Structure of and signal transduction through interleukin-4 and interleukin-13 receptors (review).

We have recently demonstrated that two different forms of IL-4R exist; classical or alternative. The classical IL-4R is predominantly expressed in hematopoietic cells and consist of IL-4R and IL-2Rgammac (gammac) chains. On the other hand, alternative form of IL-4R is predominantly expressed in non-hematopoietic cells and consists of IL-4R and IL-13Ralpha' chains. Moreover, the alternative form of IL-4R is also utilized as a functional component IL-13R complex. It has been shown that the phosphorylation and activation of JAK3 tyrosine kinase is crucial for IL-4 activation of STAT6 in hematopoietic cells. However, we have recently demonstrated that non-hematopoietic cells lack JAK3 expression. We also demonstrated that in these cells, STAT6 activation is mediated through JAK1 and JAK2 tyrosine kinases instead. Furthermore, our results show that IL-4 and IL-13 signals are transmitted through the alternative form of IL-4R in these cells. Thus, major differences exist between hematopoietic and non-hematopoietic cells with regard to structure and signal transduction through IL-4R and IL-13R systems.

Novel anti-brain tumor cytotoxins specific for cancer cells.

The vast majority of brain cancers (gliomas) express a receptor (R) for interleukin 13 (IL13). In order to achieve specific targeting of the IL13R in gliomas, we have mutagenized human (h) IL13. The mutation was made to alter IL13 interaction with the shared functional IL13/4 normal tissue receptor, but not with the glioma-associated receptor. We have thus produced hIL13.E13K (glutamic acid at position 13 changed to lysine) and fused it to derivatives of Pseudomonas exotoxin A. The hIL13.E13K-based cytotoxins are less active on normal cells and thus less toxic, and are better antitumor agents compared with the cytotoxins containing nonmutagenized hIL13.

Structure of IL-13 receptor: analysis of subunit composition in cancer and immune cells.

The structure of IL-13 receptor (IL-13R) is currently under investigation. Recently, two different human IL-13R chains, termed here IL-13R alpha and -alpha' have been cloned. We have examined various cancer and normal cell lines for the presence of mRNA for IL-13R alpha and alpha, as well as IL-4R p140 (termed beta chain) and IL-2R gamma c chains. In renal cell carcinoma, glioblastoma and ovarian carcinoma (IGROV-1) cell lines, both IL-13R alpha and alpha chains were expressed (type I IL-13R). In epidermoid, colon, ovarian adenocarcinoma (PA-1) and normal mouse fibroblast (COS7) cell lines, only IL-13R alpha' was expressed (type II IL-13R). In hematopoietic TF-1 and EBV-immortalized normal B cell lines only IL-13R alpha' but not alpha chain was expressed along with gamma c (type III or type IV IL-13R). IL-13R alpha' chain was faintly detected in human T cells. All cells expressed the IL-4Rp140 beta chain. These data provide a direct support for our model of IL-13R which consists of three different forms composed of different subunits.

Modulation of interleukin (IL)-13 binding and signaling by the gammac 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 gammac 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, gammac 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 gammac in IL-13 binding and signaling, we have transfected a renal cell carcinoma cell line with gammac 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 gammac has 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.

Receptor for interleukin 13 on AIDS-associated Kaposi's sarcoma cells serves as a new target for a potent Pseudomonas exotoxin-based chimeric toxin protein.

AIDS-associated Kaposi's sarcoma (AIDS-KS), the most common malignant complication of human immunodeficiency virus infection, is characterized by neoplastic proliferation of mesenchymal cells. AIDS-KS cells release and respond to an array of cytokines through specific plasma membrane receptors. Specific targeting of potent cytotoxic agents to cell surface receptors/antigens on Kaposi's sarcoma cells may provide effective therapy for this malignancy. We have identified a new target in the form of an interleukin 13 (IL-13) receptor that is overexpressed in the five AIDS-KS cell lines examined. Radiolabeled IL-13 cross-linked to a single protein of about Mr 70,000 in AIDS-KS cells. We utilized a chimeric cytotoxic protein composed of IL-13 and a truncated Pseudomonas exotoxin (IL13-PE38QQR), which was found to be specifically and highly cytotoxic to AIDS-KS cells, as determined by protein synthesis inhibition and clonogenic assays. IL13-PE38QQR demonstrated significant antitumor activity in a human epidermoid carcinoma xenograft model. Normal human umbilical vein-derived endothelial, lymphoid, and bone marrow precursor cells expressed low levels of IL-13 receptors, and IL-13 toxin was not cytotoxic to them. Thus, IL-13 receptor on AIDS-KS cells may represent a novel plasma membrane protein(s) that could be utilized to target therapeutic agents.

Human ovarian-carcinoma cell lines express IL-4 and IL-13 receptors: comparison between IL-4- and IL-13-induced signal transduction.

We have reported that human ovarian-carcinoma cell lines express high-affinity IL-4 receptor. Since IL-4R has been hypothesized to share a chain with IL-13R, we investigated whether ovarian cancer cells express IL-13 receptor. In the present study, we report that the ovarian-carcinoma cell lines IGROV-1 and PA-1 express varying numbers of high-affinity IL-13 receptors. Furthermore, IL-13 inhibited the binding of IL-4 on both ovarian-carcinoma cell lines, while IL-4 did not inhibit IL-13 binding on IGROV-1 cell line. IL-13 and IL-4 induced the phosphorylation of JAK1, JAK2 and Tyk2 Janus kinases in PA-1 cells. In contrast, JAK3 tyrosine kinase was expressed in PA-1 cells, but IL-4 or IL-13 did not augment its phosphorylation. In IGROV-1 cells, Tyk2 was constitutively phosphorylated and this phosphorylation was augmented by IL-4 or IL-13. JAK1 and JAK2 but not JAK3 were expressed but only JAK2 was faintly phosphorylated in response to either IL-13 or IL-4 respectively. IRS (insulin-receptor substrate)-1 and IRS-2 were also phosphorylated constitutively in both ovarian cancer cell lines examined, but only the phosphorylation of IRS-1 was augmented in response to IL-4 or IL-13. STAT6 was phosphorylated and activated in response to IL-4 and IL-13 in all cell lines examined. Our results demonstrate that ovarian cancer cell lines may express 2 types of IL-13R and the IL-13- or IL-4-induced signaling patterns may be slightly different in each type of receptor.

The IL-13 receptor structure differs on various cell types and may share more than one component with IL-4 receptor.

We have reported on the expression and characteristics of IL-13R and have demonstrated that IL-13 competes for IL-4 binding while IL-4 did not compete for the IL-13 binding on some cell types. Based on these observations, and the size of IL-13 and IL-4 cross-linked proteins, we concluded that the receptor for IL-13 is complex and shares a subunit with the receptor for IL-4. To explore the complexity of the IL-13R, a wide variety of cell types was examined for IL-13 and IL-4 binding. We report in this work that IL-4 does not always bind well to cells that bind IL-13, but the reverse is also true. We also found that IL-4 can compete more effectively for IL-13 binding than IL-13 itself. Cross-linking studies support these observations and demonstrate that 125I-labeled IL-13 bound exclusively to a single 65- to 70-kDa protein in MA-RCC and U251 cells, while in TF-1 cells it cross-linked to two membrane proteins of 65 to 70 kDa and 140 kDa. Furthermore, by using a chimeric protein composed of IL-13 and Pseudomonas exotoxin A, we observed that IL-4 neutralized the cytotoxicity of the IL-13 toxin on COS-7 cells by blocking a common form of the two cytokine receptors. We propose that the 65- to 70-kDa form of the IL-13R is the predominant common component shared between IL-13 and IL-4R. However, the primary IL-4 binding (p140) protein also participates in the formation of the IL-13R complex in some cell types. In addition, the gamma(c) or another interactive subunit may influence IL-13 binding to its receptor complex. Thus, we propose that there are at least four forms of IL-13R.

cDNA cloning and characterization of the human interleukin 13 receptor alpha chain.

We have cloned cDNAs corresponding to the human interleukin 13 receptor alpha chain (IL-13Ralpha). The protein has 76% homology to murine IL-13Ralpha, with 95% amino acid identity in the cytoplasmic domain. Only weak IL-13 binding activity was found in cells transfected with only IL-13Ralpha; however, the combination of both IL-13Ralpha and IL-4Ralpha resulted in substantial binding activity, with a Kd of approximately 400 pM, indicating that both chains are essential components of the IL-13 receptor. Whereas IL-13Ralpha serves as an alternative accessory protein to the common cytokine receptor gamma chain (gammac) for IL-4 signaling, it could not replace the function of gammac in allowing enhanced IL-2 binding activity. Nevertheless, the overall size and length of the cytoplasmic domain of IL-13Ralpha and gammac are similar, and like gammac, IL-13Ralpha is located on chromosome X.

Interleukin 13 inhibits growth of human renal cell carcinoma cells independently of the p140 interleukin 4 receptor chain.

Interleukin-13 (IL-13) is a cytokine produced primarily by activated T lymphocytes. It exerts a variety of effects on different cell types, including monocytes, B lymphocytes, mast cells, and keratinocytes. The effects of IL-13 on target cells are often similar to the effects of IL-4, which is another cytokine product of activated T lymphocytes. We recently described the expression of intermediate- to high-affinity receptors for IL-13 (IL-13R) on renal cell carcinoma (RCC) cells. In the present study, we examined the effect of IL-13 on the growth of RCC cells as measured by [3H]thymidine uptake and a clonogenic assay. In addition, we used an IL-4R-specific antibody to examine the specificity of IL-4R and IL-13R binding and function. We observed that IL-13 inhibited RCC cell proliferation by up to 50% and colony formation by up to 32% when compared with cells cultured in medium alone. A combination of IL-4 and IL-13 did not have an additive or synergistic effect on the growth of RCC cells. These cells expressed mRNA for IL-13 and secreted immunoreactive IL-13 protein in culture. The growth-inhibitory effects of IL-13 were specific, because they were not affected by antibodies to IL-4 or to the 140-kilodalton subunit of IL-4R. Furthermore, polyclonal antibodies to IL-4R failed to inhibit the binding of 125I-IL-13 to RCC cells. These results indicate that IL-13 has significant antiproliferative effects on human RCC cells, and the inhibition of IL-13 effects by anti-IL-4R antibody previously reported in lymphoid cells does not occur in RCC cells.

Receptor for interleukin (IL) 13 does not interact with IL4 but receptor for IL4 interacts with IL13 on human glioma cells.

Recently, we have demonstrated that human (h) glioma cell lines express large number of receptors (R) for interleukin 13 (IL13) (Debinski, W., Obiri, N. I., Powers, S. K., Pastan, I., and Puri, R. K. (1995) Clin. Cancer Res. 1, 1253-1258). These cells are extremely sensitive to a chimeric protein composed of hIL13 and a derivative of Pseudomonas exotoxin (PE), PE38QQR. We have found that the cytotoxicity of hIL13-PE38QQR was blocked by hIL13 but not by hIL4 on the U-251 MG and U-373 MG cells, contrary to what was observed on several adenocarcinoma cell lines. In the present study, we further explored interactions between receptor for IL13 and IL4 on glioma cells. Established human glioma cell lines, such as DBTRG MG, Hs 683, U-87 MG, SNB-19, and A-172, are very susceptible to hIL13-PE38QQR, and the action of the chimeric toxin is not blocked by hIL4 on all these cells either. Also, hIL4 is not a competitor for 125I-hIL13 binding sites on glioma cells. Of interest, a corresponding hIL4-based chimeric toxin, hIL4-PE38QQR, is poorly active or not active on all the tested glioma cell lines. When active, however, hIL4 toxin action was blocked by hIL13. hIL13 is a competitor for 125I-hIL14 binding in a competitive binding assay on glioma cells. hIL13 and hIL4 did not affect the growth of the tested glioma cell lines. Human glioblastoma multiforme explant cells exhibited similar responses to the chimeric toxins and interleukins when compared with that found in established glioma cultures. Our results suggest that the hIL13R on glioma cells is expressed in one predominant form, the form that does not interact with IL4. Thus, this type of hIL13R is apparently different from the one demonstrated previously on several adenocarcinoma cell lines.

An improved circularly permuted interleukin 4-toxin is highly cytotoxic to human renal cell carcinoma cells. Introduction of gamma c chain in RCC cells does not improve sensitivity.

We have previously demonstrated that a chimeric protein composed of human IL-4 and Pseudomonas exotoxin, termed IL4-PE4E, is cytotoxic to primary cells derived from human renal cell carcinoma (RCC). To improve the cytotoxicity of IL4-toxins such as IL4-PE4E and IL4-PE38KDEL to IL-4 receptor (IL-4R) positive tumor cells, a circularly permuted chimeric toxin was prepared by fusing a truncated PE gene encoding PE38KDEL 3' to a circularly permuted IL-4 mutant gene encoding IL4 amino acids 38-129, the linker GGNGG, and IL4 amino acids 1-37. The resulting chimeric protein, termed IL4(38-37)-PE38KDEL, was tested on five RCC cell lines and its cytotoxicity was compared to that of the native IL4-toxins IL4-PE4E and IL4-PE38KDEL. IL4(38-37)-PE38KDEL was found to be 5 to 10 times more cytotoxic to all cell cultures tested compared to either native IL4-toxin. The cytotoxic activity of IL4(38-37)-PE38KDEL was competible by excess IL-4 and was confirmed by clonogenic assay. IL4(38-37)-PE38KDEL bound to IL-4R on RCC cells with 6- to 12-fold higher affinity than IL4-PE38KDEL or IL4-PE4E. RCC tumor cells were found to lack the common gamma chain (gamma c) of the IL-4R reported to be present on immune cells. The stable transfection of RCC cells with the gamma c chain gene did not significantly change their sensitivity to IL4(38-37)-PE38KDEL. Taken together, our results indicate that the CPIL4-toxin IL4(38-37)-PE38KDEL is highly cytotoxic to human RCC cells due to increased binding affinity to IL-4R while it is not cytotoxic or slightly cytotoxic to T and B cells, monocytic cell lines, and fresh resting or activated bone marrow-derived cells. The gamma c does not seem to increase the internalization rate and/or processing of IL4-toxins in RCC cells. CPIL4-toxin may be a useful agent for the treatment of human RCC.

Targeting of interleukin-13 receptor on human renal cell carcinoma cells by a recombinant chimeric protein composed of interleukin-13 and a truncated form of Pseudomonas exotoxin A (PE38QQR).

We have previously shown that human renal cell carcinoma (RCC) cells express large numbers of interleukin-13 receptors (IL-13R), a newly described hemopoietic growth factor receptor. To target tumor cells that express IL-13R, we have produced a chimeric protein composed of human IL-13 and a derivative of Pseudomonas exotoxin A, termed PE38QQR. We report here that IL13-PE38QQR is highly cytotoxic to many human RCC cell lines. IL-13R-negative cell lines or cell lines expressing low numbers of IL-13R ( < 300 sites/cell) that include human bone marrow-derived cells were not susceptible to the cytotoxic effect of IL 13-PE38QQR. The sensitivity of RCC cells to IL13-PE38QQR correlated positively with the density of IL-13R. The cytotoxic activity of IL13-PE38QQR was competed by an excess of IL-13 in a protein synthesis inhibition assay and confirmed by a clonogenic assay. Even though IL-13 and IL-4 are homologues and IL-4R and IL-13R have been proposed to share a receptor subunit, IL-4 did not compete for the cytotoxicity mediated by IL13-toxin on RCC. IL13-PE38QQR competes for [125I]-IL-13 binding sites on RCC cells, although at a lower affinity than the wild-type recombinant cytokine. Human T-cell, B-cell, and monocytic cell lines are unresponsive to the cytotoxic action of IL13-PE38QQR. Thus, our results indicate that IL13-PE38QQR is highly cytotoxic to human RCC cells, although it is not cytotoxic to a variety of normal hematopoietic cells. IL13-PE38QQR should be further investigated preclinically for the treatment of human RCCs.

Human glioma cells overexpress receptors for interleukin 13 and are extremely sensitive to a novel chimeric protein composed of interleukin 13 and pseudomonas exotoxin.

Recently, we have demonstrated that a spectrum of human adenocarcinoma cell lines express binding sites for interleukin 13 (IL-13). These cells are killed by a chimeric protein composed of human (h) IL-13 and a derivative of Pseudomonas exotoxin, PE38QQR (Debinski et al., J. Biol. Chem., 270: 16775-16780, 1995). The cell killing was hIL-13- and hIL-4-specific, indicating that a common binding site for the two cytokines is present in several solid tumor cell lines. Herein, we report that an array of established glioma cell lines is killed by very low concentrations of hIL-13-PE38QQR, often reaching <1 ng/ml (<20 pM). Glioma cells express up to 30,000 molecules of IL-13 receptor/cell which has intermediate affinity toward hIL-13. hIL-13-PE38QQR is more active (up to 3 logs difference in cytotoxic activities) than are the corresponding chimeric toxins containing hIL-4 or hIL-6. The cytotoxic action of hIL-13-PE38QQR is blocked by an excess of hIL-13 on all cell lines studied, and it is not neutralized by hIL-4 on some of these cells. Our results show that human brain cancers richly express receptors for IL-13. Furthermore, the interaction detected previously between receptors for IL-13 and IL-4 on solid tumors cell lines is of a qualitatively different character in U-251 MG and U-373 MG glioma cells. The receptor for IL-13 may represent a new marker of brain cancers and an attractive target for anticancer therapies.

A novel chimeric protein composed of interleukin 13 and Pseudomonas exotoxin is highly cytotoxic to human carcinoma cells expressing receptors for interleukin 13 and interleukin 4.

Chimeric proteins provide a unique opportunity to target therapeutic bacterial toxins to a subset of specific cells. We have generated a new recombinant chimeric toxin composed of human interleukin 13 (hIL13) and a Pseudomonas exotoxin A (PE) mutant, PE38QQR. The hIL13-PE38QQR chimera is highly cytotoxic to cell lines derived from several human epithelial carcinomas such as adenocarcinoma of stomach, colon, and skin. The cytotoxic action of hIL13-PE38QQR, which can only occur upon internalization of ligand-receptor complex, is blocked by an excess of hIL13 but not of hIL2. This action is not solely hIL13-specific because an excess of hIL4 also blocks the cytotoxicity of hIL13-toxin. Conversely, hIL13 blocks the cytotoxicity of a hIL4-PE38QQR chimera. Binding studies showed that hIL13 displaces competitively 125I-labeled hIL4-PE38QQR on carcinoma cells. These results indicate that IL4 and IL13 compete for a common binding site on the studied human cell lines. Despite this competition, hIL4 but not hIL13 decreased protein synthesis in malignant cells susceptible to the cytotoxicity of both hIL13- and hIL4-PE38QQR. Our results suggest that a spectrum of human carcinomas express binding sites for IL13. Furthermore, hIL13 and hIL4 compete reciprocally for a form of the receptor that is internalized upon binding a ligand. Thus, cancer cells represent an interesting model for studying receptors for these two growth factors. Finally, hIL13-PE38QQR may be a useful agent in the treatment of several malignancies.

Up-regulation of intercellular adhesion molecule 1 (ICAM-1) on human renal cell carcinoma cells by interleukin-4.

We have previously demonstrated that human renal cell carcinoma (RCC) cells express high-affinity IL-4 receptors (IL-4R). To study the functions of these receptors, we have examined the effect of IL-4 on the expression of intercellular adhesion molecule-1 (ICAM-1 or CD54) on human RCC cells. Following incubation with various concentrations of IL-4, RCC cells were examined for ICAM-1 expression by flow cytometric analysis. The 2 primary RCC cell cultures and the 2 cell lines examined expressed varying basal levels of ICAM-1 on the cell surface. IL-4 treatment increased ICAM-1 expression in a time-dependent manner and maximum augmentation of ICAM-1 expression was observed after a 48 hr incubation. The increase in ICAM-1 expression was specific because anti-hIL-4 antibody blocked this effect. No enhancement of ICAM-1 expression was observed when RCC cells were incubated with IL-4 in the presence of cycloheximide, indicating that the IL-4 effect requires new protein synthesis. Up-regulation of ICAM-1 expression was also observed at the mRNA level and maximum increase in message occurred 8 hr post-IL-4 treatment. Both IL-4 and IFN-gamma also increased soluble ICAM-1 levels in WS-RCC culture supernatant. The significance of enhanced soluble and surface ICAM-1 expression was investigated by examining the lymphokine activated killer (LAK) cell-mediated lysis of IL-4-treated WS-RCC cells. LAK cells lysed WS-RCC cells very effectively, but lysis observed in target cells pre-treated with IL-4 did not correlate with the increased expression of ICAM-1 antigen. Our results indicate a previously unknown function of IL-4 on RCC and further demonstrate that IL-4R on RCC are functional.

Receptor for interleukin 13. Interaction with interleukin 4 by a mechanism that does not involve the common gamma chain shared by receptors for interleukins 2, 4, 7, 9, and 15.

Interleukin 13 (IL-13) shares many biological properties with IL-4, and although the receptor for IL-4 (IL-4R) has been characterized, the expression and structure of IL-13 receptor are unknown. We report here that human renal cell carcinoma (RCC) cells express large numbers of functional IL-13R. Human B lymphocytes and monocytes expressed a very small number of IL-13R, while resting or activated human T cells expressed little or no IL-13R. IL-4 did not compete for IL-13 binding, while IL-13 competed for IL-4 binding, even though IL-4R and IL-13R are structurally distinct on human RCC cells. IL-13 cross-linked with one major protein that is similar in size to the gamma c subunit of IL-2, -4, -7, -9, and -15 receptors but was not recognized by anti-gamma c or anti-IL-4R antibodies. IL-4, on the other hand, cross-linked with two major proteins, the smaller of which appears to be similar in size to IL-13R and gamma c, but (like the IL-13R) it did not react with anti-gamma c antibody. Although as shown in this study and in previous studies, gamma c is a functional component of IL-4R in lymphoid cells, it does not appear to be associated with IL-4R on RCC cells. Even in the absence of common gamma chain IL-4 and IL-13 were able to up-regulate intracellular adhesion molecule-1 antigen on RCC cells. These data suggest that the interaction of IL-13 with IL-4R does not involve gamma c and IL-13R itself may be a novel subunit of the IL-4R.

Characterization of interleukin-4 receptors expressed on human renal cell carcinoma cells.

We have recently reported that a variety of solid human tumor cells express high-affinity interleukin-4 receptors (IL-4R). In this study, we have compared structural characteristics of IL-4R expressed on human renal cell carcinoma cells (RCC-WS) and the lymphoid cell lines RAJI (B-cell line) and H9 (T-cell line). In crosslinking studies, the three cell types expressed a predominant 140 kDa IL-4R band. In addition, a 70 kDa band was expressed strongly in H9 cells but only faintly on RCC-WS and RAJI cells. These different species of IL-4R were not observed when crosslinking studies were performed in the presence of excess interleukin-4 (IL-4), indicating IL-4 specificity. A polyclonal anti-IL-4R antibody immunoprecipitated the two species (140 and 70 kDa) in H9 and predominantly the 140 kDa species in RCC-WS tumor cells and RAJI cells. A faint band for the 70 kDa protein was also observed. The affinity of IL-4 binding to its receptor in RCC-WS cells was similar to the binding affinity observed in H9 and RAJI cells examined. However, the RCC tumor cells and B lymphoid cells internalized IL-4R more rapidly compared to T lymphoid cells. Although IL-4R synthesis was similarly inhibited by cycloheximide in all three cell lines, IL-4R expression was more sensitive to actinomycin D inhibition on the RCC-WS and RAJI cells than on H9 cells. Our results suggest that IL-4R expressed on RCC-WS tumor cells are structurally different from those expressed on lymphoid cells because the proportions of IL-4R subunits differ in these cells. Further studies should be performed to determine the identity and functional significance of IL-4R proteins expressed on RCC and immune cells.

Expression of high-affinity IL-4 receptors on human melanoma, ovarian and breast carcinoma cells.

It has previously been shown that murine sarcoma cells express high-affinity IL-4 receptors (IL-4R) which are internalized after binding to the ligand (Puri et al., Cancer Res 1991; 51:3011-7). We have also reported that human renal cell carcinoma cells express high-affinity IL-4R, and IL-4 inhibits tumour growth in vitro (Obiri et al., J Clin Invest 1993; 91:88). In this study we investigated the expression and function of IL-4R on other human solid tumours. Human melanoma, ovarian carcinoma and breast carcinoma cell lines were assessed for the cell surface expression of IL-4R by radio-ligand receptor binding and for IL-4R gene expression by Northern blot analysis. Primary cultures of mesothelioma and neurofibrosarcoma cells were similarly investigated. Human melanoma, ovarian carcinoma and breast carcinoma cell lines expressed IL-4R on their cell surface with a dissociation constant (Kd) of 140-549 pM. These tumour lines expressed a single 4 kb species of mRNA for IL-4R. Similarly, primary cultures of mesothelioma and neurofibrosarcoma cells were positive for the IL-4R mRNA by Northern blot analysis. Fresh, non-cultured mesothelioma and neurofibrosarcoma tumour sections were also positive for the presence of IL-4R as determined by immunohistochemistry of frozen sections using anti-IL-4R antibody. In order to study possible functions of IL-4R, we evaluated the effects of IL-4 on cell growth and its effect on MHC antigen expression in the presence or absence of interferon-gamma (IFN-gamma). In tissue culture, IL-4 reduced the growth of tumour cell lines and primary cell cultures studied. IL-4 had very little effect on MHC class I antigen expression on ovarian, breast and melanoma cell lines; however, MHC class II (HLA-DR) expression was enhanced on melanoma and breast carcinoma cells. IL-4 also enhanced the IFN-gamma-induced class II expression on melanoma and breast carcinoma cells. Taken together, our observations indicate that IL-4R are expressed on a variety of human solid tumours and these receptors may be functional. IL-4 alone and in combination with IFN-gamma may play a role in host immune response against cancers.

Immunostaining of interleukin-4 receptor on human renal cell carcinoma.

Immunocytochemistry on three primary cultures of RCC tumor cells and immunohistochemistry on five frozen sections of RCC were performed by utilizing a monoclonal antibody (M-57) to human IL-4R. We found that all three RCC tumor cell cytospin preparations stained with anti-IL-4R monoclonal antibody. Tumor cells stained with IgG control did not show any staining. Similarly, five histologically proven RCC frozen sections prepared from nephrectomy specimens had moderate to intense immunoreactivity to IL-4 receptor antibody. RCC sections stained with normal mouse IgG2b showed only background type staining. Frozen section prepared from uninvolved kidney showed only nonspecific staining. A flow cytometric analysis of primary cultures of RCC tumor cells confirmed the immunohistochemical data and showed that almost all of the cells were positive for IL-4 receptor expression. These results demonstrate that human RCC express immunoreactive IL-4 receptors, which may be a target for diagnosis and therapy by an anti-IL-4 receptor antibody fused to toxins or radionuclides or alternatively by IL-4 toxins.

Expression of high affinity interleukin-4 receptors on human renal cell carcinoma cells and inhibition of tumor cell growth in vitro by interleukin-4.

Previously, Puri et al. (Puri, R. K., M. Ogata, P. Leland, G. M. Feldman, D. Fitzgerald, and I. Pastan. 1991. Cancer Res. 51:3011-3017) have demonstrated that murine sarcoma and colon adenocarcinoma cells express high affinity interleukin-4 receptors (IL-4R) which are internalized after binding to a chimeric ligand consisting of IL-4 and Pseudomonas exotoxin. In the present study, we have tested primary cultures of human renal cell carcinoma (RCC) cells, generated from tumor specimens obtained after nephrectomy, for the expression of IL-4R and their modulation by IL-4. By using iodinated IL-4 in a receptor binding assay, we observed that renal cell carcinoma cells expressed a single class of high affinity IL-4R ranging from 1,425 +/- 207 (mean +/- SEM) to 3,831 +/- 299 (mean +/- SEM) IL-4R molecules/cell with a Kd ranging from 112 +/- 11 pM to 283 +/- 71 pM. Northern blot analysis for IL-4R gene expression, performed with a cDNA probe to IL-4R, revealed that all RCC cells exhibited a single mRNA species of 4 kb. IL-4 downregulated the surface expression of IL-4R on one RCC tumor cell line. The function of IL-4R expression on RCC tumor cells was further determined by investigating the effect of IL-4 on tumor cell growth in vitro and comparing it with IL-4 effect on growth of normal fibroblast and endothelial cell lines. Tumor cell growth, as measured by [3H]thymidine incorporation, was inhibited by IL-4 from 20 to 68% in a dose-dependent manner. A neutralizing antibody to human IL-4 was able to reverse the growth inhibitory effect of IL-4. Normal human fibroblast and endothelial cell lines also expressed high affinity IL-4R, however, IL-4 did not inhibit their growth in vitro. In fact, IL-4 caused modest stimulation of their growth. Taken together, our findings can help develop strategies for the treatment of RCC in which IL-4R may be used as a target for IL-4 itself, for IL-4 toxin therapy or, alternatively, in gene therapy.