(Received for publication, November 4, 1996, and in revised form, December 19, 1996)
From the Walter and Eliza Hall Institute of Medical Research and the Cooperative Research Centre for Cellular Growth Factors and the ¶ Joint Protein Structure Laboratory of the Walter and Eliza Hall Institute and the Ludwig Institute for Cancer Research, P.O. Royal Melbourne Hospital, Victoria 3050, Australia
Interleukin-4 (IL-4) and interleukin-13 (IL-13)
are structurally and functionally related cytokines which play an
important role in the regulation of the immune response to infection.
The functional similarity of IL-4 and IL-13 can be explained, at least in part, by the common components that form their cell surface receptors, namely the IL-4 receptor -chain (IL-4R
) and the IL-13 receptor
-chain (IL-13R
). Soluble forms of the IL-4R
have also been described and implicated in modulating the effect of IL-4. In this
paper we describe the presence of a 45,000-50,000
Mr IL-13-binding protein (IL-13BP) in the serum
and urine of mice. This protein binds IL-13 with a 100-300-fold higher
affinity (KD = 20-90 pM) than does the
cloned IL-13R
(KD = 3-10 nM). In
addition to this functional difference, the IL-13BP appears to be
structurally and antigenically distinct from the IL-13R
. Finally,
unlike the cloned receptor, the IL-13BP acts as a potent inhibitor of
IL-13 binding to its cell surface receptor, raising the possibility
that it may be used to modulate the effects of IL-13 in
vivo.
Interleukin-4 (IL-4)1 and interleukin-13 (IL-13) are structurally and functionally related cytokines that share common receptor components (1-3). IL-4 and IL-13 exhibit approximately 30% amino acid sequence similarity and are encoded by genes with a similar intron/exon structure that are closely linked on human chromosome 5 and mouse chromosome 11. Both cytokines are produced by activated T-cells and act to regulate the immune response by, for example, inducing immunoglobulin class switching in B-lymphocytes to IgG1 and IgE isotypes and inhibiting the release of inflammatory mediators by macrophages (2). IL-4 but not IL-13 also regulates a variety of aspects of T-cell differentiation and function (4).
Three components of functional IL-4 and IL-13 receptors have been
cloned. These are the IL-4 receptor -chain (IL-4R
) (5), IL-13
receptor
-chain (IL-13R
) (6), and the IL-2 receptor
-chain
(IL-2R
) (7). The relationships between the IL-4 and IL-13 receptors
have been gleaned from many studies (5, 6, 8-16) and have recently
been synthesized into a simple model by Leonard and colleagues (12).
This model, which is consistent with the available data, suggests that
there are two classes of IL-4 receptors but a single class of IL-13
receptors. In the case of IL-4, binding occurs initially to the
IL-4R
, and this complex then interacts with either IL-2R
or
IL-13R
to yield a high affinity receptor capable of signal
transduction. Reciprocally, IL-13 is thought to first bind to the
IL-13R
and then to recruit IL-4R
to form a functional receptor.
IL-2R
does not appear to play a central role in IL-13 receptor
function.
In addition to cell surface IL-4R, many studies have described the
presence of a secreted form of the IL-4R
(5, 17-21). mRNA for
the cell surface and secreted forms of the IL-4R
are transcribed
from a single gene and arise by alternative splicing (5, 21). Soluble
forms of many members of the hemopoietin receptor family have been
described (22). These include the IL-2 receptor
-chain, IL-6
receptor
-chain (IL-6R
), IL-7 receptor
-chain, IL-9 receptor
-chain, GM-CSF receptor
-chain, and LIF receptor
-chain
(LIFR
). Soluble receptors have been implicated in both enhancing and
reducing the biological effect of their cognate cytokine. For example,
the complex of IL-6R
and IL-6 is capable of interacting with cell
surface gp130 to trigger a variety of biological responses (23), the
complex of IL-4R
and IL-4 inhibits the biological response of IL-4
in some settings and augments the response in other situations (24),
while the LIFR
appears to act only as an inhibitor of LIF function
(25, 26). In this study, we describe the presence of a high affinity binding protein for IL-13 in mouse serum and urine. This protein is
shown to be functionally, structurally, and antigenically distinct from
a recently cloned IL-13R
and to act as a potent inhibitor of IL-13
binding to its cell surface receptor.
Escherichia coli-expressed mouse IL-13
(R & D Systems) and mouse GM-CSF (a kind gift from Schering-Plough)
were radioiodinated using the iodine monochloride method (27, 28), and
E. coli-expressed mouse IL-4 (R & D Systems) was
radiolabeled using the Bolton-Hunter reagent (29). Recombinant soluble
mouse IL-4R was from Genzyme. FLAG peptide (DYKDDDDK) and anti-FLAG
M2 affinity gel were from Scientific Imaging Systems. Soluble mouse
IL-13R
with an N-terminal FLAG epitope tag was expressed in Chinese
hamster ovary cells using the mammalian expression vector pEF-BOS (30)
and purified on an anti-FLAG M2 affinity column by elution with FLAG
peptide. Anti-IL-13R
polyclonal antiserum was prepared by injecting
purified soluble IL-13R
into rabbits, and the rabbits were bled
after 3 months.
Aliquots of samples were
incubated with 125I-IL-13 in the presence or absence of
unlabeled IL-13 for at least 30 min at 4 °C in a final volume of 200 µl. The mixture was applied to a Superdex 200 10/30 column (Pharmacia
Biotech Inc.), equilibrated in 20 mM Tris-buffered saline
(pH 7.0) containing 0.02% (v/v) Tween 20 and 0.02% (w/v) sodium azide
(Tris-buffered saline). Samples were eluted with Tris-buffered saline
at a flow rate of 0.5 ml/min, and 0.5-ml fractions were collected and
counted in a Packard -counter.
Aliquots of samples were incubated with 125I-IL-13 or 125I-IL-4 in the presence or absence of unlabeled competitor in a final volume of 20 µl for at least 30 min at 4 °C. Then 5 µl of a 12 mM bifunctional cross-linker BS3 (Pierce) solution was added and the mixtures were incubated for 30 min at 4 °C. Samples were mixed with 8 µl of 4 × concentrated SDS sample buffer and analyzed by 13% SDS-PAGE under nonreducing conditions. The gels were dried and visualized by either autoradiography or PhosphorImager.
125I-IL-13 Saturation Binding AssayBinding of
125I-IL-13 to COS-7 cells expressing IL-13R was
performed as described previously (6). Binding of
125I-IL-13 to soluble proteins in mouse serum and urine was
determined by a gel filtration-based assay using Sephadex G-50
minicolumns (NICK columns, Pharmacia) to separate free from bound
ligand. Scatchard analyses of saturation binding isotherms were
performed using the nonlinear curve-fitting program LIGAND (31).
After SDS-PAGE, the gel,
which contained 125I-IL-13 cross-linked to either IL-13BP
from mouse urine or purified soluble IL-13R, was cut out according
to the migration positions of prestained molecular weight markers. The
gel pieces were added to 10 µl of Milli-Q purified water and 15 µl
of 0.1 M sodium phosphate buffer (pH 7.0) containing 10 mM EDTA, 0.1% SDS, 0.01% (v/v)
-mercaptoethanol and
minced into small pieces with a pair of tweezers. Then 15 µl of 0.1 M sodium phosphate buffer (pH 7.0) containing 10 mM EDTA was added to the minced gel mixtures. After 1 h of incubation at 37 °C, the mixtures were heated for 1 min at
95 °C, cooled, and then 4 µl of 10%
-octylglucoside was added.
Treatment with N-glycosidase F (0.5 unit in 2.5 µl,
Boehringer Mannheim) overnight at 37 °C completed the
deglycosylation. The deglycosylated samples were then centrifuged and
the gel pieces removed. The recovered liquid portions of the
deglycosylated samples were further digested with protease V8
(Boehringer Mannheim) at concentrations ranging from 5 to 60 µg/ml
for 3 h at 37 °C. The samples were analyzed by 15%
SDS-PAGE.
Aliquots of 12-fold concentrated mouse
urine or 3 µg/ml purified soluble IL-13R were incubated with
125I-IL-13 in the presence or absence of 0.5 µg/ml
unlabeled IL-13 followed by cross-linking as described above. The
cross-linking reactions were terminated by adding 1 M
Tris-HCl buffer (pH 7.5) to a final concentration of 40 mM.
The cross-linked samples were then mixed with 1:50 diluted control
rabbit serum or anti-IL-13R
antiserum which had been preincubated
with or without FLAG peptide (100 µg/ml) to eliminate immunoreaction
with potential anti-FLAG antibodies in the anti-FLAG-IL-13R
polyclonal antiserum. After 30 min of incubation at 4 °C, the
mixtures were added to 40 µl of 50% (v/v) protein G-Sepharose gel
slurry (Pharmacia) and incubated for 30 min at 4 °C. The samples
were centrifuged and the protein G-Sepharose beads washed with 3 × 0.5 ml of phosphate-buffered saline. For elution, the beads were
mixed with 40 µl of 2 × concentrated SDS sample buffer and
heated for 2 min at 95 °C. The supernatants were then analyzed by
13% SDS-PAGE under nonreducing conditions.
Mouse IL-13 was produced as a N-terminally FLAG-tagged fusion protein in Pichia pastoris and purified by reversed-phase high performance liquid chromatography. To prepare the IL-13 affinity support, the FLAG-tagged IL-13 was first bound to anti-FLAG antibody M2 affinity beads and then covalently linked to the M2 beads by the chemical cross-linker, BS3.
Purification and N-terminal Amino Acid Sequencing of IL-13BP270 ml of 8-fold concentrated mouse urine was incubated with 1.0 ml of IL-13 affinity beads for 5 h at room temperature. After removing unbound proteins by centrifugation, the IL-13 affinity beads were washed extensively with 20 mM phosphate-buffered saline (pH 7.0) containing 0.02% (v/v) Tween 20 and 0.02% (w/v) sodium azide (phosphate-buffered saline) followed by additional washes with 5 × 2 ml of 3-fold diluted Actisep elution medium (Sterogenes Bioseparations). The bound protein was then eluted with 6 × 2.5 ml of neat Actisep elution medium. The affinity column eluates were buffer-exchanged into phosphate-buffered saline using PD-10 columns (Pharmacia). Aliquots of fractions were analyzed by SDS-PAGE, after which the gel was silver-stained (32). Fractions were also analyzed for their ability to bind to 125I-IL-13 using the cross-linking protocol described above. The affinity-purified IL-13BP in a total volume of 12 ml (approximately 2-4 µg) was loaded onto the sample cartridge of a Hewlett Packard model G1005A protein sequencer which was operated with the routine 3 sequencer program (33).
Given the expression of transmembrane and secreted forms of many
members of the hemopoietin receptor family from alternatively spliced
transcripts or via proteolysis (22), we sought evidence for the
existence of a soluble form of the IL-13R. Using a gel filtration-based assay, mouse serum and urine were examined for the
presence of an IL-13BP. Gel filtration chromatography of
125I-IL-13 alone resulted in elution of radioactivity in
fractions 35-39 (Fig. 1,A and B).
Prior addition of mouse serum or urine to the 125I-IL-13
resulted in the presence of an additional peak of
125I-IL-13 eluting earlier in fractions 27-30 (Fig. 1,
C and D). The formation of this higher molecular
weight peak was competed for by the addition of an excess of unlabeled
IL-13 but not by unlabeled IL-4, demonstrating that the interaction
with the binding protein was specific (Fig. 1, E and
F).
In contrast to crude mouse urine, purified soluble IL-13R appeared
unable to bind IL-13 as assessed by gel filtration chromatography (data
not shown). Given the very low affinity of IL-13 for the IL-13R
and
the fast kinetic dissociation rate (6), this result was not surprising
but did suggest that the serum and urine binding proteins were either
distinct from the IL-13R
or contained other components in addition
to IL-13R
. An obvious candidate for a protein capable of interaction
with IL-13R
to generate a high affinity IL-13 receptor was the
IL-4R
. The presence of soluble IL-4R
in urine and serum has been
described in a number of previous studies (17, 18, 20), and this result
was confirmed by the cross-linking experiments described below. Despite
the presence of an IL-4 binding protein (IL-4BP) in urine and serum,
addition of purified IL-4R
to purified IL-13R
did not
recapitulate the properties of the IL-13BP found in serum and urine as
assessed by gel filtration chromatography (data not shown). Moreover,
the apparent molecular weight of the IL-13/IL-13BP complex from serum and urine (60,000-65,000) was not consistent with a complex of IL-13
with IL-13R
and IL-4R
(predicted molecular weight of
100,000).
To assess the size of the IL-13BP and its relationship to the IL-4BP,
mouse urine was fractionated on a gel filtration column (Fig.
2A). Aliquots from each fraction were then
mixed with 125I-IL-13 or 125I-IL-4 in the
presence or absence of an excess of unlabeled IL-4 or IL-13 and
subjected to cross-linking using the bifunctional reagent
BS3. The products of the cross-linking reaction were then
resolved by SDS-PAGE and visualized by autoradiography. Cross-linking
of 125I-IL-13 to unfractionated mouse urine revealed the
presence of a major band that migrated with a Mr
of approximately 60,000-65,000 (Fig. 2B). Given that
125I-IL-13 migrates with a Mr of
15,000, this would suggest that the IL-13BP has a
Mr of 45,000-50,000. Cross-linking to the
IL-13BP was specific, since it was abolished by competition with
unlabeled IL-13 (Fig. 3B) but not IL-4 (Fig.
4). The 45,000-50,000 Mr IL-13BP eluted from the gel filtration column in fractions 27 to 29, consistent with its molecular weight estimated from the cross-linking experiment. Additional apparently nonspecific lower molecular weight radioactive products were also observed in the cross-linking studies with 125I-IL-13. One of these eluted from the gel filtration
column in fractions 29 and 30, after the 45,000-50,000
Mr IL-13BP, but in a similar position to an
IL-4BP (Fig. 2C). The complexities of this interaction will
be discussed in more detail below. Cross-linking of
125I-IL-4 to the IL-4BP resulted in a species migrating
with a Mr of approximately 50,000 (Fig.
2C), suggesting that the binding protein itself had a
Mr of
35,000.
The ligand-binding specificity of the 45,000-50,000
Mr IL-13BP and purified receptor components was
examined further using cross-linking. Although binding of
125I-IL-13 to purified IL-13R was not detected by gel
filtration chromatography, an interaction was observed using
cross-linking (Fig. 3, lane 1). Consistent with previous
studies (6), this interaction was competed for by unlabeled IL-13 but
not unlabeled IL-4 (Fig. 3, lanes 2 and 3). When
cross-linking studies were performed using 125I-IL-13 and
purified IL-4R
, a product with a Mr of
50,000 was observed (Fig. 3, lane 4). Surprisingly,
cross-linking of 125I-IL-13 to IL-4R
was in competition
with unlabeled IL-4 (Fig. 4, lane 5) but not unlabeled IL-13
(Fig. 3, lane 6). Combining purified IL-13R
and IL-4R
with 125I-IL-13 resulted in the generation of species
similar in molecular weight and specificity to reactions containing
each receptor alone (Fig. 3, lanes 7-9). No
higher molecular weight complexes were observed, suggesting that
formation of ternary IL-13/IL-13R
/IL-4R
complexes or their
capture with the cross-linker occurred inefficiently in solution, even
at high concentrations of each component. Consistent with our initial
experiments (Fig. 2B), crude mouse urine contained a major
45,000-50,000 Mr IL-13BP (Fig. 3, lane
10). Cross-linking of 125I-IL-13 to this species was
in competition with unlabeled IL-13 (Fig. 3, lane 11) but
not unlabeled IL-4 (Fig. 3, lane 12). The lower molecular
weight species observed in our initial experiments (Fig. 2B,
fractions 29 and 30), was again observed. As
before, cross-linking of 125I-IL-13 to this protein was not
in competition with unlabeled IL-13 (Fig. 3, lanes 10 and
11); however, like cross-linking of 125I-IL-13
to purified IL-4R
, cross-linking to this protein was competed for by
unlabeled IL-4 (Fig. 3, lane 12). Although the addition of
purified IL-4R
to purified IL-13R
did not alter the pattern of
cross-linking observed to either component alone, we sought to
determine whether purified IL-4R
could interact with partially
purified 45,000-50,000 Mr IL-13BP from mouse
urine (Fig. 2A, fraction 27). As with the purified receptor
components, no additional effect of adding IL-4R
was observed (Fig.
3, lanes 13-15).
While interaction between IL-13 and both purified soluble IL-13R and
the 45,000-50,000 Mr binding protein was
demonstrable by cross-linking, only the interaction with the binding
protein was detectable using gel filtration chromatography. These
results suggested that the affinity of IL-13 for the binding protein
was higher than for soluble IL-13R
. To test this formally,
saturation binding experiments were performed. Scatchard
transformations revealed that, consistent with previous studies (6),
the equilibrium dissociation constant (KD) of IL-13
for the IL-13R
expressed by Chinese hamster ovary cells was
approximately 10 nM (data not shown). However, the affinity
of IL-13 for the serum and urinary binding protein was 100-300-fold
higher, with KD values ranging from 20 to 90 pM (data not shown). The difference in affinity was
confirmed in cross-linking experiments in which 125I-IL-13
was mixed with increasing concentrations of unlabeled IL-13 prior to
cross-linking to soluble IL-13R
(Fig. 4A) or urinary binding protein (Fig. 4B). Densitometric analysis of these
data demonstrated that half-maximal inhibition of binding to soluble IL-13R
occurred with approximately 100 ng/ml IL-13, a 40-fold higher
concentration than required to inhibit 50% of the cross-linking to the
45,000-50,000 Mr urinary binding protein.
The structural relationship between soluble IL-13R and the
45,000-50,000 Mr urinary IL-13BP was examined
by cross-linking 125I-IL-13 to both proteins and isolating
the resultant complexes from SDS-polyacrylamide gels. Each complex was
then subjected to exhaustive deglycosylation using
N-glycanase F and digestion with various concentrations of
Staphylococcus V8 protease. The products of these treatments
were then resolved by further SDS-PAGE. The untreated and
deglycosylated complexes of IL-13 and the soluble IL-13R
appeared
slightly larger than the corresponding complexes with the urinary
binding protein (compare Fig. 5, lane 1 with lane 10 and lane 2 with lane 9). In
addition, the products of V8 proteolysis of the two complexes were
clearly different, emphasizing the structural difference between the
IL-13R
and the 45,000-50,000 Mr urinary
IL-13BP (compare Fig. 5, lanes 3-5 with
lanes 6-8).
A rabbit polyclonal antiserum was raised against purified soluble
recombinant IL-13R. This antiserum was capable of
immunoprecipitating the cross-linked product of 125I-IL-13
with IL-13R
(Fig. 6, lane 10), while no
immunoprecipitation was observed with a preimmune rabbit serum (Fig. 6,
lane 12). Immunoprecipitation of the radioactive complex was
not inhibited by the FLAG peptide which is present in our soluble
recombinant IL-13R
(Fig. 6, lane 11) but was inhibited by
an excess of IL-13 (Fig. 6, lanes 13 and 14). In
contrast to the IL-13R
complex, the complex between the
45,000-50,000 Mr urinary IL-13BP and
125I-IL-13 was not recognized by the rabbit antiserum to
IL-13R
(Fig. 6, lanes 3-9), suggesting that
these proteins are antigenically as well as structurally and
functionally distinct.
Soluble receptors for a variety of cytokines have been described (22).
In some cases these act to augment a biological response (23, 24),
while in other situations they may inhibit the biological effect
(24-26). To gain information on whether the purified soluble IL-13R
or the urinary binding protein could influence IL-13 action we examined
their effects on binding of 125I-IL-13 to peritoneal
macrophages. Fig. 7 demonstrates that even though both
the urinary IL-13BP and the soluble IL-13R
could inhibit the binding
of 125I-IL-13 to cell surface receptors expressed by
macrophages the latter was far less efficient, raising the possibility
that the soluble IL-13BP may be used to modulate the effects of IL-13
in vivo.
The soluble IL-13BP was purified from 2.2 liters of mouse urine by
affinity chromatography on immobilized IL-13 as described under
"Experimental Procedures." Approximately 2-4 µg of the purified protein was obtained and electrophoresed as a single silver staining protein of Mr 45,000-50,000 on
SDS-polyacrylamide gel (Fig. 8A). Chemical
cross-linking of this protein incubated with 125I-IL-13
revealed a complex of Mr 60,000-65,000 (Fig.
8B) similar to that seen with crude urine preparations (Fig.
2). Moreover, chemical cross-linking of this purified preparation
incubated with unlabeled IL-13 and visualization of the products by
silver staining of the SDS-polyacrylamide gel showed that a significant proportion of the major protein band of Mr
45,000-50,000 could interact with IL-13 and shift to a band of
Mr 60,000-65,000 (Fig. 8C,
lanes 3 and 4).
The purified protein was subjected to N-terminal sequencing and
generated a single major 27-amino-acid sequence (with an initial yield
of 50 pmol), namely EIKVNPPQDFEILDPGLLGYLYLQWKP~. This sequence is
clearly different from the N-terminal amino acid sequence of the cloned
mouse IL-13R
(6). An initial search of the GenBankTM
data base identified a human expressed sequence tag (accession number
R52795[GenBank]/clone 41648) derived from an infant brain cDNA library with
an identical sequence stretch except for conservative amino acid
changes at positions 13 (L
V), 17 (L
Y), and 26 (K
Q). Moreover,
this sequence was preceded by a typical leader sequence of 27 amino
acids and signal sequence cleavage site in the expressed sequence tag
suggesting that its predicted mature amino acid sequence would start at
the same position as that identified for the IL-13BP. The 3
end of
clone 41648 (the sequence of which is represented by expressed sequence
tag R52796[GenBank]) was also deposited in the GenBankTM data base
and revealed sequences suggesting that this protein was a member of the
type I cytokine receptor family including the diagnostic sequence
WSEWS.
At least one form of the cell surface receptor complex for IL-13
is composed of the IL-4R (5) and the recently cloned IL-13R
(6).
Despite its low affinity, the specificity of the interaction between
IL-13 and the soluble IL-13R
was demonstrable by cross-linking,
since IL-13 but not IL-4 was capable of competing for binding. In the
course of the cross-linking studies, an unexpected, puzzling, and to
date unexplained observation was made. 125I-IL-13 was found
to cross-link to purified recombinant IL-4R
. This interaction was
capable of being competed for by IL-4 but not by unlabeled IL-13. The
possibility that iodination of IL-13 on tyrosine residues using the
iodine monochloride technique altered its receptor specificity was
thought to be unlikely, since a similar result was observed with IL-13
labeled on lysine residues using the Bolton-Hunter reagent.
Murine serum and urine were found to contain a protein capable of
binding IL-13. However, such a protein was not detected in human plasma
and urine (data not shown). The mouse urinary IL-13BP was characterized
in detail and shown to be structurally, antigenically, and functionally
distinct from the cloned mouse IL-13R (6). These differences could
not be explained in terms of an association of the IL-13R
and
IL-4R
in these biological fluids but rather suggested the existence
of an independent protein capable of binding IL-13. Several lines of
evidence support the notion that the mouse urinary IL-13BP was
different from the cloned mouse IL-13R
(6): (a) the size
of the binding protein was smaller than the extracellular domain of the
cloned IL-13R
, and the pattern of V8 protease digestion of the two
molecules was clearly different; (b) an antiserum raised
against the extracellular domain of the cloned IL-13R
failed to
recognize the binding protein found in urine; (c) most
dramatically, the binding protein interacted with IL-13 with a
100-300-fold higher affinity than the soluble IL-13R
(KD = 20-90 pM versus 3-10
nM) (6); and (d) N-terminal amino acid sequences of the two
proteins were different. Moreover, unlike the soluble IL-13R
, the
urinary binding protein was an efficient inhibitor of IL-13 binding to
its cell surface receptor.
These characteristics suggest that the soluble IL-13BP should be an effective inhibitor of IL-13 biological activities and it may serve to dampen the proinflammatory activities of IL-13 such as macrophage and eosinophil activation and, in humans, IgE production by B-lymphocytes (2, 3, 34).
The recent cloning of an alternate IL-13 cell surface receptor from human renal carcinoma cells by Caput et al. (35) has indicated that the IL-13BP is almost certainly a murine soluble analogue of this human receptor chain. This conclusion is based on the near identity of the first 27 amino acids of the predicted mature form of the human receptor with that of the soluble IL-13BP, the high affinity with which it binds IL-13 (KD = 250 pM), and its absolute specificity for binding IL-13 but not IL-4.
Caput et al. (35) presented evidence that their IL-13
receptor shows little alteration of binding affinity or specificity when it is co-expressed with IL-4R and, surprisingly, a diminution of IL-13-binding when it is co-expressed with
c. This
contrasts with our data with IL-13R
(6), where higher affinity
IL-13/IL-4-cross-reactive receptors were generated by co-expression
with IL-4 receptor.
The evidence of two alternative IL-13-binding receptor chains and the
possible involvement of IL-4R and
c in forming IL-13 receptor complexes points to a very complex pattern of IL-13 and IL-4
recognition and signal transduction at different cell surfaces. The
existence of a soluble IL-13 receptor adds a further layer of
complexity in the control of IL-13 responses. Issues yet to be
addressed include whether the IL-13 receptor described by Caput et al. (35) is capable of functional signaling and how the
soluble form of this receptor is generated and controlled
(e.g. by alternative splicing or proteolytic release from
the cell surface). Clearly, very careful reconstitution experiments
with all of the receptor components described above will be required to
begin to understand this complex regulatory system.
We thank Dale Cary and Naomi Sprigg for
excellent technical assistance. We are grateful to Wendy Carter and
Karen Mackwell for preparing anti-IL-13R antiserum and to animal
technicians for collecting mouse urine.