and lymph that bind IL-4 and IL-13, respectively (42– 45).
To address this possibility, we have evaluated the expression and
functional characteristics of sIL-4R␣ and IL-13R␣2 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-4R␣ for IL-4 is
considerably lower than that of sIL-13R␣2 for IL-13; IL-4/sIL4R␣ complexes rapidly dissociate while IL-13/sIL-13R␣2 complexes are very stable and have a considerably longer half-life
than uncomplexed IL-13 or sIL-13R␣2. These differences suggest
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.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
IL-4 and IL-13 are each bound by soluble receptors (sRs) that block their activity. Both of these sRs (sIL-4R␣ and sIL-13R␣2)
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-4R␣ complexes rapidly dissociate, releasing active IL-4, whereas sIL13R␣2 and IL-13 form a stable complex that has a considerably longer half-life than uncomplexed IL-13, sIL-13R␣2, IL-4, or
sIL-4R␣. Approximately 25% of sIL-13R␣2 in serum is complexed to IL-13; this percentage and the absolute quantity of sIL13R␣2 in serum increase considerably during a Th2 response. sIL-13R␣2 gene expression is up-regulated by both IL-4 and IL-13;
the effect of IL-4 is totally IL-4R␣-dependent while the effect of IL-13 is partially IL-4R␣-independent. Inhalation of an IL-13/
sIL-13R␣2 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-4R␣ predominantly sustains, increases, and diffuses the effects of IL-4, whereas sIL-13R␣2 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. The Journal of Immunology, 2007, 179: 6429 – 6438.
6430
that sIL-4R␣ 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-13R␣2 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/sIL13R␣2 complexes themselves may have proinflammatory effects
that differ from those of IL-13.
Materials and Methods
DIFFERENT FUNCTIONS OF sIL-4R␣ AND sIL-13R␣2
microtiter plate wells followed by biotin-labeled anti-IL-13 mAb (C531),
streptavidin-HRP, and luminogenic substrate. Serum levels of the IL-4/
sIL-4R␣ complex were detected by an ELISA in which microtiter plate
wells were coated with goat anti-IL-4R␣ mAb and the captured complex
was detected with biotin-anti-IL-4 mAb (BVD6-24G2.3). Total levels of
sIL-13R␣2 or sIL-4R␣ 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.
Mice
Preparation of IL-13-rich serum
BALB/c wild-type and IL-4 receptor ␣-chain-deficient mice (BALB/cJIl4ratm1) (46) were purchased from Taconic Farms and IL-4-deficient mice
(BALB/c-Il4tm2Nnt/J) (47) were purchased from The Jackson Laboratory. IL13-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-13R␣2-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-13R␣2-double deficient mice were generated
by breeding IL-13- and IL-13R␣2-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).
IL-13R␣2-deficient mice were inoculated with 200 infective Nippostrongylus
brasiliensis larvae on days 0 and 14 and injected i.p. with 10 ␮g 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.
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 antimouse CD3␧) (52). Affinity-purified goat anti-mouse IL-13R␣2 Ab was produced by immunizing a goat with mouse IL-13R␣2-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-13R␣2-mouse IgGFc fusion protein (D. Donaldson, then at Wyeth Research) coupled to cyanogen bromideactivated Sepharose. The purified Ab failed to stain mouse spleen cells more
than a nonspecific control Ab (GaKLH) but captured sIL-13R␣2 in the serum
of normal mice but not IL-13R␣2-deficient mice in a single Ab ELISA (data
not shown). Affinity-purified goat anti-mouse IL-4R␣ Ab was produced by
immunizing a goat with mouse sIL-4R␣ (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-4R␣ coupled to
cyanogen bromide-activated Sepharose. The purified Ab specifically stained
spleen cells from wild-type mice, but not IL-4R␣-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
Fc␥Rs, or interact simultaneously with Fc␥Rs and cytokine receptors because they contain a single IgG molecule and BVD4-1D11 blocks the IL-4
epitope that binds to IL-4R␣ (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-13R␣2-IgGFc and detected with biotinylated anti-IL-13 mAb (C531, which binds IL-13 that is complexed to sIL13R␣2; a gift from Centocor). Luminescence was measured with a Fluoroskan
Ascent FL microtiter plate luminometer/fluorometer (Labsystems).
Measurement of free and cytokine-complexed sIL-13R␣2 and
sIL-4R␣
Serum levels of the IL-13/sIL-13R␣2 complex were measured by ELISA
using affinity-purified goat anti-IL-13R␣2 Ab to capture the complex onto
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-13R␣2 complexes
One hundred and fifty microliters of mouse IL-13R␣2-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 (55–57) (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 ␮l containing 100 ␮M iCycler-DNA Master SYBR Green
(Roche Diagnostics), double-distilled H2O, and 4 ␮l of cDNA, which corresponds to ⬃33 ng of total RNA. The cDNA was added as the template and 5
␮l (3 mM) of the primer of interest was added to the PCR (␤-actin: 5⬘-GT
GACGTTGACATCCG-3⬘ (sense primer) and 5⬘-CAGTAACAGTCCGC
CT-3⬘ (antisense primer); 2210421G13Rik/ApoA1: 5⬘-AGGTACCACTCTGG
CAATGACCAA-3⬘ (sense primer) and 5⬘-TCTGCAGCAGTCTGTGACTT
AGCA-3⬘ (antisense primer); Vnn1: 5⬘-AAGTGTTGCTGAGTGAGG-3⬘
(sense primer) and 5⬘-TGTGCTATGAAGTCTGAGG-3⬘ (antisense primer);
Pira1: 5⬘-GAGGGTCGGGTGTATGTATTAGC-3⬘ (sense primer) and 5⬘-GC
CACAAGGAACATCAACTAAGC-3⬘ (antisense primer); IL-13R␣2: 5⬘-GG
ACTCATCAGACTATAAAGA-3⬘ (sense primer) and 5⬘-GTGTGCTCCATT
TCATTCTA-3⬘ (antisense primer); and 18S rRNA: 5⬘-GTAACCCGTTGA
ACCCCATT-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 ␤-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.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
Reagents
Worm inoculation
6
8
sIL-4Rα
sIL-13Rα 2
0
5 10 15 20 25 30
Percent Saturation
FIGURE 1. Levels of sIL-13R␣2 and sIL-4R␣ 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-13R␣2 and sIL-4R␣
and their saturation with IL-13 and IL-4, respectively, were determined.
Results
Serum sIL-13R␣2, but not serum sIL-4R␣, is partially saturated
with its ligand in naive mice
Worm infection and Ag immunization induce a greater increase
in serum concentration of sIL-13R␣2 than that of sIL-4R␣
To determine whether sIL-4R␣ and/or sIL-13R␣2 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-4R␣ concentrations
increased 2–3-fold and sIL-13R␣2 concentrations increased 7–10fold as the Th2 response peaked and then decreased back toward
baseline levels (Fig. 2). Saturation of sIL-13R␣2 with IL-13 increased to a peak of ⬃90%, whereas sIL-4R␣ 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-13R␣2
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-4R␣ and/or sIL-13R␣2
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).
Only rapid production of large amounts of IL-4 induces
detectable IL-4/sIL-4R␣ complex formation in vivo
These results raised the possibility that sIL-4R␣ does not bind IL-4
at all in vivo. To evaluate this possibility, experiments were performed that determined sIL-4R␣ 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 mas-
20
15
10
5
0
0
2
4
6
8
sIL-4Rα
sIL-13Rα
100
75
50
25
0
0
10 12 14
2
4
6
8
10 12 14
6
8
10 12 14
Days After Immunization
30
25
20
15
10
5
0
0
2
4
6
8
100
80
60
40
20
0
10 12 14
0
2
4
Day of Infection
FIGURE 2. sIL-13R␣2 and sIL-4R␣ 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-4R␣ and sIL-13R␣2 and their saturation with
their cytokine ligands were determined.
sive basophil IL-4 and IL-13 secretion over a 4-h period (19), had
little effect on serum levels of sIL-4R␣ or sIL-13R␣2 but increased
sIL-4R␣ saturation with IL-4 from 0 to ⬃8% and sIL-13R␣2 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-4R␣ and sIL-13R␣2 concentrations in serum and increased sIL-4R␣ saturation with IL-4 to nearly 50% and
sIL-13R␣2 saturation with IL-13 to nearly 100% (Fig. 4). Thus,
sIL-4R␣ 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.
Both IL-4 and IL-13 induce IL-4R␣- and Stat6-dependent
increases in serum levels of sIL-13R␣2, but only the
IL-13-induced increase is partially IL-4R␣- and
Stat6-independent
Previous studies have demonstrated that sIL-13R␣2 gene expression is up-regulated by IL-4 and IL-13 (61). To determine whether
60
40
20
0
0
4
8
12
16
Wild-type
IL-13 IL-4 - IL-13
100
75
50
25
0
0
4
8
12
16
Weeks Post-Inoculation
FIGURE 3. Cytokine dependence of the sIL-13R␣2 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-13R␣2 and their saturation
with IL-13 were determined.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
Serum levels of IL-4/sIL-4R␣ complexes and IL-13/sIL-13R␣2
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-4R␣ or sIL-13R␣2, respectively. Results of several experiments with untreated 8- to16-wkold BALB/c mice indicated that sIL-4R␣ and sIL-13R␣2 are
present in serum at similar concentrations (⬃4 –10 ng/ml; Fig. 1).
However, sIL-4R␣ was never detectably complexed with IL-4 in
naive mouse serum, whereas 15– 40% of sIL-13R␣2 was complexed with IL-13.
25
Percent Saturation
4
30
Percent Saturation
2
sReceptor (ng/ml)
0
Concentration (ng/ml)
IL-13Rα2 (ng/ml)
sIL-4Rα
sIL-13Rα 2
Percent Saturation
6431
sReceptor (ng/ml)
The Journal of Immunology
DIFFERENT FUNCTIONS OF sIL-4R␣ AND sIL-13R␣2
6432
A
sIL-4Rα
sIL-13Rα 2
Saline
IgD/Saline
*
αIgD/αIgE
0
5
10
15
20
sReceptor (ng/ml)
0
20
60
*
0
2
4
6
*
8
10
sReceptor (ng/ml)
0
25
50
B
20
40
60
80
% Saturation
*
*
2
4
6
0
25
50
*
75
100
% Saturation
C
Untreated
IL-13, 30 min
IL-13, 4 hr
IL-13 12 hr
*
0
2
4
6
8
0
20
40
60
80
% Saturation
sIL-4Rα (ng/ml)
D
Untreated
IL-13, 30 min
IL-13, 4 hr
IL-13, 12 hr
*
0
5
10
15
20
*
*
*
*
25 0
50
sIL-13Rα 2 (ng/ml)
100
% Saturation
FIGURE 5. IL-4 and IL-13 each induce increases in serum levels of
both sIL-4R␣ (A and C) and sIL-13R␣2 (B and D). BALB/c mice were
injected with 1 ␮g of IL-4 (A and B) or IL-13 (B, C, and D). Serum levels
of sIL-4R␣ and sIL-13R␣2 and their saturation of IL-4 or IL-13, respectively, were measured at the time points indicated.
and recombinant sIL-4R␣ had a longer half-life (62, 63). Our current studies demonstrate that complexes formed of natural sIL4R␣ and rIL-4 dissociate rapidly when injected into IL-4R␣-deficient mice with a half-life well under 30 min (Fig. 7A), while the
A
Wild-type, Day 1
Saline
IL-4C
*
0
2.5
5
7.5
sIL-13Rα 2 (ng/ml)
B
IL-4C Treatment
Wild-type
IL-4Rα Stat6 -
Day 0
Day 3
*
0
5
10 15 20 25 30
sIL-13Rα 2 (ng/ml)
C
IL-13 Treatment, Day 1
Wild-type
IL-4Rα Stat6 IL-13Rα 2-
*
*
*
0
5
Untreated
+ IL-13
10
sIL-13Rα 2 (ng/ml)
FIGURE 6. IL-4R␣- and Stat6-dependence of IL-4 and IL-13 up-regulation of serum sIL-13R␣2 concentration. A, BALB/c wild-type mice were
injected with saline or IL-4C that contained 2 ␮g of IL-4. Mice were bled
1 day later and serum sIL-13R␣2 levels were determined. B, Wild-type,
IL-4R␣-, and Stat6-deficient mice were injected with IL-4C that contained
2 ␮m of IL-4. Mice were bled prior to and 3 days after IL-4C injection and
serum levels of sIL-13R␣2 were determined. C, Wild-type, IL-4R␣-,
Stat6-, and IL-13R␣2-deficient mice were left untreated or injected with 1
␮g of IL-13. Mice were bled 1 day later and sIL-13R␣2 levels were
determined.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
Follow-up studies were performed to more formally investigate the
suggestion that the half-life of the IL-13/sIL-13R␣2 complex is
considerably longer than the half-life of the IL-4/sIL-4R␣ complex. Previous studies have shown that uncomplexed IL-4 has a
very short half-life (⬍15 min), whereas complexes formed of rIL-4
0
sIL-13Rα 2 (ng/ml)
Percent Saturation
IL-13/sIL-13R␣2 complexes have a longer in vivo half-life than
free IL-13 or sIL-13R␣2 while IL-4/sIL-4R␣ complexes have a
shorter in vivo half-life than free sIL-4R␣
*
0
75 100
these cytokines have similar effects on serum sIL-13R␣2 protein
levels and whether sIL-4R␣ 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-␮g dose of rIL-4, which has a very short in
vivo half-life, caused a large increase in the percentage of sIL-4R␣
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. 5A, right panel). Only a small
increase in the serum concentration of sIL-4R␣ was observed; this
returned to baseline by 12 h after IL-4 injection (Fig. 5A, left
panel). IL-4 injection had a modest effect on the serum concentration of sIL-13R␣2 and no effect on its saturation with IL-13
(Fig. 5B). Injection of a single 1-␮g dose of rIL-13 had no effect
on sIL-4R␣ levels at 2 h or on sIL-4R␣ saturation with IL-4 at any
time point, but it increased sIL-4R␣ levels by ⬃80% at 12 h (Fig.
5C). Injection of 1 ␮g of IL-13 also completely saturated sIL13R␣2 with IL-13 for ⬎12 h and caused a 2-fold increase in serum
sIL-13R␣2 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-13R␣2 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-13R␣2 (observed both 1
and 3 days later); this increase was totally IL-4R␣- and Stat6dependent (Fig. 6, A and B). In contrast, IL-13 induced considerable increases of serum sIL-13R␣2 levels in IL-4R␣- and Stat6deficient mice, although it induced larger increases in wild-type
mice (Fig. 6C).
10
Untreated
IL-4, 30 min
IL-4, 2 hr
IL-4, 12 hr
IL-13, 2 hr
*
FIGURE 4. Stimuli that cause rapid production of IL-4 and IL-13 increase serum sIL-13R␣2 and sIL-4R␣ 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 ␮g of rat anti-IgE mAb and bled 4 h later (upper panels) or injected
with saline or 10 ␮g of anti-CD3 mAb and bled 2 h later (lower panels).
Serum concentrations of sIL-4R␣ and sIL-13R␣2 and their saturation with
their cytokine ligands were determined. Asterisks (ⴱ) 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.
5
*
*
sIL-4Rα (ng/ml)
80
Percent Saturation
*
*
0
*
40
Untreated
αCD3
Untreated
IL-4, 30 min
IL-4, 2 hr
IL-4, 12 hr
The Journal of Immunology
6433
A
Total sIL-4Rα
IL-4-Saturated sIL-4Rα
Serum, 15 min
Serum, 45 min
Serum, 6 hr
Serum + IL-4, 15 min
Serum + IL-4,45 min
Serum + IL-4, 6 hr
1000
sIL-4Rα (pg/ml)
B
Free sIL-13Rα2
Free sIL-13Rα2
IL-13/sIL-13Rα2 Complex
IL-13/sIL-13Rα2 Complex
10
0
4
8
12
16
20
24
Time (hours)
C
% sIL-13Rα2 Remaining
Free IL-13Rα2
IL-13/sIL-13Rα2 Complex
100
10
1
0
5
10
15
20
Time (hours)
D
Serum
Urine
IL-13 (pg/ml)
10000
1000
Decreased renal excretion may account for the prolonged serum
half-life of IL-13/sIL-13R␣2 complexes
Because sIL-13R␣2 is excreted intact in urine (44), it seemed possible that the increased serum half-life of the IL-13/sIL-13R␣2
complex might reflect reduced urinary secretion. To evaluate this
possibility, we compared urine sIL-13R␣2 concentrations to serum
concentrations of free and IL-13-complexed sIL-13R␣2 and evaluated how urine concentrations are modified by stimuli that increase the serum concentration of free or IL-13-bound sIL-13R␣2.
In contrast to serum, which contains a mixture of free and IL-13bound sIL-13R␣2, only free sIL-13R␣2 was detected in urine (Fig.
8A). Injection of mice with 1 ␮g of rIL-13 increased the serum
concentration of the IL-13/sIL-13R␣2 complex considerably but
had little effect on either the serum or urine concentration of free
sIL-13R␣2 (Fig. 8A). In contrast, IL-4C injection of wild-type
mice (Fig. 8B) or IL-13-deficient mice (Fig. 8C) induced considerable and proportionate increases in serum and urine concentrations of free sIL-13R␣2 (Fig. 8, B and C). As expected, sIL-13R␣2
was undetectable in the serum and urine of IL-13R␣2-deficient
mice (Fig. 8B). Taken together, these observations suggest that
complex formation with IL-13 drastically reduces renal excretion
of sIL-13R␣2, which considerably increases its serum half-life.
100
10
0
200
400
600
800
Time (minutes)
FIGURE 7. Serum half-lives of free and cytokine-complexed sIL13R␣2 and sIL-4R␣. A, IL-4R␣-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/sIL4R␣ complex and total sIL-4R␣ were determined. B, IL-13/IL-13R␣2double 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-13R␣2 or IL-13/sIL-13R␣2 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-13R␣2-deficient mice, prepared as described in Materials and Methods, was injected
into nine IL-13/IL-13R␣2-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.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
Percent Remaining
100
half-life of the free sIL-4R␣ formed by dissociation of IL-4/sIL4R␣ complexes is ⬃2 h (consistent with a previous estimate of
2.3 h; Ref. 62) and was not affected by administering sIL-4R␣ as
a complex with IL-4. Two experiments that gave very similar
results were performed to evaluate the half-lives of free sIL-13R␣2
and IL-13/sIL-13R␣2 complexes in mice deficient in both IL-13
and IL-13R␣2 (Fig. 7B). Based on the last two points in the concentration curves that reflect the half-lives after injected proteins
have fully distributed, uncomplexed sIL-13R␣2 has a half-life of
⬃3.3 h and the IL-13/sIL-13R␣2 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-13R␣2 a halflife of ⬃1.3 h and IL-13/sIL-13R␣2 complex a half-life of ⬃11.1
h (Fig. 7C). A separate experiment demonstrated that the half-life
of free, natural IL-13, when injected i.v. into IL-13/IL-13R␣2double deficient mice, is ⬃20 min and that this short half-life was
associated with the loss of IL-13 in the urine (Fig. 7D). Thus,
whereas the IL-4/sIL-4R␣ complex rapidly dissociates into free
IL-4, which has an even shorter half-life, and free sIL-4R␣, which
has a somewhat longer half-life, the IL-13/sIL-13R␣2 complex is
remarkably stable and has a considerably longer half-life than either of its constituents.
DIFFERENT FUNCTIONS OF sIL-4R␣ AND sIL-13R␣2
6434
A
Serum
Complex
Total
Saline
Observed sIL-13Rα2
Expected sIL-13Rα 2
Free t1/2 = 3.3 hours
Complex t1/2 = 18 hours
Expected sIL-13Rα 2
Free t1/2 = 1.3 hours
Complex t1/2 = 11.1 hours
Urine
74%
IL-13
24%
0
15
30
45
0
15
30
45
B
BALB/c + Saline
54%
63%
0
C
25
sIL-13Rα2 (ng/ml)
BALB/c + IL-4C
IL-13Rα2 - + Saline
10
20
30
0
IL-13 - + Saline
IL-13 - + IL-4C
10
20
30
85%
98%
0
10
20
30
0
10
20
30
IL-13Rα2 (ng/ml)
The increased half-life of IL-13/sIL-13R␣2 complexes only
partially accounts for the IL-13-induced increase in serum
concentration of sIL-13R␣2 in IL-4R␣-deficient mice
The increased in vivo half-life of IL-13/sIL-13R␣2 compared with
free sIL-13R␣2 might explain the increase in serum levels of sIL13R␣2 observed in IL-13-injected Stat6- and IL-4R␣-deficient
mice; the longer half-life of the complex should cause serum levels
to increase even if there is no increase in sIL-13R␣2 production
and secretion. To test this possibility, we compared the increases in
serum sIL-13R␣2 levels in IL-4R␣-deficient mice injected with
IL-13 with the increases that would be expected if the rate of
production and secretion of sIL-13R␣2 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-13R␣2 levels was not
entirely explained by the increased half-life, even though an assumption used in calculating the expected sIL-13R␣2 levels (that
all secreted sIL-13R␣2 immediately becomes complexed with injected or secreted IL-13) maximizes the expected sIL-13R␣2 concentrations. Because differences between observed and expected
sIL-13R␣2 concentrations should reflect sIL-13R␣2 production
and secretion, these data suggest that IL-13 induces an IL-4R␣independent increase in the rate of sIL-13R␣2 production and secretion. This increase is in addition to IL-4R␣-dependent, Stat6dependent stimulation of sIL-13R␣2 production and secretion by
IL-13 (based on observations that IL-13 induces a larger increase
is sIL-13R␣2 in wild-type mice than in IL-4R␣-deficient mice and
that the IL-4C-induced increase in IL-4R␣- and Stat6-dependent
increase in sIL-13R␣2 levels induced by IL-4C is entirely IL-4R␣and Stat6-dependent) (Fig. 6).
The IL-13-induced increase in sIL-13R␣2 gene expression is
partially IL-4R␣ independent
Any increase in sIL-13R␣2 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
15
10
5
0
0
4
8
12
Time (hours)
FIGURE 9. Increased half-life does not entirely explain the IL-13-induced increase in sIL-13R␣2 concentration in IL-13-treated IL-4R␣-deficient mice. IL-4R␣-deficient BALB/c mice were injected with 1 ␮g of
IL-13, and total sIL-13R␣2 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-13R␣2 secretion and an increase in serum
sIL-13R␣2 half-life from 3.3 h for free sIL-13R␣2 to 18 h for IL-13/sIL13R␣2 complex (open squares, based on the last segment of each curve in
Fig. 7C) or an increase in serum sIL-13R␣2 half-life from 1.3 h for free
sIL-13R␣2 to 11.1 h for the IL-13/sIL-13R␣2 complex (gray triangles,
based on the entire curves (dashed lines) in Fig. 7C).
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-13R␣2 mRNA levels in wild-type and
IL-13-deficient mice and IL-13 effects on sIL-13R␣2 mRNA levels in wild-type and IL-4R␣-deficient mice. IL-4-induced similar
substantial increases in sIL-13R␣2 mRNA levels in wild-type and
A
Wild-type
*
IL-13 -
Vehicle
IL-4C
*
0
1
2
3
Relative IL-13R α2 mRNA
B
Vehicle
IL-13
Wild-type
IL-4Rα -
*
0
2
4
6
Relative IL-13R α2 mRNA
C
Vehicle
IL-13
IL-13 + sIL-13Rα2
*
0
5
10
15
20
Relative IL-13R α2 mRNA
FIGURE 10. IL-4R␣ dependence of IL-4- and IL-13-induced increases
in steady-state levels of IL-13R␣2 mRNA. A, Wild-type and IL-13-deficient BALB/c mice were injected i.v. with vehicle or IL-4C (2 ␮g IL-4/10
␮g of BVD4-11D11 in 200 ␮l of saline) and sacrificed 3 days later. Levels
of sIL-13R␣2 mRNA relative to mRNA levels for a housekeeping gene
(18S RNA) were determined by real-time PCR. B, Wild-type and IL-4R␣deficient BALB/c mice were injected with vehicle or 2 ␮g of IL-13 and
sacrificed 1 day later. Levels of sIL-13R␣2 mRNA relative to 18S RNA
levels were determined by real-time PCR. C, IL-4R␣-deficient BALB/c
mice were injected i.v. with vehicle, 3 ␮g of IL-13, or 3 ␮g of IL-13 plus
9 ␮g of sIL-13R␣2-Fc and sacrificed 1 day later. Levels of sIL-13R␣2
mRNA relative to 18S RNA were determined by real-time PCR.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 8. Complex formation with IL-13 blocks the renal clearance of
sIL-13R␣2. 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-13R␣2 concentration and IL-13/sIL-13R␣2 complex concentration were determined. Percentages shown in this and other panels
indicate the urine concentration relative to serum concentration of total
sIL-13R␣2. B, BALB/c wild-type and IL-13R␣2-deficient mice were injected with saline or IL-4C. Blood and urine was collected 1 day later and
serum and urine total sIL-13R␣2 concentration and IL-13/sIL-13R␣2 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-13R␣2 concentration and IL-13/sIL-13R␣2 complex concentration were determined.
20
The Journal of Immunology
6435
IL-13 (pg/ml)
3.5M MgCl2
A
PBS
Wild-type Serum
IL-13-/sIL-13Rα 2 - Serum
1000
100
7
6
5
4
pH
FIGURE 11. IL-13/sIL-13R␣2 complexes are stable at acid pH. One
hundred and fifty microliters of IL-13R␣2-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.
IL-13/sIL-13R␣2 complexes are acid stable and may suppress
IL-13-induced gene expression
The considerably increased in vivo half-life of sIL-13R␣2 complexed with IL-13 as compared with free IL-13 suggests that IL13/sIL-13R␣2 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 sIL13R␣2 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-13R␣2-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).
As an alternative, we considered the possibility that intact IL13/sIL-13R␣2 complexes might have a biological activity. To
screen for this, we increased the serum levels of IL-13/sIL-13R␣2
complexes by infecting mice with N. brasiliensis, saturated serum
sIL-13R␣2 with IL-13 by adding recombinant mouse IL-13 to the
serum, and removed the excess, free IL-13 by adsorption with
sIL-13R␣2-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/sIL13R␣2-double deficient mice. Extracts from the lungs of both sets
2.20
2.14
0.11
1
10
100
Normalized
Gene Expression
B
Anti-IL-13Rα 2 Absorbed
Mock Absorbed
1.98
Pira1
2.42
Vnn1
ApoA1
1.34
10
100
Normalized
Gene Expression
FIGURE 12. Effect of IL-13/sIL-13R␣2 complexes on pulmonary gene
expression. A, IL-13/sIL-13R␣2-double deficient mice were inoculated i.t.
daily on three consecutive days with 50 ␮l of PBS, serum from N. brasiliensis-infected wild-type, or serum from N. brasiliensis-infected IL-13/sIL13R␣2 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, ␤-actin. Results are pooled from the two identical experiments.
Differences between values for lungs inoculated with serum that contained
IL-13 and sIL-13R␣2 are all significantly different from values for lungs
inoculated with IL-13/sIL-13R␣2 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-13R␣2-containing serum vs IL-13/sIL-13R␣2deficient serum. B, A similar experiment was performed in which IL-13/
sIL-13R␣2 double-deficient mice were inoculated i.t. with serum from N.
brasiliensis-infected wild-type mice that had been adsorbed with anti-IL13R␣2 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.
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-13R␣2
complexes lack IL-13 activity. To determine whether the expression of any lung genes was induced by IL-13/sIL-13R␣2 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
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
IL-13-deficient mice (Fig. 10A), 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-13R␣2 mRNA levels in wild-type
mice and a considerably smaller, but still statistically significant,
increase in sIL-13R␣2 mRNA levels in IL-4R␣-deficient mice
(Fig. 10B). This result was reproduced in a second experiment and
IL-13 enhancement of sIL-13R␣2 mRNA levels was not observed
when injected IL-13 was mixed with an IL-13 antagonist (a sIL13R␣2-IgGFc fusion protein), demonstrating that the IL-4R␣-independent effect of IL-13 is not due to a contaminant in our IL-13
preparation (Fig. 10C). Thus, IL-13 can increase steady-state sIL13R␣2 mRNA levels and rates of sIL-13R␣2 secretion by signaling through an IL-4R␣-containing receptor (presumably the type 2
IL-4R) and by signaling through a second distinct pathway.
Pira1
Vnn1
ApoA1
6436
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-4R␣-dependent activation of Stat6. The increase in sIL-13R␣2
concentration is considerably greater than the increase in sIL-4R␣
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-13R␣2 complex as compared with that of free sIL-13R␣2. This
increase in serum half-life is most likely caused by a marked decrease
in the urinary excretion of sIL-13R␣2 when it is complexed by IL-13.
This decrease in renal clearance may reflect a loss of the ability of
sIL-13R␣2 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-13R␣2 complex); changes in shape
and charge may also hinder glomerular filtration.
No similar increase in half-life is seen for IL-4/sIL-4R␣ complexes; in fact, these complexes rapidly break down to release free
sIL-4R␣ and free IL-4 (which is rapidly used or eliminated). These
distinctions reflect in part a difference in the in vivo affinity of
sIL-4R␣ and sIL-13R␣2 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-4R␣ affinity for IL-4 (Kd of ⬃70 pM) and IL13R␣2 for IL-13 (Kd of ⬃50 pM) (65). Subsequent studies, however,
revealed that the Kd for IL-4/sIL-4R␣ 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-4R␣ have a stronger agonist effect than an equal quantity of
free IL-4. The ease with which these complexes dissociate suggests
that sIL-4R␣ 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-
4R␣ 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-4R␣ can also amplify
responses to low doses of IL-13, possibly by stabilizing complexes
produced between IL-13 and cell membrane IL-13R␣1 before
these complexes can bind to cell membrane IL-4R␣ to form a
signaling complex (67). In contrast, the high stability of IL-13/
sIL-13R␣2 complexes and our failure to find any evidence that
saturated IL-13/sIL-13R␣2 complexes have IL-13 agonist activity
in vivo suggest that sIL-13R␣2 has a purely antagonist effect toward IL-13 and that the large amount of sIL-13R␣2 secreted during the course of a Th2 response confines IL-13 activity to the site of
IL-13 secretion. The higher affinity of sIL-13R␣2 than that of the cell
membrane form of the same molecule for IL-13 (68) additionally
suggests that sIL-13R␣2 may effectively limit the binding of IL-13 to
cell membrane IL-13R␣2, 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-13R␣2 complexes, however,
seems at odds with the hypothesis that sIL-13R␣2 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-13R␣2 complexes might be expected to have a short in vivo halflife. Instead, the in vivo half-life of IL-13/sIL-13R␣2 complexes is
increased 4 –5-fold as compared with the half-life of free sIL-13R␣2.
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-13R␣2 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-13R␣2 complex dissociation.
We also evaluated the possibility that IL-13/sIL-13R␣2 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-13R␣2 complexes, and serum from N. brasiliensis-infected mice deficient in both IL-13 and
sIL-13R␣2 to stimulate pulmonary gene expression when inoculated i.t. into mice deficient in both IL-13 and sIL-13R␣2. To
assure that serum from N. brasiliensis-infected wild-type mice
contained IL-13/sIL-13R␣2 complexes but no free IL-13 or sIL13R␣2, we added recombinant mouse IL-13 to this serum to fully
saturate sIL-13R␣2 and then absorbed the serum repeatedly with
sIL-13R␣2-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-13R␣2 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-13R␣2.
A subsequent experiment in which serum from N. brasiliensisinfected wild-type mice was absorbed with anti-IL-13R␣2 Ab-agarose or mock-absorbed before inoculating it i.t. into IL-13/IL13R␣2-double deficient mice identified IL-13/sIL-13R␣2
complexes as the serum component responsible for Vnn1 and
Pira1 up-regulation, but not ApoA1 down-regulation.
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be decreased by a factor ⬍2 in the lungs of mice inoculated with
IL-13/sIL-13R␣2 complex-enriched serum as compared with mice
inoculated with serum that lacked IL-13 and sIL-13R␣2. 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. 12A). An
additional experiment was performed to determine whether these
changes in gene expression are stimulated by IL-13/sIL-13R␣2 complexes as opposed to a different serum molecule that is dependent on
IL-13 and/or IL-13R␣2. Serum from N. brasiliensis-infected wildtype mice was absorbed with anti-IL-13R␣2 Ab-agarose until it no
longer had sIL-13R␣2 or IL-13 detectable by ELISA or was mockabsorbed and then used to inoculate IL-13/IL-13R␣2-double deficient
mice i.t. Increases in Pira1 and Vnn1 gene expression were induced
by the mock-absorbed as compared with the anti-IL-13R␣2-absorbed
serum, whereas mice inoculated with either serum expressed similar
levels of ApoA1 mRNA (Fig. 12B). Thus, IL-13/sIL-13R␣2 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-13R␣2 appears to suppress pulmonary ApoA1
expression.
DIFFERENT FUNCTIONS OF sIL-4R␣ AND sIL-13R␣2
The Journal of Immunology
that modifies classical Th2 inflammation; and 4) provides a marker for
an IL-4R␣-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-13R␣2-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.
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In addition to demonstrating biologically important differences
between the properties of sIL-4R␣ and sIL-13R␣2 and a possible
function for IL-13/sIL-13R␣2 complexes, our studies provide evidence for IL-4R␣-independent signaling by IL-13. Although all of
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DIFFERENT FUNCTIONS OF sIL-4R␣ AND sIL-13R␣2