©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization and Comparison of the Interleukin 13 Receptor with the Interleukin 4 Receptor on Several Cell Types (*)

(Received for publication, September 26, 1994; and in revised form, November 17, 1994)

Natalio Vita Sylvie Lefort Patrick Laurent Daniel Caput Pascual Ferrara (§)

From the From Sanofi Recherche, Labège Innopole, BP 137, 31676 Labège CEDEX, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 approx 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.


INTRODUCTION

Recently, we (1) and others (2) described the cloning of the cDNA for interleukin 13 (IL-13)(^1), 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/FcRII 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 approx130 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.


MATERIALS AND METHODS

Reagents

Recombinant human IL-4 was obtained from ICN Biomedicals (Costa Mesa, CA), and recombinant human IL-13 was produced and purified in our laboratory as previously described (1) . Dulbecco's modified essential medium, RPMI 1640, fetal calf serum, and phosphate-buffered saline were from Life Technologies, Inc. (Paisley, Scotland). Phenyl-Sepharose and Sephacryl HR-100 were obtained from Pharmacia LKB Biotechnology Inc. (Uppsala, Sweden), and Ultrogel AcA 54 gel was from IBF (Villeneuve-la-Garenne, France). NaI and I-labeled IL-4 (55-85 µCi/µg) were from Amersham (Buckinghamshire, United Kingdom). All other chemicals were reagent grade.

Cell Culture

NALM6 and TF-1 cells were kindly provided by Dr. R. Callard (London, UK), and by Dr. P. Manoni (Marseille, France), respectively. All of the other cell lines were purchased from the American Type Culture Collection. The cell lines were cultured in RPMI 1640 containing 10% fetal calf serum, 2 mM glutamine, penicillin (100 units/ml) at 37 °C in a humidified atmosphere containing 5% CO(2).

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(2).

Construction of the IL-13-GYGY-encoding Plasmid and Site-directed Mutagenesis

The 284-base pair-long AlwNI-PpuMI IL-13 coding fragment was purified on 2% low melting agarose gel and then inserted between the HindIII and BamHI sites of the pSE-1 vector(19) . Two synthetic oligonucleotides were used to ligate the DNA fragment to the vector. The first, 5`-GACCTGCTCTTACATTTAAAGAAACTTTTTCGCGAGGGACGGTTCAACGGCTACGGCTACTGATGAG-3`, reconstitutes the C-terminal region of IL-13 from the PpuMI site and adds (sequence written in bold) the tetrapeptide Gly-Tyr-Gly-Tyr two-stop codon and a G, which correspond to the first nucleotide of a BamHI cloning site, respectively. The second oligonucleotide, 5`-AGCTTGCCGCCACCATGGCGCTTTTGTTGACCACGGTCATTGCTCTCACTTGCCTTGGCGGCTTTGCCTCCCCAGGCC-3`, corresponds to the region of the initiation codon ATG (A in position 57 of the previously described cDNA sequence(1) ), in which the 14 first nucleotides were replaced by a ``Kozak consensus sequence'' (20) (sequence written in bold) to improve the translation and to reconstitute a HindIII cloning site. The ligation product was transformed into Escherichia coli NM522 strain by electroporation. The nucleotide sequence of the recombinant plasmid obtained, pSE1.IL-13-GYGY, was controlled by DNA sequencing(21) .

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.

Production and Purification of [Phe]IL-13-GYGY

COS-7 cells (4 times 10^7) were transfected with 112 µg of pSE1.[Phe]IL-13-GYGY as described by Seed and Arufo (22) . The transfected cells were cultured for 3 days, after which the culture medium containing the secreted recombinant protein was collected. The conditioned medium (600 ml) was filtered through 0.2-µm Millipore filters and concentrated to 100 ml using an Amicon Diaflo YM-5 ultrafiltration membrane.

(NH(4))(2)SO(4) (1 M final concentration) was added to the concentrated material, and then it was loaded onto a phenyl-Sepharose CL-4B column (1.0 times 8.0 cm) equilibrated in 25 mM Tris-HCl, 1 M (NH(4))(2)SO(4) and washed with the same buffer until the UV absorbance returned to base-line values. Proteins were stepwise eluted using decreasing concentrations of (NH(4))(2)SO(4) 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 times 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(4))(2)SO(4)) were pooled and further purified by gel filtration on a Sephacryl HR-100 column (1.6 times 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) .

Radiolabeling of [Phe]IL-13-GYGY and Binding Experiments

I-Labeled [Phe]IL-13-GYGY was prepared by the method of Greenwood et al.(25) using a limiting amount of chloramine T (26) to minimize oxidative damage. The reaction mixture contained 10 µl of 0.3 M potassium phosphate buffer, pH 7.4 (P(i) buffer), 1 mCi of NaI, 4 µg of protein (20 µl), and 8 µl of a solution of chloramine T (60 µg/ml) in 50 mM potassium phosphate buffer, pH 7.4. After 8 min at room temperature, 10 µl of a solution containing 1.15 mg/ml tyrosine in P(i) buffer were added, and the reaction mixture was diluted with 200 µl of 50 mM potassium phosphate buffer, pH 7.4, containing 0.1% (w/v) of bovine serum albumin (elution buffer). The radiolabeled cytokine was separated from aggregation products and free iodine on an Ultrogel AcA-54 column (0.7 times 45 cm) equilibrated with the elution buffer. The specific radioactivity of the labeled IL-13, estimated as described(27) , usually ranged between 80 and 120 µCi/µg. An identical protocol (with NaI instead of NaI) was used to produce ``cold'' iodinated I-labeled [Phe]IL-13-GYGY.

For binding experiments on COS-3 cells expressing the recombinant IL-4R and/or the chain, 5 times 10^5 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(3) (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) .

IL-13 Bioassay

The biological activity of IL-13 was measured by an in vitro colorimetric assay on B9.1.3 cells, a subclone derived from the B9 cell line(30) , which was selected for its IL-13 dependence. (^2)The data from triplicated determinations were fitted by maximum likelihood using a 4-parameter logistic weighted non-linear regression method. IC values were evaluated by the Fisher's test of comparison of parameters and considered different when p < 0.05.


RESULTS

Purification and Characterization of [Phe]IL-13-GYGY

Recombinant [Phe]IL-13-GYGY was purified from COS-3-conditioned medium using a two-step protocol, hydrophobic interaction chromatography followed by gel filtration chromatography. The purified recombinant [Phe]IL-13-GYGY was characterized by amino acid analysis, N-terminal sequencing, and mass spectrometry. The amino acid composition showed that the expected extra Phe, Gly, and Tyr residues are present in the purified [Phe]IL-13-GYGY (not shown). The N-terminal sequence analysis showed the expected sequence(1) , indicating that the recombinant modified IL-13 was correctly processed during secretion by COS-3 cells. The [Phe]IL-13-GYGY has a molecular mass of 12768 ± 3 by laser desorption time of flight mass spectrometry, close to the predicted M(r) of 12765.9, confirming the overall integrity of the molecule.

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 (box), I-labeled [Phe]IL-13-GYGY (bullet), or I-labeled IL-13 (circle) were tested in parallel. Each point represents the mean of triplicates.



Binding ofI-Labeled [Phe]IL-13-GYGY andI-Labeled IL-4 to Different Cell Lines

The results of binding experiments with I-labeled [Phe]IL-13-GYGY or I-labeled IL-4 on a variety of cell lines are shown in Table 1. Monocytic (U937 and THP-1), erythroleukemia (TF-1), keratinocyte (A431), and fibroblast (COS-3) cell lines express high affinity IL-13 binding sites. Even though in these cells IL-4 binding sites are also present, the binding characteristics are different because in some of these cells (THP-1 and COS-3) IL-13 fully competed with IL-4, and in the others (U937, TF-1 and A431) IL-13 only partially displaced the binding of iodinated IL-4. IL-13 specific binding was always fully displaced by IL-4 (results not shown). The B-lymphoblastic (Daudi), the pre-B (NALM-6), and the gibbon lymphoma (MLA-144) cell lines only displayed IL-4 specific binding.



Binding Experiments on COS-3 and A431 Cells

The receptors for IL-4 and IL-13 present on COS-3 cells, in which IL-4 and IL-13 completely displace each other, were characterized in displacement and saturation experiments. The competition of I-labeled IL-4 by unlabeled IL-4 and IL-13 showed, for IL-4, an IC of 50 ± 8 pM, and for IL-13, an IC of 500 ± 46 pM (Fig. 2A). Scatchard plots generated from saturation experiments done with I-labeled IL-4 showed the presence of only one class of binding sites with a K(d) of 30 ± 5 pM and 2200 ± 210 receptors/cell (Fig. 2B).


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 (box) 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 (box), IL-13 (), [Phe]IL-13-GYGY (circle), and cold labeled I-labeled [Phe]IL-13-GYGY (bullet). 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(d) = 300 ± 45 pM and 2500 ± 190 receptors/cell) (Fig. 2D). It should be noted that the K(d) 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 (box) or IL-13 (). B, Scatchard plot analysis from saturation isotherm data of the specific binding of I-labeled IL-4 (box). The saturation experiment with labeled IL-4 was also performed after preincubation of the cells with 100 nM of unlabeled IL-13 (circle). C, competitive displacement of I-labeled [Phe]IL-13-GYGY binding by IL-4 (box) 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(d) = 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(d) = 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).

Affinity Cross-linking of the IL-13 and IL-4 Receptors on COS-3 and A431 Cells

The molecular nature of the IL-4/IL-13 receptors was also investigated by cross-linking studies using both ligands, I-labeled IL-4 and I-labeled [Phe]IL-13-GYGY, on COS-3 and A431 cells.

The results are shown in Fig. 4. Three main broad bands (approx70, 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 (approx45 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 (approx70 and 220 kDa) on COS-3 cells and one (approx70 kDa) on A431 cells were consistently detected; a faint labeled complex, approx45 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-4bulletIL-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) .



Binding Experiments on Transfected COS-3 Cells

The relationship of the IL-13 receptor present on COS-3 cells with the other known components of the IL-4 receptor was explored in binding experiments on COS-3 cells expressing recombinant IL-4R and/or the chain molecules.

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.''




DISCUSSION

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(d) approx300 pM, while the IL-4 also binds to a single class of binding site with an affinity of approx20 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, (^3)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-4bulletIL-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-4bulletIL-13 receptor complex.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 33-61-39-96-00; Fax: 33-61-39-86-37.

(^1)
The abbreviations used are: IL, interleukin; chain, chain subunit of the IL-2 receptor.

(^2)
C. Labit-LeBouteiller, R. Astruc, A. Minty, and J. B. Lupker, manuscript in preparation.

(^3)
N. Vita, S. Lefort, P. Laurent, D. Caput, and P. Ferrara, unpublished results.


ACKNOWLEDGEMENTS

We thank M. Goncalves for help with the cross-linking experiments, C. Cassan, J. Capdevielle, and J. C. Guillemot for the amino acid and sequence analysis, C. Labit and F. Jamme for production of [Phe]IL-13-GYGY in COS cells, and R. Reeb for help with the culture of the cells. We also thank Dr. K. Sugamura for the cDNA of the human chain, Dr. J. M. Lelias for the cDNAs for human and simian IL-4R, and Dr D. Shire, M. Magazin, and A. Minty for critical reading of the manuscript and stimulating suggestions and discussions.


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