The innate antiviral response upregulates IL-13 receptor α2 in bronchial fibroblasts.

BACKGROUND: IL-13 is key mediator of allergic inflammation in asthmatic patients. We have previously shown that the decoy receptor IL-13 receptor (IL-13R) α2 attenuates responses of fibroblasts to IL-13. Because the expression of IL-13Rα2 can be regulated by IFN-γ, a type II interferon, we hypothesized that innate antiviral responses characterized by type I interferon expression can also induce IL-13Rα2 expression. OBJECTIVE: We sought to induce an innate antiviral response in primary fibroblasts using exposure to double-stranded RNA (dsRNA) and to examine the expression and function of IL-13Rα2. METHODS: Primary human fibroblasts were cultured from endobronchial biopsy specimens obtained from healthy or asthmatic volunteers and challenged with dsRNA. Upregulation of IL-13Rα2 mRNA was measured by using real-time quantitative PCR, and cell-surface IL-13Rα2 protein expression was measured by using fluorescence-activated cell sorting. Eotaxin release was determined by means of ELISA. RESULTS: Direct treatment with IFN-ß led to an upregulation of IL-13Rα2. Exposure to dsRNA rapidly induced IFN-ß mRNA in fibroblasts, and this was followed by significant induction of IL-13Rα2 mRNA and cell-surface protein expression, which was dependent on de novo protein synthesis. A neutralizing antibody to the IFN-α/ß receptor blocked cell-surface expression of IL-13Rα2 in the presence of dsRNA. Pretreatment of fibroblasts with dsRNA led to attenuation of IL-13-stimulated eotaxin production. However, the presence of an IL-13Rα2 neutralizing antibody restored IL-13-stimulated eotaxin production in dsRNA-treated cells. CONCLUSION: IFN-ß induces IL-13Rα2 expression, leading to a consequential suppression of responsiveness to IL-13. These data suggest cross-talk between TH1 and TH2 pathways and point to an immunomodulatory role for IL-13Rα2 in human bronchial fibroblasts during viral infection.

Cytoplasmic tail of IL-13Ralpha2 regulates IL-4 signal transduction.

IL (interleukin)-4 and IL-13 are key cytokines in the pathogenesis of allergic inflammatory disease. IL-4 and IL-13 share many functional properties as a result of their utilization of a common receptor complex comprising IL-13Ralpha1 (IL-13 receptor alpha-chain 1) and IL-4Ralpha. The second IL-13R (IL-13 receptor) has been identified, namely IL-13Ralpha2. This has been thought to be a decoy receptor due to its short cytoplasmic tail and its high binding affinity for IL-13 but not IL-4. IL-13Ralpha2 exists on the cell membrane, intracellularly and in a soluble form. Recent reports revealed that membrane IL-13Ralpha2 may have some signalling capabilities, and a soluble form of IL-13Ralpha2 can be generated in the presence of environmental allergens such as DerP. Interestingly, IL-13Ralpha2 has also been shown to regulate both IL-13 and IL-4 response in primary airway cells, despite the fact that IL-13Ralpha2 does not bind IL-4. The regulator mechanism is still unclear but the physical association of IL-13Ralpha2 with IL-4Ralpha appears to be a key regulatory step. These results suggest that the cytoplasmic tail of IL-13Ralpha2 may interfere with the association or activation of signalling molecules, such as JAK1 (Janus kinase 1), on IL-4Ralpha and thus prevents downstream signal cascade. The receptor has more complicated functions than a simple decoy receptor. In this review, we discuss newly revealed functions of IL-13Ralpha2.

The association of the cytoplasmic domains of interleukin 4 receptor alpha and interleukin 13 receptor alpha 2 regulates interleukin 4 signaling.

Interleukin-4 (IL-4) and Interleukin-13 (IL-13), key cytokines in the pathogenesis of allergic inflammatory disease, mediate their effects via a receptor composed of IL-13Rα1 and IL-4Rα. A third (decoy) receptor called IL-13Rα2 regulates interleukin signaling through this receptor complex. We employed a variety of biophysical and cell-based techniques to decipher the role of this decoy receptor in mediating IL-4 signaling though the IL-4Rα-IL-13Rα1 receptor complex. Surface plasmon resonance (SPR) analysis showed that IL-13Rα2 does not bind IL-4, and does not affect binding of IL-4 to IL-4Rα. These results indicate that the extracellular domains of IL-4Rα and IL-13Rα2 are not involved in the regulation of IL-4 signaling by IL-13Rα2. We next used a two-hybrid system to show that the cytoplasmic domains of IL-4Rα and IL-13Rα2 interact, and that the secondary structure of the IL-13Rα2 intracellular domain is critical for this interaction. The cellular relevance of this interaction was next investigated. BEAS-2B bronchial epithelial cells that stably express full length IL-13Rα2, or IL-13Rα2 lacking its cytoplasmic domain, were established. Over expression of IL-13Rα2 attenuated IL-4 and IL-13 mediated STAT6 phosphorylation. IL-13Rα2 lacking its cytoplasmic domain continued to attenuate IL-13-mediated signaling, but had no effect on IL-4-mediated STAT6 signaling. Our results suggest that the physical interaction between the cytoplasmic domains of IL-13Rα2 and IL-4Rα regulates IL-4 signaling through the IL-4Rα-IL-13Rα1 receptor complex.

Effect of IL-13 receptor alpha2 levels on the biological activity of IL-13 variant R110Q.

BACKGROUND: IL-13 is a key cytokine associated with the asthmatic phenotype. IL-13 signals via its cognate receptor, a complex of IL-13 receptor (IL-13R) alpha 1 chain with IL-4 receptor alpha; however, a second protein, IL-13Ralpha2, also binds IL-13. Recently a polymorphic variant of IL-13 (R110Q) has been shown to be associated with atopy. OBJECTIVE: To investigate the binding properties of this IL-13 variant to its cognate receptors. METHODS: We used surface plasmon resonance to measure the binding kinetics of R110Q to its receptors. Primary human fibroblasts were grown from endobronchial biopsies obtained from volunteers. Receptor levels were measured by fluorescence-activated cell sorting. RESULTS: There was no significant difference in the binding of R110Q with soluble human IL-13Ralpha1 compared with IL-13 (32 +/- 5 nmol/L and 36 +/- 7 nmol/L, respectively; P = .625). However, a small but significant difference was observed in the binding of R110Q to soluble human IL-13Ralpha2 compared with IL-13 (840 +/- 87 pmol/L and 1.1 +/- .05 nmol/L, respectively; P = .04). We observed that primary human lung fibroblasts expressed different levels of IL-13Ralpha2. Eotaxin release from fibroblasts expressing low IL-13Ralpha2 levels was significantly higher in response to R110Q compared with IL-13. This was not evident in cells that had high baseline IL-13Ralpha2 levels. CONCLUSION: These results suggest that relatively small changes in functional properties of a ligand combined with variation in receptor levels in vivo can result in significant differences in responsiveness. CLINICAL IMPLICATIONS: Expression of R110Q and low IL-13Ralpha2 levels can result in important biological differences that may have clinical relevance in an atopic environment.

IL-13 receptor alpha 2: a regulator of IL-13 and IL-4 signal transduction in primary human fibroblasts.

BACKGROUND: IL-13 and IL-4 share many functional properties as a result of their use of a common receptor complex comprising IL-13 receptor alpha 1 (IL-13Ralpha1) and IL-4 receptor alpha (IL-4Ralpha). The nonsignaling receptor IL-13 receptor alpha 2 (IL-13Ralpha2) binds IL-13 with high affinity and specificity and is believed to be a decoy receptor for IL-13. OBJECTIVE: We sought to examine the inhibitory effects of soluble and membrane-bound IL-13Ralpha2 on IL-13- and IL-4-mediated effects. METHODS: Primary human fibroblasts were grown from endobronchial biopsy specimens obtained from volunteers. Upregulation of IL-13Ralpha2 mRNA was measured by means of RT-PCR, and the level of surface expression was measured by means of FACS. RESULTS: We found that a recombinant soluble form of IL-13Ralpha2 blocked the effects of IL-13, but not IL-4, in fibroblasts in vitro. However, we found that the transmembrane form of IL-13Ralpha2 could attenuate both IL-13 and IL-4 responses, even though the response to TNF-alpha was unaffected. Furthermore, we found that IL-13Ralpha2 became associated with IL-4Ralpha in the presence of IL-4. Addition of a blocking antibody to the extracellular domain of IL-13Ralpha2 restored responses of both IL-13 and IL-4. CONCLUSION: The ability of IL-13Ralpha2 to regulate IL-4 was previously unrecognized in primary airway cells. These data reveal a novel role for IL-13Ralpha2 as a negative regulator of both IL-13 and IL-4 signaling in human bronchial fibroblasts. CLINICAL IMPLICATIONS: It appears that IL-13Ralpha2 might be a powerful suppressor of TH2-mediated responses and thus represents a potential therapeutic target for the treatment of asthma.

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.

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.