(Received for publication, September 26, 1994; and in revised form, November 17, 1994)
From the
We describe here the characterization of the interleukin (IL) 13
receptor and a comparison with the IL-4 receptor on different cell
types. Several, but not all, of the IL-4 receptor-positive cells showed
specific IL-13 binding, which was always completely displaced by IL-4.
In the IL-13 receptor-positive cells, the IL-13 either completely or
partially displaced the labeled IL-4. Further characterization of the
IL-13 receptor in two cell lines, COS-3 and A431, representative of the
groups of complete and partial displacement of IL-4 by IL-13,
respectively, showed that the IL-13 binds with high affinity (K
300 pM) to both cells
and that the number of binding sites is, in COS-3 cells, equivalent to
that for IL-4 and, in A431 cells, is smaller than that for IL-4.
Cross-linking of labeled IL-13 yielded, on COS-3 cells, two
affinity-labeled complexes of 220 and 70 kDa, and on A431 cells, one
complex of 70 kDa; labeled IL-4 yielded on both cells the same pattern
of three complexes of 220, 145, and 70 kDa. Altogether, these results
suggest that the IL-13 receptor may be constituted by a subset of the
IL-4 receptor complex associated with at least one additional protein.
Recently, we (1) and others (2) described the
cloning of the cDNA for interleukin 13 (IL-13)(), a cytokine
secreted by activated T cells. IL-13 regulates inflammatory and immune
responses. On peripheral blood monocytes, for example, it inhibits the
production of inflammatory cytokines induced by
lipopolysaccharide(1) , induces the production of the IL-1
receptor antagonist(3) , and modulates the expression of cell
surface proteins, like CD14, class II MHC antigens(4) , and
mannose receptor(5) , relevant to the function of these cells.
It also inhibits human immunodeficiency virus production by infected
tissue-cultured differentiated macrophages(6) . On B cells,
another target for IL-13, it acts on different stages of maturation.
For example, on resting B cells, it enhances the expression of
CD23/Fc
RII and class II MHC antigens(7, 8) , it
stimulates, in combination with anti-Ig or anti-CD40 antibodies, B cell
proliferation, and it induces IgE synthesis(8) . IL-13 plays a
role also in the regulation of proliferation and differentiation of
primitive hematopoietic progenitor cells(9) . Non-hematopoietic
cells, such as fibroblast, endothelial, or epidermal cells, are also
targets for IL-13(10, 11) . These biological
activities are also displayed by IL-4, another pleiotropic T-cell
cytokine that shows limited amino acid sequence homology, about 25%,
with IL-13. The similar biological activities of IL-4 and IL-13 may
result from the use of the same receptor, but the differences between
the biological effects of these cytokines on, for example T cells,
suggests an overlapping but not identical population of receptors. In
fact, Zurawski et al.(12) recently showed that the
receptors for IL-4 and IL-13 are structurally related. Furthermore, it
has been shown that a mutated IL-4 (13) that blocks the
biological activity of IL-4 also antagonizes IL-13
activity(12, 14) , adding support for shared
component(s) important for signal transduction between both receptors.
Two proteins have been described as components of the high affinity
IL-4 receptor, a glycoprotein of
130 kDa (IL-4R) (15, 16) that when expressed in COS-7 cells binds IL-4
with a K
of 50-100
pM(12) , and the
chain of the IL-2 receptor
(
chain) (17, 18) , that when associated to the
IL-4R results in a 2-3-fold increase in affinity for IL-4 (17) and that participates in some of the IL-4-mediated signal
transduction events(18) .
The direct binding of IL-13, which
may help to a better understanding of its receptor and the
identification of potential target cells, has not been reported,
probably because the strong conditions needed to iodinate the single
tyrosine, Tyr, on the IL-13 molecule markedly reduces its
binding capacity. To circumvent this difficulty, we mutated the
Tyr
to Phe and added, at the C terminus, a motif
Gly-Tyr-Gly-Tyr to obtain new targets for iodination. This recombinant
protein ([Phe
]IL-13-GYGY), was expressed in
COS-7 cells, purified to homogeneity, and radiolabeled using a
classical chloramine T method with no significant modification in its
binding characteristics.
We describe here the screening of IL-13 and
IL-4 receptors on cell lines of various types and the analysis of the
IL-13 and IL-4 receptors by displacement, saturation, and cross-linking
experiments on two cell lines, A431 and COS-3, which displayed
different IL-13 binding properties. Finally, using COS-3 cells
transfected with the cDNAs for the IL-4R and the chain, we have
studied the relationships between these proteins and the IL-13 binding
site.
The human epidermoid
carcinoma cell line A431 (ATCC CRL 1555) and the simian fibroblast-like
cell line COS-3 (kindly provided by Dr. M. Yaniv, Paris) were
maintained in monolayer cultures in Dulbecco's modified essential
medium supplemented with 10% fetal calf serum at 37 °C in a
humidified atmosphere containing 5% CO.
The Tyr in position 43 of the polypeptide
chain of IL-13 was replaced by Phe using the site-directed mutagenesis
kit from Amersham. The recombinant plasmid obtained, pSE1.
[Phe]IL-13-GYGY, was controlled as described
above.
(NH)
SO
(1 M final
concentration) was added to the concentrated material, and then it was
loaded onto a phenyl-Sepharose CL-4B column (1.0
8.0 cm)
equilibrated in 25 mM Tris-HCl, 1 M (NH
)
SO
and washed with the
same buffer until the UV absorbance returned to base-line values.
Proteins were stepwise eluted using decreasing concentrations of
(NH
)
SO
in the same buffer. The
fractions containing [Phe
]IL-13-GYGY were
identified by high pressure liquid chromatography on a Brownlee BU 300
column (2.1
100 mm) eluted with a 30-70% linear gradient
of acetonitrile, 0.1% trifluoroacetic acid. Purified Chinese hamster
ovary-derived IL-13 (1) was used as standard. Fractions
containing [Phe
]IL-13-GYGY (eluted with 600
mM (NH
)
SO
) were pooled and
further purified by gel filtration on a Sephacryl HR-100 column (1.6
100 cm) equilibrated and eluted with 50 mM ammonium
acetate, pH 6.8. The purified material was characterized by
SDS-polyacrylamide gel electrophoresis, amino acid composition,
sequence analysis(23) , and laser desorption time of flight
mass spectrometry on a Lasermat spectrometer (Finnigan, San Jose, CA)
as described(24) .
For
binding experiments on COS-3 cells expressing the recombinant IL-4R
and/or the chain, 5
10
cells were transfected
with 2 µg of the recombinant expression plasmids, as described
above. The expression vector used was pSE1(19) , the human and
monkey IL-4R cDNA (15, 16) were cloned from U937 and
COS-3 cells, respectively, and the cDNA coding for the human
chain (pSRG1) (17) was a gift of Dr. Sugamura (Sendai, Japan).
The expression of the recombinant
chain on the membrane of the
transfected cells was determined by immunofluorescence using a rabbit
antibody anti-
chain obtained in our laboratory.
For binding experiments, the adherent cell lines (A431, COS-3, or transfected COS-3 cells) were seeded in 6-well plates (Falcon) and cultured as described above. Binding experiments with the non-adherent cell lines were carried out as previously described(28) .
Saturation
experiments were done in RPMI containing 20 mM HEPES, 3 mg/ml
bovine serum albumin, and 0.1% NaN (binding buffer) (1 ml)
with
I-labeled IL-4 or
I-labeled
[Phe
]IL-13-GYGY over a range from 5 pM to 2 nM for 2 h at room temperature. Then, the buffer was
aspirated, cell monolayers were washed twice with binding buffer, and 2
ml of 1 M NaOH were added to solubilize the cells for
quantification of the bound radioactivity. Nonspecific binding was
defined as binding in the presence of a 500-fold excess of unlabeled
IL-4 or IL-13.
Similar conditions were used for the displacement
experiments with 70 pM of I-labeled IL-4 or 300
pM of
I-labeled
[Phe
]IL-13-GYGY as ligands. Binding data derived
from saturation and competition experiments were analyzed with the
computerized nonlinear curve fitting described by Munson and
Rodbard(29) .
Cross-linking experiments of I-labeled IL-4 or
I-labeled
[Phe
]IL-13-GYGY on A431 and COS-3 cell lines
were done with disuccinimidyl suberate (Pierce) and analyzed on
SDS-polyacrylamide gel electrophoresis as previously
described(28) .
The mutation of Tyr to Phe and the presence of the
tetrapeptide Gly-Tyr-Gly-Tyr at the C-terminal end of the IL-13 did not
significantly modify its biological activity in the B9.1.3
proliferation assay (Fig. 1). More important, the modifications
resulted in a protein that was easily radiolabeled by the standard
chloramine-T method. The in vitro biological activity of the
labeled protein was reduced, at the most by 40%, after some
iodinations, but the binding characteristics were not significantly
modified, as described below. Under similar labeling conditions, the
natural IL-13 was not labeled, and when stronger conditions were used,
the resulting labeled molecule was biologically inactive (Fig. 1), and it bound very poorly to the tested cells (results
not shown).
Figure 1:
Growth-promoting activity of IL-13,
[Phe]IL-13-GYGY, and labeled
[Phe
]IL-13-GYGY on B9.1.3 cells. Dilutions of
stock solutions at 1 µg/ml purified IL-13 (
),
[Phe
]IL-13-GYGY (
),
I-labeled [Phe
]IL-13-GYGY
(
), or
I-labeled IL-13 (
) were tested in
parallel. Each point represents the mean of
triplicates.
Figure 2:
Binding analysis of radiolabeled IL-4 and
IL-13 to COS-3 cells. A, competitive displacement of I-labeled IL-4 binding by IL-4 (
) or IL-13
(
). B, Scatchard plot analysis from saturation isotherm
data of the specific binding of
I-labeled IL-4. C, competitive displacement of
I-labeled
[Phe
]IL-13-GYGY binding by IL-4 (
),
IL-13 (
), [Phe
]IL-13-GYGY (
), and
cold labeled
I-labeled
[Phe
]IL-13-GYGY (
). D, Scatchard
plot analysis from saturation isotherm data of the specific binding of
I-labeled [Phe
]IL-13-GYGY. The
competitive displacements of
I-labeled IL-4 (70
pM) or
I-labeled
[Phe
]IL-13-GYGY (300 pM) binding were
done with increasing concentrations of cold cytokines for 2 h at room
temperature. Bound and unbound fractions were determined as described
under ``Materials and Methods.'' Each point represents the mean of triplicates. Curve fitting was performed as
described under ``Materials and
Methods.''
Similar displacement and saturation
experiments were done on the COS-3 cells with I-labeled
[Phe
]IL-13-GYGY as tracer. In competition
experiments, unlabeled IL-4 totally displaced the
I-labeled [Phe
]IL-13-GYGY
(IC
= 30 ± 7 pM), and IL-13, as
expected, also completely displaced the labeled IL-13 (IC
= 400 ± 25 pM). On these cells, we also
used [Phe
]IL-13-GYGY and the cold labeled
I-labeled [Phe
]IL-13-GYGY as
competitors, and, as shown in Fig. 2C, no major
differences in IC
were observed (IC
=
380 ± 40 pM and 550 ± 45 pM,
respectively) for these two modified IL-13 molecules.
The Scatchard
analysis of the saturation curves done with the labeled IL-13 showed a
single class of binding sites (K = 300
± 45 pM and 2500 ± 190 receptors/cell) (Fig. 2D). It should be noted that the K
for the
I-labeled
[Phe
]IL-13-GYGY obtained in the saturation
experiments is in agreement with the IC
obtained for the
IL-13 and the cold labeled IL-13 in the competition binding analysis.
These results suggest that the affinity of the iodinated IL-13 was not
significantly modified and that the reduced biological activity
observed after some iodinations may be due to modifications that
resulted in an impaired receptor activation.
A431 cell receptors for
IL-4 and IL-13 were also characterized since on these cells the IL-13
was not able to completely displace the labeled IL-4. The binding of I-labeled IL-4 to A431 cells was displaced by IL-4 and
IL-13 in a dose-dependent manner (Fig. 3A). The
displacement curves shown by the two cytokines are different. IL-4 was
able to fully compete with
I-labeled IL-4 (IC
= 20 ± 3 pM) whereas IL-13 displaced with
high affinity (IC
= 300 ± 30 pM)
only 70% of the bound
I-labeled IL-4. Higher
concentrations of IL-13 (>100 nM) did not displace the
remaining 30% of the IL-4 binding. These results are in line with the
presence of at least two binding sites for IL-4 of which only one is
shared with IL-13, as previously described in binding experiments on
TF-1 cells(12) .
Figure 3:
Binding analysis of radiolabeled IL-4 and
IL-13 to A431 cells. A, competitive displacement of I-labeled IL-4 binding by IL-4 (
) or IL-13
(
). B, Scatchard plot analysis from saturation isotherm
data of the specific binding of
I-labeled IL-4
(
). The saturation experiment with labeled IL-4 was also
performed after preincubation of the cells with 100 nM of
unlabeled IL-13 (
). C, competitive displacement of
I-labeled [Phe
]IL-13-GYGY binding
by IL-4 (
) or IL-13 (
). D, Scatchard plot
analysis from saturation isotherm data of the specific binding of
I-labeled [Phe
]IL-13-GYGY. Each point represents the mean of triplicates. Experimental
conditions are identical to those described in Fig. 2.
These results on A431 cells were confirmed
in saturation experiments using I-labeled IL-4 as a
tracer. As shown in Fig. 3B, the Scatchard plots
derived from the saturation isotherms show that IL-13 was able to
compete with only a fraction (about 70%) of the total binding sites
occupied by IL-4. The cells preincubated with saturating concentrations
(100 nM) of unlabeled IL-13 showed, when compared with the
control, a reduction in the number of binding sites without a
significant modification of the affinity (K
= 16 ± 2 pM).
Similar displacement and
saturation experiments were done on the A431 cells with I-labeled [Phe
]IL-13-GYGY as
tracer. In competition experiments, unlabeled IL-4 totally displaced
the
I-labeled [Phe
]IL-13-GYGY
binding and with higher affinity (IC
= 15 ±
2 pM) than IL-13 (IC
= 350 ± 20
pM) (Fig. 3C).
In saturation experiments (Fig. 3D), a single class of binding sites (K = 260 ± 15 pM) was
found. As expected, the number of specific binding sites for IL-13 was
lower (205 ± 18 receptors/cell) than the number of binding sites
for IL-4 (310 ± 25 receptors/cell).
The results are shown in Fig. 4. Three
main broad bands (70, 145, and 220 kDa) were consistently and
specifically cross-linked to
I-labeled IL-4 on COS-3 and
A431 cells, and a fourth band (
45 kDa) was detected in some of the
cross-linking experiments (Fig. 4, A and B, lanesa). When the
I-labeled
[Phe
]IL-13-GYGY was cross-linked, only two bands
(
70 and 220 kDa) on COS-3 cells and one (
70 kDa) on A431 cells
were consistently detected; a faint labeled complex,
45 kDa, was
also detected in some of the experiments (Fig. 4, A and B, lanesd). The 145 kDa have been
previously assigned to the IL-4 cross-linked to the cloned
IL-4R(31) . The nature of the protein that yields the 70-kDa
complex is not clear (32, 33, 34) . Recently,
a low affinity IL-4 receptor, with a molecular weight compatible with
this complex, has been described(36) . But NALM-6 cells
possessing only the low affinity IL-4 receptor do not bind IL-13
(results not shown); thus, this protein is not sufficient to constitute
an IL-13 receptor. The 220-kDa complex observed on COS-3 and A431
cells, when
I-labeled IL-4 was used as a tracer, may
result from the chemical cross-linking of the two membrane proteins
detected (70- and 145-kDa complexes). Alternatively, this band may
result from a new component of these receptors. This high molecular
weight band was also detected in the cross-linking experiments with
labeled IL-13 on COS-3 cells, suggesting that the IL-4R subunit, even
if it does not bind IL-13 directly, may be close to the 70-kDa IL-13
binding protein. The absence of this band in the cross-linking
experiments on A431 may be related, either to the low number of
receptors or to a different structure of the proteins that do not allow
the cross-linking. Unexpectedly, IL-13 was able to displace labeled
IL-4 from the IL-4
IL-4R 145-kDa complex in both cell lines (Fig. 4, A and B, lanesc),
as does IL-4 (Fig. 4, A and B, lanes
b).
Figure 4:
Characterization of IL-4 and IL-13
receptors by affinity cross-linking. COS-3 cells (A) or A431
cells (B) were incubated for 2 h at room temperature with 70
pM of I-labeled IL-4 (lanesa-c) or 300 pM of
I-labeled
[Phe
]IL-13-GYGY(lanesd-f) in the absence or in the presence of a
500-fold excess of unlabeled IL-4 (lanesb and e) or IL-13 (lanesc and f).
Cross-linking of the ligands to the receptors was done with 2.5 mM disuccinimidyl suberate, and then the cells were solubilized and
the proteins were subjected to electrophoresis. The radioautographs
were obtained as previously
described(28) .
The displacement of I-labeled IL-4 (70 pM) by IL-4 and IL-13 from
COS-3 cells transfected with an expression plasmid with or without the
cDNA for the
chain are shown in Fig. 5A. Both
IL-4 and IL-13 can compete with the labeled IL-4 when large amount of
recombinant
chain, as detected by immunofluorescence analysis
(results not shown), are present on the membrane of the transfected
COS-3 cells. Similar results were obtained when
I-labeled
[Phe
]IL-13-GYGY (300 pM) was used as
tracer. Again, the presence of recombinant
chain molecules in the
membrane of the COS-3 cells did not prevent the displacement of IL-13
by IL-4 (Fig. 5B). Thus, these results suggest that the
chain, if shared by both receptors, is not responsible for the
cross-competition between IL-4 and IL-13 in the binding experiments.
Furthermore, they also show that the
chain is not able to bind
either IL-4, as previously described(17) , or IL-13.
Figure 5:
High affinity binding of radiolabeled IL-4
and IL-13 to COS-3 cells expressing high levels of recombinant
chain. COS-3 cells were transfected either with the cDNA for the
chain of the IL-2 receptor (openbars) or mock
transfected (solidbars) as described under
``Materials and Methods,'' and the binding of
I-labeled IL-4 (A) or
I-labeled
[Phe
]IL-13-GYGY (B) was tested in the
absence or in the presence of a 500-fold excess of unlabeled IL-4 and
IL-13.
To
investigate whether the binding sites of IL-4 and IL-13 overlap we did
binding experiments with saturating concentrations of I-labeled IL-4,
I-labeled
[Phe
]IL-13-GYGY, and a mixture of both tracers.
The results are shown in Fig. 6. On control COS-3 cells the
binding of labeled IL-4 or IL-13 was not additive, and similar results
were observed on COS-3 cells expressing recombinant
chain. The
binding analysis of COS-3 cell transfected with the cDNA coding for the
human or simian IL-4R alone or cotransfected with the cDNA for the
chain showed that, as expected, the specific binding for IL-4 was
significantly increased in COS-3 cells expressing recombinant IL-4R,
but the IL-13 specific binding remained unaltered. No increase in the
specific binding was observed when labeled IL-4 and IL-13 were added
simultaneously. When the
chain was expressed with the IL-4R (from
human or monkey), a slight increase of
I-labeled IL-4
binding was observed. Since the
chain does not bind IL-4
directly, these results are attributed to an increase in IL-4R affinity
toward IL-4 induced by the
chain(17) . The expression of
both the IL-4R and the
chain did not result in increased specific
binding when both cytokines were added simultaneously.
Figure 6:
High
affinity binding of radiolabeled IL-4 and IL-13 to COS-3 cells
expressing high levels of recombinant IL-4R and/or chain. COS-3
cells were mock transfected or transfected with the cDNA for the
chain of the IL-2 receptor alone or co-transfected with the human or
simian IL-4R. Binding experiments were performed with 500 pM of
I-labeled IL-4, 2 nM of
I-labeled [Phe
]IL-13-GYGY, or 500
pM of
I-labeled IL-4 and 2 nM of
I-labeled [Phe
]IL-13-GYGY as
tracers in triplicate as described under ``Materials and
Methods.''
The purpose of this study was to characterize the receptor
for IL-13 and to compare it with that for IL-4, since these two
cytokines share many biological activities and, as recently proposed by
Zurawski et al.(12) , may also share receptor
subunits. The almost complete inactivation of the IL-13 after
iodination of the single tyrosine, Tyr, present in the
molecule prompted us to produce a recombinant protein in which the Tyr
was replaced by Phe and the motif Gly-Tyr-Gly-Tyr was added to the C
terminus. The resulting protein was biologically active and easily
radiolabeled without significant modification of the binding
characteristics.
The screening of several cell types for IL-13 and
IL-4 receptors revealed that several, but not all, of the IL-4R
positive cells were IL-13R positive. The binding of IL-13 was always
displaced by IL-4, but in the IL-13R positive cells the binding of
labeled IL-4 was either partially displaced, as described previously by
Zurawski et al.(12) , or completely displaced by cold
IL-13. To further explore the relationship between the IL-13 and IL-4
receptors, we characterized them in two cell lines, COS-3, in which
IL-13 completely displaced the binding of IL-4, and A431, in which
IL-13 partially displaced IL-4. The saturation and displacement
experiments showed that on both cell lines IL-13 binds to a single
class of binding sites with a K
300
pM, while the IL-4 also binds to a single class of binding
site with an affinity of
20 pM.
In the cross-linking experiments on COS-3 cells, the labeled IL-4 consistently yielded three labeled complexes of 220, 145, and 70 kDa, while the labeled IL-13 yielded only two complexes of 220 and 70 kDa. On A431 cells, the patterns were similar to those obtained on COS-3 cells, but the 220-kDa complex was not detected when the cross-linking was done with labeled IL-13. Work is in progress to investigate if this absence is related to the low number of receptors present on A431 cells and/or to differences in the structure of the complex that impeded cross-linking.
The 145- and 70-kDa complexes have been previously described in cross-linking experiments with labeled IL-4. The 145-kDa complex has been assigned to the IL-4 cross-linked to the cloned IL-4R(31) , and the 70-kDa complex has been assigned to the IL-4 cross-linked either to a modified IL-4R (32, 33) or to a different protein participating in the IL-4 receptor complex(34) . The fact that the IL-13 does not label the IL-4R but yielded the 70-kDa complex suggests that different binding proteins are involved in the 145- and 70-kDa complexes.
The displacement of labeled IL-4 by IL-13 from the
145-kDa complex was unexpected since the cloned IL-4R, when expressed
in COS cells, does not bind IL-13. A possible explanation could be that
the binding of IL-13 results in a conformational change of the IL-4R
protein that still allows binding but not cross-linking of IL-4. In
line with this speculation, preliminary results showed that the binding
of iodinated IL-13 to COS-3 cells is completely inhibited by an IL-4R
monoclonal antibody, ()suggesting that the IL-4R may be
closely associated with the IL-13 binding site. It is tempting to
speculate that the 220-kDa complex may result from the cross-linking of
the labeled cytokines with both proteins involved in the 70- and the
145-kDa complexes.
It has been suggested that the cross-competition
of IL-13 and IL-4 in binding experiments may result from the use of
shared components, present in limiting amounts, between the two
receptors(12, 13) . Our results in COS-3 cells
transfected either with the cDNAs for the IL-4R and/or the chain,
the two known components of the IL-4 receptor, showed that these two
proteins do not bind IL-13 and that, even when the recombinant proteins
are present in large amounts in the cell membrane, IL-4 and IL-13 can
compete with each other for the binding sites. Thus, these two proteins
most probably are not the shared subunits responsible for the
cross-competition of the two cytokines in the binding experiments.
These results suggest that, if the proposed model (12) is
correct, another chain, part of the IL-4 receptor complex, may be
shared with the yet to be identified IL-13R.
An alternative model,
in line with our results and those already reported, is that the IL-13
receptor is constituted by the IL-4R complex associated with another
protein(s), the IL-13 binding subunit(s). Overlapping binding sites for
IL-4 and IL-13 in the IL-4IL-13 receptor complex can explain the
cross-competition of both cytokines in the binding experiments. The
relative amount of the IL-13 binding subunit(s) with respect to the
IL-4R will determine whether the IL-13 completely or partially
displaces the IL-4 from the receptor. Furthermore, since the IL-4R is
necessary but not sufficient to constitute the IL-13R, this model
predicts that all cells that bind IL-13 will bind IL-4, but not all the
cells that bind IL-4 will bind IL-13. If this is the case, some
regulatory activities of the IL-4 will not be shared by IL-13; thus,
the in vivo role of the two cytokines in normal or
pathological situations may be different. Finally, the iodinated IL-13
described here may facilitate the cloning of the IL-13 binding
subunit(s) that will be necessary for the reconstitution and
elucidation of the molecular structure of the IL-4
IL-13 receptor
complex.