Reconstitution of a Functional Human Type II IL-4/IL-13 Receptor in Mouse B Cells: Demonstration of Species Specificity

This information is current as Ryan P. Andrews, Lillian Rosa Rosa, Michael O. Daines and of September 28, 2021. Gurjit K. Khurana Hershey J Immunol 2001; 166:1716-1722; ; doi: 10.4049/jimmunol.166.3.1716 http://www.jimmunol.org/content/166/3/1716 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Reconstitution of a Functional Human Type II IL-4/IL-13 Receptor in Mouse B Cells: Demonstration of Species Specificity1

Ryan P. Andrews, Lillian Rosa Rosa, Michael O. Daines, and Gurjit K. Khurana Hershey2

IL-13 is a Th2-derived pleiotropic that recently was shown to be a key mediator of allergic . IL-13 mediates its effects via a complex receptor system, which includes the IL-4R ␣-chain, IL-4R␣, and at least two other cell surface , IL-13R␣1 and IL-13R␣2, which specifically bind IL-13. IL-13 has been reported to have very limited effects on mouse B cells. It was unclear whether this was due to a lack of receptor expression, a disproportionate relative expression of the receptor com- ponents, or an additional subunit requirement in B cells. To determine the requirements for IL-13 signaling in murine B cells, we examined IL-13-dependent Stat6 activation and CD23 induction in the murine line, A201.1. A201.1 cells responded to Downloaded from murine IL-4 via the type I IL-4R, but were unresponsive to IL-13, and did not express IL-13 receptor. B220؉ splenocytes also failed to signal in response to IL-13 and did not express IL-13 receptor. We transfected A201.1 cells with human IL-4R␣, IL-13R␣1, or both. Transfectants expressing either human IL-4R␣ or human IL-13R␣1 alone were unable to respond or signal to IL-13. Thus, human IL-13R␣1 could not combine with the endogenous murine IL-4R␣ to generate a functional IL-13R. However, cells transfected with both human IL-4R␣ and IL-13R␣1 responded to IL-13. Thus, the relative lack of IL-13 respon- ␣

siveness in murine B cells is due to a lack of receptor expression. Furthermore, the heterodimeric interaction between IL-4R and http://www.jimmunol.org/ IL-13R␣1 is species specific. The Journal of Immunology, 2001, 166: 1716–1722.

nterleukin-13 is an immunoregulatory pleiotropic cytokine Although IL-4 and IL-13 have many overlapping functions, they secreted predominantly by activated Th2 cells (1). IL-4 and also have distinct roles. In parasitic models, IL-13, but I IL-13 are related that belong to the same ␣ helix not IL-4, was necessary for the Th2-dependent expulsion of Nip- superfamily, and their respective are located on postrongyloides brasiliensis (12). Furthermore, IL-13 has recently 5q31 only 12 kb apart (2). Although they share only 25% homol- been shown to be a key mediator of allergic asthma independent of ogy, IL-13 shares many functional properties with IL-4, including IL-4 in mouse models whereby IL-13 blockade prevented - the up-regulation of MHC class II and CD23 Ags on monocytes (1, induced airway inflammation (13, 14). Tissue-specific overexpression by guest on September 28, 2021 2). IL-13 mediates its effects via a complex receptor system that of IL-13 in the lungs of transgenic mice resulted in several features includes IL-4R␣ (IL-4R ␣-chain) and at least two other cell sur- found in an asthmatic airway, including airway inflammation, mucus face proteins, IL-13R␣1 and IL-13R␣2, which specifically bind hypersecretion, hyperplasia, deposition of Charcot-Leyden IL-13 (3–8). IL-13R␣1 binds IL-13 with low affinity by itself, but crystals, increased nonspecific airways hyperreponsiveness, and sub- when paired with IL-4R␣, it binds IL-13 with high affinity and epithelial fibrosis and airway remodeling (15). forms a functional IL-13R that signals (6). Consistent with the fact In human B cells, human IL-13 and IL-4 have similar effects, that IL-4 and IL-13 share a common receptor component, IL-4R␣, including modulating surface Ag expression and inducing class they also share some common signaling pathways. Studies in Stat6- switching to IgG4 and IgE in combination with CD40:CD40 li- deficient mice have revealed that IL-13 signaling utilizes the Janus gand costimulation (16, 17). In contrast, mouse IL-13 has been kinase-Stat pathway and specifically Stat6 (9, 10). In contrast, IL- reported to have no effects on mouse B cells (2). However, several 13R␣2 by itself binds IL-13 with high affinity, but does not signal lines of indirect evidence exist for IL-13 actions on mouse B cells. (11). Thus, the receptor complexes for IL-4 and IL-13 are intertwined IL-13-deficient mice had depressed levels of serum IgE (18), and systems that are most likely regulated at multiple levels, including by IL-13 transgenic mice on the IL-4 null background had elevated differential levels of expression of the various components and/or by levels of serum IgE (19). Furthermore, administration of rIL-13 to preferential association of certain components. mice resulted in enhanced Ab production, although IgE was not enhanced (20). Thus, both overexpression and the absence of IL-13 had an impact on Ig levels, supporting a role for IL-13 on B Division of Pulmonary Medicine, Allergy, and Clinical Immunology, Department of cell function. However, the effects of IL-13 on the regulation of Pediatrics, Children’s Hospital Medical Center, Cincinnati, OH 45229 IgE production in murine in vivo systems may be indirect. These Received for publication April 6, 2000. Accepted for publication November 2, 2000. observations led us to investigate IL-13 responsiveness in murine The costs of publication of this article were defrayed in part by the payment of page B cells. Mouse B cells express IL-4R␣ and readily respond to charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. murine IL-4, and since IL-13 utilizes the same signaling pathways 1 This work was supported in part by National Institutes of Health/National Institute as IL-4, IL-13 responsiveness in mouse B cells is most likely reg- of Child Health and Human Development Grant P30HD2887 and National Institutes ulated at the level of IL-13R expression. Several possibilities ex- of Health Grant RO1A146652-01A1. isted for the relative unresponsiveness of mouse B cells to IL-13 2 Address correspondence and reprint requests to Dr. Gurjit K. Khurana Hershey, (2, 21): 1) a relative overexpression of IL-13R␣2 compared with Division of Pulmonary Medicine, Allergy, and Clinical Immunology, Children’s Hos- ␣ pital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229. E-mail address: IL-13R 1 may result in a cell being unresponsive to IL-13 since [email protected] the nonsignaling, high affinity IL-13R␣2 would bind the available

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 The Journal of Immunology 1717 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. Characterization of IL-13 responsiveness in A201.1. A, A201.1 cells were incubated in the absence or presence of murine IL-4 or IL-13 at the indicated doses for 48 h and then assayed for CD23 or Ia expression by flow cytometry (solid line, autofluorescence; dotted line, unstimulated cells; bold line, stimulated cells). B, A201.1 cells were incubated in the absence (solid line) or presence (bold line) of 10 ng/ml murine IL-4 or 50 ng/ml murine IL-13 for the indicated time intervals and then assayed for CD23 expression by flow cytometry. In the top two histograms, the cells are unstimulated (dotted line represents autofluorescence; bold line represents CD23 staining). C, A201.1 cells were incubated in media alone or media containing 50 ng/ml IL-4 or 100 ng/ml IL-13 for 15 min. Cells were then lysed and Stat6 was immunoprecipitated. Phosphorylated Stat6 was detected by immunoblotting with an anti-phosphotyrosine Ab (top strip). The nitrocellulose was stripped and reprobed with Stat6 to demonstrate equal loading of the lanes (bottom strip). Each experiment was performed a minimum of three times, and a representative experiment is shown.

IL-13 and prevent its association with IL-13R␣1; 2) mouse B cells ␮g/ml streptomycin (BioWhittaker), and 50 ␮M 2-ME (Sigma, St. Louis, may express little or no IL-13R␣1; and 3) a functional IL-13R in MO). Recombinant human and mouse IL-4 and human IL-13 were pur- B cells may require an additional yet undefined component(s). In chased from R&D Systems (Minneapolis, MN). Murine IL-13, rabbit anti- murine IL-13R␣2 IgG, and Ba/F3.mIL-13R␣2 cells (Ba/F3 cells stably this manuscript, we addressed each of these possibilities, and pro- expressing mouse IL-13R␣2) were kindly provided by Debra Donaldson vide evidence for the mechanism of relative unresponsiveness in (Genetics Institute, Cambridge, MA). Anti-Stat6 rabbit polyclonal Ab was mouse B cells. a generous gift from Ulrike Schindler (Tularik, San Francisco, CA). Anti- CD40, anti-␮, and anti-I-Ad (MKD6) were gifts from Fred Finkelman (University of Cincinnati, Cincinnati, OH). FITC-coupled anti-murine Materials and Methods CD23 Ab was purchased from PharMingen (San Diego, CA). Biotinylated Cells and reagents anti-human IL-13R␣1 was purchased from Diaclone Research (Besancon, France). Anti-murine IL-4R␣ (code 1688-01) and anti-human IL-4R␣ A201.1 murine B cells, a gift from Gregg Milligan (Children’s Hospital (code 80-3285-01) were purchased from Genzyme Diagnostics (Cam- Medical Center, Cincinnati, OH), are derived from the parent line A20. The 32 ϩ ϩ ϩ Ϫ Ϫ Ϫ bridge, MA); both Abs are blocking Abs. [␥- P]ATP was purchased from cells are B220 , IgG ,Ia , IgA , IgM , and IgD and were originally NEN (Boston, MA). FITC-conjugated goat anti-rabbit IgG was purchased derived from a BALB/c mouse. Cells were maintained in complete RPMI from Southern Biotechnology Associates (Birmingham, AL). 1640 (cRPMI),3 consisting of RPMI 1640 (Life Technologies, Grand Is- land, NY) supplemented with 10% FBS (Life Technologies), 2 mM L- cDNA constructs and expression vectors glutamine (BioWhittaker, Walkersville, MD), 100 U/ml penicillin and 100 Human IL-13R␣1 cDNA, kindly provided by Debra Donaldson (Genetics Institute), was subcloned into the mammalian expression vector pCEP4 3 Abbreviation used in this paper: cRPMI, complete RPMI 1640. (Invitrogen, Carlsbad, CA). Human IL-4R␣ cDNA, obtained from John 1718 SPECIES SPECIFICITY OF THE TYPE II IL-4/IL-13 RECEPTOR COMPONENTS

Ryan (Virginia Commonwealth University, Richmond, VA), was sub- Spleen cell suspensions were prepared from BALB/c mice, reconsti- cloned into the mammalian expression vector pREP9 (Invitrogen). tuted in cold PBS with 2% FBS, and stained with FITC-conjugated RA3- 6B2 anti-mouse B220 Ab (PharMingen) in the presence of anti-FcR Ab Transfection 2.4G2 (PharMingen) for 30 min on ice. Cells were then washed in cold ϩ Ϫ ϫ 6 PBS with 2% FBS and sorted into B220 and B220 populations using a A total of 5 10 A201.1 cells was washed, resuspended in RPMI 1640 FACSvantage instrument (Becton Dickinson). containing 20 ␮g of uncut pREP9.human IL-4R␣ and/or pCEP4.humanIL- ␣ ␣ For double staining of splenocytes for B220 and IL-13R 2 expression, 13R 1, and pulsed with a Genepulser II electroporation device (Bio-Rad, ϫ 6 ␮ 3.5 10 splenocytes prepared from BALB/c mice were washed with Melville, NY) set at 960 F and 200 V. After electroporation, cells were PBS, and then incubated in the presence of 0.2 ␮g of anti-murine IL- grown for 24 h in 10 ml cRPMI and then selected for resistance to neo- ␣ ␮ 13R 2 and anti-FcR Ab 2.4G2 (PharMingen) for 30 min on ice. Cells were mycin (G418 sulfate; BioWhittaker) at 1000 g/ml and/or hygromycin at then washed with PBS and incubated in the presence of PE-conjugated 400 ␮g/ml for 12–21 days, respectively. Cell populations were screened by Ј DNL-1.9 anti-mouse B220 Ab (PharMingen) and goat F(ab )2 anti-rabbit flow cytometry for CD23 surface expression in response to stimulation IgG FITC (Southern Biotechnology Associates) for an additional 30 min with human IL-4 (10 ng/ml) for 48 h and/or by staining with anti-IL- on ice. Then cells were washed and analyzed on a FACScalibur instrument. 13R␣1-FITC Ab. Positive transfectant pools were cloned by limiting dilution. Immunoprecipitation and immunoblotting EMSA A201.1 cells (2 ϫ 107) were pelleted by centrifugation at 20,000 ϫ g at ϫ 6 4°C and reconstituted in IP-lysis buffer (50 mM Tris, pH 8, 150 mM NaCl, A201.1 cells (2.5 10 ) were stimulated with murine or human IL-4 (10 1% Nonidet P-40, 1 mM PMSF, 10 ␮g/ml leupeptin, 10 ␮g/ml aprotinin, ng/ml), or murine IL-13 (50 ng/ml) in cRPMI for 15 min, pelleted by 5 mM iodoacetamide, 1 mM sodium orthovanadate, 20 mM NaF, and 1 centrifugation at 10,000 ϫ g, and reconstituted in lysis buffer (10 mM mM EDTA). Cells were incubated on ice for 30 min, and lysates were HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1.5 mM MgCl , 0.2% Non- 2 clarified by centrifugation at 20,000 ϫ g for 20 min at 4°C. Soluble Stat6 idet P-40, 1 mM DTT, and 0.5 mM PMSF). Lysates were centrifuged at Downloaded from was immunoprecipitated with anti-Stat6 polyclonal Ab, followed by pro- 10,000 ϫ g for 5 min at 4°C, and supernatants containing the cytoplasmic tein A/G PLUS agarose (Santa Cruz Biotechnology), as previously de- extracts were removed. Pelleted nuclei were reconstituted in nuclear extract scribed (22). Briefly, precipitates were washed three times in IP-lysis buffer (20 mM HEPES, pH 7.9, 420 mM NaCl, 0.1 mM EDTA, 1.5 mM buffer, reconstituted in Laemmli buffer, and resolved by electrophoresis on MgCl , 25% glycerol, 1 mM DTT, and 0.5 mM PMSF). Nuclei were lysed 2 10% SDS-polyacrylamide gels. for 15 min at 4°C and centrifuged at 20,000 ϫ g for 15 min at 4°C, and Proteins were transferred to nitrocellulose membranes and blocked supernatants were removed as nuclear extracts. overnight in block solution (20 mM Tris, pH 7.4, 150 mM NaCl, 3.1% After quantitation of by CoomassiePlus (Pierce, Rockford, IL), BSA, and 0.1% polyethylene glycol 20,000). Membranes were probed with http://www.jimmunol.org/ 5 ␮g of nuclear extracts was reconstituted in TE buffer (10 mM Tris-Cl, pH ϫ anti-phosphotyrosine mAb PY20 (Transduction Laboratories, Lexington, 7.4, and 1 mM EDTA). Equal volumes of 2 EMSA reaction buffer (24 KY) or anti-Stat6 polyclonal Ab. Bound Abs were detected by incubation mM HEPES, pH 7.9, 8 mM Tris, 50 mM KCl, 10 mM MgCl , 24% glyc- 2 with anti-mouse or anti-rabbit IgG Abs conjugated to HRP (Transduction erol, 0.08 ␮ g/ml poly(dI-dC), 2 mM EDTA, and 2 mM DTT) were added, Laboratories), followed by ECL using ECL substrate (Amersham, Arling- and the reaction mixtures were incubated for 10 min on ice. Reactions were ton Heights, IL). incubated with 0.2 ng of Stat6 probe (Santa Cruz Biotechnology, Santa Cruz, CA) end labeled with [␥-32P]ATP for an additional 10 min on ice. A 100-fold excess of unlabeled nucleotide (20 ng) was used in cold compe- tition samples, and 1 ␮l of anti-Stat6 polyclonal Ab was added to supershift samples. Extracts were incubated on ice for an additional hour and then electrophoresed on 5% polyacrylamide gels in 0.5ϫ TBE. Bands were by guest on September 28, 2021 visualized by autoradiography. Flow cytometry and cell sorting A201.1 cells (5 ϫ 105) were washed in cold PBS with 1% FBS and stained with FITC-conjugated anti-mouse CD23 Ab (PharMingen) or biotinylated anti-human IL-13R␣1 Ab in the presence of anti-FcR Ab 2.4G2 (PharM- ingen) for 30 min on ice. In the case of IL-13R␣1 staining, cells were then washed and incubated in the presence of streptavidin-PE (Southern Bio- technology Associates). Cells were washed in cold PBS with 1% FBS and analyzed on a FACScan instrument (Becton Dickinson, San Jose, CA).

FIGURE 3. A, Analysis of IL-4 and IL-13 signaling in B220ϩ and B220Ϫ splenocytes. Splenocytes isolated from a BALB/c mouse were sorted into B220ϩ and B220Ϫ populations and stimulated for 15 min with media alone, 10 ng/ml murine IL-4, or 50 ng/ml murine IL-13. Nuclear FIGURE 2. Analysis of IL-13R␣2 expression on A201.1. A201.1 (top) extracts were then analyzed for Stat6 activation by EMSA. A201.1 cells or Ba/F3.mIL-13R␣2 (Ba/F3 cells stably expressing mouse IL-13R␣2) were used as a positive control. The EMSA results are representative of (bottom) cells were stained with either rabbit IgG (solid line) or rabbit three separate experiments. B, Analysis of IL-13R␣2 expression on B220ϩ anti-mouse IL-13R␣2 (bold line), followed by FITC-conjugated goat anti- and B220Ϫ splenocytes. Splenocytes isolated from a BALB/c mouse were rabbit IgG, and then analyzed by flow cytometry. The dotted lines represent stained for both B220 and IL-13R␣2. IL-13R␣2 expression is illustrated autofluorescence. The histograms depicted are representative of three sep- for the B220ϩ and B220Ϫ gated populations. The histograms depicted are arate experiments. representative of three separate experiments. The Journal of Immunology 1719

FIGURE 4. Analysis of IL-4 and IL-13 signaling in A201.1 cells stably expressing human IL-4R␣. Untrans- fected A201.1 or A201.1 cells stably transfected with human IL-4R␣ cDNA were stimulated with media alone, murine IL-4 (10 ng/ml), human IL-4 (10 ng/ml), murine IL-13 (200 ng/ml), or human IL-13 (200 ng/ml) for 15 min. Nuclear extracts were then analyzed for Stat6 activation by EMSA. The EMSA results are rep- resentative of three separate experiments.

Results (Fig. 2). Ba/F3 cells transfected with IL-13R␣2 demonstrated A201.1 cells do not respond or signal to IL-13 staining with an anti-IL-13R␣2 Ab, but the A201.1 cells did not

stain above background. Thus, the absence of an IL-13 signal or Downloaded from A201.1 is a mouse B cell line, which expresses IL-2R␥c (data not response in A201.1 cells is most likely due to only negligible or shown) and readily responds to murine IL-4, as evidenced by the absent expression of IL-13R␣1. induction of CD23 and class II MHC surface expression after 48 h

(Fig. 1). However, the cells did not respond to murine IL-13 at any ϩ dose (Fig. 1A) even after prolonged treatment with IL-13 up to B220 mouse splenocytes are unresponsive to IL-13 96 h (Fig. 1B). The cells also did not respond to IL-13 at super- Our data demonstrate that A201.1 cells are unresponsive to IL-13. physiologic (10 ␮g/ml) doses (data not shown). To determine We next wanted to determine whether primary mouse B lympho- http://www.jimmunol.org/ whether IL-13 was capable of transducing a signal in A201.1 cells, cytes behaved in a similar fashion. We isolated splenocytes from we examined Stat6 activation. Murine IL-4 induced Stat6 activa- a BALB/c mouse, sorted the cells into B220ϩ and B220Ϫ popu- tion, but murine IL-13 treatment did not result in Stat6 phosphor- lations, and then tested the cells for the ability to respond to IL-13 ylation (Fig. 1C). (Fig. 3A). As expected, both the B220ϩ and B220Ϫ cells re- sponded to IL-4. In contrast, IL-13-induced Stat6 activation was ␣ IL-13R 2 is not expressed on A201.1 cells detected only in the B220Ϫ population. The B220ϩ population was IL-13R␣2 binds IL-13 with high affinity, but does not transduce a unresponsive to IL-13. ϩ Ϫ

signal (11), thus a relative abundance of this receptor compared We next examined B220 and B220 splenocytes for IL- by guest on September 28, 2021 with the signaling IL-13R␣1 would lead to a cell being unrespon- 13R␣2 expression to determine whether IL-13R␣2 expression sive to IL-13. We analyzed A201.1 cells for IL-13R␣2 expression might be responsible for the lack of IL-13 response. However, like and found that A201.1 cells do not express detectable IL-13R␣2 the A201.1 cells, the B220ϩ cells did not express detectable levels

FIGURE 5. Analysis of human IL-13R␣1 surface expression and IL-13 signaling in transfectants. A, Untransfected A201.1 (shaded histograms) or cells transfected with human IL-4R␣ and human IL-13R␣1(top) or human IL-13R␣1 alone (bottom) were stained with biotinylated anti-human IL-13R␣1 Ab, followed by streptavidin-PE (open histograms), and analyzed by flow cytometry. B, Untransfected A201.1, or cells transfected with either human IL-4R␣, human IL-13R␣1, or both were incubated in the presence of media alone (lane 1), 10 ng/ml murine IL-4 (lane 2), 10 ng/ml human IL-4 (lane 3), or 50 ng/ml murine IL-13 (lane 4) for 15 min. Identical results were also obtained with human IL-13 (data not shown). Nuclear extracts were then analyzed for Stat6 activation by EMSA. Each experiment was performed a minimum of five times, and a representative experiment is shown. 1720 SPECIES SPECIFICITY OF THE TYPE II IL-4/IL-13 RECEPTOR COMPONENTS

FIGURE 7. Blockade of human, but not murine, IL-4R␣ inhibits IL-13 signaling in double transfectants. A201.1 cells stably transfected with both human IL-4R␣ and human IL-13R␣1 were incubated in the presence of 50 ␮g/ml anti-human IL-4R␣ (H) or anti-murine IL-4R␣ (M) for 2 h. Cells were then stimulated in the presence of media alone, 0.1 ng/ml murine IL-4, 0.1 ng/ml human IL-4, or 50 ng/ml murine IL-13 for 15 min, and then nuclear extracts were analyzed for Stat6 activation by EMSA. The EMSA results are representative of three separate experiments..

induce Stat6 activation in transfectants (Fig. 4). In addition, IL-13

had no effect on the IL-4 response in the transfectants. Downloaded from

Stable transfection of A201.1 with human IL-4R␣ and IL-13R␣1, but neither one alone, renders them responsive to IL-13 We next examined whether transfection of A201.1 cells with hu- man IL-13R␣1 would render them responsive to IL-13. Untrans- ␣ fected A201.1 or cells stably expressing human IL-4R were sta- http://www.jimmunol.org/ bly transfected with human IL-13R␣1, and surface expression was confirmed by flow cytometry using a biotinylated anti-human IL- 13R␣1 Ab (Fig. 5A). The single transfectants expressing human IL-13R␣1 and the double transfectants expressing both human IL- 13R␣1 and human IL-4R␣ both expressed human IL-13R␣1on the cell surface. We then analyzed the transfectants for the ability to signal in response to IL-13 (Fig. 5B). No appreciable Stat6 ac- tivation was detected after IL-13 stimulation in cells transfected

FIGURE 6. Analysis of IL-13-dependent CD23 induction in transfec- with either human IL-13R␣1 or human IL-4R␣. Thus, human IL- by guest on September 28, 2021 tants. Untransfected A201.1, or cells transfected with either human IL- 13R␣1 is incapable of associating with the endogenous murine 4R␣, human IL-13R␣1, or both were incubated in the presence of media IL-4R␣ to generate a functional IL-13R. In Fig. 5, mouse IL-13 alone (dotted lines), 10 ng/ml murine IL-4 (left side, solid lines), 10 ng/ml human IL-4 (left side, bold lines), or 50 ng/ml murine IL-13 (right side, was used, but identical results were obtained with human IL-13 as bold lines) for 48 h. Cells were then assayed for CD23 expression by flow expected since IL-13 is not species specific (data not shown). In cytometry. Each experiment was performed a minimum of five times, and contrast, cells transfected with both human IL-13R␣1 and IL-4R␣ a representative experiment is shown. were capable of transducing a signal in response to IL-13, as ev- idenced by Stat6 activation. Densitometric analysis revealed that Stat6 activation was 1.8-fold stronger with either human or murine IL-4, than with murine or human IL-13. This was not surprising of IL-13R␣2. In contrast, the B220Ϫ cells did express IL-13R␣2 since human IL-13R␣1 expression was relatively low in the double (Fig. 3B). transfectants (Fig. 5A). To verify that the double transfectants were responsive to IL-13, IL-13 signaling is not apparent in the presence of transfected we also examined IL-13-dependent induction, specifically ␣ human IL-4R in A201.1 CD23 expression (Fig. 6). Untransfected A201.1, or transfectants Unlike IL-13, which is not species specific, IL-4 is species specific expressing either IL-13R␣1 or IL-4R␣ remained unresponsive to and does not cross-react between human and mouse. A201.1 cells IL-13. However, in cells expressing both human IL-13R␣1 and respond to murine IL-4, but not human IL-4. They gain the ability human IL-4R␣, IL-13 treatment resulted in the induction of CD23 to respond to human IL-4 only after they are successfully trans- expression. fected with the human IL-4R␣ (Fig. 4). Since IL-13 is not species ␣ ␣ specific, it remained possible that the endogenous IL-4R␣-chain Human IL-13R 1 associates with human IL-4R , but not ␣ preferentially binds IL-2R␥c, and thus is not available to associate murine IL-4R to form a functional IL-13R with IL-13R␣1 and form a functional IL-13R. We investigated Since A201.1 cells expressing human IL-13R␣1 were not respon- whether overexpressing human IL-4R␣, which associates with the sive to IL-13, our data supported that the interaction between IL- endogenous murine IL-2R␥c to form a functional human IL-4R, 13R␣1 and IL-4R␣ to generate a functional receptor complex was would render the cells responsive to IL-13 by enabling more en- species specific. To test this directly, we pretreated A201.1 cells dogenous murine IL-4R␣ to be available for association with IL- stably transfected with both human IL-13R␣1 and IL-4R␣ with 13R␣1. A201.1 cells transfected with human IL-4R␣ expressed either anti-human IL-4R␣ or anti-murine IL-4R␣ to block either ϳ1800 receptors/cell by Scatchard analysis (data not shown) and the transfected human IL-4R␣ or the endogenous murine IL-4R␣. gained the ability to respond to human IL-4. However, even in the Both Abs recognize their cognate IL-4R␣ and block IL-4 binding. presence of excess IL-4R␣, IL-13, either human or mouse, did not We then treated the cells with IL-13 and analyzed them for Stat6 The Journal of Immunology 1721

absence of an IL-13 response provides evidence that IL-13R␣1is absent or negligibly expressed on A201.1 mouse B cells. However, IL-13-deficient mice had depressed levels of serum IgE (18), and IL-13 transgenic mice on the IL-4 null background had elevated levels of serum IgE (19), supporting a role for IL-13 on mouse B cells in vivo. Based on our data, we propose that mouse B cells do not express functional IL-13R at baseline, but that receptor ex- pression can be induced at certain stages of B cell development or in specific B cell subsets. Alternatively, since B220Ϫ splenocytes do express IL-13R and signal in response to IL-13, the role for IL-13 in regulating IgE production may be indirect. Studies to define which factors may regulate IL-13R expression are currently underway. A201.1 cells transfected with human IL-13R␣1 alone remained unresponsive to IL-13. Thus, human IL-13R␣1 cannot associate with the endogenous murine IL-4R␣. Transfection with both hu- man IL-4R␣ and IL-13R␣1 rendered cells responsive to IL-13 (Fig. 8). Thus, the interaction between IL-4R␣ and IL-13R␣1is

completely species specific. We went on to confirm the species Downloaded from specificity by demonstrating that Ab blockade of human IL-4R␣, FIGURE 8. Schematic illustration of the association of the type II IL- but not mouse IL-4R␣ inhibited IL-13 signaling in the double 4R/IL-13R complexes in A201.1 transfectants. transfectants. The open reading frame of human IL-13R␣1 has 81% nucleotide and 76% amino acid identity with murine IL- 13R␣1 (3, 5). Thus, the epitopes on IL-13R␣1 for binding IL-4R␣

activation by EMSA (Fig. 7). Blockade of the endogenous mouse must not be conserved between human and mouse. This is sur- http://www.jimmunol.org/ IL-4R␣ completely inhibited the cellular response to murine IL-4, prising since the epitope on mouse IL-2R␥c for associating with but had no effect on IL-13-dependent Stat6 activation. In contrast, human or mouse IL-4R␣ to generate a signaling type I IL-4R is blockade of the human IL-4R␣ inhibited signaling to both human conserved (26). Murine IL-2R␥c can complex with either human IL-4 and IL-13. Since the anti-IL-4R␣ Abs do not recognize IL- or murine IL-4R␣ to create a functional type I IL-4R. In contrast, 13R␣1, the inhibition of IL-13 signaling by anti-human IL-4R␣ is our data establish that this is not the case for IL-13R␣1, and pro- due to steric hinderance, preventing the formation of a functional vide novel insights into the possible contact residues that must not receptor complex. Thus, the interaction between IL-4R␣ and IL- be conserved between human and mouse. Exploitation of this spe- 13R␣1 is completely species specific. cies specificity will aid in the identification of the epitope required for the IL-13R␣1/IL-4R␣ interaction. Studies to identify these key by guest on September 28, 2021 Discussion epitopes are underway. Pharmaceuticals targeted specifically to the Asthma is a major health concern in the United States and world- target residues would allow specific inhibition of IL-13 function wide. It is the most common chronic disease of childhood (23) and while leaving IL-4 signaling intact, and may prove beneficial in the its incidence is on the rise (24). Considerable effort is taking place treatment of atopic disorders. to delineate the mediators of allergic inflammation and specifically asthma. IL-13 has recently been shown to be a key mediator of Acknowledgments allergic asthma (13–15). Delineating the molecular mechanisms by We are grateful to Drs. Fred Finkelman, Timothy Weaver, and Jeffrey which IL-13R interact and transduce cellular signals resulting in Whitsett for critical review of this manuscript and to Drs. Debra Donald- allergy-promoting activities are key steps to understanding the son, Fred Finkelman, and Ulrike Schindler for their generosity. We thank pathogenesis of atopic disorders. In this study, we have defined the Connie Petitt for excellent secretarial support. requirements for IL-13 signaling in mouse B cells, and provide evidence that the interaction between the IL-13R complex com- References ponents is species specific. 1. Minty, A., P. Chalon, J.-M. Derocq, X. Dumont, J.-C. Guillemot, M. Kaghad, IL-13 has two cognate receptors, IL-13R␣1 and IL-13␣2 (3–8). C. Labit, P. Leplatois, P. Liauzen, B. Miloux, et al. 1993. -13 is a new ␣ ␣ human regulating inflammatory and immune responses. Nature 362: IL-13R 1 associates with IL-4R to form a high affinity signaling 248. receptor complex. 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