©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Primary Binding Subunit of the Human Interleukin-4 Receptor Is Also a Component of the Interleukin-13 Receptor (*)

Sandra M. Zurawski (1), Pascale Chomarat (2), Odile Djossou (2), Christine Bidaud (2), Andrew N. J. McKenzie (1)(§), Pierre Miossec (2), Jacques Banchereau (2), Gerard Zurawski (1)(¶)

From the (1) Department of Molecular Biology, DNAX Research Institute of Cellular and Molecular Biology, Palo Alto, California 94304-1104 and (2) Laboratory for Immunological Research, Schering-Plough France, Dardilly 69571, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Interleukin (IL)-13 elicits a subset of the biological activities of the related IL-4. The basis of this functional similarity is that their specific cell-surface receptors (called IL-13R and IL-4R) are distinct, yet are complex and share a common subunit(s). The IL-4R primary binding subunit (called IL-4R) does not by itself bind IL-13. We show that the ability of IL-13 to partially compete for IL-4 binding to some human cell types depended on co-expression of IL-4R and IL-13R. However, IL-13 binding was always associated with IL-4 binding. Hyperexpression of IL-4R on cells expressing both IL-4R and IL-13R decreased their binding affinity for IL-4, abrogated the ability of IL-13 to compete for IL-4 binding, and yet had no effect on IL-13R properties. Anti-human IL-4R monoclonal antibodies which blocked the biological function and binding of IL-4 also blocked the function and binding of IL-13. These data show that IL-4R is a secondary component of IL-13R.


INTRODUCTION

Adjacent genes encode the T cell-derived cytokines IL-13 ()and IL-4 (Morgan et al., 1992; McKenzie et al., 1993a). These proteins are distantly related at the amino acid sequence level and elicit similar proliferative responses and morphological changes on many cell types, including monocytes, B cells, endothelial cells, and natural killer cells. However, unlike IL-4, IL-13 has no effect on T cells (reviewed in Zurawski and de Vries(1994)). The overlap of these biological functions appears to be due to the specific high affinity cell surface receptors for IL-4 and IL-13 being distinct multisubunit complexes which share at least one component. The direct evidence to support this notion is 1) IL-13 can partially compete for IL-4 binding to a human cell line that responds to both IL-13 and IL-4, but cannot compete for IL-4 binding to a human T-cell line or to cells expressing the 130-kDa IL-4R protein (Zurawski et al., 1993a); and 2) a mutant human IL-4 antagonist protein, which binds to IL-4R with an affinity equal to that of hIL-4 and binds 100-fold less well than hIL-4 to IL-4R, also inhibits the biological action of IL-13 (Zurawski et al., 1993a; Aversa et al., 1993). In addition, the complex nature of IL-4R has been inferred from observations of IL-4R-associated proteins (Foxwell et al., 1989; Galizzi et al., 1990a; Harada et al., 1990) and heterogeneity in IL-4 binding (Foxwell et al., 1989; Fernandez and Vitetta, 1990, 1991) and activation (Rigley et al., 1991) studies. Recently, the IL-2 receptor subunit (IL-2R) has been identified as a component of human and mouse IL-4R that functions in signal transduction and slightly enhances the ability of IL-4R to bind IL-4 (Russell et al., 1993; Kondo et al., 1993, 1994).

In this work, we have further studied the ability of IL-13 to cross-compete for IL-4 binding on human and monkey cell types and utilized for the first time direct IL-13 binding analyses to characterize human IL-13R properties. In addition, we have used anti-human IL-4R mAbs to show that IL-4R is a component of IL-13R.


EXPERIMENTAL PROCEDURES

Cell Culture and Reagents

Purified hIL-4 (10 units/mg), produced in Escherichia coli (Le et al., 1988), was obtained from Schering-Plough Research (Kennilworth, NJ). Purified hIL-13 and mIL-13 were prepared as described by McKenzie et al. (1993b). The soluble extracellular domain of hIL-4R (called hIL-4R) was derived from Cos-7 cells transfected with a plasmid obtained by DraIII digestion of the full-length hIL-4R cDNA (Galizzi et al., 1990b) and encoded a 200-amino acid protein. hIL-4R was purified from transfected cell culture supernatants by hIL-4-Affi-Gel 10 chromatography as described by Galizzi et al. (1990a). Purified phytohemagglutinin (PHA) was from Wellcome (Dartford, Great Britain). The fluorescein isothiocyanate-conjugated F(ab`) fragment of goat anti-mouse Ig was from Grub (Vienna, Austria). Phycoerythrin-conjugated streptavidin (streptavidin-PE) was from Becton Dickinson (Mountain View, CA). Jijoye cells, a Burkitt lymphoma; Jy, an Epstein-Barr virus-transformed human B-cell line; Cos-7, a monkey kidney fibroblast cell line; NALM6, a pre-B human cell line (Hurwitz et al., 1979); L cells, a fibroblast cell line; and NS1, a myeloma cell line, were from ATCC. Human monocytes used in the binding studies were prepared as described by de Waal Malefyt et al.(1993). Monocytes used for the cell surface expression studies were purified in a standard chamber by counterflow centrifugal elutriation (J2-21 M elutriation system, Beckman Instruments) from peripheral blood mononuclear cells isolated by Ficoll-Hypaque centrifugation. This monocyte preparation was >90-95% pure, as determined by flow cytometry and May Grünwald Giemsa staining. Jijoye, NS1, L cells, tonsil B cells, and monocytes were cultured in complete culture medium RPMI 1640 (Life Technologies, Inc.) supplemented with 10% fetal calf serum, 2 mML-glutamine, 50 µg/ml gentamycin (Schering-Plough). Other cells were cultured as described by Zurawski et al. (1993a). Geneticin G418 was from Life Technologies, Inc., or from Schering-Plough and was used at 1 mg/ml. Where not specified, chemicals were from Sigma.

Transfection and Fluorescence-activated Sorting of L Cells

The neomycin resistance gene was introduced into a mammalian expression vector, pME18s (Kitamura et al., 1991), containing the hIL-4R cDNA (pME18s neo hIL-4R). L cells, plated at 10 cells/10-cm plate the previous day, were harvested by trypsinization and washed once with RPMI 1640, 10% fetal calf serum and resuspended at 10 cells/ml in RPMI 1640. 25 µg of linearized (KpnI endonuclease) plasmid (pME18s neo hIL-4R or pME18s neo) DNA were added to 0.8 ml of cell suspension (10 cells/ml) in a 0.4-cm electroporation cuvette (Bio-Rad). Electroporation was performed using a gene pulser (Bio-Rad) at 960 mF and 250 V. Transfectants were selected with G418. After 1 week of selection, 4 10 cells were harvested in phosphate-buffered saline (PBS, 10 mM sodium phosphate, 150 mM NaCl, 3.6 mM KCl, pH 7.4) with 0.5 mM EDTA and washed by centrifugation, and the pellet was resuspended at 10 cells/ml in PBS, 0.5 mM EDTA, 0.1% bovine serum albumin (PEB buffer). The cell suspension was incubated (4 °C) with 0.1 µM biotinylated IL-4 in the absence or presence (nonspecific) of 4 µM unlabeled hIL-4. After 90 min of incubation at 4 °C, cells were washed twice by centrifugation with PEB buffer and further incubated with a 1/5 dilution of streptavidin-PE for 30 min at 4 °C (10 cells/ml). Then, samples were washed twice by centrifugation and resuspended at 10 cells/ml in PEB. Fluorescence-activated cell sorting (FACS) was on a FACS 440 (Becton Dickinson) equipped with a 5-W argon laser running at 488 nm, 0.5 W. The 4% brightest stained cells were collected at each sort and cultured in G418-containing culture medium until the next sort. After four cycles of sorting, cells were cloned at 0.5 cell/well in 96-well plates. One clone (C70), selected among 300, expressed 500,000 sites/cells, as measured by I-hIL-4 binding.

Generation of Monoclonal Anti-IL-4R Antibodies (mAbs)

Four BALB/C mice (Iffa Credo, Les Oncins, France) were immunized intraperitoneally with 10 µg of hIL-4R in Freund's complete adjuvant, as described previously (Garrone et al., 1991). At 3-week intervals, booster immunizations (2 10 µg, 1 50 µg) were given in Freund's incomplete adjuvant. Immune mouse spleen cells (3 10 cells) were fused with NS1 cells (ratio 5:1) using 50% polyethylene glycol 1500 (Boehringer Mannheim) in 50 mM Hepes, pH 7.0. Then 20 96-well plates were seeded at 2 10 spleen B cells/well (100 µl) in RPMI 1640 medium containing 0.1 mM hypoxanthine, 58 µM azaserine, 0.15 mg/ml oxaloacetate, 50 µg/ml pyruvate, 0.2 IU/ml insulin, 10% hybridoma cloning efficiency factor (EGS, Interchim), 20% fetal calf serum, 2 mML-glutamine, and 40 µg/ml gentamycin. Hybridomas secreting antibodies specific for hIL-4R were screened for their ability to stain L cells transfected with hIL-4R by FACscan analysis, and cloned by limiting dilution. Positive hybridomas were produced in ascites from pristane-treated BALB/c mice, and mAbs were purified by an anion exchange chromatography (DEAE 5PW, Waters Associates, Millipore Corp., Milford, MA). The mAb isotype was determined with the mouseTyper Isotyping kit (Bio-Rad).

Inhibition of I-hIL-4 Binding to Cells

hIL-4 was labeled with NaI using a Tejedor-Ballesta method at the specific radioactivity of 2000-4000 cpm/mmol, as described earlier (Cabrillat et al., 1987) or as described below for labeling mIL-13. Binding on Jijoye cells and PHA blasts was performed as described (Galizzi et al., 1990a). Briefly, 3 10 cells were incubated in RPMI 1640, 1% bovine serum albumin at 4 °C in the presence of increasing concentrations of sera, hybridoma supernatant, or purified mAbs for 30 min. Then, 50 pM of I-hIL-4 was added with or without 10 µM cold hIL-4. After 4 h, samples (500 µl total) were washed twice by centrifugation with 2 ml of binding medium at 4 °C and radioactivity of the cell pellet was determined in a gamma counter. Binding on all other cell types was similar, but with the modifications previously (Zurawski and Zurawski, 1992) noted.

Biotinylation and Cross-competition between mAbs for Binding to Cell Lines

1 mg of mAb was biotinylated as described by Bayer (1980). 5 10 cells (C70) were incubated with unlabeled mAb in PBS, 1% bovine serum albumin, 0.1% azide (PBA), 15 min at 4 °C. Cells were washed twice with 200 µl of PBA and stained with 200 ng of the biotinylated anti-hIL-4R mAbs for 15 min. After washing twice, cells were incubated with 100 µl (1/5 dilution in PBA) of streptavidin-PE, washed twice, and analyzed by FACS.

Immunoprecipitation and Western blot Analysis

I-labeled hIL-4R was prepared at a specific radioactivity of 3300 Ci/mmol by incubating 1 µg of hIL-4R with 0.5 mCi of NaI and 25 µl of chloramine T (10 µg/mg protein) in 150 µl of PBS for 5 min. Free I was separated from bound by PD10 gel permeation (Pharmacia, Uppsala, Sweden). 10 µg of anti-mouse IgG (Sigma) in 100 µl of PBS was coated on polyvinyl microplates (96 wells, Dynatech) at 4 °C, overnight. Plates were then saturated 1 h at 25 °C with 300 µl of 10% fetal calf serum, PBS. After washing with PBS (four times), wells were filled with 100 µl (10 µg/ml) of anti-hIL-4R mAbs, 4 h at 4 °C. Wells were incubated with 3 nMI-hIL-4R in PBS, 10% fetal calf serum (100 µl). After 12 h, wells were washed, and radioactivity was extracted with SDS-gel buffer and counted. Samples were also analyzed by electrophoresis to confirm the presence of hIL-4R.

Proliferation of PHA Blasts

To test the effects of anti-hIL-4R mAbs on PHA blast proliferation, peripheral blood mononuclear cells purified from normal blood were cultured at 1 10 cells/ml with 1 µg/ml PHA in complete RPMI 1640 medium. After 4 days, proliferation of cells was measured by [H]deoxythymidine incorporation as described previously (Garonne et al., 1991).

Biological Assays on B Lymphocytes

Functional properties of anti-hIL-4R mAbs were also studied on B lymphocytes purified from tonsils as described previously (Defrance et al., 1987). Study of CD23 and surface IgM expression or proliferation assays were previously described (Garonne et al., 1991).

Purification of Proteins and Radiolabeling of mIL-13

E. coli-derived hIL-4, hIL-4.Y124D, hIL-13, and mIL-13 were purified as described previously (van Kimmenade et al., 1988; McKenzie et al., 1993b). mIL-13 (5 µg) was radiolabeled to a specific activity of 1525 Ci/mmol with 2.5 mCi of NaI (IMS30, Amersham) in 100 µl of PBS and 5 µg of Iodogen (Pierce) for 5 min followed by PD-10 gel permeation in RPMI 1640, 2% bovine serum albumin, 0.01% NaN, 20 mM Hepes.

Affinity Labeling of IL-4R and IL-13R Proteins

Chemical cross-linking was performed essentially as described by Kitamura et al.(1991). Cells were incubated with 2 nMI-hIL-13 or 1 nMI-hIL-4 in the presence or absence of a 100-fold excess of unlabeled hIL-13 or hIL-4. After 2 h at 4 °C, the cells were collected by centrifugation, washed once in PBS containing 200 mM bis-sulfosuccinimidyl suberate (BS, Pierce), then incubated in this buffer for 30 min at 4 °C. Following centrifugation the cell pellet was solubilized in 1% Triton X-100, 500 mM Na EDTA, 100 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 1 µg/ml pepstatin A, 10 µg/ml antipapain, and analyzed on 7.5% gel SDS-polyacrylamide gel electrophoresis followed by autoradiography.

Determination of hIL-6 Production by Human Monocytes

Monocytes were activated in 96-well plates for 48 h with lipopolysaccharide (LPS, 1 µg/ml), alone or in combination with hIL-4 (0.001-100 units/ml) and/or hIL-13 (0.0001-100 ng/ml), or anti-IL-4R mAbs (20 µg/ml). After incubation, cell-free supernatants were collected and frozen at -20 °C until IL-6 levels were determined. Levels of hIL-6 were measured by specific enzyme-linked immunosorbent assay (sensitivity: 600 pg/ml) using two rat mAbs (39C3 for IL-6 coating and biotinylated 13A5 for IL-6 detection) kindly provided by Dr. J. S. Abrams (Abrams et al., 1992).

Immunofluorescence Analysis of Monocyte Cell Surface Markers

After 48 h of culture, monocytes (3 10 cells/bag) were harvested from Teflon bags. For fluorescence staining, cells were incubated with 10 µl of fluorescein isothiocyanate-labeled mAbs: CD14, HLA-DR, HLA-DQ (Becton Dickinson), or 10 µl of PE-labeled mAb CD23 (Serotec). After washes, the labeled cell samples were analyzed by flow cytometry on FACscan.


RESULTS

Characteristics of IL-4R on Various Cell Types

Competitive I-hIL-4 displacement studies have defined three classes of IL-4 binding (Zurawski et al., 1993a). 1) Cells expressing only human IL-4R bind hIL-4 and the biologically inactive hIL-4 Tyr Asp mutant protein (hIL-4.Y124D) with a similar affinity (K10M). This type of hIL-4 binding is not competed for by hIL-13. 2) hIL-4-responsive human SP-B21 T cells bind hIL-4.Y124D at this same affinity, but bind hIL-4 100-fold more avidly. This type of hIL-4 binding is also not competed for by hIL-13. 3) IL-4R on hIL-4- and IL-13-responsive human pre-myeloid erythroleukemic TF-1 cells are like those on SP-B21 cells, except that hIL-13 can partially (a maximum of 65%) compete for I-hIL-4 binding. We have extended these studies to other cell types which are known to bind hIL-4. Fig. 1A shows that IL-4R on monkey kidney Cos-7 cells had properties identical to those described above for TF-1 cells. Human monocytes, which respond similarly to both hIL-4 and IL-13 (McKenzie et al., 1993b; Minty et al., 1993; de Waal Malefyt et al., 1993; Herbert et al., 1993), had fewer IL-4R, but hIL-13 competed for I-hIL-4 binding similarly to that observed for IL-4R on TF-1 and Cos-7 cells (Fig. 1B). The Epstein-Barr virus-transformed human Jy B-cell line had IL-4R with properties identical to SP-B21 cells, except that hIL-13 competed for only 20% of the bound I-hIL-4 (Fig. 1C). Collectively, these hIL-4 binding data establish that high affinity IL-4R on various cell types are similar and that hIL-4.Y124D typically binds to such IL-4R 100-fold less avidly than hIL-4. In contrast, the ability of hIL-13 to compete for I-hIL-4 binding varies dramatically with cell type. Simplistically, these data suggest that there are subtypes of high affinity IL-4R (i.e. those which can also bind IL-13 and those which cannot bind IL-13). To explore this notion, we developed a radiolabeling protocol for IL-13 which permitted direct examination of IL-13R.


Figure 1: Competition displacement of I-hIL-4 binding to various cell types. Various amounts of unlabeled hIL-4 (), hIL-4.Y124D (), and hIL-13 () were incubated for 2 h at 4 °C with 2 10MI-hIL-4; I-hIL-4 bound to cells was then determined. Panel A, Cos-7 cells, maximum was 5810 ± 314 cpm, minimum was 93 ± 51 cpm, 23% of the bound counts were not competed for by hIL-13. Panel B, monocytes, maximum was 702 ± 66 cpm, minimum was 73 ± 16 cpm, 23% of the bound counts were not competed for by hIL-13. Panel C, Jy cells, maximum was 3790 ± 161 cpm, minimum was 270 ± 25 cpm, 79% of the bound counts were not competed for by hIL-13. Arrows on the abscissa indicate the dose required to displace 50% of the maximal counts displaced (IC). Error bars represent standard deviation of the mean (S.D., n = 2).



Properties of I-IL-13 Binding to Cos-7 Cells

IL-4R on Cos-7 cells are of the type where IL-13 partially competes for I-hIL-4 binding, inferring the existence of IL-13R on these cells. This was demonstrated directly by the ability of I-mIL-13 to bind specifically to these cells (Fig. 2A). Specific I-mIL-13 binding to Cos-7 cells saturated at 5 10M (no specific binding was observed above 10MI-mIL-13 due to high nonspecific binding). Scatchard analysis of the data was most consistent with a single class of I-mIL-13 binding site with K10M (Fig. 2A). Kinetic analysis revealed that I-mIL-13 associated with Cos-7 cells relatively slowly (t 60 min, Fig. 2B).


Figure 2: Binding of I-mIL-13 to Cos-7 cells. Panel A, Various amounts of labeled mIL-13, together with () or without (), a 500-fold excess of unlabeled hIL-13 were added to cells and incubated for 2 h at 4 °C; I-mIL-13 bound to cells was then determined. Analysis of specific binding () by the program Ligand (Munson and Rodbard, 1980) indicated the presence of 1000 binding sites with a single affinity class of K 1.4 10M. Panel B, 10MI-mIL-13 was added to cells at 4 °C and aliquots were removed at various times and I-mIL-13 bound to cells was then determined. The inset shows a natural log plot of the data with [HR], bound I-mIL-13 at time t; [HReq], bound I-mIL-13 at equilibrium.



Characteristics of IL-13R on Various Cell Types

Competitive I-mIL-13 displacement studies on Cos-7 cells, TF-1 cells, and human monocytes were remarkably similar. These data (Fig. 3, A-C) revealed that hIL-13 binds to these cells slightly more avidly than does mIL-13 (K= 2 10Mversus 6 10M) and that hIL-4 is a potent (K= 1 10M) and complete competitor of I-mIL-13 binding. Cos-7 had 1000 IL-13R/cell, while TF-1 and monocytes had 70 IL-13R/cell. In similar experiments, no specific I-mIL-13 binding was detected on NALM6 cells, a human pre B-cell line (Hurwitz et al., 1979) on which I-hIL-4 binding was not inhibited by up to 10M IL-13 (data not shown). There was an absolute correlation between the existence of the IL-4R subtype which interacts with IL-13 and the presence of IL-13R as detected by IL-13 binding.


Figure 3: Competition displacement of I-mIL-13 binding to various cell types. Various amounts of unlabeled hIL-4 (), hIL-13 (), and mIL-13 () were incubated for 2 h at 4 °C with 10MI-mIL-13; I-mIL-13 bound to cells was then determined. Panel A, TF-1 cells, maximum was 496 ± 30 cpm, minimum was 243 ± 20 cpm. Panel B, monocytes, maximum was 202 ± 16 cpm, minimum was 80 ± 12 cpm. Panel C, Cos-7 cells, maximum was 1388 ± 71 cpm, minimum was 367 ± 32 cpm. Using similar conditions, no specific binding to NALM6 cells was detected. Arrows on the abscissa indicate IC. Error bars represent S.D. (n = 2).



Effects of IL-4R Hyperexpression on IL-4R and IL-13R

To further explore the notion that a subtype of IL-4R can bind IL-13, we took advantage of a transient expression system in Cos-7 cells. Previous studies demonstrated that binding of hIL-4 to Cos-7 cells hyperexpressing hIL-4R is not competed for by IL-13 (Zurawski et al., 1993a). With the availability of a direct probe for IL-13 binding, we could investigate the effects of expressing various amounts of hIL-4R on a cell type which expresses both subtypes of IL-4R. Increasing hIL-4R expression on Cos-7 cells resulted in an enhanced total bound hIL-4, concomitant with decreased apparent affinity of binding by hIL-4 (Fig. 4A). This result is expected since additional hIL-4R contributes to binding hIL-4, but its affinity for hIL-4 is markedly less than that of the endogenous IL-4R. Of interest was the observation that these changes in IL-4R were associated with a decreased maximal ability of IL-13 to compete for binding by hIL-4, without concomitant changes in the potency of IL-13 competition or changes in number and affinity of IL-13R (Fig. 4, B and C). These observations are consistent with a model in which the ability of IL-13 to compete for binding of IL-4 to IL-4R is determined by the relative abundance of IL-4R subtypes and show that the IL-4R subtype which interacts with IL-13 on Cos-7 cells is not limited by availability of IL-4R.


Figure 4: Effect of hyperexpression of hIL-4R on the properties of IL-4R and IL-13R on Cos-7 cells. Panel A, various amounts of unlabeled hIL-4 were incubated for 2 h at 4 °C with 10MI-hIL-4; I-hIL-4 bound to 10 cells was then determined. Cells (10) were untransfected () or transfected with 5 µg (), 15 µg (), or 40 µg () hIL-4R expression plasmid. Panel B, various amounts of unlabeled mIL-13 (circles) or unlabeled hIL-13 (squares) were incubated with 10MI-hIL-4, and I-hIL-4 bound to cells was then determined. Cells were untransfected (, ) or transfected with 5 µg (,), 15 µg (,), or 40 µg (,) hIL-4R expression plasmid. Panel C, various amounts of unlabeled hIL-13 were incubated with 10MI-mIL-13, and I-mIL-13 bound to cells was then determined. Cells were untransfected () or transfected with 5 µg (), or 40 µg () hIL-4R expression plasmid. Arrows on the abscissa indicate IC.



IL-13R Cross-linking Studies

Cross-linking studies with I-hIL-4 and various human cell lines reveal a predominant 130-140-kDa hIL-4R protein (reviewed in Harada et al. (1992a)). These same studies suggested that other proteins of 80 and 65 kDa could sometimes be found associated with hIL-4R, and other studies show that the 64-kDa human IL-2R protein is a component of IL-4R (Russell et al., 1993; Kondo et al., 1993, 1994). We attempted to identify protein components specific to IL-13R by cross-linking I-mIL-13 bound to TF-1 and Cos-7 cells. On both cell types, I-mIL-13 affinity-labeled a predominant protein of 65 kDa (assuming a 1:1 complex with 11.6-kDa mIL-13) and this affinity-labeled species was not detected in the presence of excess unlabeled hIL-13 or hIL-4 (Fig. 5). This property correlates absolutely with the binding properties of IL-13R and contrasts with parallel experiments utilizing I-hIL-4 as the affinity probe. On Cos-7 cells, I-hIL-4 predominantly labeled a 130-140-kDa species and minor 65-kDa species which were partially competed for by unlabeled hIL-13 and completely competed for by unlabeled hIL-4 (Fig. 5). On TF-1 cells, I-hIL-4 labeled 130-140-kDa, 95-kDa, and 65-kDa species which were competed for by both unlabeled hIL-13 and hIL-4 (Fig. 5). Taken together, these data reveal significant differences between IL-4R and IL-13R and tentatively identified a 65-kDa IL-13R protein(s) as the primary binding protein for IL-13. Further such work with other cell types should reveal whether the 65-kDa protein is specific to IL-13R.


Figure 5: Affinity labeling of IL-4R and IL-13R proteins on Cos-7 and TF-1 cells. Cells were incubated for 2 h at 4 °C with I-hIL-13 or I-hIL-4 in the presence or absence of excess unlabeled hIL-13 or hIL-4, then washed and incubated with cross-linking agent. Solubilized cells were then subjected to SDS-polyacrylamide gel electrophoresis and autoradiography. Positions of the cross-linked receptor subunits are indicated by arrows. The high molecular mass band (>200 kDa) may represent a higher order complex.



Production and Characterization of Anti-IL-4R Monoclonal Antibodies

We developed mAbs against hIL-4R to aid in the further study of IL-4R and IL-13R. A 200-amino acid extracellular portion of hIL-4R (hIL-4R) was used to immunize mice. We subsequently derived several hybridomas which produced mAbs reactive in FACS analysis to an L cell line stably expressing hIL-4R (C70), but were not reactive to the parental L cell line. The mAbs varied in their reactivity to hIL-4R as judged by blotting, precipitation, inhibition of I-hIL-4 binding, and cross-competition (). shows that three of the mAbs were effective inhibitors of I-hIL-4 binding to monocytic (monocytes), B-cell (Jijoye), and T-cell (PHA blasts) types. As expected from their ability to inhibit binding of hIL-4, these mAbs also abolished IL-4-induced biological effects, such as proliferation of B and T cells and induction on B cells of low affinity IgE receptor (FcII) and surface IgM (Fig. 6), as described previously (Defrance et al., 1987; Garonne et al., 1991).


Figure 6: Inhibition of hIL-4-induced biological effects by anti-hIL-4R mAbs. Panel A, peripheral blood mononuclear cells were preactivated for 4 days with 1 µg/ml PHA and viable blasts were recultured for 3 days with 5 10M hIL-4 and various concentrations of mAbs s103 (), s456 (), and s924 (). Panel B, purified tonsil B cells were cultured for 3 days with 5 µg/ml insolubilized anti-IgM and 5 10M hIL-4 and various concentrations of the above mAbs. The response was measured by uptake of [H]thymidine. Panels C and D, purified tonsil B cells were cultured for 48 h with 2.5 10M hIL-4 and various concentrations of the above mAbs. The response was measured by staining cells with specific anti-CD23 (Panel C) or anti-IgM (Panel D) antibodies together with fluorescein isothiocyanate-labeled anti-mouse IgG and FACscan analysis.



Anti-IL-4R Monoclonal Antibodies Inhibit IL-13 Binding

The above and previous data show that, although hIL-4R alone cannot bind IL-13, there appears to be an absolute correlation between IL-4R and IL-13R. We utilized the anti-hIL-4R mAbs to investigate a possible role for hIL-4R in IL-13R. Two anti-hIL-4R mAbs tested were equipotent inhibitors of both I-hIL-4 binding and I-mIL-13 binding to TF-1 cells (Fig. 7). These data revealed that hIL-4R, which itself cannot bind IL-13, is an important component for the binding of IL-13 by IL-13R.


Figure 7: Displacement by anti-hIL-4R mAbs of I-mIL-13 and I-hIL-4 binding to TF-1 cells. Various amounts of unlabeled hIL-4 (), mIL-13 (), and mAbs s456 (), s103 (), or o361 (*) were incubated for 2 h at 4 °C with Panel A, 10MI-hIL-4 or Panel B, 10MI-mIL-13, and radioligand bound to cells was then determined.



Anti-IL-4R Monoclonal Antibodies Inhibit IL-13 Action

IL-13 and IL-4 elicit diverse effects on monocytic cells (McKenzie et al., 1993b; Herbert et al., 1993; de Waal Malefyt et al., 1993; Doherty et al., 1993) including inhibition of LPS-stimulated synthesis of pro-inflammatory cytokines such as IL-6 and regulation of cell surface marker expression such as CD23, CD16, and MHC class II. We first assessed the effect of anti-IL-4R mAbs on IL-6 production by LPS-activated human monocytes cultured in the presence of IL-13. Fig. 8shows that the two blocking anti-IL-4R mAbs tested reversed the hIL-13-induced inhibition of hIL-6 production by monocytes. This effect was not observed with a control mAb, the nonblocking anti-IL-4R mAb s697 (Fig. 8), or with an anti-IL-4 mAb (data not shown).


Figure 8: Reversal by anti-hIL-4R mAbs of hIL-13 inhibition of monocyte hIL-6 production. LPS-activated human monocytes were incubated alone or with hIL-13 and with and without mAbs for 48 h. Supernatants were then collected and levels of hIL-6 produced were determined. Similar experiments (not shown) established that 5 ng/ml hIL-13 elicits a 50% maximal response and 50 ng/ml elicits a 90% maximal response. Also, blocking anti-hIL-4 mAb and non blocking anti-hIL-4R mAb s697 (both at 20 µg/ml) had no effect on hIL-13 action (data not shown).



We also investigated whether anti-IL-4R mAbs reversed the hIL-13 effects on the expression of the monocyte cell-surface markers CD23, CD14, and MHC class II. Fig. 9shows that the anti-IL-4R mAbs s103 and s924 reversed in a dose-dependent fashion the hIL-13- and hIL-4-induced increase of CD23 expression on monocytes. No inhibition of CD23 induction was seen with the nonblocking anti-IL-4R mAb s697. Furthermore, an anti-hIL-4 mAb blocked CD23 expression induced by hIL-4 but did not block CD23 expression induced by hIL-13 (data not shown). Similar results were obtained with anti-IL-4R mAbs when we investigated the expression of CD14 and MHC class II molecules on monocytes in the presence of hIL-4 or hIL-13 (Fig. 10). In addition, we have previously shown that s460 reverses hIL-13 inhibition of the growth of a leukemic human B-cell precursor (Renard et al., 1994). Together, these data show that IL-4R is a functionally important component of IL-13R.


Figure 9: Reversal by anti-hIL-4R mAb of hIL-4- and hIL-13-directed enhancement of monocyte CD23 expression. Human monocytes were cultured with LPS in the presence of 50 units/ml hIL-4 or 50 ng/ml hIL-13 with and without various amounts (indicated in micrograms/ml in the parentheses) of the anti-hIL-4R mAb s103. After 48 h the cells were stained with phycoerythrin-labeled CD23 antibody and analyzed by FACscan flow cytometry. Parallel experiments (not shown) revealed that mAb s924 had similar though slightly less potent effects and that the nonblocking anti-hIL-4R mAb s697 and blocking anti-hIL-4 mAb had no effect on hIL-13 action.




Figure 10: Reversal by anti-hIL-4R mAb of hIL-13-directed changes of monocyte CD14, HLA-DR, and HLA-DQ expression. Human monocytes were cultured with LPS in the presence and absence (medium control, left panels) of 50 ng/ml hIL-13 with (shaded histogram in center and right panels) and without (unshaded histograms) 20 µg/ml of the anti-hIL-4R mAb s456. After 48 h the cells were stained with fluorescein isothiocyanate-labeled CD14, HLA-DR, and HLA-DQ antibodies and analyzed by FACscan flow cytometry. Parallel experiments (not shown) revealed that mAb s103 had effects similar to those of s456 on CD14 and HLA-DR expression and that these effects were proportionally greater at suboptimal levels of hIL-13.




DISCUSSION

In this work, we have further studied the structures and relatedness of IL-4R and IL-13R. When probed with hIL-4 and the antagonist hIL-4.Y124D, the properties of IL-4R on various human cell types were remarkably similar. In all cases, hIL-4.Y124D bound to IL-4R 100-fold less avidly than did hIL-4. We have previously observed that the cloned hIL-4R protein alone binds both hIL-4 and hIL-4.Y124D with the lesser affinity characteristic of hIL-4.Y124D binding to IL-4R (Zurawski et al., 1993a). These data imply that an additional subunit associates with hIL-4R to enhance its affinity for hIL-4 and that this association facilitates signaling and involves interaction with the Y124D residue of hIL-4. The hIL-2R subunit appears to associate with hIL-4R, although co-expression of hIL-4R and hIL-2R did not enhance the affinity of hIL-4R to the extent expected from the above studies (Russell et al., 1993). IL-2R plays similar affinity-enhancing and signaling roles in IL-4R and IL-2R (where it interacts with IL-2R or IL-2R; reviewed in Minami et al.(1993)). Additionally, mutations in similar locations, but different side chains (Gln in IL-2, Tyr in IL-4) within the C-terminal -helix of IL-2 and IL-4 abrogate interaction with IL-2R (Zurawski et al., 1993a, 1993b).

In contrast to the homogeneity of hIL-4R that we observed on various cell types probed with hIL-4 or hIL-4.Y124D, the ability of IL-13 to compete for hIL-4 binding to these same cells varied widely, indicating that this is not a general property of IL-4R. The simplest notion accounting for the above data is the existence of two IL-4R subtypes, only one of which binds IL-13. This concept of two IL-4R subtypes was supported by our observations of IL-13R using radiolabeled mIL-13 as a probe. IL-13R were detected only on those cell types on which IL-13 effectively competed for IL-4 binding. Importantly, IL-13 binding to IL-13R was always fully competed for by hIL-4. These data suggest a model where one type of IL-4R, the complex of IL-4R + IL-2R, cannot bind IL-13, while the other type of IL-4R is IL-13R.

Two studies permitted us to define the composition of IL-13R. First, a cross-linking study revealed a 65-kDa protein that was specifically associated with IL-13R. It is most likely that this 65-kDa affinity-labeled protein is the primary binding subunit of IL-13R (which we shall here refer to as IL-13R). The 65-kDa protein is distinct from hIL-2, since hIL-2R and IL-4R are present on cells such as human SP-B21 T cells which do not bind IL-13 (data not shown). Second, we observed that certain anti-hIL-4R mAbs exhibited equipotent inhibition of IL-4 and IL-13 binding and biological activity. These data show that hIL-4R is a component of IL-13R. Thus IL-13R is composed of IL-4R + IL-13R.

Our characterization of IL-13R accounts for a number of biological and receptor binding observations. Biological studies (reviewed in Zurawski and de Vries(1994)) have revealed that IL-13 elicits only a subset of the responses of IL-4. This occurs since IL-4R (as IL-4R + IL-2R) appears on a large number of cell types, while IL-13R, whose presence determines the ability of a cell to bind IL-13, has a more restricted distribution. The remarkably similar cellular responses to IL-4 and IL-13 (Zurawski and de Vries, 1994) reflect the action of the intracellular domain of the shared IL-4R in signaling (Harada et al., 1992b). The hIL-4.Y124D antagonist acts against responses to both IL-4 and IL-13 (Zurawski et al., 1993a) since it binds normally to hIL-4R, which is a component of both IL-4R and IL-13R. The ability of IL-13 to compete for IL-4 binding varies widely with cell type because the prevalence of IL-4R, IL-2R, and IL-13R varies. We demonstrated this directly by showing that increasing levels of hIL-4R decreased the maximal ability, but not the potency, of IL-13 to compete for hIL-4 binding. This and the concomitant decrease in the affinity of hIL-4 binding (due to increased ratio of IL-4R to IL-2R) had no effect on the numbers or binding properties of IL-13R.

It is possible that IL-13R contains IL-2R as well as IL-4R + IL-13R; however, recent studies (Matthews et al., 1994) show that IL-13 responses in B cells from two X-SCID patients with characterized IL-2R mutations do not respond to IL-2, but respond normally to IL-13. These same B cells respond normally to IL-4, suggesting that IL-4 can signal via binding to IL-13R.

The anti-hIL-4R mAbs which we developed will be important reagents for further characterizations of IL-4R abundance and distribution. Since some recognize distinct epitopes, they can be used in solid phase assays for the detection of hIL-4R in biological fluids (not shown). Such soluble forms of IL-4R have been detected in mouse sera (Fernandez and Vitetta, 1990, 1991; Fanslow et al., 1990) and may be indicators of disease states such as allergy and graft rejection.

  
Table: Properties of anti-hIL-4Ra mAbs


  
Table: Inhibition of hIL-4 binding to cells by anti-hIL-4R mAbs



FOOTNOTES

*
The work of the DNAX Research Institute and the Laboratory for Immunological Research is supported by Schering-Plough. 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.

§
Current address: Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK.

To whom correspondence should be addressed: DNAX Research Institute of Cellular and Molecular Biology, Palo Alto, CA 94304-1104. Tel.: 415-496-1255; Fax: 415-496-1214.

The abbreviations used are: IL, interleukin; hIL, human interleukin; mIL, mouse interleukin; Ig, immunoglobulin; LPS, lipopolysaccharide; mAb, monoclonal antibody; PHA, phytohemagglutinin; R, receptor; R, primary binding component of the receptor; PE, phycoerythrin; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorting; MHC, major histocompatibility complex.


ACKNOWLEDGEMENTS

We thank Felix Vega, Jr., for purification of proteins, Nithya Rajan and Shane Tareni (S-P Research for providing mAbs, Rene de Waal Malefyt for providing monocytes, Gregorio Aversa for the Jy cell line, Sem Saeland for the NALM6 cell line, and Jean-Pierre Galizzi and Smina Ait-Yahia for their contributions to the generation of anti-IL-4R mAbs.


REFERENCES
  1. Abrams, J. S., Roncarolo, M. G., Yssel, H., Andersson, U., Gleich, G. J., and Silver, J. E.(1992) Immunol. Rev. 127, 5-24 [Medline] [Order article via Infotrieve]
  2. Aversa, G., Punnonen, J., Cocks, B. G., de Waal Malefyt, R., Vega, F., Jr., Zurawski, S. M., Zurawski, G., and de Vries, J. E.(1993) J. Exp. Med. 178, 2213-2218 [Abstract]
  3. Cabrillat, H., Galizzi, J. P., Djossou, O., Arai, N., Yokota, T., Arai, K., and Banchereau, J.(1987) Biochem. Biophys. Res. Commun. 149, 995-1001 [Medline] [Order article via Infotrieve]
  4. Defrance, T., Vanbervliet, B., Aubry, J. P., Takebe, Y., Arai, N., Miyajima, A., Yokota, T., Lee, F., Arai, K., de Vries, J. E., and Banchereau J.(1987) J. Immunol. 139, 1135-1141 [Abstract/Free Full Text]
  5. de Waal Malefyt, R., Figdor, C. G., Huijbens, R., Mohan-Peterson, S., Bennett, B., Culpepper, J., Dang, W., Zurawski, G., and de Vries, J. E. (1993) J. Immunol. 151, 6370-6381 [Abstract/Free Full Text]
  6. Doherty, T. M., Kastelein, R., Menon, S., Andrade, S., and Coffman, R. L.(1993) J. Immunol. 151, 7151-7160 [Abstract/Free Full Text]
  7. Fanslow, W. C., Sims, J. E., Sassenfeld, H., Morrissey, P. J., Gillis, S., Dower, S. K., and Widmer, M. B.(1990) Science 248, 739-742 [Medline] [Order article via Infotrieve]
  8. Fernandez, B. R., and Vitetta, E. S.(1990) Proc. Natl. Acad. Sci. U. S. A. 87, 4202-4206 [Abstract]
  9. Fernandez, B. R., and Vitetta, E. S.(1991) J. Exp. Med. 174, 673-681 [Abstract]
  10. Foxwell, B. M., Woerly, G., and Ryffel, B.(1989) Eur. J. Immunol. 19, 1637-1641 [Medline] [Order article via Infotrieve]
  11. Galizzi, J. P., Castle, B., Djossou, O., Harada, N., Cabrillat, H., Yahia, S. A., Barrett, R., Howard, M., and Banchereau, J. (1990a) J. Biol. Chem. 265, 439-444 [Abstract/Free Full Text]
  12. Galizzi, J. P., Zuber, C. E., Harada, N., Gorman, D. M., Djossou, O., Kastelein, R., Banchereau, J., Howard, M., and Miyajima, A. (1990b) Int. Immunol. 2, 669-675 [Medline] [Order article via Infotrieve]
  13. Garonne, P., Djossou, O., Galizzi, J. P., and Banchereau, J.(1991) Eur. J. Immunol. 21, 1365-1369 [Medline] [Order article via Infotrieve]
  14. Harada, N., Castle, B. E., Gorman, D. M., Itoh, N., Schreurs, J., Barrett, R. L., Howard, M., and Miyajima, A.(1990) Proc. Natl. Acad. Sci. U. S. A. 87, 857-861 [Abstract]
  15. Harada, N., Howard, M., and Miyajima, A. (1992a) in IL-4: Structure and Function (Spits, H., ed) pp. 33-54, CRC Press, Boca Raton, FL
  16. Harada, N., Yang, G., Miyajima, A., and Howard, M. (1992b) J. Biol. Chem. 267, 22752-22758 [Abstract/Free Full Text]
  17. Herbert, J. M., Savi, P., Laplace, M. C., Lale, A., Dol, F., Dumas, A., Labit, C., and Minty, A.(1993) FEBS Lett. 328, 268-270 [CrossRef][Medline] [Order article via Infotrieve]
  18. Hurwitz, R., Hozier, J., LeBien, T., Minowada, J., Gajl-Peczalska, Kubonishi, I., and Kersey, J.(1979) Int. J. Cancer 23, 174-180 [Medline] [Order article via Infotrieve]
  19. Kitamura, T., Sato, N., Arai, K., and Miyajima, A.(1991) Cell 66, 1165-1174 [Medline] [Order article via Infotrieve]
  20. Kondo, M., Takeshita, T., Ishii, N., Nakamura, M., Watanabe, S., Arai, K., and Sugamura, K.(1993) Science 262, 1874-1877 [Medline] [Order article via Infotrieve]
  21. Kondo, M., Takeshita, T., Higuchi, M., Nakamura, M., Sudo, T., Nishikawa, S., and Sugamura, K.(1994) Science 263, 1453-1454 [Medline] [Order article via Infotrieve]
  22. Laemmli, U. K.(1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  23. Le, H. V., Ramanathan, L., Labdon, J. E., Mays-Ichinco, C., Syto, R., Arai, N., Hoy, P., Takebe, Y., Nagabhushan, T. L., and Trotta, P. P. (1988) J. Biol. Chem. 263, 10817-10823 [Abstract/Free Full Text]
  24. Matthews, D. J., Clark, P. A., Herbert, J., Morgan, G., Armitage, R. J., Kinnon, C., Minty, A., Grabstein, K. H., Caput, D., and Callard, R. (1995) Blood 85, 38-42 [Abstract/Free Full Text]
  25. McKenzie, A. N. J., Li, X., Largaespada, D. A., Sato, A., Kaneda, A., Zurawski, S. M., Doyle, E. L., Milatovich, A., Francke, U., Copeland, N. G., Jenkins, N. A., and Zurawski, G. (1993a) J. Immunol. 150, 5436-5444 [Abstract/Free Full Text]
  26. McKenzie, A. N. J., Culpepper, J. A., de Waal Malefyt, R., Briere, F., Punnonen, J., Aversa, G., Sato, A., Dang, W., Cocks, B. G., Menon, S., de Vries, J. E., Banchereau, J., and Zurawski, G. (1993b) Proc. Natl. Acad. Sci. U. S. A. 90, 3735-3739 [Abstract]
  27. Minami, Y., Kono, T., Miyazaki, T., and Taniguchi, T.(1993) Annu. Rev. Immunol. 11, 245-268 [CrossRef][Medline] [Order article via Infotrieve]
  28. Minty, A., Chalon, P., Derocq, J. M., Dumont, X., Guillemot, J. C., Kaghad, M., Labit, C., Leplatois, P., Liauzun, P., Miloux, B. Minty, C., Casellas, P., Loison, G., Lupker, J., Shire, D., Ferrard, P., and Caput, D.(1993) Nature 362, 248-250 [CrossRef][Medline] [Order article via Infotrieve]
  29. Morgan, J. G., Dolganov, G. M., Robbins, S. E., Hinton, L. M., and Lovett, M.(1992) Nucleic Acids Res. 20, 5173-5179 [Abstract]
  30. Munson, P. J., and Rodbard, D.(1980) Anal. Biochem. 107, 220-239 [Medline] [Order article via Infotrieve]
  31. Renard, N., Duvert, V., Banchereau, J., and Saeland, S.(1994) Blood 84, 2253-2260 [Abstract/Free Full Text]
  32. Rigley, K. P., Thurstan, S. M., and Callard, R. E.(1991) Int. Immunol. 3, 197-203 [Abstract]
  33. Russell, S. M., Keegan, A. D., Harada, N., Nakamura, Y., Noguchi, M., Leland, P., Friedmann, M. C., Miyajima, A., Puri, R. K., Paul, W. E., and Leonard, W. J.(1993) Science 262, 1880-1883 [Medline] [Order article via Infotrieve]
  34. van Kimmenade, A., Bond, M. W., Schumacher, J. H., Laquoi, C., and Kastelein, R. A.(1988) Eur. J. Biochem. 173, 109-114 [Abstract]
  35. Zurawski, G., and de Vries, J. E.(1994) Immunol. Today 15, 19-26 [CrossRef][Medline] [Order article via Infotrieve]
  36. Zurawski, S. M., and Zurawski, G.(1992) EMBO J. 11, 3905-3910 [Abstract]
  37. Zurawski, S. M., Vega, F., Jr., Huyghe, B., and Zurawski, G. (1993a) EMBO J. 12, 2663-2670 [Abstract]
  38. Zurawski, S. M., Vega, F., Jr., Doyle, E. L., Huyghe, B., Flaherty, K., McKay, D. B., and Zurawski, G. (1993b) EMBO J. 12, 5113-5119 [Abstract]

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