Mechanism of action of interleukin-13 antagonist (IL-13E13K) in cells expressing various types of IL-4R

As interleukin (IL)-13 and IL-4 play a major role in various diseases including asthma, allergy, and malignancies, it is desirable to generate a molecule that blocks the effects of both cytokines. We previously generated a human IL-13 mutant (IL-13E13K), which is a powerful antagonist of IL-13, blocking the biological activities of IL-13. We now show that IL-13E13K also competitively inhibits signaling and biological activities of IL-4 through type II and partially through type III IL-4 receptor (R) system. IL-13E13K completely blocked the IL-4-induced phosphorylation of STAT6 and IL-4-dependent protein synthesis in cells expressing type II and partially type III IL-4R but not type I IL- 4R. Consistent with the inhibition of biological activities, IL-13E13K inhibited IL-4 binding to type II IL-4R-expressing cells but not to type I IL-4R-expressing cells. The inhibition efficiency of IL-4 binding by IL-13E13K was relatively lower compared to wtIL-13 even though IL-13E13K bound to IL-13Ralpha1 positive cells with a similar affinity to wtIL-13. These results indicate that Glu13 in IL-13 associates with IL-4Ralpha, and mutation to lysine decreases its binding ability to IL-4Ralpha chain. IL-13E13K binds to IL- 13Ralpha1, which is shared by both IL-13R and IL-4R systems. Consequently, IL-13E13K inhibits IL-4 binding to these cells and prevents heterodimer formation between IL-13Ralpha1 and IL-4Ralpha chains. This interference by IL-13E13K blocks the biological activities of not only IL-13 but also partially of IL-4. Thus, IL-13E13K may be a useful agent for the treatment of diseases such as asthma, allergic rhinitis, and cancer, which are dependent on signaling through both IL-4 and IL-13 receptors.     

Lysophosphatidic acid induces interleukin-13 (IL-13) receptor alpha2 expression and inhibits IL-13 signaling in primary human bronchial epithelial cells

Interleukin-13 (IL-13), a Th2 cytokine, plays a pivotal role in pathogenesis of bronchial asthma via IL-13 receptor alpha1 (IL-13Ralpha1) and IL-4 receptor alpha (IL-4Ralpha). Recent studies show that a decoy receptor for IL-13, namely IL-13Ralpha2, mitigates IL-13 signaling and function. This study provides evidence for regulation of IL-13Ralpha2 production and release and IL-13-dependent signaling by lysophosphatidic acid (LPA) in primary cultures of human bronchial epithelial cells (HBEpCs). LPA treatment of HBEpCs in at imedependent fashion increased IL-13Ralpha2 gene expression without altering the mRNA levels of IL-13Ralpha1 and IL-4Ralpha. Pretreatment with pertussis toxin (100 ng/ml, 4 h) or transfection of c-Jun small interference RNA or an inhibitor of JNK attenuated LPA-induced IL-13Ralpha2 gene expression and secretion of soluble IL-13Ralpha2. Overexpression of catalytically inactive mutants of phospholipase D (PLD) 1 or 2 attenuated LPA-induced IL-13Ralpha2 gene expression and protein secretion as well as phosphorylation of JNK. Pretreatment of HBEpCs with 1 microM LPA for 6 h attenuated IL-13-but not IL-4-induced phosphorylation of STAT6. Transfection of HBEpCs with IL-13Ralpha2 small interference RNA blocked the effect of LPA on IL-13-induced phosphorylation of STAT6. Furthermore, pretreatment with LPA (1 microM, 6 h) attenuated IL-13-induced eotaxin-1 and SOCS-1 gene expression. These results demonstrate that LPA induces IL-13Ralpha2 expression and release via PLD and JNK/AP-1 signal transduction and that pretreatment with LPA down-regulates IL-13 signaling in HBEpCs. Our data suggest a novel mechanism of regulation of IL-13Ralpha2 and IL-13 signaling that may be of physiological relevance to airway inflammation and remodeling.     

TNF-alpha and IL-4 regulate expression of IL-13 receptor alpha2 on human fibroblasts

Two interleukin 13 receptors (IL-13Rs) have been identified as IL-13Ralpha1 and IL-13Ralpha2. IL-13Ralpha1 is composed of a heterodimer consisting of IL-13Ralpha1 and IL-4 receptor alpha (IL-4Ralpha) as a signaling subunit. In contrast, IL-13Ralpha2 is known as a decoy receptor for IL-13. In this study, we investigated the expression of IL-13Rs on human fibroblasts. IL-13Ralpha2 was significantly up-regulated after stimulation with tumor necrosis factor-alpha (TNF-alpha) and/or IL-4. In contrast, IL-13Ralpha1 was constitutively detectable and was not up-regulated. After the induction of IL-13alpha2 by IL-4, STAT6 phosphorylation through IL-13Ralpha1 by IL-13 was inhibited. We also detected large intracellular pools of IL-13Ralpha2 in fibroblasts quantitatively. Furthermore, mobilization of the IL-13Ralpha2 protein stores from the cytoplasm to the cell surface was prevented by an inhibitor of protein transport, brefeldin-A. These results indicate that TNF-alpha and IL-4 synergistically up-regulate the expression of IL-13Ralpha2 decoy receptor on human fibroblasts by inducing gene expression and mobilizing intracellular receptors, and thus may down-regulate the IL-13 signaling.     

The negative-feedback regulation of the IL-13 signal by the IL-13 receptor alpha2 chain in bronchial epithelial cells

Bronchial asthma is a complex disease characterized by airway inflammation involving Th2 cytokines. Among Th2 cytokines, the significance of IL-13 in the pathogenesis of bronchial asthma has recently emerged. Particularly, the direct action of IL-13 on bronchial epithelial cells (BECs) is critical for generation of airway hyperresponsiveness. IL-13 has two binding units; the IL-13 receptor alpha1 chain transduces the IL-13 signal comprising a heterodimer with the IL-4R alpha chain, whereas the IL-13 receptor alpha2 chain (IL-13Ralpha2) is thought to act as a decoy receptor. However, it remains obscure how expression of these molecules is regulated in each cell. In this article, we analyzed the expression of these components in BECs. Either IL-4 or IL-13 induced intracellular expression of IL-13Ralpha2 in BECs, which was STAT6-dependent and required de novo protein synthesis. IL-13Ralpha2 expressed on the cell surface as a monomer inhibited the STAT6-dependent IL-13 signal. Furthermore, expression of IL-13Ralpha2 was induced in lung tissues of ovalbumin-induced asthma model mice. Taken together, our results suggested the possibility that IL-13Ralpha2 induced by its ligand is transferred to the cell surface by an unknown mechanism, and it down-regulates the IL-13 signal in BECs, which functions as a unique negative-feedback system for the cytokine signal.     

IL-4 receptor alpha is an important modulator of IL-4 and IL-13 receptor binding: implications for the development of therapeutic targets

IL-4 is a key cytokine associated with allergy and asthma. Induction of cell signaling by IL-4 involves interaction with its cognate receptors, a complex of IL-4Ralpha with either the common gamma-chain or the IL-13R chain alpha1 (IL-13Ralpha1). We found that IL-4 bound to the extracellular domain of IL-4Ralpha (soluble human (sh)IL-4Ralpha) with high affinity and specificity. In contrast with the sequential mechanism of binding and stabilization afforded by IL-4Ralpha to the binding of IL-13 to IL-13Ralpha1, neither common gamma-chain nor IL-13Ralpha1 contributed significantly to the stabilization of the IL-4:IL-4Ralpha complex. Based on the different mechanisms of binding and stabilization of the IL-4R and IL-13R complexes, we compared the effects of shIL-4Ralpha and an IL-4 double mutein (R121D/Y124D, IL-4R antagonist) on IL-4- and IL-13-mediated responses. Whereas IL-4R antagonist blocked responses to both cytokines, shIL-4Ralpha only blocked IL-4. However, shIL-4Ralpha stabilized and augmented IL-13-mediated STAT6 activation and eotaxin production by primary human bronchial fibroblasts at suboptimal doses of IL-13. These data demonstrate that IL-4Ralpha plays a key role in the binding affinity of both IL-13R and IL-4R complexes. Under certain conditions, shIL-4Ralpha has the potential to stabilize binding IL-13 to its receptor to augment IL-13-mediated responses. Thus, complete understanding of the binding interactions between IL-4 and IL-13 and their cognate receptors may facilitate development of novel treatments for asthma that selectively target these cytokines without unpredicted or detrimental side effects.     

IL-13 induces airways hyperreactivity independently of the IL-4R alpha chain in the allergic lung

The potent spasmogenic properties of IL-13 have identified this molecule as a potential regulator of airways hyperreactivity (AHR) in asthma. Although IL-13 is thought to primarily signal through the IL-13Ralpha1-IL-4Ralpha complex, the cellular and molecular components employed by this cytokine to induce AHR in the allergic lung have not been identified. By transferring OVA-specific CD4(+) T cells that were wild type (IL-13(+/+) T cells) or deficient in IL-13 (IL-13(-/-) T cells) to nonsensitized mice that were then challenged with OVA aerosol, we show that T cell-derived IL-13 plays a key role in regulating AHR, mucus hypersecretion, eotaxin production, and eosinophilia in the allergic lung. Moreover, IL-13(+/+) T cells induce these features (except mucus production) of allergic disease independently of the IL-4Ralpha chain. By contrast, IL-13(+/+) T cells did not induce disease in STAT6-deficient mice. This shows that IL-13 employs a novel component of the IL-13 receptor signaling system that involves STAT6, independently of the IL-4Ralpha chain, to modulate pathogenesis. We show that this novel pathway for IL-13 signaling is dependent on T cell activation in the lung and is critically linked to downstream effector pathways regulated by eotaxin and STAT6.     

IL-13 receptor alpha2 selectively inhibits IL-13-induced responses in the murine lung

IL-13 is a critical cytokine at sites of Th2 inflammation. In these locations it mediates its effects via a receptor complex, which contains IL-4Ralpha and IL-13Ralpha1. A third, high-affinity IL-13 receptor, IL-13Ralpha2, also exists. Although it was initially felt to be a decoy receptor, this has not been formally demonstrated and the role(s) of this receptor has recently become controversial. To define the role(s) of IL-13Ralpha2 in IL-13-induced pulmonary inflammation and remodeling, we compared the effects of lung-targeted transgenic IL-13 in mice with wild-type and null IL-13Ralpha2 loci. We also investigated the effect of IL-13Ralpha2 deficiency on the OVA-induced inflammatory response. In this study, we show that in the absence of IL-13Ralpha2, IL-13-induced pulmonary inflammation, mucus metaplasia, subepithelial fibrosis, and airway remodeling are significantly augmented. These changes were accompanied by increased expression and production of chemokines, proteases, mucin genes, and TGF-beta1. Similarly, an enhanced inflammatory response was observed in an OVA-induced phenotype. In contrast, disruption of IL-13Ralpha2 had no effect on the tissue effects of lung-targeted transgenic IL-4. Thus, IL-13Ralpha2 is a selective and powerful inhibitor of IL-13-induced inflammatory, remodeling, and physiologic responses in the murine lung.     

Functional importance of regional differences in localized gene expression of receptors for IL-13 in murine gut

IL-13 induces a STAT6-dependent hypercontractility of intestinal smooth muscle that is mediated by binding to the IL-13Ralpha1 component of the type 2 IL-4R that is linked to STAT6. IL-13 also binds to the IL-13Ralpha2 that is not linked to STAT6 and functions to limit the effects of IL-13 in vivo. In this study we assessed the contributions of regional and cellular differences in the distribution of the IL-13R components to the physiological regulation of smooth muscle function in wild-type mice and mice deficient in STAT6 or IL-13Ralpha2. The expression of IL-13 and IL-13Ralpha2 was higher in colon than in small intestine. Laser capture microdissection of specific cell types revealed that the expression of IL-13Ralpha2 was higher in the smooth muscle layer compared with levels in the epithelial cells of the mucosa. In contrast, there was a uniform distribution of IL-13alpha1 in smooth muscle, epithelia, and myenteric neurons. The significant hypercontractility of smooth muscle in mice deficient in IL-13Ralpha2, but not in STAT6, shows the physiological importance of IL-13 binding to IL-13Ralpha2. The pronounced differences in the expression of IL-13Ralpha2 suggest that the gut has developed sophisticated mechanisms for controlling the physiological and pathophysiological activities of IL-13.     

Inhibition of NF-kappaB activation reduces the tissue effects of transgenic IL-13

IL-13 is a major Th2 cytokine that is capable of inducing inflammation, excessive mucus production, airway hyperresponsiveness, alveolar remodeling, and fibrosis in the murine lung. Although IL-13 through its binding to IL-4Ralpha/IL-13Ralpha1 uses the canonical STAT6-signaling pathway to mediate these tissue responses, recent studies have demonstrated that other signaling pathways may also be involved. Previous studies from our laboratory demonstrated that IL-13 mediates its tissue effects by inducing a wide variety of downstream genes many of which are known to be regulated by NF-kappaB. As a result, we hypothesized that NF-kappaB activation plays a critical role in the pathogenesis of IL-13-induced tissue alterations. To test this hypothesis, we compared the effects of transgenic IL-13 in mice with normal and diminished levels of NF-kappaB activity. Three pharmacologic approaches were used to inhibit NF-kappaB including 1) PS1145, a small molecule inhibitor of IkappaBalpha kinase (IKK2), 2) antennapedia-linked NF-kappaB essential modulator-binding domain (NBD) peptide (wild-type NBD), and 3) an adenoviral construct expressing a dominant-negative version of IKK2. We also crossed IL-13-transgenic mice with mice with null mutations of p50 to generate mice that overproduced IL-13 in the presence and absence of this NF-kappaB component. These studies demonstrate that all these interventions reduced IL-13-induced tissue inflammation, fibrosis and alveolar remodeling. In addition, we show that both PS1145 and wild-type NBD inhibit lung inflammatory and structural cell apoptosis. PS1145 inhibits caspase activation and up-regulates inhibitor of apoptosis protein cellular-inhibitor of apoptosis protein 1 (c-IAP-1). Therefore, NF-kappaB is an attractive target for immunotherapy of IL-13-mediated diseases.     

Kinetic analysis of the interleukin-13 receptor complex

Interleukin (IL)-13 is a key cytokine associated with the asthmatic phenotype. It signals via its cognate receptor, a complex of IL-13 receptor alpha1 chain (IL-13Ralpha1) with IL-4Ralpha; however, a second protein, IL-13Ralpha2, also binds IL-13. To determine the binding contributions of the individual components of the IL-13 receptor to IL-13, we have employed surface plasmon resonance and equilibrium binding assays to investigate the ligand binding characteristics of shIL-13Ralpha1, shIL-13Ralpha2, and IL-4Ralpha. shIL-13Ralpha1 bound IL-13 with moderate affinity (K(D) = 37.8 +/- 1.8 nm, n = 10), whereas no binding was observed for hIL-4Ralpha. In contrast, shIL-13Ralpha2 produced a high affinity interaction with IL-13 (K(D) = 2.49 +/- 0.94 nm n = 10). IL-13Ralpha2 exhibited the binding characteristics of a negative regulator with a fast association rate and an exceptional slow dissociation rate. Although IL-13 interacted weakly with IL-4Ralpha on its own (K(D) > 50 microm), the presence of hIL-4Ralpha significantly increased the affinity of shIL-13Ralpha1 for IL-13 but had no effect on the binding affinity of IL-13Ralpha2. Detailed kinetic analyses of the binding properties of the heteromeric complexes suggested a sequential mechanism for the binding of IL-13 to its signaling receptor, in which IL-13 first binds to IL-13Ralpha1 and this then recruits IL-4Ralpha to stabilize a high affinity interaction.     

IL-11 receptor alpha in the pathogenesis of IL-13-induced inflammation and remodeling

IL-13 is a major stimulator of inflammation and tissue remodeling at sites of Th2 inflammation. In Th2-dominant inflammatory disorders such as asthma, IL-11 is simultaneously induced. However, the relationship(s) between IL-11 and IL-13 in these responses has not been defined, and the role(s) of IL-11 in the genesis of the tissue effects of IL-13 has not been evaluated. We hypothesized that IL-11, signaling via the IL-11Ralpha-gp130 receptor complex, plays a key role in IL-13-induced tissue responses. To test this hypothesis we compared the expression of IL-11, IL-11Ralpha, and gp130 in lungs from wild-type mice and transgenic mice in which IL-13 was overexpressed in a lung-specific fashion. We simultaneously characterized the effects of a null mutation of IL-11Ralpha on the tissue effects of transgenic IL-13. These studies demonstrate that IL-13 is a potent stimulator of IL-11 and IL-11Ralpha. They also demonstrate that IL-13 is a potent stimulator of inflammation, fibrosis, hyaluronic acid accumulation, myofibroblast accumulation, alveolar remodeling, mucus metaplasia, and respiratory failure and death in mice with wild-type IL-11Ralpha loci and that these alterations are ameliorated in the absence of IL-11Ralpha. Lastly, they provide insight into the mechanisms of these processes by demonstrating that IL-13 stimulates CC chemokines, matrix metalloproteinases, mucin genes, and gob-5 and stimulates and activates TGF-beta1 via IL-11Ralpha-dependent pathways. When viewed in combination, these studies demonstrate that IL-11Ralpha plays a key role in the pathogenesis of IL-13-induced inflammation and remodeling.     

Expression of human IL-13 receptor alpha2 extracellular domain in Pichia pastoris

Interleukin-13 receptor alpha2 (IL-13Ralpha2) binds IL-13 with high affinity and plays an important role in IL-13 signaling as a decoy receptor. We expressed the extracellular domain of human IL-13Ralpha2 (1-313) in methylotrophic yeast Pichia pastoris. SDS-PAGE analysis by PAS staining and Western blot analysis detected the product of the extracellular domain of human IL-13Ralpha2 as glycoprotein from P. pastoris. The yield of purified extracellular domain of human IL-13Ralpha2 was 2mg from 1L of culture. From CD analysis, the 2D structure of the purified IL-13Ralpha2 showed the typical beta-sheet. ELISA of the purified IL-13Ralpha2 detected the binding activity for human IL-13. Thus, it was found that the active extracellular domain of human IL-13Ralpha2 was expressed from P. pastoris.     

A novel mechanism by which interferon-gamma can regulate interleukin (IL)-13 responses. Evidence for intracellular stores of IL-13 receptor alpha -2 and their rapid mobilization by interferon-gamma.

Interleukin (IL)-13 mediates its activities via a complex receptor system. Interleukin-13 receptor alpha-1 chain (IL-13Ralpha1) binds IL-13 with low affinity, but does not signal. However, when IL-13Ralpha1 combines with IL-4 receptor alpha (IL-4Ralpha), a signaling high affinity receptor complex for IL-13 is generated. In contrast, IL-13Ralpha2 alone binds IL-13 with high affinity, but does not signal and has been postulated to be a decoy receptor. Herein, we investigated the cellular localization of IL-13Ralpha2 and the regulation of its expression by confocal microscopy and flow cytometry in primary and cultured cells. Our results demonstrate that IL-13Ralpha2 is largely an intracellular molecule, which is rapidly mobilized from intracellular stores following treatment with interferon (IFN)-gamma. Up-regulation of IL-13Ralpha2 surface expression in response to IFN-gamma was rapid, did not require protein synthesis, and resulted in diminished IL-13 signaling. These results provide the first evidence that the IL-13Ralpha2 is predominantly an intracellular molecule and demonstrate a novel mechanism by which IFN-gamma can regulate IL-13 responses.     

Characterization of the interaction between interleukin-13 and interleukin-13 receptors

Interleukin-13 (IL-13) possesses two types of receptor: the heterodimer, composed of the IL-13Ralpha1 chain (IL-13Ralpha1) and the IL-4Ralpha chain (IL-4Ralpha), transducing the IL-13 signals; and the IL-13Ralpha2 chain (IL-13Ralpha2), acting as a nonsignaling "decoy" receptor. Extracellular portions of both IL-13Ralpha1 and IL-13Ralpha2 are composed of three fibronectin type III domains, D1, D2, and D3, of which the last two comprise the cytokine receptor homology modules (CRHs), a common structure of the class I cytokine receptor superfamily. Thus far, there has been no information about the critical amino acids of the CRHs or the role of the D1 domains of IL-13Ralpha1 and IL-13Ralpha2 in binding to IL-13. In this study, we first built the homology modeling of the IL-13.hIL-13 receptor complexes and then predicted the amino acids involved in binding to IL-13. By incorporating mutations into these amino acids, we identified Tyr-207, Asp-271, Tyr-315, and Asp-318 in the CRH of human IL-13Ralpha2, and Leu-319 and Tyr-321 in the CRH of human IL-13Ralpha1, as critical residues for binding to IL-13. Tyr-315 in IL-13Ralpha2 and Leu-319 in IL-13Ralpha1 are positionally conserved hydrophobic amino acid residues. Furthermore, by using D1 domain-deleted mutants, we found that the D1 domain is needed for the expression of IL-13Ralpha2, but not IL-13Ralpha1, and that the D1 domain of IL-13Ralpha1 is important for binding to IL-13, but not to IL-4. These results provide the basis for a precise understanding of the interaction between IL-13 and its receptors.