Polymorphisms in IL-4R alpha correlate with airways hyperreactivity, eosinophilia, and Ym protein expression in allergic IL-13-/- mice

The development of airways hyperreactivity in allergic IL-13(-/-) mice is controversial and appears to correlate with the number of times that the original 129 x C57BL/6 founder strain has been crossed to the BALB/c background. In this investigation, we compared allergic responses in founder IL-13(-/-) mice crossed for either 5 (N5) or 10 (N10) generations to BALB/c mice. Whereas allergic N5 IL-13(-/-) mice developed airways hyperreactivity, tissue eosinophilia, elevated IgE, and pulmonary expression of Ym proteins, these processes were attenuated in N5 IL-13(-/-) mice treated with an IL-4-neutralizing Ab, and in N10 IL-13(-/-) mice. These data showed that IL-4 was more effective in regulating allergic responses in N5 IL-13(-/-) mice than in N10 IL-13(-/-) mice. To elucidate the mechanism associated with these observations, we show by restriction and sequence analysis that N5 IL-13(-/-) mice express the C57BL/6 form of IL-4Ralpha and N10 IL-13(-/-) mice express the BALB/c form. Despite the near identical predicted molecular mass of these isoforms, IL-4Ralpha from N5 IL-13(-/-) mice migrates with a slower electrophoretic mobility than IL-4Ralpha from N10 IL-13(-/-) mice, suggesting more extensive posttranslational modification of the N5 form. The Thre(49)Ile polymorphism in the extracellular domain of BALB/c IL-4Ralpha has been demonstrated to disrupt N-linked glycosylation of Asn(47) and increase the dissociation rate of the IL-4Ralpha/IL-4 interaction. Collectively, these data show that polymorphisms in IL-4Ralpha, which have been shown to affect the interaction with IL-4, correlate with the ability of IL-4 to regulate allergic responses in IL-13(-/-) mice.     

Integrated signals between IL-13, IL-4, and IL-5 regulate airways hyperreactivity

In this investigation, we have examined the integrated relationship between IL-13, IL-4, and IL-5 for the development of airways hyperreactivity (AHR) in a model of asthma in BALB/c mice. Sensitization and aeroallergen challenge of both wild-type (WT) and IL-13 gene-targeted (IL-13-/-) mice induced allergic disease that was characterized by pulmonary eosinophilia and AHR to beta-methacholine. Although these responses in IL-13-/- mice were heightened compared with WT, they could be reduced to the level in nonallergic mice by the concomitant neutralization of IL-4. Mice in which both IL-4 and IL-13 were depleted displayed a marked reduction in tissue eosinophils, despite the development of a blood eosinophilia. Similar neutralization of IL-4 in WT mice only partially reduced AHR with no effect on tissue eosinophilia. In addition, neutralization of IL-5 in IL-13-/- mice, but not in WT mice, inhibited AHR, suggesting that tissue eosinophilia is linked to the mechanism underlying AHR only in the absence of IL-13. Additionally, mucus hypersecretion was attenuated in IL-13-/- mice, despite the persistence of AHR. Taken together, our data suggest both a modulatory role for IL-13 during sensitization and a proinflammatory role during aeroallergen challenge. The latter process appears redundant with respect to IL-4.     

Distinct signal transduction processes by IL-4 and IL-13 and influences from the Q551R variant of the human IL-4 receptor alpha chain

Background  

Although IL-4 and IL-13 share the IL-13 receptor, IL-13 exhibits unique functions. To elicit the cellular basis of these differences, signal transduction processes have been compared. Additionally, the role of the IL-4 receptor alpha (IL-4Rα) variant Q551R was investigated.     

Distinct signal transduction processes by IL-4 and IL-13 and influences from the Q551R variant of the human IL-4 receptor alpha chain

Background

Pseudomonas aeruginosa and Burkholderia cepacia infections of cystic fibrosis patients' lungs are often resistant to conventional antibiotic therapy. Protegrins are antimicrobial peptides with potent activity against many bacteria, including P. aeruginosa. The present study evaluates the correlation between protegrin-1 (PG-1) sensitivity/resistance and protegrin binding in P. aeruginosa and B. cepacia.

Methods

The PG-1 sensitivity/resistance and PG-1 binding properties of P. aeruginosa and B. cepacia were assessed using radial diffusion assays, radioiodinated PG-1, and surface plasmon resonance (BiaCore).

Results

The six P. aeruginosa strains examined were very sensitive to PG-1, exhibiting minimal active concentrations from 0.0625–0.5 μg/ml in radial diffusion assays. In contrast, all five B. cepacia strains examined were greater than 10-fold to 100-fold more resistant, with minimal active concentrations ranging from 6–10 μg/ml. When incubated with a radioiodinated variant of PG-1, a sensitive P. aeruginosa strain bound considerably more protegrin molecules per cell than a resistant B. cepacia strain. Binding/diffusion and surface plasmon resonance assays revealed that isolated lipopolysaccharide (LPS) and lipid A from the sensitive P. aeruginosa strains bound PG-1 more effectively than LPS and lipid A from resistant B. cepacia strains.

Conclusion

These findings support the hypothesis that the relative resistance of B. cepacia to protegrin is due to a reduced number of PG-1 binding sites on the lipid A moiety of its LPS.     

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.     

V75R576 IL-4 receptor alpha is associated with allergic asthma and enhanced IL-4 receptor function

Asthma is a complex polygenic disease. Many studies have implicated the importance of IL-4R alpha in the development of allergic inflammation and its gene has been implicated in the genetics of asthma and atopy. In this study, we examined the functional consequences of two of the human IL-4R alpha allelic variants that have been found to associate with asthma and atopy. We examined the effects of each variant alone and in combination on IL-4-dependent gene induction. We found that neither the Q576R nor the I75V variants affected IL-4-dependent CD23 expression. However, the combination of V75R576 resulted in expression of an IL-4R alpha with enhanced sensitivity to IL-4. We next examined the genetics of five of the known IL-4R alpha allelic variants in asthmatic and nonatopic populations. Strikingly, the association of V75/R576 with atopic asthma was greater than either allele alone and the association of R576 with atopic asthma was dependent on the coexistence of V75. A haplotype analysis revealed a single IL-4R alpha haplotype that was associated with allergic asthma, VACRS, further confirming the importance of the V75 and R576 combination in the genetics of asthma. This is the first report demonstrating that a functional alteration in IL-4R alpha requires the coexistence of two naturally occurring single nucleotide polymorphisms (snps) in combination; neither snp alone is sufficient. These data illustrate the importance of studying snps in combination, because the functional significance of a given snp may only be evident in a specific setting of additional snps in the same or different genes.     

IL-4 inhibits IL-2 synthesis and IL-2-induced up-regulation of IL-2R alpha but not IL-2R beta chain in CD4+ human T cells.

There is growing evidence to suggest a regulatory role of IL-4 in the immune system affecting both proliferation and lymphokine production. In the present work we have analyzed the effect of IL-4 on IL-2 and IFN-gamma synthesis by stimulating CD4+ human T cells (+10% accessory cells) with Con A in the presence of several doses (1 to 100 U/ml) of human rIL-4. The results showed an impaired IL-2 and IFN-gamma synthesis in the presence of IL-4. This inhibition was dose dependent and was evident only when IL-4 was added in the first 2 h of culture. Moreover, the external addition of IL-2 did not revert the inhibitory effect of IL-4 on IL-2 and IFN-gamma synthesis induced by Con A. We have also analyzed the effect of IL-4 on the expression of both alpha- and beta-chains of the IL-2R. Although the expression of IL-2R alpha mRNA was not modified after 6 h in culture in the presence of IL-4, a decrease was observed at 24 and 48 h. The addition of rIL-2 showed that the inhibition in IL-2R alpha expression could be explained by an impairment in the up-regulatory signal transmitted through the IL-2R. In addition to this, IL-4 did not modify the IL-2R beta mRNA expression at 6 and 24 h although a decreased expression was observed at 48 h which could be explained by the defective IL-2 production. The differential effect of IL-4 on the up-regulatory effect of IL-2 in the expression of IL-2R alpha and IL-2R beta suggest the existence of different regulatory mechanisms acting on the expression of both chains.     

Essential role of NKT cells producing IL-4 and IL-13 in the development of allergen-induced airway hyperreactivity

Using natural killer T (NKT) cell-deficient mice, we show here that allergen-induced airway hyperreactivity (AHR), a cardinal feature of asthma, does not develop in the absence of V(alpha)14i NKT cells. The failure of NKT cell-deficient mice to develop AHR is not due to an inability of these mice to produce type 2 T-helper (Th2) responses because NKT cell-deficient mice that are immunized subcutaneously at non-mucosal sites produce normal Th2-biased responses. The failure to develop AHR can be reversed by the adoptive transfer of tetramer-purified NKT cells producing interleukin (IL)-4 and IL-13 to Ja281(-/-) mice, which lack the invariant T-cell receptor (TCR) of NKT cells, or by the administration to Cd1d(-/-) mice of recombinant IL-13, which directly affects airway smooth muscle cells. Thus, pulmonary V(alpha)14i NKT cells crucially regulate the development of asthma and Th2-biased respiratory immunity against nominal exogenous antigens. Therapies that target V(alpha)14i NKT cells may be clinically effective in limiting the development of AHR and asthma.     

Level of expression of IL-13R alpha 2 impacts receptor distribution and IL-13 signaling

IL-13, a critical cytokine for allergic inflammation, exerts its effects through a complex receptor system including IL-4Ralpha, IL-13Ralpha1, and IL-13Ralpha2. IL-4Ralpha and IL-13Ralpha1 form a heterodimeric signaling receptor for IL-13. In contrast, IL-13Ralpha2 binds IL-13 with high affinity but does not signal. IL-13Ralpha2 exists on the cell surface, intracellularly, and in soluble form, but no information is available regarding the relative distributions of IL-13Ralpha2 among these compartments, whether the compartments communicate, and how the relative expression levels impact IL-13 responses. Herein, we investigated the distribution of IL-13Ralpha2 in transfected and primary cells, and we evaluated how the total level of IL-13Ralpha2 expression impacted its distribution. Our results demonstrate that the distribution of IL-13Ralpha2 is independent of the overall level of expression. The majority of the IL-13Ralpha2 protein existed in intracellular pools. Surface IL-13Ralpha2 was continually released into the medium in a soluble form, yet surface expression remained constant supporting receptor trafficking to the cell surface. IL-13Ralpha2 inhibited IL-13 signaling proportionally to its level of expression, and this inhibition could be overcome with high concentrations of IL-13.     

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.     

Critical role for IL-13 in the development of allergen-induced airway hyperreactivity

Airway hyperresponsiveness to a variety of specific and nonspecific stimuli is a cardinal feature of asthma, which affects nearly 10% of the population in industrialized countries. Eosinophilic pulmonary inflammation, eosinophil-derived products, as well as Th2 cytokines IL-13, IL-4, and IL-5, have been associated with the development of airway hyperreactivity (AHR), but the specific immunological basis underlying the development of AHR remains controversial. Herein we show that mice with targeted deletion of IL-13 failed to develop allergen-induced AHR, despite the presence of vigorous Th2-biased, eosinophilic pulmonary inflammation. However, AHR was restored in IL-13(-/-) mice by the administration of recombinant IL-13. Moreover, adoptive transfer of OVA-specific Th2 cells generated from TCR-transgenic IL-13(-/-) mice failed to induce AHR in recipient SCID mice, although such IL-13(-/-) Th2 cells produced high levels of IL-4 and IL-5 and induced significant airway inflammation. These studies definitively demonstrate that IL-13 is necessary and sufficient for the induction of AHR and that eosinophilic airway inflammation in the absence of IL-13 is inadequate for the induction of AHR. Therefore, treatment of human asthma with antagonists of IL-13 may be very effective.     

Suppressive effect of IL-4 on IL-13-induced genes in mouse lung

Although IL-4 signals through two receptors, IL-4R alpha/common gamma-chain (gamma(c)) and IL-4R alpha/IL-13R alpha1, and only the latter is also activated by IL-13, IL-13 contributes more than IL-4 to goblet cell hyperplasia and airway hyperresponsiveness in murine asthma. To determine whether unique gene induction by IL-13 might contribute to its greater proasthmatic effects, mice were inoculated intratracheally with IL-4 or IL-13, and pulmonary gene induction was compared by gene microarray and real-time PCR. Only the collagen alpha2 type VI (Ca2T6) gene and three small proline-rich protein (SPRR) genes were reproducibly induced > 4-fold more by IL-13 than by IL-4. Preferential IL-13 gene induction was not attributable to B cells, T cells, or differences in cytokine potency. IL-4 signaling through IL-4R alpha/gamma(c) suppresses Ca2T6 and SPRR gene expression in normal mice and induces these genes in RAG2/gamma(c)-deficient mice. Although IL-4, but not IL-13, induces IL-12 and IFN-gamma, which suppress many effects of IL-4, IL-12 suppresses only the Ca2T6 gene, and IL-4-induced IFN-gamma production does not suppress the Ca2T6 or SPRR genes. Thus, IL-4 induces genes in addition to IL-12 that suppress STAT6-mediated SPRR gene induction. These results provide a potential explanation for the dominant role of IL-13 in induction of goblet cell hyperplasia and airway hyperresponsiveness in asthma.     

Effects of common atopy-associated amino acid substitutions in the IL-4 receptor alpha chain on IL-4 induced phenotypes

The human IL-4 receptor alpha chain gene (IL4R) is highly polymorphic and controversial reports have been published with respect to the association of different single nucleotide polymorphisms (SNPs) with atopy markers. Here we analyzed the functional and associational relevance of common IL4R coding SNPs. Transfection of B cell lines expressing the IL-4R variant V75+R576 did not result in enhanced IL-4 induced CD23 expression compared to cell lines expressing the wild type IL-4R alpha chain. Transfection of the IL-4R variant P503 into a murine T cell line did not influence IL-4 induced T-cell proliferation compared to wild type constructs. Analysis of six IL4R coding SNPs (I75V, E400A, C431R, S436L, S503P, Q576R) and common haplotypes (frequency 0.05%) in blood donors (n=300) did not indicate a significant association with elevated serum IgE level. Moreover, the most informative IL4R coding SNPs (I75V, C431R, Q576R) and related two- and three-point haplotypes (frequency 0.05%) were analyzed in a second, extended group of blood donors (n=689). Again, no significant association with elevated serum IgE was detectable. We conclude that common coding SNPs in the IL4R gene are unlikely to contribute significantly to increased IgE levels and variations outside the coding region may influence atopy susceptibility.     

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.     

IL-4 and IL-13 form a negative feedback circuit with surfactant protein-D in the allergic airway response

The innate immune molecule surfactant protein-D (SP-D) plays an important regulatory role in the allergic airway response. In this study, we demonstrate that mice sensitized and challenged with either Aspergillus fumigatus (Af) or OVA have increased SP-D levels in their lung. SP-D mRNA and protein levels in the lung also increased in response to either rIL-4 or rIL-13 treatment. Type II alveolar epithelial cell expression of IL-4Rs in mice sensitized and challenged with Af, and in vitro induction of SP-D mRNA and protein by IL-4 and IL-13, but not IFN-gamma, suggested a direct role of IL-4R-mediated events. The regulatory function of IL-4 and IL-13 was further supported in STAT-6-deficient mice as well as in IL-4/IL-13 double knockout mice that failed to increase SP-D production upon allergen challenge. Interestingly, addition of rSP-D significantly inhibited Af-driven Th2 cell activation in vitro whereas mice lacking SP-D had increased numbers of CD4(+) cells with elevated IL-13 and thymus- and activation-regulated chemokine levels in the lung and showed exaggerated production of IgE and IgG1 following allergic sensitization. We propose that allergen exposure induces elevation in SP-D protein levels in an IL-4/IL-13-dependent manner, which in turn, prevents further activation of sensitized T cells. This negative feedback regulatory circuit could be essential in protecting the airways from inflammatory damage after allergen inhalation.     

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.     

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.     

Sequence motifs in IL-4R alpha mediating cell-cycle progression of primary lymphocytes

IL-4 signaling through the IL-4Ralpha chain regulates the development and proliferation of the Th2 lineage of effector CD4(+) T cells. Analyses of the IL-4R in factor-dependent cell lines led to the development of two apparently conflicting models of the primary structural determinants of IL-4R-mediated proliferative signaling. In one model, proliferation was dependent on the first conserved tyrosine in the cytoplasmic tail (Y1), while in the second, proliferation was independent of cytoplasmic tyrosines. We found that in activated primary T cells, mutation of only the Y1 residue resulted in a modest decrease in IL-4-induced S phase entry, a further decrease in cell-cycle completion, and a complete failure of IL-4 to induce p70S6 kinase phosphorylation. Consistent with a role for the PI3K/mammalian target of rapamycin pathway in mediating cytokine acceleration of G(2)/M transit, pretreatment of activated T cells with rapamycin resulted in only a modest decrease in IL-4-induced S phase entry, but a total block of cell-cycle completion. Strikingly, IL-4Ralpha chains that lacked all cytoplasmic tyrosines were competent to signal for STAT5 phosphorylation, mediated efficient S phase entry, and promoted cell-cycle progression. The ability of tyrosine-deficient IL-4Rs to mediate proliferative signaling and STAT phosphorylation was absolutely dependent on the presence of an intact ID-1 region. These findings show that IL-4Ralpha lacking cytoplasmic tyrosine residues is competent to induce ID-1-dependent proliferation, and indicate that IL-4 can promote G(2)/M progression via activation of the mammalian target of rapamycin pathway initiated at the Y1 residue.     

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.