Distinct structural requirements for interleukin-4 (IL-4) and IL-13 binding to the shared IL-13 receptor facilitate cellular tuning of cytokine responsiveness.
ABSTRACT: Both interleukin-4 (IL-4) and IL-13 can bind to the shared receptor composed of the IL-4 receptor alpha chain and the IL-13 receptor alpha1 chain (IL-13Ralpha1); however, the mechanisms by which these ligands bind to the receptor chains are different, enabling the principal functions of these ligands to be different. We have previously shown that the N-terminal Ig-like domain in IL-13Ralpha1, called the D1 domain, is the specific and critical binding unit for IL-13. However, it has still remained obscure which amino acid has specific binding capacity to IL-13 and why the D1 domain acts as the binding site for IL-13, but not IL-4. To address these questions, in this study we performed mutational analyses for the D1 domain, combining the structural data to identify the amino acids critical for binding to IL-13. Mutations of Lys-76, Lys-77, or Ile-78 in c' strand in which the crystal structure showed interaction with IL-13, and those of Trp-65 and Ala-79 adjacent to the interacting site, resulted in significant impairment of IL-13 binding, demonstrating that these amino acids generate the binding site. Furthermore, mutations of Val-35, Leu-38, or Val-42 at the N-terminal beta-strand also resulted in loss of IL-13 binding, probably from decreased structural stability. None of the mutations employed here affected IL-4 binding. These results demonstrate that the D1 domain of IL-13Ralpha1 acts as an affinity converter, through direct cytokine interactions, that allows the shared receptor to respond differentially to IL-4 and IL-13.
Project description:IL-13 and IL-4 are central T helper 2 (Th2) cytokines in the immune system and potent activators of inflammatory responses and fibrosis during Th2 inflammation. Recent studies using Il13ra1(-/-) mice have demonstrated a critical role for IL-13 receptor (IL-13R) alpha1 in allergen-induced airway responses. However, these observations require further attention especially because IL-4 can induce similar lung pathology to IL-13, independent of IL-13, and is still present in the allergic lung. Thus, we hypothesized that IL-13Ralpha1 regulates IL-4-induced responses in the lung. To dissect the role of IL-13Ralpha1 and the type I and II IL-4Rs in experimental asthma, we examined lung pathology induced by allergen, IL-4, and IL-13 challenge in Il13ra1(-/-) mice. We report that IL-13Ralpha1 is essential for baseline IgE production, but Th2 and IgE responses to T cell-dependent antigens are IL-13Ralpha1-independent. Furthermore, we demonstrate that increased airway resistance, mucus, TGF-beta, and eotaxin(s) production, but not cellular infiltration, are critically dependent on IL-13Ralpha1. Surprisingly, our results identify a CCR3- and IL-13Ralpha1-independent pathway for lung eosinophilia. Global expression profiling of lungs from mice stimulated with allergen or IL-4 demonstrated that marker genes of alternatively activated macrophages are differentially regulated by the type I and type II IL-4R. Taken together, our data provide a comprehensive mechanistic analysis of the critical role by which IL-13Ralpha1 mediates allergic lung pathology and highlight unforeseen roles for the type II IL-4R.
Project description:Interleukin-13 is a cytokine important for development of T helper cell type 2 (Th2) responses and plays a critical role in asthma and allergy. The IL-13 Receptor alpha2 (IL-13Ralpha2) is a receptor for IL-13 lacking canonical Jak/STAT signaling functions. Here we present the crystal structure along with a mutational and biophysical analysis of the IL-13/IL-13Ralpha2 complex. While retaining a similar mode of IL-13 binding to its related signaling receptor, IL-13Ralpha1, IL-13Ralpha2 uses peripheral receptor residues unused in the IL-13/IL-13Ralpha1 complex to generate a larger and more complementary interface for IL-13. This results in a four orders of magnitude increase in affinity, to the femtomolar level, compared to IL-13Ralpha1. Alanine scanning mutagenesis of the IL-13 interface reveals several common "hotspot" residues important for binding to both receptors, but also identifies a prominent IL-13Ralpha2-specific contact. These results provide a framework for development of receptor subtype-selective IL-13 antagonists and indicate a decoy function for IL-13Ralpha2.
Project description:Interleukin (IL)-4 and -13 are related cytokines sharing functional receptors. IL-4 signals through the type I (IL-4Ralpha/common gamma-chain [gammac]) and the type II (IL-4Ralpha/-13Ralpha1) IL-4 receptors, whereas IL-13 utilizes only the type II receptor. In this study, we show that mouse bone marrow-derived macrophages and human and mouse monocytes showed a much greater sensitivity to IL-4 than to IL-13. Lack of functional gammac made these cells poorly responsive to IL-4, while retaining full responsiveness to IL-13. In mouse peritoneal macrophages, IL-4 potency exceeds that of IL-13, but lack of gammac had only a modest effect on IL-4 signaling. In contrast, IL-13 stimulated greater responses than IL-4 in fibroblasts. Using levels of receptor chain expression and known binding affinities, we modeled the assemblage of functional type I and II receptor complexes. The differential expression of IL-4Ralpha, IL-13Ralpha1, and gammac accounted for the distinct IL-4-IL-13 sensitivities of the various cell types. These findings provide an explanation for IL-13's principal function as an "effector" cytokine and IL-4's principal role as an "immunoregulatory" cytokine.
Project description:Allergic airway inflammation is characterized by marked in situ changes in gene and protein expression, yet the role of microRNAs (miRNAs), a new family of key mRNA regulatory molecules, in this process has not yet been reported. Using a highly sensitive microarray-based approach, we identified 21 miRNAs with differential expression between doxycycline-induced lung-specific IL-13 transgenic mice (with allergic airway inflammation) and control mice. In particular, we observed overexpression of miR-21 and underexpression of miR-1 in the induced IL-13 transgenic mice compared with control mice. These findings were validated in two independent models of allergen-induced allergic airway inflammation and in IL-4 lung transgenic mice. Although IL-13-induced miR-21 expression was IL-13Ralpha1 dependent, allergen-induced miR-21 expression was mediated mainly independent of IL-13Ralpha1 and STAT6. Notably, predictive algorithms identified potential direct miR-21 targets among IL-13-regulated lung transcripts, such as IL-12p35 mRNA, which was decreased in IL-13 transgenic mice. Introduction of pre-miR-21 dose dependently inhibited cellular expression of a reporter vector harboring the 3'-untranslated region of IL-12p35. Moreover, mutating miR-21 binding sites in IL-12p35 3'-untranslated region abrogated miR-21-mediated repression. In summary, we have identified a miRNA signature in allergic airway inflammation, which includes miR-21 that modulates IL-12, a molecule germane to Th cell polarization.
Project description:BACKGROUND: Interleukin 4 (IL-4) is a key regulator of the immune system and an important factor in the development of allergic hypersensitivity. Together with interleukin 13 (IL-13), IL-4 plays an important role in exacerbating allergic and asthmatic symptoms. For signal transduction, both cytokines can utilise the same receptor, consisting of the IL-4Ralpha and the IL-13Ralpha1 chain, offering an explanation for their overlapping biological functions. Since both cytokine ligands share only moderate similarity on the amino acid sequence level, molecular recognition of the ligands by both receptor subunits is of great interest. IL-4 and IL-13 are interesting targets for allergy and asthma therapies. Knowledge of the binding mechanism will be important for the generation of either IL-4 or IL-13 specific drugs. RESULTS: We present a structure/function analysis of the IL-4 ligand-receptor interaction. Structural determination of a number of IL-4 variants together with in vitro binding studies show that IL-4 and its high-affinity receptor subunit IL-4Ralpha interact via a modular protein-protein interface consisting of three independently-acting interaction clusters. For high-affinity binding of wild-type IL-4 to its receptor IL-4Ralpha, only two of these clusters (i.e. cluster 1 centered around Glu9 and cluster 2 around Arg88) contribute significantly to the free binding energy. Mutating residues Thr13 or Phe82 located in cluster 3 to aspartate results in super-agonistic IL-4 variants. All three clusters are fully engaged in these variants, generating a three-fold higher binding affinity for IL-4Ralpha. Mutagenesis studies reveal that IL-13 utilizes the same main binding determinants, i.e. Glu11 (cluster 1) and Arg64 (cluster 2), suggesting that IL-13 also uses this modular protein interface architecture. CONCLUSION: The modular architecture of the IL-4-IL-4Ralpha interface suggests a possible mechanism by which proteins might be able to generate binding affinity and specificity independently. So far, affinity and specificity are often considered to co-vary, i.e. high specificity requires high affinity and vice versa. Although the binding affinities of IL-4 and IL-13 to IL-4Ralpha differ by a factor of more than 1000, the specificity remains high because the receptor subunit IL-4Ralpha binds exclusively to IL-4 and IL-13. An interface formed by several interaction clusters/binding hot-spots allows for a broad range of affinities by selecting how many of these interaction clusters will contribute to the overall binding free energy. Understanding how proteins generate affinity and specificity is essential as more and more growth factor receptor families show promiscuous binding to their respective ligands. This limited specificity is, however, not accompanied by low binding affinities.
Project description:IL-13 is a central mediator of airway responsiveness and mucus expression in patients with allergic airway inflammation, and IL-13 is currently a therapeutic target for asthma. However, little is known about how IL-13 regulates human CD4(+) T-cell lineages because IL-13 receptor (IL-13R) ?1, a subunit of IL-13R, has not previously been reported to exist on human T cells.We sought to determine whether human CD4(+) T(H)17 cells express IL-13R?1 and whether IL-13 regulates T(H)17 cytokine production.Naive human CD4(+) cells were isolated from whole blood, activated with anti-CD3 and anti-CD28, and polarized to T(H)1, T(H)2, T(H)17, or induced regulatory T cells in the presence of IL-13 (0-10 ng/mL). Cell supernatants, total RNA, or total protein was examined 4 days after T(H)17 polarization.T(H)17 cells, but not T(H)0, T(H)1, T(H)2, or induced regulatory T cells, expressed IL-13R?1. IL-13 attenuated IL-17A production, as well as expression of retinoic acid-related orphan receptor, runt-related transcription factor-1, and interferon regulatory factor 4 in T(H)17-polarized cells. IL-13 neither inhibited IFN-? production from T(H)1 cells nor inhibited IL-4 production from T(H)2 cells. Furthermore, attenuation of IL-17A production only occurred when IL-13 was present within 24 hours of T-cell activation or at the time of restimulation.IL-13R?1 is expressed on human CD4(+) T(H)17 cells, and IL-13 attenuates IL-17A production at polarization and restimulation. Although IL-13 is an attractive therapeutic target for decreasing symptoms associated with asthma, these results suggest that therapies inhibiting IL-13 production could have adverse side effects by increasing IL-17A production.
Project description:The interleukin 4 receptor (IL-4R) is a central mediator of T helper type 2 (T(H)2)-mediated disease and associates with either the common gamma-chain to form the type I IL-4R or with the IL-13R alpha1 chain (IL-13Ralpha1) to form the type II IL-4R. Here we used Il13ra1-/- mice to characterize the distinct functions of type I and type II IL-4 receptors in vivo. In contrast to Il4ra-/- mice, which have weak T(H)2 responses, Il13ra1-/- mice had exacerbated T(H)2 responses. Il13ra1-/- mice showed much less mortality after infection with Schistosoma mansoni and much more susceptibility to Nippostrongylus brasiliensis. IL-13Ralpha1 was essential for allergen-induced airway hyperreactivity and mucus hypersecretion but not for fibroblast or alternative macrophage activation. Thus, type I and II IL-4 receptors exert distinct effects on immune responses.
Project description:INTRODUCTION:A unique anti-interleukin (IL)-13 monoclonal antibody, RPC4046, was generated on the basis of differential IL-13 receptor (R) blockade as assessed in a murine asthma model; the safety, tolerability, pharmacokinetics, and pharmacodynamics of RPC4046 were evaluated in a first-in-human study. METHODS:Anti-IL-13 antibodies with varying receptor blocking specificity were evaluated in the ovalbumin-induced murine asthma model. A randomized, double-blind, placebo-controlled, dose-escalation first-in-human study (NCT00986037) was conducted with RPC4046 in healthy adults and patients with mild to moderate controlled asthma. RESULTS:In the ovalbumin model, blocking IL-13 binding to both IL-13Rs (IL-13Rα1 and IL-13Rα2) inhibited more asthma phenotypic features and more fully normalized the distinct IL-13 gene transcription associated with asthma compared with blocking IL-13Rα1 alone. In humans, RPC4046 exposure increased dose-dependently; pharmacokinetics were similar in healthy and asthmatic subjects, and blockade of both IL-13Rs uniquely affected IL-13 gene transcription. A minority of participants (28%) had antidrug antibodies, which were transient and appeared not to affect pharmacokinetics. Adverse event profiles were similar in healthy and asthmatic subjects, without dose-related or administration route differences, systemic infusion-related reactions, or asthma symptom worsening. Adverse events were mild to moderate, with none reported as probably related to RPC4046 or leading to discontinuations. Non-serious upper respiratory tract infections were more frequent with RPC4046 versus placebo. CONCLUSION:RPC4046 is a novel anti-IL-13 antibody that blocks IL-13 binding to both receptors and more fully blocks the asthma phenotype. These results support further investigation of RPC4046 for IL-13-related allergic/inflammatory diseases (e.g., asthma and eosinophilic esophagitis). FUNDING:AbbVie Inc. sponsored the studies and contributed to the design and conduct of the studies, data management, data analysis, interpretation of the data, and in the preparation and approval of the manuscript.
Project description:Signaling of the cytokine interleukin-6 (IL-6) via its soluble IL-6 receptor (sIL-6R) is responsible for the proinflammatory properties of IL-6 and constitutes an attractive therapeutic target, but how the sIL-6R is generated in vivo remains largely unclear. Here, we use liquid chromatography-mass spectrometry to identify an sIL-6R form in human serum that originates from proteolytic cleavage, map its cleavage site between Pro-355 and Val-356, and determine the occupancy of all O- and N-glycosylation sites of the human sIL-6R. The metalloprotease a disintegrin and metalloproteinase 17 (ADAM17) uses this cleavage site in vitro, and mutation of Val-356 is sufficient to completely abrogate IL-6R proteolysis. N- and O-glycosylation were dispensable for signaling of the IL-6R, but proteolysis was orchestrated by an N- and O-glycosylated sequon near the cleavage site and an N-glycan exosite in domain D1. Proteolysis of an IL-6R completely devoid of glycans is significantly impaired. Thus, glycosylation is an important regulator for sIL-6R generation.
Project description:Interleukin-13 is a Th2-associated cytokine responsible for many pathological responses in allergic asthma including mucus production, inflammation, and extracellular matrix remodeling. In addition, IL-13 is required for immunity to many helminth infections. IL-13 signals via the type-II IL-4 receptor, a heterodimeric receptor of IL-13R?1 and IL-4R?, which is also used by IL-4. IL-13 also binds to IL-13R?2, but with much higher affinity than the type-II IL-4 receptor. Binding of IL-13 to IL-13R?2 has been shown to attenuate IL-13 signaling through the type-II IL-4 receptor. However, molecular determinants that dictate the specificity and affinity of mouse IL-13 for the different receptors are largely unknown. Here, we used high-density overlapping peptide arrays, structural modeling, and molecular docking methods to map IL-13 binding sequences on its receptors. Predicted binding sequences on mouse IL-13R?1 and IL-13R?2 were in agreement with the reported human IL-13 receptor complex structures and site-directed mutational analysis. Novel structural differences were identified between IL-13 receptors, particularly at the IL-13 binding interface. Notably, additional binding sites were observed for IL-13 on IL-13R?2. In addition, the identification of peptide sequences that are unique to IL-13R?1 allowed us to generate a monoclonal antibody that selectively binds IL-13R?1. Thus, high-density peptide arrays combined with molecular docking studies provide a novel, rapid, and reliable method to map cytokine-receptor interactions that may be used to generate signaling and decoy receptor-specific antagonists.