Modulation of Interleukin (IL)-13 Binding and Signaling by the γc Chain of the IL-2 Receptor*

Interleukin (IL)-13 and IL-4 are cytokine products of TH2 cells which exert similar effects in a variety of cell types. We recently described IL-13R expression on human renal cell and colon carcinoma cells and demonstrated that γc is not a component of IL-13R or IL-4R systems in these cells. In lymphoid cells such as B cells and monocytes, which respond to IL-13, γc is a component of IL-4R but does not appear to be a component of IL-13R. Furthermore, while significant IL-13 binding is observed on carcinoma cells, IL-13 barely binds these lymphoid cells and the binding characteristics are different. To better understand the role of γc in IL-13 binding and signaling, we have transfected a renal cell carcinoma cell line with γc and examined IL-13 and IL-4 binding and signaling. IL-13 binding as well as IL-13 and IL-4 signaling through the JAK-STAT signaling pathway were severely inhibited. This inhibition was paralleled by a loss of expression of one of the IL-13R chains and intercellular cell adhesion molecule-1. Thus, although γchas been shown to enhance IL-4 binding and function in some cell types, its influence on IL-13R function in tumor cells appear to be largely negative.

Interleukin-13 (IL-13) 1 and IL-4 are pleiotropic immune regulatory cytokines, which are predominantly produced by activated lymphocytes. Both cytokines mediate similar effects on B cells and monocytes; however, IL-13 has not been shown to have direct effects on T cells (1)(2)(3). The effects of both cytokines are mediated by cell surface receptors (R) that are specific for each of these ligands (4,5). The receptor for IL-4 has been extensively investigated and its structure appears to vary with cell types. We have proposed three models for the structure of the IL-4R complex (6). However, despite their structural differences, all of the identified forms of the IL-4R complex are quite effective in transducing signals in response to IL-4 binding.
Unlike the IL-4 receptor system, the receptor for IL-13 has not been well characterized. We have demonstrated that RCC cells express a large number of IL-13 binding sites and that these cells respond to IL-13 (4,7). Cross-linking studies indicate that the IL-13R is predominantly composed of a ϳ65-70-kDa protein (4). RCC cells also express IL-4R, and 125 I-IL-4 cross-links to IL-4R␤ (the p140 chain of IL-4R) and a 65-70-kDa IL-4-binding protein. The inhibition of IL-4 binding to both proteins by IL-13 supports the notion that IL-4 and IL-13 receptors are structurally and functionally interrelated (4,(7)(8)(9)(10). We recently proposed several models to account for the complex interactions among IL-13, IL-4, and the receptors for these ligands as they are expressed in different cell types. We proposed that IL-13R shares IL-4R␤ (p140) and IL-4R␣ (p70) with the IL-13R system in certain cell types (11). We had also predicted that IL-13 binds to a heterodimer consisting of two ϳ70-kDa proteins, one of which binds IL-13 alone (we termed this IL-13R␣) while the other binds IL-13 as well as IL-4 (we termed this IL-13R␣Ј) (7,11). The genes for a murine IL-13binding protein, its human homologue, and a second human IL-13-binding protein were subsequently identified and characterized (12)(13)(14). The two human IL-13R genes encode 65-70-kDa proteins with different IL-13 binding characteristics. One of these (corresponding to our IL-13R␣Ј) requires IL-4R␤ to bind and transduce IL-13 signal (14), while the other (corresponding to our IL-13R␣) can bind IL-13 in the absence of IL-4R␤, but its role in IL-13 signaling remains unclear (13). Whether other as yet unidentified proteins are required to constitute a functional high affinity IL-13R remains to be resolved. The IL-2R␥ chain termed ␥ c is shared by receptors for IL-4, IL-7, IL-9, and IL-15 on immune cells (15)(16)(17)(18). It was hypothesized that IL-2R␥ c is also a component of the IL-13R system (15). However, we have reported that an anti-␥ c antibody did not immunoprecipitate any 125 I-IL-13-bound protein from RCC (4) or colon carcinoma (6) cell lysates, indicating that the ␥ c protein may not be directly involved in IL-13 binding. The ␥ c protein was not expressed in these cells. However, whether ␥ c affects IL-13 binding in cells that normally express it is not known. It is also not known whether ␥ c affects IL-13R structure and signal transduction.
In this study, we have examined the effect of the ␥ c chain on IL-13 and IL-4 binding and signaling. We have transfected ML-RCC cells with the ␥ c cDNA and examined its influence on certain biological responses of these cells to IL-13 and IL-4. We present evidence that ␥ c severely decreased the IL-13 binding capacity of these cells and prevented the expression of the ␣ chain of the IL-13 receptor as well as ICAM-1. Furthermore, although ␥ c had no significant effect on the binding of IL-4 to its receptors, its presence altered IL-13 and IL-4 signaling pathways in these cells in several ways, including the inhibition of IL-13-and IL-4-induced STAT-6 activation. * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

EXPERIMENTAL PROCEDURES
Cytokines and Reagents-Recombinant human IL-13 (rhIL-13) was expressed in Escherichia coli and purified as described previously (4). Recombinant human IL-4 was kindly provided by Dr. Michael Widmer of Immunex Corp., Seattle, WA. Antibodies to JAKs 1 and 2 and biotinanti-phosphotyrosine (4G10) were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Antibodies to ␥ c , JAK3, Tyk2, and STATs 3, 4, and 6 were purchased from Santa Cruz (Santa Cruz, CA). Antibodies to STAT1 and STAT5 were provided by Dr. David Finbloom (Center for Biologics Evaluation and Research, Food and Drug Administration (CBER, FDA), Bethesda, MD).
Cells-The ML-RCC (synonym: MA-RCC) renal cell carcinoma cell line was established in our laboratory as described previously (19) from primary surgical tissues and was maintained in HEPES-buffered Dulbecco's modified Eagle's medium with high glucose supplemented with glutamine plus 10% fetal calf serum and antibiotics (100 units/ml penicillin and 100 g/ml streptomycin).
Transfection of ML-RCC Cells-ML-RCC cells (3 ϫ 10 5 ) were plated in 12-well tissue culture plates and cultured overnight. They were then transfected with a mixture of cDNAs encoding the genes for the ␥ c subunit of the IL-2 receptor or neomycin transferase or both. Transfection was effected by the calcium phosphate method using the PROFEC-TANT kit (Promega) according to the manufacturer's instruction. Neomycin-resistant transfectants were selected by incubating the transfected cells in 0.8 mg/ml active G418 for 10 days. Selected cells were examined by Northern blot analysis to identify clones containing the ␥ c gene in addition to the neomycin resistance gene. Two such clones isolated from the ML-RCC cell line were identified as ML␥c. Clones transfected with the neomycin resistance but not the ␥ c gene (ML neo ) were used as negative controls for the double transfectants. Additional negative control transfectants (i.e. neomycin-resistant, ␥ c (Ϫ)) were generated by using a recombination deficient retroviral vector to introduce the neomycin resistance gene into the target ML-RCC cells. This was achieved by infecting target cells with supernatant generated by the amphotropic retroviral vector-producing PA317 cell line (20), kindly provided by Dr. Carolyn Wilson (CBER, FDA). Identical results were observed when all negative control and parental cell lines were used in all tests. Similarly the two ␥ c -positive clones (ML␥ c ) were identical in their responses to all tests performed.
Northern Blot Analysis-Cells were harvested from culture with Versene (Bio-Whittaker, Walkersville, MD), washed, and total RNA isolated in a one-step procedure using Trizol (Life Technologies, Inc.) according to manufacturer's instructions. Aliquots of total RNA were electrophoresed through agarose (0.8%)/formaldehyde denaturing gel, transferred to a nylon membrane (S & S Nytran; Schleicher & Schuell) by capillary action and immobilized by ultraviolet cross-linking (Stratagene, Inc., La Jolla, CA). The membrane was then prehybridized overnight at 42°C and hybridized with a 32 P-labeled cDNA probe for ␥ c at 42°C for 18 h. The membrane was subsequently exposed to an X-AR film (Eastman Kodak Co.) to obtain an autoradiograph. To evaluate the amount of RNA loaded per lane, the blots were stripped and reprobed with 32 P-labeled GAPDH cDNA.
Radioreceptor Binding Assay-rhIL-13 and rhIL-4 were labeled with 125 I (Amersham Research Products) by using IODO-GEN reagent (Pierce) according to the manufacturer's instructions. The specific activity of the radiolabeled cytokines was estimated to range from 20 to 80 Ci/g of protein for 125 I-IL-4 and 40 -120 Ci/g for 125 I-IL-13. Binding experiments were performed as described previously (4). Typically, 1 ϫ 10 6 cells were incubated at 4°C for 3-5 h with 125 I-IL-13 (100 -500 pM) or 125 I-IL-4 (100 -500 pM) in the presence or absence of increasing concentrations (up to 1000 nM) of unlabeled IL-13 or IL-4. The data were analyzed with the LIGAND (21) program to determine the number of binding sites/cell as well as binding affinity.
Flow Cytometry-ML␥c RCC cells as well as ML neo control cells were cultured at 1 ϫ 10 5 cells/ml in medium containing IL-4 or IL-13 (0 -10 ng/ml) over a 48-h period. Cells were washed and incubated with a fluorescein isothiocyanate (FITC)-conjugated anti-human ICAM-1 antibody (Immunotech/Coulter) at a concentration of 40 g/ml in FACS staining buffer (phosphate-buffered saline containing 0.1% fetal bovine serum, 2.5 mM EDTA, and 0.1% sodium azide) at 4°C for 60 min. Control cells were incubated in FACS staining buffer alone or FITCconjugated IgG 1 (isotype control antibody). The cells were subsequently washed and fluorescence data collected on a FACScan/C32 equipment (Becton Dickinson, San Jose, CA). The data were analyzed with a LYSIS II software program, and fluorescence intensity was expressed as mean channel number on a 256-channel/10 4 log scale.
Affinity Cross-linking Studies-Cells (5-10 ϫ 10 6 ) were incubated with 125 I-IL-13 in the presence or absence of excess IL-13 or IL-4 for 2 h at 4°C. The bound ligand was cross-linked to its receptor with disuccinimidyl suberate (Pierce) at a final concentration of 2 mM for 30 min. Cells were lysed in lysis buffer consisting of 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 0.02 mM leupeptin, 5.0 M trypsin inhibitor, 10 mM benzamidine HCl, 1 mM phenanthroline, iodoacetamide, 50 mM aminocaproic acid, 10 g/ml pepstatin, and 10 g/ml aprotinin. The cell lysates were precleared and cleared by boiling in sample buffer containing 2-mercaptoethanol and analyzed by electrophoresis through an 8% SDS-polyacrylamide gel. The gel was subsequently dried and autoradiographed. In some instances, the receptor-ligand complex was captured from the precleared total cell lysate on protein A-Sepharose beads previously incubated with specific antibodies. The complex was washed in lysis buffer, boiled in sample buffer, and analyzed by SDS-PAGE. Immunoprecipitation and Immunoblotting-ML-RCC cells (2 ϫ 10 7 ) were incubated with 250 ng/ml IL-13 or 50 ng/ml IL-4 or left untreated as control for 10 min at 37°C. The reactions were quenched by addition of 10 ml of PBS containing 1 mM sodium vanadate, 25 mM NaF, 10 mM sodium pyrophosphate, and 1 mM EDTA. The cells were lysed at 4°C with 1% Nonidet P-40, 300 mM NaCl, 50 mM Tris (pH 7.4), leupeptin (10 mg/ml), aprotinin (10 mg/ml), phenylmethylsulfonyl fluoride (1 mM), 1 mM sodium vanadate, 25 mM NaF, 10 mM sodium pyrophosphate, and 1 mM EDTA. The cell lysates were precleared by incubation with protein A-Sepharose beads (Sigma). They were then incubated with protein A-Sepharose beads that had been preincubated with primary antibody for 2 h at 4°C. The resulting complex was washed five times in lysis buffer, resuspended in loading buffer (NOVEX), and separated through 8% SDS-polyacrylamide gel. Separated proteins were transferred to PVDF membrane (NOVEX) for 1 h. The blots were washed three times in TBS-T buffer (20 mM Tris-HCl, 137 mM NaCl, 0.1% Tween 20), followed by 1 h of incubation in horseradish peroxidase-conjugated goat anti-rabbit or strepto-avidin (Amersham), depending on the nature of the primary antibody, followed by three washes. The blots were incubated with RENAISSANCE™ chemiluminescence substrate mixture according to the manufacturer's instructions (NEN Life Science Products) for 1 min and exposed to x-ray film for 1-30 min.
Electrophoretic Mobility Shift Assay (EMSA)-After treatment with/ without IL-13 (250 ng/ml) or IL-4 (50 ng/ml), cells were washed in cold PBS and solubilized with cold whole cell extraction buffer (1 mM MgCl 2 , 20 mM HEPES, pH 7.0, 10 mM KCl, 300 mM NaCl, 0.5 mM dithiothreitol, 0.1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na 3 VO 4 , and 20% glycerol). DNA-binding proteins were identified by EMSA by using bandshift kit from Pharmacia Biotech Inc. Briefly, 30 g of protein samples were incubated for 20 min at room temperature with 1 ng of 32 P-labeled oligonucleotide probe consisting of double-stranded GRR (5Ј-AGCATGTTTCAAGGATTTGAGATGTATTTCCCAGAAAAG-3Ј) of the promoter of the FcR␥1 gene in 20 l of binding buffer (10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 0.5 mM dithiothreitol, 10% glycerol, 0.05% Nonidet P-40, 0.05 mg/ml poly(dI-dC)⅐(dI-dC)). A 200-fold excess of cold GRR probe was added as a competitor. Before loading, 10 ϫ loading dye was added to each sample and separated in a 5% nonreducing polyacrylamide gel at 150 V for 2.5 h. Gels were dried for 2 h and autoradiographed overnight at room temperature. For the supershift assay, cell lysates were preincubated with preimmune rabbit serum as a control or STAT1, 3, 4, 5, and 6 antibodies on ice for 30 min, standard EMSA was then performed as described above.

RESULTS
Transfection of IL-2R ␥ Chain into RCC Cells-Two ␥ c mRNA-positive (ϩ) clones of ML-RCC (ML␥ c ) were selected from five neomycin-resistant (ML neo ) clones. As shown in Fig.  1A, alternatively spliced ␥ c mRNA (16) was detected in ML␥ c but not in ML neo cells. Both of the ML␥ c clones were tested in all experiments shown, and both yielded identical results. Similarly, all the negative control clones (ML neo ) yielded identical results in tests performed. To confirm that the ␥ c mRNA being transcribed was also being properly translated into its protein in ML␥ c cell lines, we examined total cell lysates from these ␥ c mRNA ϩ cell lines for the presence of ␥ c protein by immunoprecipitation and Western blot analysis. The results indicate that ML␥ c cells expressed the ␥ c protein as detected by anti-␥ c antibody, whereas the control and untransfected parental cell lines were negative (Fig. 1B). We have also demonstrated ␥ c protein expression on the surface of these ␥ c mRNA ϩ cells by flow cytometry (22). Following ␥ c transfection, the ML␥ c clones took on a different morphology, appearing smaller and more elongated than the neomycin-resistant control cell lines (data not shown).
Association of Transfected ␥ c Chain with IL-13R in ML-RCC Cells-Since ␥ c modulated the binding of IL-13 to its plasma membrane receptors, it was important to determine whether the ␥ c protein expressed in ML-RCC cells transfected with the ␥ c cDNA is associated with IL-13 receptor proteins. We crosslinked 125 I-IL-13 to its receptors on ␥ c -transfected and control cells. The cell lysate from each group was subsequently incubated with anti-␥ c antibody, and the resulting antigen-antibody complex was analyzed by SDS-PAGE. The gel was overexposed (5 days) to ensure visualization of the IL-13/␥ c band. The results ( Fig. 1C) indicate the formation of an 125 I-IL-13⅐IL-13R complex of 58 -65 kDa in ML␥ c cells. Furthermore, the ␥ c protein could be immunoprecipitated from this band in ML␥ c as a faint ϳ60-kDa band ( Fig. 1C) but not from the control ML neo cell line (data not shown).
Effect of ␥ c on IL-13 and IL-4 Binding to Their Receptors-Since ␥ c has previously been shown to increase the binding affinity of certain cytokine ligands to their receptor (15)(16)(17)(18), we examined its effect on IL-13 binding to ML␥ c and ML neo cells by performing 125 I-IL-13 equilibrium binding assays. The results (Table I, Fig. 2, A and B) show that the neoR ϩ control RCC cells (ML neo ) significantly bound 125 I-IL-13, and this binding was specific as it was displaced by increasing concentrations of the unlabeled ligand (Fig. 2). However, introduction of the ␥ c chain FIG. 1. A and B, expression of ␥ c mRNA and protein in ML␥ c cells. Ten micrograms of total RNA from ML-RCC cells transfected with ␥ c and/or neomycin phosphotransferase cDNA as described under "Experimental Procedures" was fractionated over formaldehyde denaturing agarose gel, transferred to a nylon membrane support, and hybridized with a 32 P-labeled ␥ c cDNA probe as described previously (4). Blots were stripped and rehybridized with 32 P-labeled cDNA probe for GAPDH (lower panel, A). ML␥ c and ML neo cells (10 ϫ 10 6 ) were lysed at 4°C with 1% Nonidet P-40 containing protease inhibitors as described under "Experimental Procedures." Proteins in the cell lysate were separated by SDS-PAGE and transferred to a PVDF membrane (NOVEX) for Western blotting using a ␥ c specific antibody. Detection was achieved by chemiluminescence using RENAISSANCE reagents (NEN Life Science Products). Molecular size standards are shown on the left of the gel (B). C, affinity cross-linking and immunoprecipitation. ML␥ c and ML neo cells (7 ϫ 10 7 cells/ml) were incubated with 5 nM 125 I-IL-13 for 2 h at 4°C. 125 I-IL-13 was cross-linked to its receptor with disuccinimidyl suberate (2 mM), and total cell lysate was prepared from the cells, precleared in protein A-Sepharose beads. Some lysates were incubated with protein A-Sepharose beads previously incubated with ␥ c -specific antibody. The immunoprecipitated complex (IP) as well as the 125 I-IL-13⅐IL-13R complex were analyzed by SDS-PAGE as described (4).  into these cells resulted in a drastic reduction of 125 I-IL-13 binding. For example, assuming a 1:1 ratio of radiolabeled ligand to cell surface receptor, an analysis of the 125 I-IL-13 binding data with the LIGAND program (21) indicates that neoR ϩ control ML neo cell lines express 480 Ϯ 86 ϫ 10 3 (mean Ϯ S.D.) IL-13 binding sites/cell, and the number of binding sites/ cell on the parental cell line, ML-RCC was determined to be 351 Ϯ 90 ϫ 10 3 . However, in the ␥ c -transfected ML␥ c , 125 I-IL-13 binding was dramatically reduced (by up to 2 log orders) to 4080 Ϯ 1890 binding sites/cell. It is interesting that, in addition to reducing the level of ligand binding, ␥ c induced high affinity 125 I-IL-13 binding to RCC cells (Fig. 2B). For example, ML and ML neo cells bound 125 I-IL-13 with a dissociation constant (K d ) ranging from 7.5 to 20.5 nM (mean Ϯ S.D. ϭ 13.4 Ϯ 4.6 nM), but in ML␥ c , the K d value (mean Ϯ S.D.) was 0.23 nM Ϯ 0.14 (n ϭ 4) ( Table I). In some experiments, analysis of the binding data with the LIGAND program (21) indicated a concurrent expression of a lower affinity IL-13R (K d ϭ 7.5 nM) (Fig. 2B).
Since IL-4R expression is much more limited in ML-RCC cells, we compared the effect of ␥ c on IL-4 binding in ML neo and ML␥ c cells by a direct binding assay using a saturating concentration of 125 I-IL-4 and 125 I-IL-13. Our results (Fig. 2C) show that in comparison to the profound decrease in IL-13R expression induced by ␥ c , IL-4R expression is not significantly affected.
Modulation of IL-13R␣ and IL-13R␣Ј mRNA Expression by ␥ c -Since ␥ c inhibited IL-13 binding and IL-13R␣ and IL-13R␣Ј chains were recently cloned, we were interested to examine whether one or both chains were modulated by the presence of ␥ c in ML-RCC. By Northern analysis, we found that IL-13R␣ mRNA is expressed in ML neo but not in ML␥ c cells (Fig. 3,  upper panel). The expression of IL-13R␣Ј mRNA was reduced in ML␥ c in comparison to ML neo (Fig. 3, middle panel). When we normalized IL-13R␣Ј mRNA band to the GAPDH mRNA band in each cell type for comparison, we observed that IL-13R␣Ј expression is about 3 times higher in ML neo than in ML␥ c .
Modulation of ICAM-1 Expression by ␥ c -Since ␥ c modulated IL-13 binding and appeared to alter the morphology of ␥ ctransfected cells, we further investigated its role in ML-RCC cells by examining the effect of IL-13 and IL-4 on the surface expression of ICAM-1 molecules. Consistent with our previous findings (4, 23), the control ML neo cell line had considerable baseline expression of ICAM-1 (Fig. 4A, left panel), which was significantly enhanced by IL-13 or IL-4 treatment. However, transfection of ␥ c inhibited the basal level of ICAM-1 expression in ML␥ c and neither IL-13 nor IL-4 could reverse this inhibition (Fig. 4A, right panel). Additionally, we examined the effect of ␥ c on ICAM-1 mRNA expression by Northern analysis. Our results show that in the ␥ c -positive ML␥ c cells (Fig. 4B, right lane) ICAM-1 mRNA is not expressed, but it is expressed in the ␥ c -negative ML neo and the parental ML-RCC cells (two left lanes).
Effect of ␥ c on the Phosphorylation of JAK Kinase by IL-13 and IL-4 -It has been shown that a number of cytokines including IL-13 and IL-4 can induce phosphorylation and activation of the Janus family of kinase (24 -27). We recently demonstrated that colon carcinoma cells express JAK1, JAK2, and Tyk2 (6,24) and that these kinases are phosphorylated by IL-4 and IL-13. JAK3 was not expressed and was not phosphoryl- Cell-bound radioactivity was determined with a ␥ counter and the data analyzed with the LIGAND program (21). Binding data are shown in A, while a Scatchard plot of the data is shown in B. IL-4R and IL-13R expression in control (ML neo ) and ␥ c -transfected (ML␥ c ) cells was also analyzed by direct binding to 2 nM 125 I-IL-4 or 125 I-IL-13. Nonspecific binding was determined by including excess unlabeled IL-4 or IL-13 in the binding tube and was subtracted from total binding values to obtain specific binding data (C). ated in these cells (24). In the present study, we examined the impact of the presence of ␥ c chain on the IL-13-and IL-4induced phosphorylation of JAK tyrosine kinase in ML-RCC cell lines. As shown in Fig. 5A, IL-4 induced the phosphorylation of JAK1 and JAK2 tyrosine kinase in ML cells. JAK1 kinase was weakly phosphorylated in response to IL-13, but in contrast to our previous observation in colon carcinoma cells (24), IL-13 did not induce phosphorylation of JAK2 in ML-RCC cells. Tyk2 kinase was constitutively phosphorylated, and this level of phosphorylation was not enhanced by either interleukin. In the ␥ c (ϩ) ML␥ c cells, JAK1 was constitutively phosphorylated and the level of phosphorylation was not affected by IL-4 or IL-13 treatment. JAK2 was weakly phosphorylated in unstimulated as well as in IL-13-or IL-4-treated groups and Tyk2 kinase was not detected. As previously observed in colon carcinoma cells (24), JAK3 kinase was neither expressed nor phosphorylated in ML neo and ML␥ c cells, although it was expressed and phosphorylated in the EBV-B cell line used as a positive control (Fig. 5B and Ref. 24). Thus, in ␥ c -transfected ML␥c cells, Tyk2 kinase was not expressed and IL-4 and IL-13 could not enhance or induce the phosphorylation of JAK1 and JAK2 otherwise seen in control ML cells.
Effect of ␥ c on the IL-13-and IL-4-mediated Activation of STAT Protein-We next investigated the effect of ␥ c expression on STAT activation by performing EMSA on lysates from these cells. As shown in Fig. 6A, in ␥ c -negative cells (ML and ML neo ), NF (nuclear factor)-IL-4 was activated and bound to the labeled GRR probe in response to IL-13 or IL-4. However, in the ␥ cpositive ML␥ c cells, NF-IL-4 was not activated in response to IL-13 or IL-4. We ascertained the specificity of the assay by incubating the labeled probe with a 200-fold excess unlabeled probe before running it on the gel. No NF-IL-4/GRR bands were observed in lanes containing these samples.
To confirm the identity of the STAT protein which was activated in response to IL-4 and IL-13 in ML and ML neo RCC cells, we next performed the supershift assay. The results (Fig. 6B) show that the binding of NF-IL-4 in the IL-4-treated ML group is not affected by preincubation in normal serum or in antibodies specific for STATs 1, 3, 4, or 5, whereas the band representing the bound NF-IL-4 was not visible when the lysate was preincubated in anti-STAT6 antibody. These results indicate that NF-IL-4 corresponds to STAT6 and the other STATs were not activated in response to IL-4 in ML cells. In the ␥ c -positive cells (ML␥ c ), no STAT protein was activated in response to IL-4.
To further investigate the mechanism underlying the ␥ c inhibition of STAT6 activation, we examined the phosphorylation of STAT6 protein from ␥ c (ϩ) and ␥ c (Ϫ) ML-RCC cells in response to IL-13 and IL-4 by immunoprecipitation and immunoblotting. As shown in Fig. 6C, while IL-13 and IL-4 treatment induced FIG. 3. Effect of ␥ c on IL-13R␣ and IL-13R␣ expression. Total RNA was isolated from ML␥ c and ML neo cells, and 10 g from each preparation was examined for the presence of mRNA for the two known chains of IL13R (13,14) by Northern blot analysis. The cDNA for these genes, provided by Dr. Warren Leonard (NHLBI, NIH, Bethesda, MD) and Dr. P. Ferrara (Sanofi, Labege Cedex, France) were labeled with 32 P and used as probes. The filter was subsequently stripped and reprobed for GAPDH (bottom panel).

FIG. 4.
Effect of ␥ c on ICAM-1 expression. ML␥ c RCC cells containing ␥ c cDNA as well as ␥ c -control ML neo cells were cultured at 1 ϫ 10 5 cells/ml in medium containing IL-4 or IL-13 (10 ng/ml) over a 48-h period. Cells (4 -6 ϫ 10 5 /sample) were washed and examined for surface ICAM-1 expression by flow cytometry as described under "Experimental Procedures." Fluorescence intensity (log 10 ) expressed as mean channel number (MCN) is shown on the x axis. Cells cultured in medium only and stained with FITC-conjugated IgG 1 (isotype control) were used as controls (A). ICAM-1 mRNA expression was examined in ␥ c -positive ML␥ c and in ␥ c -negative ML neo control cells by Northern analysis (B). Ten micrograms total mRNA was electrophoresed through formaldehyde/agarose denaturing gel and analyzed for ICAM-1 mRNA expression as described previously (23). STAT6 phosphorylation in the parental ML and the control neoR ϩ transfectant (ML neo ) cell lines, STAT6 phosphorylation was not induced in the ␥ c (ϩ) ML␥ c cell line (left panel). These results indicate that ␥ c inhibited the IL-4-and IL-13-mediated activation of STAT6 by inhibiting its phosphorylation. DISCUSSION In our initial characterization of IL-13 binding, we noted that while IL-13 was able to displace or inhibit IL-4 binding to RCC cells, which lack ␥ c expression, it did not inhibit IL-4 binding in Raji B and MLA144 T lymphoid cells in which ␥ c is highly expressed. Furthermore, we noted that B cells and monocytes which are highly responsive to IL-13 express very few IL-13R (4). This led us to further explore the role of ␥ c in IL-13 binding. In this report, we have demonstrated that the ␥ c protein can exert a profound influence on the binding of IL-13 to its receptors and on the signal transduction pathways of IL-13 and IL-4 following ligand binding. Expression of ␥ c in ML-RCC profoundly inhibited IL-13 binding, IL-13-induced signaling, and ICAM-1 expression. Although IL-4 binding was not affected by ␥ c expression, IL-4-induced signaling was also impaired in the ␥ c -transfected cell line and IL-4 failed to induce ICAM-1 expression in these cells.
Our results regarding the expression of ␥ c in cells that coexpress IL-4R and IL-13R demonstrate that ␥ c expression is disruptive to the expression of the ␣ chain of the IL-13R while diminishing IL-13R␣Ј expression, and may be responsible for the reduction in IL-13 binding. On a broader scale, this may explain why ␥ c -positive cells such as B cells, monocytes, and TF-1 cell lines, which are very responsive to IL-13, have been shown to express low levels of IL-13R. Similarly, the failure of IL-13 to compete for the binding of IL-4 on MLA 144 and Raji B lymphoblastoid cell line (4) otherwise seen in other cell types (4,8,10,25) may be due to the high ␥ c content of these cells and the consequent deficiency in IL-13R␣ expression.
It is of interest that the ␥ c protein affected IL-13R rather than IL-4R in this manner because, although previous studies have shown that ␥ c is a natural component of IL-4R in a number of cell types, it has not been identified as a natural component of IL-13R in any cell type examined thus far (11). In a study carried out with COS-7 cells, the inhibition of IL-13 but not IL-4 binding by ␥ c was demonstrated (13). However, the biologic consequences of the observation were not addressed. Our results demonstrate that ␥ c has the potential to physically interact with components of native IL-13 signaling pathway and exert a downstream effect. Consistent with this concept, ␥ c expression altered the pattern of intracellular signaling in response to IL-13. For example, IL-13 has been shown to cause the phosphorylation and activation of JAK1 and Tyk2 tyrosine kinase in hematopoietic cells and JAK1, JAK2, and Tyk2 in non-hematopoietic cells (6, 24 -31). However, in the current study, phosphorylation of JAK2 and Tyk2 was not seen in ␥ c -transfected ML␥ c cells. Similarly, IL-13 failed to induce the phosphorylation of JAK-1 kinase in ␥ c -transfected cells, although IL-13 induced the phosphorylation of JAK1 in control cells. Tyk2 was constitutively phosphorylated in control cells, and IL-13 did not modulate its phosphorylation. In ␥ c -transfected cells, neither constitutive nor IL-13-induced phosphorylation of Tyk2 was observed. These results suggest that the expression of ␥ c leads to an inhibition of IL-13-mediated activation of JAK kinase in ML-RCC cell line and that IL-13R␣ may play a pivotal role in these signaling events.
The activation of JAK kinase has been shown to cause the phosphorylation and activation of STAT proteins. We therefore evaluated phosphorylation and activation of STAT proteins in control and ␥ c -transfected ML␥ c cells as well as the effects of IL-13 on these events. It is of interest to note that IL-13 phosphorylated and activated STAT6 protein in control cells but not in ␥ c -transfected ML␥ c cells. These data suggest that ␥ c expression and the accompanying disruption of IL-13R␣ and/or diminution of IL-13R␣Ј expression inhibited not only the proximal events that result from IL-13 activation but the distal/ terminal IL-13 and IL-4 signaling events as well. The results also suggest that IL-13R␣ and IL-13R␣Ј may be essential for the transduction of certain IL-4 and IL-13 signals supporting previous suggestions that the signal pathways for both cytokines converge at one or more points.
The reason for the profound decrease in IL-13 binding to ␥ c -transfected ML cells is not completely known. However, our results suggest that IL13R␣ is predominantly essential for IL-13 binding and its apparent displacement by ␥ c in the IL-13R complex may be related to the drastic reduction in IL-13 binding. This structural modification of the IL-13R complex by ␥ c may also have affected the affinity of its binding to IL-13. In this study, we evaluated IL-13 binding by 125 I-IL-13 displacement assays and our results suggest that the affinity of IL-13 binding is lower in ML neo than in ML␥ c . However, in other studies, 2 we have proposed a different model for the analysis of IL-13 binding to these cells, which may be more appropriate for addressing the apparent cluster distribution of IL-13R on these cells. While ␥ c may have favored high affinity IL-13 binding, it may also have disrupted necessary interactions between the different chains of IL-4R and IL-13R through the sequestration of IL-4R␤ by ␥ c .
The loss of IL-4 and IL-13 signaling in ML␥ c may also have resulted from a disruption of necessary interactions between the intracellular domains of the receptor for IL-4 and IL-13. It was recently shown that the IL-4R␤ protein undergoes homodimerization for IL-4 signaling and that ␥ c is not required for the phosphorylation and activation of STAT6 protein (32,33). Although there is no direct evidence that the different intracellular domains of IL-4R and IL-13R interact, IL-13 has been shown to phosphorylate IL-4R␤ (24). Thus it is quite likely that the introduction of ␥ c interferes with the assembly of a functional IL-4R complex, resulting in an impaired intracellular signaling by IL-4 and IL-13. It is also possible that the introduction of ␥ c may have activated phosphatases whose activities could have resulted in the disruption of IL-13 and IL-4 signaling. Additional studies are necessary to address these possibilities.
We next examined the effect of ␥ c on the functional response of ML-RCC cells to IL-4 and IL-13 stimulation. Surprisingly, in ␥ c -transfected ML␥ c cells, there was a distinct change in morphology accompanied by a drastic diminution of ICAM-1 expression, and neither interleukin reversed these changes. The mechanism(s) underlying these changes is not clear but our results suggest that the disruption of IL-13R␣ is directly or indirectly involved in a structural modification of the cytoskeletal framework of the cell to alter its shape and size. Whether this morphological effect is related to the loss of ICAM-1 expression is not clear. Alternatively, the introduction of ␥ c into the cells may have turned off ICAM-1 gene expression and cellular responsiveness to IL-13 and IL-4. Additional studies are needed to improve our understanding of these events.
In summary, we have provided evidence for a novel role for ␥ c protein in ML-RCC cells. Not only did this protein inhibit the binding of IL-13 to its receptor but, more importantly, it also inhibited intracellular signaling induced by IL-13 and IL-4. In addition, ␥ c modified cellular function in these cells by inhibiting constitutive IL-13R␣ and ICAM-1 expression, and this inhibition was not restored by IL-4 or IL-13. Our results suggest that an abnormal expression of ␥ c may have significant disease implications with or without IL-13 and IL-4 involvement and that additional studies are needed to better understand the role of ␥ c in tumor cell function. FIG. 6. Effect of ␥ c on IL-13-and IL-4-induced STAT phosphorylation and activation in ML-RCC cells. A, after treatment with medium, IL-13 (250 ng/ml), or IL-4 (50 ng/ml), cells (ML, ML␥ c , ML neo ) were washed in cold PBS and solubilized with cold whole cell extraction buffer. DNA binding activity was determined by EMSA. Briefly, 30 g of protein samples were incubated for 20 min at room temperature with 1 ng of 32 P-labeled GRR probe in 20 l of binding buffer. A 200-fold excess of cold GRR probe was added as a competitor. For the supershift assay (B), cell lysates were preincubated with preimmune rabbit serum as a control or STAT1, 3, 4, 5, and 6 antibodies on ice for 30 min; standard EMSA was then performed as described above. C, following IL-13 and IL-4 treatment, cells were lysed in lysing buffer and STAT6 protein was immunoprecipitated (IP) with specific antibody. The complex was separated by SDS-PAGE and transferred onto PVDF membrane. STAT6 phosphorylation was detected by anti-phosphotyrosine antibody (4G10) (upper panel). STAT6 protein expression was confirmed by reprobing the stripped blot with STAT6 antibody (lower panel).