Interleukin-13 Receptor Α2 Research Articles

Blockade of interleukin-13-mediated cell activation by a novel inhibitory antibody to human IL-13 receptor α1

Interleukin-13 (IL-13) is a cytokine with a crucial role in the development of allergic asthma. The IL-13 receptor shares the IL-4Rα subunit with the IL-4R system, but contains as a specific component the IL-13Rα1 chain. Blocking signal release by IL-13 without affecting IL-4 function is a potentially interesting therapeutical option for the treatment of asthma. Employing genetic immunization, we generated a set of novel monoclonal antibodies to the IL-13Rα1 receptor that proved very specific and efficient inhibitors of human IL-13 activity. Receptor binding antibodies were identified by their specific reactivity with both human monocytes and a murine pro-B cell line overexpressing human IL-13Rα1 by flow cytometry and cell ELISA. A luciferase reporter cell system based on STAT6-mediated promoter activation in murine Ba/F3 cells was employed to screen the antibodies for IL-13 antagonistic properties. Inhibitory antibody effects were quantified by interference with IL-13-dependent proliferation of TF-1 cells. The capability of blocking IL-13-driven responses of primary, inflammation-relevant cells was tested by Western blot analysis of STAT6 tyrosine phosphorylation and expression of 15-lipoxygenase in monocytes from fresh blood. The most potent inhibitory antibody identified, GM1E7, inhibited IL-13-driven gene activation and cell proliferation in immune cell lines with IC 50 values in the low nanomolar range. Both short-term (STAT6 activation) and long-term (15-LO induction) responses of primary human blood cells to IL-13 were almost entirely blocked, whereas IL-4 effects remained virtually unaffected. GM1E7 is superior to available agents interfering with IL-13 activity in terms of specificity and efficiency and offers potential novel therapeutic perspectives for the treatment of allergic asthma.

Gene expression profiling identifies IL‐13 receptor α2 chain as a therapeutic target in prostate tumor cells overexpressing adrenomedullin

Human adrenomedullin (AM) is a 52 amino acid peptide, which shares homology with the calcitonin gene-related peptide. Overexpression of AM in the prostate carcinoma cell line PC-3 results in growth inhibition with a 20% (for human AM) and 35% (for rat AM) increase in doubling time compared to parental or mock-transfected cells. We demonstrate by gene expression profiling that AM overexpression results in the dysregulation of approximately 100 genes. Examples of such genes include many involved in the formation of the cytoskeleton, cell adhesion and the extracellular matrix, as well as regulators of the cell cycle and apoptosis, cytokines and transcription factors. Several genes related to cell growth arrest, such as GADD45, IGF-BP6 and RUNX-3, are upregulated by AM. Interestingly, interleukin-13 receptor alpha 2 (IL-13R alpha 2) transcripts were significantly increased in clones overexpressing AM, which was confirmed by semiquantitative RT-PCR analysis. In addition, PC-3 cells treated with AM showed an overexpression of IL-13R alpha 2, which was abolished when cells were preincubated with an anti-AM blocking antibody. When PC-3 cells overexpressing AM and the IL-13R alpha 2 were treated with the highly specific IL13-PE38 cytotoxin, which binds to this receptor, a concentration-dependent inhibition of protein synthesis was observed. The IC(50) (concentration of cytotoxin inhibiting protein synthesis by 50%) ranged from 1 to 4 ng/ml. This cytotoxicity was specific as it was neutralized by the excess of IL-13 and confirmed by clonogenic assays. This study describes a novel AM-induced mechanism of tumor sensitization through the upregulation of functional IL-13R alpha 2 chain, an ideal target for the highly specific recombinant chimeric cytotoxin IL13-PE38.

Upregulation of Interleukin-13 and Its Receptor in a Murine Model of Bleomycin-Induced Scleroderma

Background: Interleukin-13 (IL-13) has been implicated in the pathogenesis of fibrotic conditions. Previously, a murine model for scleroderma has been established by repeated local injections of bleomycin. This animal model enabled us to study local expression and production of IL-13 in skin lesions during disease progression. Methods: Dermal sclerosis (DSc) was induced by repeated subcutaneous injections of bleomycin (1 mg/ml) in C3H/HeJ mice. IL-13 and IL-4 expressions were examined by RT-PCR, ELISA and immunohistochemistry. Results: RT-PCR showed that both IL-4 and IL-13 mRNA levels in skin lesions were increased and peaked after 4 weeks of bleomycin treatment. Quantification by densitometry revealed up to 4.2- and 1.9-fold increases, respectively. Immunohistochemical localization showed in skin lesions expression of IL-13 on infiltrating inflammatory cells, including mononuclear cells and possibly mast cells, increased with DSc progression. IL-13 protein production was also significantly increased. In skin lesions, IL-13 receptor (IL-13R) α2 expression was augmented mainly in the infiltrating mononuclear cells after 4 weeks of bleomycin exposure. IL-13Rα2, but not IL-13Rα1, mRNA was upregulated in the whole skin after 4 weeks. On the contrary, mRNA expression of IL-13Rα1 and IL- 13Rα2 was significantly altered in the cultured fibroblasts derived from bleomycin-treated skin. Conclusion: These data demonstrate that in skin lesions levels of IL-13 as well as its receptor increase in parallel with DSc progression, suggesting that IL-13 promotes the progression of cutaneous fibrosis/sclerosis in the murine model of bleomycin-induced scleroderma.

Immunopathogenesis of schistosomiasis.

In schistosomiasis mansoni, the chronic egg-induced granulomatous response in the liver and intestines may eventually cause extensive tissue scarring and development of portal hypertension. Indeed, much of the morbidity and mortality associated with this disease is directly attributable to the deposition of connective tissue elements in affected tissues. Elucidating the mechanisms that regulate the severity of schistosomiasis has been a major research objective over the past several years. Research conducted with DNA microarrays as well as investigations with a variety of gene knock-out mice have been particularly helpful in achieving this goal. A notable accomplishment in the past few years was the identification of interleukin-13 (IL-13) and the IL-13 receptor complex as central regulators of disease progression in schistosomiasis. Liver fibrogenesis is severely decreased in infected IL-13-deficient mice as well as in wildtype animals treated with IL-13 antagonists. In contrast, IL-13 effector function increases dramatically in IL-13 receptor alpha2 (IL-13Ralpha2)-deficient mice. These mice develop severe hepatic fibrosis, fail to downregulate granuloma formation in the chronic phase of S. mansoni infection, and succumb to the disease at an accelerated rate; thus, identifying the 'decoy' IL-13 receptor as a critical life sustaining 'off' switch for tissue damaging egg-induced inflammation.

Analysis of interleukin-13 receptor alpha2 expression in human pediatric brain tumors.

Compared with normal brain tissue cells, human malignant glioma cells express higher levels of interleukin-13 receptor (IL-13R). However, whether this receptor is expressed in situ has not been carefully examined. With IL-13R-targeted cytotoxin (IL13-PE38QQR, comprising IL-13 and a mutated form of Pseudomonas exotoxin [PE]) being tested in three Phase I/II clinical trials for the treatment of adult human glioma, and with pediatric studies being planned, the authors set out to analyze pediatric brain tumor tissue specimens for the expression of IL-13R. Using in situ hybridization and immunohistochemical staining, the authors examined 58 pediatric brain tumor specimens for expression of the predominant IL-13 binding and internalizing protein (IL-13Ralpha2) chain at the mRNA and protein levels. Overall, approximately 83% of pediatric brain tumor samples expressed IL-13Ralpha2. One hundred percent (11 of 11) high-grade astrocytoma, 79% (26 of 33) low-grade astrocytoma, 67% (4 of 6) medulloblastoma, and 67% (2 of 3) ependymoma samples were positive for IL-13Ralpha2. Among IL-13Ralpha2-positive samples, 88% (42 of 48 samples) had positive expression in > or = 50% of all tumor fields. The results obtained using both assays were consistent with each other. The current study established that pediatric brain tumor specimens expressed the IL-13Ralpha2 chain. Because the IL-13Ralpha2 chain is a major binding component of the IL-13R complex, these results suggest that the targeting of IL-13R may represent a useful approach for the treatment of pediatric brain tumors.

Up-regulation of interleukin-13 receptor α1 on human keratinocytes in the skin of psoriasis and atopic dermatitis

Background: Interleukin (IL)-13 is a pleiotropic cytokine, which shares many biological functions with IL-4. The receptor subunits of IL-13 consist of IL-4Rα, IL-13Rα1 and IL-13Rα2. The regulatory mechanisms of the IL-13Rα expression in the keratinocytes of certain skin disease have not been known. Objective: To clear the roles of IL-13 and the regulatory mechanisms of its receptor in atopic dermatitis (AD) and psoriasis. Method: The expression of IL-13Rα1 in the skin of AD and psoriasis was investigated by immunohistochemistry. The regulation of IL-13Rα mRNA in the skin and human primary keratinocyte (HPK) was investigated by quantitative PCR. The secretion of IL-6 and RANTES from HPK was measured by ELISA. Results: The expression of IL-13Rα1 was more prominent on the suprabasal keratinocytes in the skin of AD and striking increase of staining was observed on all layers of keratinocyte in the skin of psoriasis. The mRNA of IL-13Rα1, but not of IL-13Rα2 was overexpressed in both skin of AD and psoriasis. In vitro experiment using HPK demonstrated that IFN-γ, IL-13 but not IL-4 could up-regulate the mRNA expression of IL-13Rα1. In contrast, IL-13Rα2 mRNA expression was up-regulated by IFN-γ plus IL-4. Furthermore, the stimulation of HPK with IFN-γ plus IL-13 and/or IL-4 resulted in significant enhancement of IL-6 and RANTES secretion. Conclusion: These findings indicate that IL-4 and IL-13 have different regulatory effects on the expression of IL-13Rα1 and α2, and the overexpression of IL-13Rα1 may play some roles in the pathogenesis of chronic stage of AD or psoriasis.

The interleukin-13 receptor α2 chain: an essential component for binding and internalization but not for interleukin-13–induced signal transduction through the STAT6 pathway

AbstractThe interleukin-13 receptor (IL-13R) complex is composed of 2 different chains, IL-13Rα1 (also known as IL-13Rα′) and IL-13Rα2 (also known as IL-13Rα). For a functional IL-13 receptor, the IL-13Rα1 chain forms a productive complex with the primary IL-4 binding protein (IL-4Rα also known as IL-4Rβ). However, the function of the IL-13Rα2 chain is not clear even though this chain binds IL-13 with high affinity. This study demonstrates that IL-13Rα2 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-13Rα2 chain; (2) a recombinant chimeric fusion protein comprising IL-13 and a mutated form ofPseudomonas exotoxin (termed IL13-PE38QQR or IL-13 toxin) is specifically cytotoxic to IL-13Rα2–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-13Rα2–transfected cells. IL-13 efficiently caused activation of STAT6 protein in cells transfected with the IL-13Rα1 and IL-4Rα chains, and IL-13Rα2 inhibited this activation. Taken together, these observations indicate that internalization of IL-13Rα2 is signal independent and that this property of IL-13Rα2 can be exploited for receptor-directed cancer therapy.

Identification of a Novel Type I Cytokine Receptor CRL2 Preferentially Expressed by Human Dendritic Cells and Activated Monocytes

From human dendritic cell (DC) cDNA library, we identified a novel type I cytokine receptor, designated as cytokine receptor-like molecule 2 (CRL2). CRL2 cDNA encoded a 371-residue type I transmembrane protein with an extracellular domain of 210 residues and an intracellular domain of 119 residues. Its extracellular domain contains conserved cysteine residues and WAS-like motif in place of the hallmark of WSXWS motif present in other type I cytokine receptors. The intracellular domain contained a membrane-proximal “box 1” motif and conserved tyrosine residue potentially as a binding site for signal transducing molecules. CRL2 protein shares significant homology with common cytokine receptor (γc) and interleukin-13 receptor α1 chain. Northern blot analysis showed that CRL2 was restrictedly expressed by spleen and peripheral blood leukocytes, and abundantly expressed by HL-60 cells. RT-PCR analysis demonstrated that CRL2 was preferentially expressed by DC and monocytes. Interestingly, CRL2 expression was up-regulated when monocytes were activated by LPS. These indicate that CRL2 may be involved in the biological functions of DC and monocytes. The Ba/F3 transfectants of CRL2 was retrovirally established with the expressed FLAG-tagged CRL2 in the size of approximately 48 kD, which could be efficiently immunoprecipitated. We also prepared CRL2Ig fusion protein. The identification of its ligand and involvement of signal transduction will help to elucidate its potential function.

Structural Characterization of the Human Interleukin-13 Receptor α1 Gene Promoter

Human cancer cells have been found to express a large number of IL-13 receptors. We have previously shown that mRNA encoding one of these receptors, IL-13Rα1, is increased in cisplatin-resistant cells and is upregulated in tumor cells cultured with cisplatin. To understand the molecular mechanism of IL-13Rα1 gene expression, we cloned approximately 52 kbp of the IL-13Rα1 gene and sequenced the first exon and about 1 kbp of the upstream DNA. The first exon is 211 bp and contains 88 bp of coding sequence, while the first intron is about 13 kbp in length. The promoter region, which is GC rich, was found to lack both TATA and CCAAT boxes. Transient expression assays revealed that transcription of the IL-13Rα1 gene was significantly higher in cisplatin-resistant cells than in parental, cisplatin-sensitive cells. Deletion analysis of the IL-13Rα1 promoter identified a 70-bp core promoter region upstream of the transcription initiation site. Electrophoretic gel mobility shift assays showed that a synthetic IL-13Rα1 oligonucleotide (nt −40 to nt −15) bound a nuclear factor from cisplatin-resistant cells to a significantly greater degree than the equivalent factor from parental cells. This oligonucleotide was found to contain a palindromic sequence with a BstEII recognition site at its center. This palindromic sequence functions to mediate upregulation of IL-13Rα1 promoter in cisplatin-resistant cells and deletion or disruption of this sequence also resulted in severe reduction of the promoter activity. These findings suggest that IL-13Rα1 expression is upregulated at the transcriptional level in cisplatin-resistant cells. The characterization of both the IL-13Rα1 promoter and the transcription factors binding to it may contribute to our understanding of IL-13Rα1 regulation in cancer cells.