SEARCH:   Advanced

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Kawakami, K. Articles by Puri, R. K. Search for Related Content PubMed PubMed Citation Articles by Kawakami, K. Articles by Puri, R. K. Related Collections Immunobiology Signal Transduction Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 1 May 2001, Vol. 97, No. 9, pp. 2673-2679 IMMUNOBIOLOGY The interleukin-13 receptor 2 chain: an essential component for binding and internalization but not for interleukin-13-induced signal transduction through the STAT6 pathway Koji Kawakami, Jun Taguchi, Takashi Murata, and Raj K. Puri From the Laboratory of Molecular Tumor Biology, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, National Institutes of Health, Bethesda, MD.     Abstract Top Abstract Introduction Materials and methods Results Discussion References The interleukin-13 receptor (IL-13R) complex is composed of 2 different chains, IL-13R1 (also known as IL-13R') and IL-13R2 (also known as IL-13R). For a functional IL-13 receptor, the IL-13R1 chain forms a productive complex with the primary IL-4 binding protein (IL-4R also known as IL-4R). However, the function of the IL-13R2 chain is not clear even though this chain binds IL-13 with high affinity. This study demonstrates that IL-13R2 can undergo internalization after binding to ligand without causing activation of its signaling pathways. These conclusions were drawn on the basis of (1) internalization of 125I-IL-13 in Chinese hamster ovarian (CHO-K1) and T98G glioblastoma cells transiently transfected with the IL-13R2 chain; (2) a recombinant chimeric fusion protein comprising IL-13 and a mutated form of Pseudomonas exotoxin (termed IL13-PE38QQR or IL-13 toxin) is specifically cytotoxic to IL-13R2-transfected CHO-K1 cells in a gene dose-dependent manner, whereas cells transfected with vector alone were not sensitive; and (3) IL-13 did not cause activation of signal transduction and activation of transcription 6 (STAT6) in IL-13R2-transfected cells. IL-13 efficiently caused activation of STAT6 protein in cells transfected with the IL-13R1 and IL-4R chains, and IL-13R2 inhibited this activation. Taken together, these observations indicate that internalization of IL-13R2 is signal independent and that this property of IL-13R2 can be exploited for receptor-directed cancer therapy. (Blood. 2001;97:2673-2679) © 2001 by The American Society of Hematology.     Introduction Top Abstract Introduction Materials and methods Results Discussion References Interleukin-13 (IL-13) and IL-4 are related multifunctional cytokines with similar biological activities on human B cells and monocytes.1-7 The cognate receptors for these cytokines are complex and have been shown to share 2 chains with each other.8-12 The IL-4 receptor system is well characterized and has been shown to be composed of a primary IL-4 binding protein, IL-4R (also known as IL-4R).13 This chain forms a heterodimer with either the IL-2Rc chain (type I IL-4R) or IL-13R1 (also known as IL-13R') (type II IL-4R) for signaling.11,14-17 In some situations, all 3 chains (IL-4R, IL-2Rc, and IL-13R1) may constitute the IL-4R complex; however, whether all 3 chains are simultaneously required for IL-4 function is not known. In contrast, the receptor for IL-13 is less well characterized. We have studied the structure of IL-13R in various cell types and reported that IL-13 binds to 2 isoforms of an approximate 65-kDa protein in human renal cell carcinoma cells.8-12 One of these proteins also binds IL-4.8,14 On the basis of the binding characteristics, cross-linking, and displacement of radiolabeled IL-4 and IL-13 in various cell types, we hypothesized that, as is the case for the IL-4R system, IL-13R may also be composed of 3 different types.9-12 More recently, 2 different chains of the IL-13R system have been cloned. The murine and human IL-13R1 chain was first cloned.14,18 This chain can bind IL-13 with low affinity, but, when coupled with the IL-4R chain, the heterodimer binds IL-13 with high affinity and mediates IL-13-induced signaling.14,18 The second chain of the IL-13R, termed IL-13R2 (also known as IL-13R), has also been cloned from a human renal cell carcinoma cell line (Caki-1). This chain shares approximately 50% homology with the IL-5R at the DNA level. It contains a very short intracellular domain and binds IL-13 with high affinity.19 On the basis of the above studies, we have proposed that the type I IL-13R complex is composed of both chains of IL-13R (IL-13R1 and IL-13R2) and IL-4R chain. Although IL-13 binds to all 3 chains, only the IL-13R1 and IL-4R chains form a productive complex. Because of this arrangement, IL-13 binds to these cells strongly. Only IL-13, not IL-4, is able to displace the binding of 125I-IL-13. In the type II IL-13R system, IL-13R2 is not present, and the IL-13R1 chain forms a complex with the IL-4R chain. In these cells both interleukins compete for the binding of their radiolabeled cognaters.12 The structure of the type III IL-13R is similar to that of type II receptors except that these cells also express the IL-2Rc chain. Although it appears that the IL-2Rc chain does not bind IL-13, it does affect IL-13 binding and function in some cell types.20,21 After binding to their receptors, both IL-4 and IL-13 signal through phosphorylation-dependent activation of Jak kinases and signal transduction and activator of transcription (STAT) protein.11,17,22 In particular, STAT6 is activated in response to both IL-4 and IL-13.17,22,23 In the IL-4R system, activation of STAT6 protein was seen in cells transfected with IL-13R1 chain and IL-4R chain; however, no activation was observed in cells transfected with the IL-13R2 chain alone or when transfected with IL-4R chain.15 Whether IL-13 induces STAT6 activation by similar combination of receptor subunits is not known. It is also not known whether the IL-13R2 chain plays a major role in IL-13 functions. It has been reported that the extracellular domain of IL-13R2 is secreted in the plasma and urine of mice. However, in the context of human physiology, this protein has not been identified.24,25 To determine the function of IL-13R2, this chain was transfected either alone or in combination with other known chains of the IL-4R and IL-13R systems in Chinese hamster ovary (CHO-K1) cells and T98G glioblastoma cells. Transfectants were studied to determine whether (1) IL-13R is internalized after binding to 125I-IL-13, (2) the intracellular domain of IL-13R2 plays a role in ligand internalization, (3) IL-13 can transmit signals in cells that are transfected with this chain, and (4) a chimeric protein composed of IL-13 and a mutated form of Pseudomonas exotoxin (termed IL13-PE38QQR)26,27 is cytotoxic to cells that are transfected with the IL-13R2 chain. Our studies demonstrate, for the first time, that IL-13R2 can be internalized after binding to IL-13 without inducing intracellular signaling through the STAT6 pathway.     Materials and methods Top Abstract Introduction Materials and methods Results Discussion References Recombinant cytokines and toxins Recombinant human IL-131,5 was produced and purified to homogeneity in our laboratory (B. H. Joshi and R.K.P., unpublished results).28 Recombinant IL13-PE38QQR was also produced and purified in our laboratory (Joshi et al, unpublished results). The cpIL4-toxin IL438-37-PE38KDEL, containing the circularly permuted IL-4 mutant in which amino acids 38-129 were linked to amino acids 1-37 via a GGNGG linker and then fused to truncated toxin PE38KDEL, consisting of amino acids 253-364 and 381-608 of PE followed by KDEL, was expressed in Escherichia coli and purified as described previously.29,30 Cell lines CHO-K1 and human glioblastoma multiforma cell lines (T98G) were purchased from the American Type Culture Collection (Rockville, MD). CHO-K1 cells were cultured in a modified Eagle minimum essential medium (AMEM), and T98G cells were cultured in Eagle minimum essential medium (EMEM) containing 10% fetal bovine serum (Biowhittaker, Walkersville, MD), 1 mmol HEPES, 1 mmol L-glutamine, 100 µg/mL penicillin, and 100 µg/mL streptomycin (Biowhittaker). Plasmids, mutagenesis, and transient transfection of DNA Complementary DNAs (cDNAs) of human IL-4R chain,13 IL-13R2 chain,19 and IL-13R1 chain14 were cloned into pME18S mammalian expression vector [pIL4R, pIL13R (2), and pIL13R' (1)]. The IL-13R deletion mutants 335L, 338R, or 343Y were constructed by the polymerase chain reaction (PCR), using Taq DNA polymerase with the primer 5'-CCGCTCGAGATGGCTTTCGTTTGCTTGGCTATCGG-3' and 3'-GCTCTAGATCAACCGGTTACAAATATAACTAATATTAAG-5' (335L) or 3'-GCTCTAGATCACAAAAGCAGACCGGTTACAAATATAAC-5' (338R) or 3'-GCTCTAGATCAGGTGTTTGGCTTACGCAAAAG-5' (343Y), each containing an in-frame stop codon. The cDNAs were subcloned into the expression vector pCIneo, using the XhoI and XbaI sites. Individual plasmid DNA or a combination of multiple plasmid DNAs (6 µg/60-mm dish or 12 µg/100-mm culture dish) were transfected into semiconfluent cells, using GenePORTER transfection reagent (Gene Therapy Systems, San Diego, CA) according to the manufacturer's instructions. Briefly, cells (1 × 106/60-mm dish or 3 × 106/100-mm dish) were incubated with the DNA-GenePORTER mixture for 5 hours in Dulbecco modified Eagle medium (DMEM; Biowhittaker). Then DMEM containing 20% fetal bovine serum (FBS) was added, and incubation was continued. Twenty-four hours after transfection, the medium was changed to AMEM (CHO-K1) or EMEM (T98G) with 10% FBS, and cells were incubated for and an additional 24 hours. Radioreceptor binding assay Recombinant human IL-13 was labeled with 125I (Amersham, Arlington Heights, IL), using IODO-GEN reagent (Pierce, Rockford, IL) as previously described.8 The specific activity of the radiolabeled IL-13 was estimated to be 12.7 µCi/µg protein. For binding experiments, 5 × 105 cells in 100 µL binding buffer (RPMI 1640 containing 0.2% human serum albumin and 10 mmol HEPES) were incubated with 200 pmol 125I-IL-13 with or without 40 nmol unlabeled IL-13 at 4°C for 2 hours. Cell-bound 125I-IL-13 was separated from unbound by centrifugation through a phthalate oil gradient, and radioactivity was determined with a gamma counter (Wallac, Gaithersburg, MD). For the displacement assay, T98G cells were incubated with 125I-IL-13 (200 pmol) with or without increasing concentration (up to 100 nmol) of IL-13, as described above. IL-13R internalization Internalization assays were performed as described before.21 CHO-K1 or T98G cells with or without transfected IL-13R chain were incubated in binding buffer containing 0.2 nmol chloroquine at 37°C for 5 minutes to prevent degradation of internalized 125I-IL-13. The cells were then washed, and 2 × 107 cells were incubated with 0.5 nmol 125I-IL-13 at 4°C for 2 hours. At various time intervals, 2 duplicate sets of 50 µL aliquots were taken. One set was incubated with glycine buffer (final pH = 2.0) for 10 minutes on ice. The suspension was then centrifuged through a mixture of phthalate oils, and the radioactivity in the cell pellet (acid-resistant or internalized) and in the supernatant (surface-bound plus dissociated) was determined with a gamma counter. The other set of 50-µL aliquots was directly centrifuged through phthalate oils, and the radioactivity observed in the supernatants was used for dissociated 125I-IL-13 values. Surface-bound 125I-IL-13 was determined by subtracting dissociated 125I-IL-13 values from surface-bound plus dissociated values. Protein synthesis inhibition assay The cytotoxic activity of IL-13 toxin or IL-4 toxin was tested as previously described.27 Typically, 104 cells were cultured in leucine-free medium with or without various concentrations of IL13-PE38QQR or IL438-37-PE38KDEL for 20 to 22 hours at 37°C. Then 1 µCi of [3H]leucine (NEN Research Products, Boston, MA) was added to each well and incubated for an additional 4 hours. Cells were harvested, and radioactivity incorporated into cells was measured by a beta plate counter (Wallac). Electrophoretic mobility shift assay After incubation with IL-13 (250 ng/mL) for 10 minutes, cells were washed with cold extraction buffer (1 mg/mL leupeptin, 5 mg/mL pepstatin A, 2 mg/mL aprotinin, 20 mmol HEPES pH 7.0, 10 mmol KCl, 300 mmol NaCl, 0.5 mmol dithiothreitol [DTT], 0.1% NP-40, 1 mmol phenylmethylsulfonyl fluoride, 1 mmol Na3VO4, and 20% glycerol). DNA protein interactions were assessed by electrophoretic mobility shift assay (EMSA), using a Bandshift kit (Pharmacia, Piscataway, NJ). Briefly, 50 µg sample proteins was incubated in 20 µL binding buffer [10 mmol Tris-HCl (pH 7.5), 50 mmol NaCl, 0.5 mmol DTT, 10% glycerol, 0.05% NP-40, 0.05 mg/mL poly (dI-dC)2] for 20 minutes at room temperature with 1 ng 32P-labeled double-stranded oligonucleotide probe SBE1. SBE1 is a STAT-binding element (5'-gatcGCTCTTCTTCCCAGGAACTCAATG-3'; 3'-CGAGAAG AAGGGTCCTTGAGTTACagct-5') that is from the region flanking the transcription start site of the human sIL-1R antagonist gene that is necessary for response to IL-13.31 Ten times concentrated loading dye (2 µL) was added to samples that were then applied to a 4% nonreducing polyacrylamide gel and run at 150 V for 2.5 hours. Gels were dried for 2 hours and autoradiographed overnight at room temperature. For the supershift assay, antihuman STAT6 (S-20) rabbit polyclonal immunoglobulin G (Santa Cruz Biotechnology, Santa Cruz, CA) was included in the reaction mixture before electrophoresis.     Results Top Abstract Introduction Materials and methods Results Discussion References 125I-IL-13 binding on CHO-K1 or T98G cells transfected with IL-13R2, IL-13R1, and IL-4R chains To demonstrate ligand binding and directly examine the subunit structure of IL-13R, cDNAs of various human IL-13R chains were introduced into the CHO-K1 cell line, and binding studies were performed. As shown in Figure 1A, 125I-IL-13 was bound at a higher level to IL-13R2-transfected CHO-K1 cells as compared to cells transfected with IL-131 or IL-4R chains. A 200-fold mole per liter excess of IL-13 but not IL-4 inhibited this binding completely, indicating specific 125I-IL-13 binding (Figure 1A,B). IL-13R1 or IL-4R single-chain transfectants showed a slight increase in binding of radiolabeled ligand over vector-transfected controls. The binding of 125I-IL-13 in control cells was also inhibited although only partially by IL-4 (Figure 1B). Cotransfection of IL-13R2 chain with IL-4R or IL-13R1 chain did not induce a significant increase in 125I-IL-13 binding. In contrast, cotransfection of IL-13R2 chain along with IL-13R1 and IL-4R chains resulted in 125I-IL-13 binding that was significantly increased to maximum levels. These data suggest that IL-13R2 bind IL-13 with the greatest avidity and that this chain may not directly interact with IL-13R1 and IL-4R with respect to ligand binding. View larger version (20K): [in this window] [in a new window]   Figure 1. 125I-IL-13 binding to CHO-K1 and T98G cells transfected with the IL-13R2, IL-13R1, IL-4R, and c chains. cDNA for various receptor chains (6 µg/chain) was transfected into cells (1 × 106) by using GenePORTER reagent for 48 hours. For the IL-13 binding assay, 1 × 106 transfected cells were incubated with 200 pmol/L of 125I-IL-13 with or without a 200-fold molar excess of unlabeled IL-13 (A) or control CHO-K1 and IL-13R2-transfected cells with IL-13 or IL-4 (B). Binding assays were performed in 2 separate experiments. Cell bound radioactivity was determined as described in "Materials and methods." The binding characteristics and affinity of IL-13R on T98G cells transfected with IL-13R1 and 2 chain was also analyzed. As shown in Figure 2A, unlike control CHO-K1 cells (Figure 1A,B), vector-transfected T98G cells exhibited little 125I-IL-13 binding; however, cells transfected with IL-13R2 chain showed more than 15-fold higher IL-13 binding when compared to the control cells. Similar to CHO-K1 cells, the binding of 125I-IL-13 was inhibited by excess of IL-13 but not by IL-4. T98G cells transfected with IL-13R1 did not show appreciable binding to 125I-IL-13. To determine the binding affinity of 125I-IL-13 to its receptors in IL-13R2-transfected T98G cells, Scatchard analysis was performed, using the LIGAND program. This program allowed fitting of the data to a one-site model. As shown in Figure 2B and C, IL-13 bound to IL-13R2-transfected T98G cells with strong affinity (Kd = 1.12 nmol/L), and the receptor number was estimated to be approximately 30 580 sites/cell. These results suggest that IL-13R2 chain binds IL-13 with high affinity. View larger version (27K): [in this window] [in a new window]   Figure 2. 125I-IL-13 binding to T98G cells transfected with IL-13R1 or IL-13R2 chains. cDNA for receptor chains (6 µg/chain) was transfected into T98G cells (1 × 106) by using GenePORTER reagent for 48 hours. For the IL-13 binding assay, 1 × 106 cells were incubated with 200 pmol/L of 125I-IL-13 with or without a 200-fold molar excess of unlabeled IL-4 or IL-13 (A). Displacement curve (B) and Scatchard data (C) with T98G cells transfected with IL-13R2 chain were analyzed by the LIGAND program. Internalization of IL-13R on CHO-K1 and T98G cells transfected with the IL-13R2 chain To determine whether the IL-13R2 chain when expressed alone can be internalized after binding to its ligand, IL-13, we examined the rate of internalization and disappearance of surface-bound IL-13 using 125I-IL-13. As shown in Figure 3, CHO-K1 and T98G cells transfected with IL-13R2 chain bound IL-13, and the level of surface-bound 125I-IL-13 steadily declined with time in both CHO-K1 cells and T98G cells. Concurrently, the level of 125I-IL-13 internalized increased in these cells. In CHO-K1 cells transfected with the IL-13R2 chain, the level of internalization was higher compared to control vector-transfected cells. IL-13R2 chain-transfected cells showed an approximately 35% higher internalization level at 90 minutes when compared to control CHO-K1 cells (Figure 3A). Interestingly, control CHO-K1 cells also showed internalization of IL-13, indicating that these cells express functional IL-13R and that hamster cells bind human IL-13. However, in T98G cells, vector-transfected control cells showed no internalization, and 100% of the IL-13 remained surface bound even after 2 hours of incubation at 37°C. However, IL-13R2 chain transfectants showed a remarkable level of internalization that reached to maximum of approximately 80% of total bound ligand at 120 minutes (Figure 3B). These results suggest that, on binding to ligand 125I-IL-13, the IL-13R2 chain is internalized at a high rate and IL-13R2 is required for internalization. View larger version (20K): [in this window] [in a new window]   Figure 3. Internalization of 125I-IL-13 in CHO-K1 and T98G cells. CHO-K1 (A) and T98G (B) cells were incubated with 0.5 nmol 125I-IL-13 at 4°C for 2 hours. Then the temperature was raised to 37°C, and, at various time intervals, 2 duplicate sets of 50 µL aliquots were taken. One set was incubated with glycine buffer (pH = 2.0) for 10 minutes. The mixture was then centrifuged through phthalate oils, and the radioactivity in the cell pellet (internalized) and in the supernatant (surface-bound plus dissociated) was determined with a gamma counter. The other set of 50 µL aliquots was directly centrifuged through phthalate oils, and the radioactivity observed in the supernatant was used for dissociated 125I-IL-13 values. Surface-bound 125I-IL-13 was determined by subtracting dissociated 125I-IL-13 values from (surface-bound plus dissociated) values. Data are expressed as a percentage of total IL-13 bound at time 0. Open circles, surface IL-13 bound on control cells; closed circles, surface-bound on IL-13R2-transfected cells; open diamonds, internalization in control cells; and closed diamonds, internalization in IL-13R2-transfected cells. Values are the mean of 2 independent experiments. When not shown, standard deviations are smaller than the symbol. Cytotoxicity of IL13-PE38QQR on CHO-K1 cells transfected with the IL-13R2, IL-13R1, and IL-4R chains To further confirm whether the IL-13R chain is internalized after binding to ligand, the cytotoxicity of a recombinant IL13-PE38QQR targeting IL-13R was assessed. IL13-PE38QQR binds to IL-13R and is internalized by endocytosis, subsequently causing apoptotic and/or necrotic cell death through the inhibition of new protein synthesis.32,33 Thus, cytotoxicity observed in transfected cells indicates receptor internalization. CHO-K1 cells were transfected with the IL-13R2 chain in the absence or presence of other IL-13R chains, and sensitivity to IL13-PE38QQR was determined. As shown in Figure 4A, when CHO-K1 cells were transfected with IL-13R2 alone, IL13-PE38QQR was highly cytotoxic to these cells. The 50% inhibitory concentration (IC50; IL-13 toxin concentration causing 50% inhibition of protein synthesis) ranged between 7 and 10 ng/mL. When CHO-K1 cells were transfected with the IL-13R1 chain alone, no cytotoxicity was observed even when 1000 ng/mL IL-13 toxin was added (data not shown). However, when CHO-K1 cells were transfected with the IL-4R chain, 25% cytotoxicity was seen at 100 ng/mL IL-13 toxin (Figure 4A). Similar to binding data (Figure 1), when the 2 chain was cotransfected with the IL-13R1 or IL-4R chains, the cytotoxicity was similar to that seen with 2 chain transfection alone (Figure 4B). However, when all 3 chains (IL-4R, IL-13R, and 2) were cotransfected, cytotoxic activity was slightly increased compared to 21 or 2 IL-4R transfectants (IC50 of 10 ng/mL compared to 30 ng/mL in 12 IL-4R or 12 transfectants, respectively). Cells transfected with the 1 and IL-4R chains showed little sensitivity to IL13-PE38QQR. View larger version (33K): [in this window] [in a new window]   Figure 4. Cytotoxicity of IL-13 toxin and IL-4 toxin to CHO-K1 cells transfected with various chains of IL-13R. CHO-K1 cells were transfected with various receptor chains and then IL-13 toxin- or IL-4 toxin-mediated cytotoxicity was determined by protein synthesis inhibition assay. CHO-K1 transfected with IL-13R2 (closed circles) or IL-4R (open circles) chains (A); cells were transfected with various combinations of receptor chains, control (closed circles), 1 2 (open circles), 2 IL4R (closed diamonds), 1 IL4R (open diamonds), 12 IL4R (closed triangles) (B); cells transfected with increasing concentrations of DNA for IL-13R2 chain, 0 µg (closed circles), 1 µg (open circles), 2 µg (closed diamonds), 3 µg (open diamonds), 4 µg (closed triangles), 5 µg (open triangles), 6 µg (closed squares) (C); transfected with 6 µg cDNA of IL-132 chain and incubated with various concentrations of IL13-PE38QQR (closed circles) or IL438-37-PE38KDEL (open circles) (D); or transfected with control (open circles), IL4R (closed triangles), c (open triangles), IL4Rc (closed squares). The results are represented as means ± SD of quadruplicate determinations, and the assay was repeated several times. We next examined whether sensitivity to IL-13 toxin was IL-13R2 concentration dependent. To address this point, we transfected CHO-K1 cells with various amounts of IL-13R2 cDNA and examined the cytotoxicity of IL13-PE38QQR. As shown in Figure 4C, increasing protein synthesis inhibition was observed as the amounts of cDNA used for transfection increased. When more than 3 µg IL-13R2 cDNA was transfected in CHO-K1 cells, the protein synthesis was inhibited by 50% with less than 100 ng/mL IL13-PE38QQR. At the highest concentration of DNA (6 µg), maximum sensitivity to IL13-PE38QQR was observed (IC50 = 4 ng/mL). To confirm whether the cytotoxicity of IL13-PE38QQR was mediated through IL-13R, IL-13R2-transfected cells were tested for sensitivity to IL-4 toxin, IL438-37-PE38KDEL as well as IL13-PE38QQR. As shown in Figure 4D, CHO-K1 cells transfected with IL-13R2 chain were highly sensitive to IL-13 toxin but not to IL438-37-PE38KDEL even when up to 1000 ng/mL recombinant toxin was used. These results correspond to our previous findings, indicating that the IL-4R system does not utilize the IL-13R2 chain.9-11,15 IL-4 toxin was cytotoxic to CHO-K1 cells when transfected with IL-4R alone or in combination with c chain. However, control cells and cells transfected with c chain alone were not killed by IL-4 toxin (Figure 4E). Role of intracellular domain of IL-13R2 in ligand internalization To examine the role of the intracellular domain of the IL-13R2 chain in the internalization process, we made deletions in the intracellular domain of 2 chain (Figure 5A). After transient transfection of these deletion mutant genes in CHO-K1 cells, we performed IL-13 binding and internalization studies. As shown in Figure 5B, deletion of the part of transmembrane domain and whole intracellular domain (335L mutant) caused a significant decrease in IL-13 binding. In addition, this deletion mutant showed no sensitivity to IL-13-PE38QQR similar to control cells (Figure 5C). These results indicate that the part of transmembrane domain and/or all of intracellular domain of IL-13R2 are required for IL-13 internalization. However, deletions of part or complete intracellular domain alone (338R or 343Y) showed no change in IL-13 binding or sensitivity to IL-13 toxin. Therefore, it was concluded that amino acids between L335 and Y343 in the IL-13R2 chain might be involved in IL-13 binding and/or internalization. View larger version (20K): [in this window] [in a new window]   Figure 5. 125I-IL-13 binding and cytotoxicity of IL-13 toxin to CHO-K1 cells transfected with IL-13R2 deletion mutants. Schematic representation of the wild-type and mutant IL-13R2 chains. EC, extracellular domain; TM, transmembrane domain; IC, intracellular domain (A). cDNA for various mutant receptor chains (6 µg/chain) was transfected into cells (5 × 105) by using GenePORTER reagent for 48 hours. For IL-13 binding assay, 5 × 105 cells were incubated with 200 pmol/L 125I-IL-13 with or without a 200-fold molar excess of unlabeled IL-13. Binding assays were performed in 2 separate experiments. Cell-bound radioactivity was determined as described in "Materials and methods" (B). CHO-K1 cells were transfected with IL-13R2 and its mutants, and then IL13-PE38QQR cytotoxicity was determined by protein synthesis inhibition assay. The results are represented as means ± SD of quadruplicate determinations (C). Activation of STAT6 in response to IL-13 in IL-13R chain transfectants To demonstrate whether the IL-13R2 chain signals after binding IL-13 and whether IL-13R1 and IL-4R by themselves or heterodimers with the IL-13R2 chain are biologically functional, we analyzed STAT6 activation in response to IL-13 in various IL-13R chain transfectants. As shown in Figure 6A (lane 1), no STAT6 activation was observed in CHO-K1 cells transfected with the IL-13R2 chain. However, when cells were transfected with 1 or IL-4R chains, low-level activation was observed (Figure 6A, lanes 2 and 3). When the 1 chain was cotransfected with the IL-4R chain, the STAT6 activation was induced to its highest level (Figure 6A, lane 8). Cotransfection of the 2 chain with the IL-4R chain or 1 chain did not induce STAT6 activation. In fact, 2 chain transfection abrogated IL-4R or 1-induced STAT6 activation (Figure 6A, lanes 5 and 6). In addition, when 1, IL-4R, 1c, and IL-4Rc were cotransfected with the 2 chain, the STAT6 activation was decreased (Figure 6A, lanes 11-13). CHO-K1 cells transfected with all 4 chains (21IL-4Rc) appeared to show similar STAT6 activation compared to 1 IL-4R transfectants (Figure 6A, lane 15). View larger version (82K): [in this window] [in a new window]   Figure 6. Modulation of STAT6 activation by IL-13 and the IL-13R2 chain. CHO-K1 cells were transfected with various receptor chains and incubated with IL-13 for 10 minutes, solubilized with cold whole-cell extraction buffer, and 50 µg sample protein was incubated for 20 minutes with 1 ng 32P-labeled SBE1 probe in binding buffer. DNA-protein interaction was analyzed by SDS-PAGE analysis (A). CHO-K1 cells were transfected with increasing amounts (0 to 10.5 µg) of IL-13R2 cDNA along with fixed amounts of DNA for 1 IL4R (6 µg) chains (B) or 1 IL4Rc (C). For supershift assay, protein extract from 1 IL4R-transfected CHO-K1 cells was incubated with antihuman STAT6 rabbit polyclonal immunoglobulin G before electrophoresis (D). To further study the impact of the IL-13R2 chain on STAT6 activation, we transfected CHO-K1 cells with the 1 and IL-4R chains along with various amounts of 2 chain cDNA. As shown in Figure 6B, the extent of STAT6 activation in response to IL-13 was gradually decreased as the amount of 2 chain cDNA transfected increased. We also assessed the effect of the IL-13R2 chain in IL-13-induced STAT6 activation in CHO-K1 cells transfected with IL-4R, IL-13R1, and c chains. As shown in Figure 6C, introduction of the IL-13R2 chain at very high concentration can still inhibit IL-13-induced STAT6 activation as seen in IL-4R and IL-13R1 transfectants. The presence of c chain had no effect on this inhibition. To confirm whether the IL-13-induced SBE-1-binding complex contains STAT6, an antibody supershift assay was performed. The STAT6 activation that was induced by IL-13 in 1 IL-4R heterodimer transfectants showed a substantial supershift by anti-STAT6 antibody (Figure 6D, lane 2). These data confirm that SBE-1 binding complexes induced by IL-13 contain the STAT6 molecule.     Discussion Top Abstract Introduction Materials and methods Results Discussion References The major goal of our study was to investigate whether the IL-13R2 chain can internalize IL-13 ligand by itself and whether this chain can induce signal transduction through STAT pathways. In this report, we demonstrate that the IL-13R2 chain can bind with high affinity to IL-13 and can promote internalization of ligand, but it cannot induce signaling via the STAT6 pathway. Although we cannot rule out the possibility that IL-13 may signal through different signaling cascades, our results suggest an interesting dissociation between internalization and signal transduction by IL-13R. This phenomenon is similar to that observed for the IL-6 receptor system in which IL-6 signal transducer gp130 is internalized without inducing signal transduction through the JAK/STAT pathway.34,35 The IL-13R2 chain affects a decrease in the level of IL-13-induced STAT6 activation in cells cotransfected with the IL-13R1 and IL-4R chains or IL-13R1, IL-4R, and c chain. This decrease in signaling may be due in part to the "stealing" of IL-13 from other components of the receptor, thus making less IL-13 available for binding and signaling through type II IL-13 receptors. The internalization of ligand on IL-13R2 transfection alone was also confirmed by cytotoxicity assays that utilize IL-13 cytotoxin. CHO-K1 cells transfected with the IL-13R2 chain were very sensitive to recombinant IL13-PE38QQR cytotoxin in a dose- and IL-13R2 concentration-dependent manner. However, IL-4 toxin was not cytotoxic, indicating specificity of the internalization. When IL-13R2 chain was cotransfected with IL-13R1 and IL-4R chains, a slightly higher cytotoxicity was observed. These results agree with the binding studies and further indicate that IL-13 toxin is internalized through the IL-13R2 chain that, when combined with the IL-13R1 and IL-4R chains, produces an additive effect. It is of interest that about 25% protein synthesis inhibition occurs at 100 ng/mL and higher concentrations of IL13-PE38QQR in CHO-K1 cells transfected with the IL-4R chain alone. Because CHO-K1 cells express low levels of specific IL-13R, transfection of the IL-4R chain may form functional type II IL-13 complex, resulting in internalization of IL13-PE38QQR. It has been demonstrated that a di-leucine motif in the intracellular domain of type I cytokine receptor systems (eg, IL-6R,35,36 granulocyte colony-stimulating factor receptor,37 epidermal growth factor receptor,38 growth hormone receptor,39 and human insulin receptor40) plays an essential role in the internalization of ligand. In the IL-13R2 chain, there is no intracellular domain L-L motif; however, 3 residues, L335, L336, and L337, are present at the carboxy terminus of the transmembrane domain.19 Whether these leucine residues interact with other amino acids and contribute to the internalization of IL13-IL-13R complex is not known. To study this, we produced 3 deletion mutants in the intracellular domain of the IL-13R2 chain, and the significance of different intracellular mutants was examined by binding and internalization studies. From these studies, it was concluded that amino acids between L335 and Y343 play a role in IL-13 binding and/or internalization. We found that when CHO-K1 cells were transduced with IL-13R1 or IL-4R chain cDNA, a modest activation of STAT6 was observed. It is possible that CHO-K1 cells naturally express IL-4R and IL-13R1 chains and on introduction of either of these chains form a functional IL-13R complex. To address this issue, we performed reverse transcriptase (RT)-PCR analysis of these chains. Although we did not find expression of these chains by sensitive RT-PCR assay (data not shown), we do not rule out the possibility of their expression because (1) CHO-K1 cells specifically bind IL-13 and (2) primers used in our study were designed to amplify human receptor chain RNA. The specific primers for Chinese hamster may be needed to reverse transcribe and detect hamster receptor chains. Alternatively, the IL-13R1 and IL-4R chains can homodimerize on binding to IL-13 albeit at low levels and can activate STAT6. Homodimerization of IL-4R has been shown to activate STAT6 on stimulation with IL-4.15 In conclusion, we have reconstituted a functional IL-13R by transfecting various components of the IL-13R system. For the first time we provide experimental evidence that the IL-13R2 chain is a functional component of an IL-13R system that promotes binding and internalization of ligand. In addition, the IL-13R2 chain may interact with the 1 or IL-4R chains, or both, as it inhibits the effect of IL-13 on STAT6 activation in 1- or IL-4R-transfected cells. Since IL-13R2 is not involved in type II or type III IL-13R systems commonly expressed on normal immune and some nonimmune cells, our observations indicate that the IL-13R2 chain can be a potential target for receptor-directed immunotherapy or gene therapy for cancer or inflammatory diseases in which the IL-13R is involved. In addition, transfer of the IL-13R2 chain gene may make cells more susceptible to the cytotoxic effect of IL-13 toxin.     Acknowledgments We thank Drs S. Rafat Husain and Bharat H. Joshi for labeling IL-13 and for providing IL13-PE38QQR, and Dr Yasuo Oshima and Ms Pamela Dover for helpful suggestions. We also thank Drs Elizabeth Shores and Donald Fink for critical reading of this manuscript.     Footnotes Submitted February 2, 2000; accepted November 29, 2000. K.K. and J.T. contributed equally to this paper. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Raj K. Puri, Laboratory of Molecular Tumor Biology, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, National Institutes of Health, Bldg 29B, Rm 2NN10, 29 Lincoln Dr MSC 4555, Bethesda, MD 20892; e-mail: puri{at}cber.fda.gov.     References Top Abstract Introduction Materials and methods Results Discussion References 1. Minty A, Chalon P, Dorocq J-M, et al. Interleukin-13 is a new human lymphokine regulating inflammatory and immune responses. Nature. 1993;362:248-250[CrossRef][Medline] [Order article via Infotrieve]. 2. Zurawski G, de Vries JE. Interleukin-13, an interleukin-4-like cytokine that acts on monocytes and B cells but not on T cells. Immunol Today. 1994;15:19-26[CrossRef][Medline] [Order article via Infotrieve]. 3. de Waal Malefyt R, Figdor CG, Huijbens R, et al. Effects of IL-13 on phenotype, cytokine production, and cytotoxic function of human monocytes: comparison with IL-4 and modulation by IFN- or IL-10. J Immunol. 1993;151:6370-6381[Abstract]. 4. de Waal Malefyt R, Figdor CG, de Vries JE. Effects of interleukin 4 on monocyte functions: comparison to interleukin 13. Res Immunol. 1993;144:629-633[CrossRef][Medline] [Order article via Infotrieve]. 5. McKenzie ANJ, Culpepper JA, de Waal Malefyt R, et al. Interleukin 13, a novel T cell-derived cytokine that regulates human monocytes and B cell function. Proc Natl Acad Sci U S A. 1993;90:3735-3739[Abstract/Free Full Text]. 6. Defrance T, Carayon P, Billian G, et al. Interleukin 13 is a B cell stimulating factor. J Exp Med. 1994;179:1325-1333. 7. Cooks BG, de Waal Malefyt R, Galizzi J-P, de Vries JE. IL-13 induces proliferation and differentiation of human B-cells activated by the CD40 ligand. Int Immunol. 1993;5:657-663[Abstract/Free Full Text]. 8. Obiri NI, Debinski W, Leonard WJ, Puri RK. Receptor for interleukin 13: interaction with interleukin 4 by a mechanism that does not involve the common  chain shared by receptors for interleukins 2, 4, 7, 9, and 15. J Biol Chem. 1995;270:8797-8804[Abstract/Free Full Text]. 9. Murata T, Obiri NI, Debinski W, Puri RK. Structure of IL-13 receptor: analysis of subunit composition in cancer and immune cells. Biochem Biophys Res Commun. 1997;238:90-94[CrossRef][Medline] [Order article via Infotrieve]. 10. Murata T, Obiri NI, Puri RK. Human ovarian carcinoma cell lines express IL-4 and IL-13 receptors: comparison between IL-4- and IL-13-induced signal transduction. Int J Cancer. 1997;70:230-240[CrossRef][Medline] [Order article via Infotrieve]. 11. Murata T, Obiri NI, Puri RK. Structure of and signal transduction through interleukin-4 and interleukin-13 receptors. Int J Mol Med. 1998;1:551-557[Medline] [Order article via Infotrieve]. 12. Obiri NI, Leland P, Murata T, Debinski W, Puri RK. The IL-13 receptor structure differs on various cell types and may share more than one component with IL-4 receptor. J Immunol. 1997;158:756-764[Abstract]. 13. Idzerda RL, March CJ, Mosley B, et al. Human interleukin 4 receptor confers biological responsiveness and defines a novel receptor superfamily. J Exp Med. 1990;171:861-873[Abstract/Free Full Text]. 14. Aman MJ, Tayebi N, Obiri NI, Puri RK, Modi WS, Leonard WJ. cDNA cloning and characterization of the human interleukin 13 receptor  chain. J Biol Chem. 1996;271:29265-29270[Abstract/Free Full Text]. 15. Murata T, Taguchi J, Puri RK. Interleukin-13 receptor ' but not a chain:  functional component of interleukin-4 receptors. Blood. 1998;91:3884-3891[Abstract/Free Full Text]. 16. Russell SM, Keegan AD, Harada N, et al. Interleukin-2 receptor gamma chain: a functional component of the interleukin-4 receptor. Science. 1993;262:1880-1883[Abstract/Free Full Text]. 17. Murata T, Noguchi PD, Puri RK. Receptors for interleukin-4 do not associate with the common c chain, and IL-4 induces the phosphorylation of JAK2 tyrosine kinase in human colon carcinoma cells. J Biol Chem. 1995;270:30829-30836[Abstract/Free Full Text]. 18. Hilton DJ, Zhang J-G, Metcalf D, Alexander WS, Nicola NA, Willson TA. Cloning and characterization of a binding subunit of the interleukin 13 receptor that is also a component of the interleukin 4 receptor. Proc Natl Acad Sci U S A. 1996;93:497-501[Abstract/Free Full Text]. 19. Caput D, Laurent P, Kaghad M, et al. Cloning and characterization of a specific interleukin (IL)-13 binding protein structurally related to the IL-5 receptor  chain. J Biol Chem. 1996;271:16921-16926[Abstract/Free Full Text]. 20. Obiri NI, Murata T, Debinski W, Puri RK. Modulation of interleukin (IL)-13 binding and signaling by the c chain of the IL-2 receptor. J Biol Chem. 1997;272:20251-20258[Abstract/Free Full Text]. 21. Kuznetsov VA, Puri RK. Kinetic analysis of high affinity forms of interleukin (IL)-13 receptors: suppression of IL-13 binding by IL-2 receptor  chain. Biophys J. 1999;77:154-172[Medline] [Order article via Infotrieve]. 22. Murata T, Noguchi PD, Puri RK. IL-13 induces phosphorylation and activation of JAK2 janus kinase in human colon carcinoma cell lines. J Immunol. 1996;156:2972-2978[Abstract]. 23. Schindler C, Darnell JE Jr. Transcriptional responses to polypeptide ligands: the JAK-STAT pathway. Annu Rev Biochem. 1995;64:621-651[Medline] [Order article via Infotrieve]. 24. Zhang J-G, Hilton DJ, Willson TA, et al. Identification, purification, and characterization of a soluble interleukin (IL)-13-binding protein. J Biol Chem. 1997;272:9474-9480[Abstract/Free Full Text]. 25. Gauchat J-F, Schlagenhauf E, Feng N-P, et al. A novel 4-kb interleukin-13 receptor  mRNA expressed in human B, T, and endothelial cells encoding an alternate type-II interleukin-4/interleukin-13 receptor. Eur J Immunol. 1997;27:971-978[Medline] [Order article via Infotrieve]. 26. Debinski W, Obiri NI, Pastan I, Puri RK. A novel chimeric protein composed of interleukin 13 and Pseudomonas extoxin is highly cytotoxic to human carcinoma cells expressing receptors for interleukin 13 and interleukin 4. J Biol Chem. 1995;270:16775-16780[Abstract/Free Full Text]. 27. Puri RK, Leland P, Obiri NI, et al. Targeting of interleukin-13 receptor on human renal carcinoma cells by a recombinant chimeric protein composed of interleukin-13 and a truncated form of Pseudomonas exotoxin A (PE38QQR). Blood. 1996;87:4333-4339[Abstract/Free Full Text]. 28. Oshima Y, Joshi BH, Puri RK. Conversion of interleukin-13 into a high affinity agonist by a single amino acid substitution. J Biol Chem. 2000;275:14375-14380[Abstract/Free Full Text]. 29. Kreitman RJ, Puri RK, Pastan I. A circularly permuted recombinant interleukin 4 toxin with increased activity. Proc Natl Acad Sci U S A. 1994;91:6889-6893[Abstract/Free Full Text]. 30. Kreitman RJ, Puri RK, Pastan I. Increased antitumor activity of circularly permuted interleukin 4-toxin in mice with interleukin 4 receptor-bearing human carcinoma. Cancer Res. 1995;55:3357-3363[Abstract/Free Full Text]. 31. Ohmori Y, Smith MF Jr, Hamilton TA. IL-4-induced expression of the IL-1 receptor antagonist gene is mediated by Stat6. J Immunol. 1996;157:2058-2065[Abstract]. 32. Kawakami K, Leland P, Puri RK. Structure, function, and targeting of interleukin 4 receptors on human head and neck cancer cells. Cancer Res. 2000;60:2981-2987[Abstract/Free Full Text]. 33. Keppler-Hafkemeyer A, Kreitman RJ, Pastan I. Apoptosis induced by immunotoxins used in the treatment of haematologic malignancies. Int J Cancer. 2000;87:86-94[CrossRef][Medline] [Order article via Infotrieve]. 34. Murakami M, Hibi M, Nakagawa N, et al. IL-6-induced homodimerization of gp130 and associated activation of a tyrosine kinase. Science. 1993;260:1808-1810[Abstract/Free Full Text]. 35. Thiel S, Behrmann I, Dittrich E, et al. Internalization of the interleukin 6 signal transducer gp130 does not require activation of the Jak/STAT pathway. Biochem J. 1998;330:47-54. 36. Dittrich E, Haft CR, Muys L, Heinrich PC, Graeve L. A di-leucine motif and an upstream serine in the interleukin-6 (IL-6) signal transducer gp130 mediate ligand-induced endocytosis and down-regulation of the IL-6 receptor. J Biol Chem. 1996;271:5487-5494[Abstract/Free Full Text]. 37. Hunter MG, Avalos BR. Deletion of a critical internalization domain in the G-CSFR in acute myelogenous leukemia preceded by severe congenital neutropenia. Blood. 1999;93:440-446[Abstract/Free Full Text]. 38. Kil SJ, Hobert M, Carlin C. A leucine-based determinant in the epidermal growth factor receptor juxtamembrane domain is required for the efficient transport of ligand-receptor complexes to lysosomes. J Biol Chem. 1999;274:3141-3450[Abstract/Free Full Text]. 39. Govers R, Kerkhof PV, Schwartz AL, Strous GJ. Di-leucine-mediated internalization of ligand by a truncated growth hormone receptor is independent of the ubiquitin conjugation system. J Biol Chem. 1998;273:16426-16433[Abstract/Free Full Text]. 40. Hamer I, Haft CR, Paccaud J-P, Maeder C, Taylor S, Carpentier J-L. Dual role of a dileucine motif in insulin receptor endocytosis. J Biol Chem. 1997;272:21685-21691[Abstract/Free Full Text]. © 2001 by The American Society of Hematology.   CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: M. Wills-Karp and F. D. Finkelman Untangling the Complex Web of IL-4- and IL-13-Mediated Signaling Pathways Sci. Signal., December 23, 2008; 1(51): pe55 - pe55. [Abstract] [Full Text] [PDF] N. M. Heller, X. Qi, I. S. Junttila, K. A. Shirey, S. N. Vogel, W. E. Paul, and A. D. Keegan Type I IL-4Rs Selectively Activate IRS-2 to Induce Target Gene Expression in Macrophages Sci. Signal., December 23, 2008; 1(51): ra17 - ra17. [Abstract] [Full Text] [PDF] B. H. Joshi, P. Leland, A. Calvo, J. E. Green, and R. K. Puri Human Adrenomedullin Up-regulates Interleukin-13 Receptor {alpha}2 Chain in Prostate Cancer In vitro and In vivo: A Novel Approach to Sensitize Prostate Cancer to Anticancer Therapy Cancer Res., November 15, 2008; 68(22): 9311 - 9317. [Abstract] [Full Text] [PDF] A. Mozaffarian, A. W. Brewer, E. S. Trueblood, I. G. Luzina, N. W. Todd, S. P. Atamas, and H. A. Arnett Mechanisms of Oncostatin M-Induced Pulmonary Inflammation and Fibrosis J. Immunol., November 15, 2008; 181(10): 7243 - 7253. [Abstract] [Full Text] [PDF] I. S. Junttila, K. Mizukami, H. Dickensheets, M. Meier-Schellersheim, H. Yamane, R. P. Donnelly, and W. E. Paul Tuning sensitivity to IL-4 and IL-13: differential expression of IL-4R{alpha}, IL-13R{alpha}1, and {gamma}c regulates relative cytokine sensitivity J. Exp. Med., October 27, 2008; 205(11): 2595 - 2608. [Abstract] [Full Text] [PDF] T. Shimamura, T. Fujisawa, S. R. Husain, M. Kioi, A. Nakajima, and R. K. Puri Novel Role of IL-13 in Fibrosis Induced by Nonalcoholic Steatohepatitis and Its Amelioration by IL-13R-Directed Cytotoxin in a Rat Model J. Immunol., October 1, 2008; 181(7): 4656 - 4665. [Abstract] [Full Text] [PDF] G. P. Katsoulotos, M. Qi, J. C. Qi, K. Tanaka, W. E. Hughes, T. J. Molloy, R. Adachi, R. L. Stevens, and S. A. Krilis The Diacylglycerol-dependent Translocation of Ras Guanine Nucleotide-releasing Protein 4 inside a Human Mast Cell Line Results in Substantial Phenotypic Changes, Including Expression of Interleukin 13 Receptor {alpha}2 J. Biol. Chem., January 18, 2008; 283(3): 1610 - 1621. [Abstract] [Full Text] [PDF] T. Zheng, W. Liu, S.-Y. Oh, Z. Zhu, B. Hu, R. J. Homer, L. Cohn, M. J. Grusby, and J. A. Elias IL-13 Receptor {alpha}2 Selectively Inhibits IL-13-Induced Responses in the Murine Lung J. Immunol., January 1, 2008; 180(1): 522 - 529. [Abstract] [Full Text] [PDF] Y. Zhao, D. He, J. Zhao, L. Wang, A. R. Leff, E. Wm. Spannhake, S. Georas, and V. Natarajan Lysophosphatidic Acid Induces Interleukin-13 (IL-13) Receptor {alpha}2 Expression and Inhibits IL-13 Signaling in Primary Human Bronchial Epithelial Cells J. Biol. Chem., April 6, 2007; 282(14): 10172 - 10179. [Abstract] [Full Text] [PDF] M. Kioi, S. Seetharam, and R. K. Puri N-linked glycosylation of IL-13R{alpha}2 is essential for optimal IL-13 inhibitory activity FASEB J, November 1, 2006; 20(13): 2378 - 2380. [Abstract] [Full Text] [PDF] K. Kawakami, M. Terabe, M. Kioi, J. A. Berzofsky, and R. K. Puri Intratumoral Therapy with IL13-PE38 Results in Effective CTL-Mediated Suppression of IL-13R{alpha}2-Expressing Contralateral Tumors Clin. Cancer Res., August 1, 2006; 12(15): 4678 - 4686. [Abstract] [Full Text] [PDF] K. Kawakami, M. Terabe, M. Kawakami, J. A. Berzofsky, and R. K. Puri Characterization of a Novel Human Tumor Antigen Interleukin-13 Receptor {alpha}2 Chain. Cancer Res., April 15, 2006; 66(8): 4434 - 4442. [Abstract] [Full Text] [PDF] G. Zhou and B. Roizman Construction and properties of a herpes simplex virus 1 designed to enter cells solely via the IL-13{alpha}2 receptor PNAS, April 4, 2006; 103(14): 5508 - 5513. [Abstract] [Full Text] [PDF] L. F. Alves Oliveira, E. C. Moreno, G. Gazzinelli, O. A. Martins-Filho, A. M. S. Silveira, A. Gazzinelli, L. C. C. Malaquias, P. LoVerde, P. M. Leite, and R. Correa-Oliveira Cytokine Production Associated with Periportal Fibrosis during Chronic Schistosomiasis Mansoni in Humans Infect. Immun., February 1, 2006; 74(2): 1215 - 1221. [Abstract] [Full Text] [PDF] A. Kelly-Welch, E. M. Hanson, and A. D. Keegan Interleukin-13 (IL-13) Pathway Sci. Signal., July 19, 2005; 2005(293): cm8 - cm8. [Abstract] [Full Text] [PDF] P. V. Beum, H. Basma, D. R. Bastola, and P.-W. Cheng Mucin biosynthesis: upregulation of core 2 {beta}1,6 N-acetylglucosaminyltransferase by retinoic acid and Th2 cytokines in a human airway epithelial cell line Am J Physiol Lung Cell Mol Physiol, January 1, 2005; 288(1): L116 - L124. [Abstract] [Full Text] [PDF] N. M. Heller, S. Matsukura, S. N. Georas, M. R. Boothby, P. B. Rothman, C. Stellato, and R. P. Schleimer Interferon-{gamma} Inhibits STAT6 Signal Transduction and Gene Expression in Human Airway Epithelial Cells Am. J. Respir. Cell Mol. Biol., November 1, 2004; 31(5): 573 - 582. [Abstract] [Full Text] [PDF] K. Kawakami, M. Kawakami, and R. K. Puri Nitric Oxide Accelerates Interleukin-13 Cytotoxin-Mediated Regression in Head and Neck Cancer Animal Model Clin. Cancer Res., August 1, 2004; 10(15): 5264 - 5270. [Abstract] [Full Text] [PDF] C. Jakubzick, E. S. Choi, K. J. Carpenter, S. L. Kunkel, H. Evanoff, F. J. Martinez, K. R. Flaherty, G. B. Toews, T. V. Colby, W. D. Travis, et al. Human Pulmonary Fibroblasts Exhibit Altered Interleukin-4 and Interleukin-13 Receptor Subunit Expression in Idiopathic Interstitial Pneumonia Am. J. Pathol., June 1, 2004; 164(6): 1989 - 2001. [Abstract] [Full Text] [PDF] Y. Trieu, X.-Y. Wen, B. F. Skinnider, M. R. Bray, Z. Li, J. O. Claudio, E. Masih-Khan, Y.-X. Zhu, S. Trudel, J. A. McCart, et al. Soluble Interleukin-13R{alpha}2 Decoy Receptor Inhibits Hodgkin's Lymphoma Growth in Vitro and in Vivo Cancer Res., May 1, 2004; 64(9): 3271 - 3275. [Abstract] [Full Text] [PDF] C Jakubzick, E S Choi, S L Kunkel, H Evanoff, F J Martinez, R K Puri, K R Flaherty, G B Toews, T V Colby, E A Kazerooni, et al. Augmented pulmonary IL-4 and IL-13 receptor subunit expression in idiopathic interstitial pneumonia J. Clin. Pathol., May 1, 2004; 57(5): 477 - 486. [Abstract] [Full Text] [PDF] K. Kawakami, M. Kawakami, and R. K. Puri Specifically targeted killing of interleukin-13 (IL-13) receptor-expressing breast cancer by IL-13 fusion cytotoxin in animal model of human disease Mol. Cancer Ther., February 1, 2004; 3(2): 137 - 147. [Abstract] [Full Text] [PDF] M. Booth, J. K. Mwatha, S. Joseph, F. M. Jones, H. Kadzo, E. Ireri, F. Kazibwe, J. Kemijumbi, C. Kariuki, G. Kimani, et al. Periportal Fibrosis in Human Schistosoma mansoni Infection Is Associated with Low IL-10, Low IFN-{gamma}, High TNF-{alpha}, or Low RANTES, Depending on Age and Gender J. Immunol., January 15, 2004; 172(2): 1295 - 1303. [Abstract] [Full Text] [PDF] M. Kawakami, K. Kawakami, J. L. Kasperbauer, L. L. Hinkley, M. Tsukuda, S. E. Strome, and R. K. Puri Interleukin-13 Receptor {alpha}2 Chain in Human Head and Neck Cancer Serves as a Unique Diagnostic Marker Clin. Cancer Res., December 15, 2003; 9(17): 6381 - 6388. [Abstract] [Full Text] [PDF] K. J. Ishii, K. Kawakami, I. Gursel, J. Conover, B. H. Joshi, D. M. Klinman, and R. K. Puri Antitumor Therapy with Bacterial DNA and Toxin: Complete Regression of Established Tumor Induced by Liposomal CpG Oligodeoxynucleotides plus Interleukin-13 Cytotoxin Clin. Cancer Res., December 15, 2003; 9(17): 6516 - 6522. [Abstract] [Full Text] [PDF] C. Jakubzick, E. S. Choi, B. H. Joshi, M. P. Keane, S. L. Kunkel, R. K. Puri, and C. M. Hogaboam Therapeutic Attenuation of Pulmonary Fibrosis Via Targeting of IL-4- and IL-13-Responsive Cells J. Immunol., September 1, 2003; 171(5): 2684 - 2693. [Abstract] [Full Text] [PDF] A.-h. Wu and W. C. Low Molecular cloning and identification of the human interleukin 13 alpha 2 receptor (IL-13Ra2) promoter Neuro-oncol, July 1, 2003; 5(3): 179 - 187. [Abstract] [PDF] C. Jakubzick, E. S. Choi, S. L. Kunkel, B. H. Joshi, R. K. Puri, and C. M. Hogaboam Impact of Interleukin-13 Responsiveness on the Synthetic and Proliferative Properties of Th1- and Th2-Type Pulmonary Granuloma Fibroblasts Am. J. Pathol., May 1, 2003; 162(5): 1475 - 1486. [Abstract] [Full Text] [PDF] M. G. Chiaramonte, M. Mentink-Kane, B. A. Jacobson, A. W. Cheever, M. J. Whitters, M. E.P. Goad, A. Wong, M. Collins, D. D. Donaldson, M. J. Grusby, et al. Regulation and Function of the Interleukin 13 Receptor {alpha} 2 During a T Helper Cell Type 2-dominant Immune Response J. Exp. Med., March 17, 2003; 197(6): 687 - 701. [Abstract] [Full Text] [PDF] N. Wood, M. J. Whitters, B. A. Jacobson, J. Witek, J. P. Sypek, M. Kasaian, M. J. Eppihimer, M. Unger, T. Tanaka, S. J. Goldman, et al. Enhanced Interleukin (IL)-13 Responses in Mice Lacking IL-13 Receptor {alpha} 2 J. Exp. Med., March 17, 2003; 197(6): 703 - 709. [Abstract] [Full Text] [PDF] K. Kawakami, M. Kawakami, and R. K. Puri IL-13 Receptor-Targeted Cytotoxin Cancer Therapy Leads to Complete Eradication of Tumors with the Aid of Phagocytic Cells in Nude Mice Model of Human Cancer J. Immunol., December 15, 2002; 169(12): 7119 - 7126. [Abstract] [Full Text] [PDF] A.-L. Andrews, J. W. Holloway, S. M. Puddicombe, S. T. Holgate, and D. E. Davies Kinetic Analysis of the Interleukin-13 Receptor Complex J. Biol. Chem., November 22, 2002; 277(48): 46073 - 46078. [Abstract] [Full Text] [PDF] C. Jakubzick, S. L. Kunkel, B. H. Joshi, R. K. Puri, and C. M. Hogaboam Interleukin-13 Fusion Cytotoxin Arrests Schistosoma mansoni Egg-Induced Pulmonary Granuloma Formation in Mice Am. J. Pathol., October 1, 2002; 161(4): 1283 - 1297. [Abstract] [Full Text] [PDF] J. L. Lordan, F. Bucchieri, A. Richter, A. Konstantinidis, J. W. Holloway, M. Thornber, S. M. Puddicombe, D. Buchanan, S. J. Wilson, R. Djukanovic, et al. Cooperative Effects of Th2 Cytokines and Allergen on Normal and Asthmatic Bronchial Epithelial Cells J. Immunol., July 1, 2002; 169(1): 407 - 414. [Abstract] [Full Text] [PDF] B. H. Joshi, K. Kawakami, P. Leland, and R. K. Puri Heterogeneity in Interleukin-13 Receptor Expression and Subunit Structure in Squamous Cell Carcinoma of Head and Neck: Differential Sensitivity to Chimeric Fusion Proteins Comprised of Interleukin-13 and a Mutated Form of Pseudomonas Exotoxin Clin. Cancer Res., June 1, 2002; 8(6): 1948 - 1956. [Abstract] [Full Text] [PDF] K. Kawakami, M. Kawakami, P. Leland, and R. K. Puri Internalization Property of Interleukin-4 Receptor {alpha} Chain Increases Cytotoxic Effect of Interleukin-4 Receptor-targeted Cytotoxin in Cancer Cells Clin. Cancer Res., January 1, 2002; 8(1): 258 - 266. [Abstract] [Full Text] [PDF] K. Blease, C. Jakubzick, J. M. Schuh, B. H. Joshi, R. K. Puri, and C. M. Hogaboam IL-13 Fusion Cytotoxin Ameliorates Chronic Fungal-Induced Allergic Airway Disease in Mice J. Immunol., December 1, 2001; 167(11): 6583 - 6592. [Abstract] [Full Text] [PDF] K. Kawakami, M. Kawakami, B. H. Joshi, and R. K. Puri Interleukin-13 Receptor-targeted Cancer Therapy in an Immunodeficient Animal Model of Human Head and Neck Cancer Cancer Res., August 1, 2001; 61(16): 6194 - 6200. [Abstract] [Full Text] [PDF] K. Kawakami, F. Takeshita, and R. K. Puri Identification of Distinct Roles for a Dileucine and a Tyrosine Internalization Motif in the Interleukin (IL)-13 Binding Component IL-13 Receptor alpha 2 Chain J. Biol. Chem., June 29, 2001; 276(27): 25114 - 25120. [Abstract] [Full Text] [PDF] This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Kawakami, K. Articles by Puri, R. K. Search for Related Content PubMed PubMed Citation Articles by Kawakami, K. Articles by Puri, R. K. Related Collections Immunobiology Signal Transduction Social Bookmarking                   What's this?

	Copyright © 2001 by American Society of Hematology         Online ISSN: 1528-0020