Inhibition of arginase I activity by RNA interference attenuates IL-13-induced airways hyperresponsiveness

Increased arginase I activity is associated with allergic disorders such as asthma. How arginase I contributes to and is regulated by allergic inflammatory processes remains unknown. CD4+ Th2 lymphocytes (Th2 cells) and IL-13 are two crucial immune regulators that use STAT6-dependent pathways to induce allergic airways inflammation and enhanced airways responsiveness to spasmogens (airways hyperresponsiveness (AHR)). This pathway is also used to activate arginase I in isolated cells and in hepatic infection with helminths. In the present study, we show that arginase I expression is also regulated in the lung in a STAT6-dependent manner by Th2-induced allergic inflammation or by IL-13 alone. IL-13-induced expression of arginase I correlated directly with increased synthesis of urea and with reduced synthesis of NO. Expression of arginase I, but not eosinophilia or mucus hypersecretion, temporally correlated with the development, persistence, and resolution of IL-13-induced AHR. Pharmacological supplementation with l-arginine or with NO donors amplified or attenuated IL-13-induced AHR, respectively. Moreover, inducing loss of function of arginase I specifically in the lung by using RNA interference abrogated the development of IL-13-induced AHR. These data suggest an important role for metabolism of l-arginine by arginase I in the modulation of IL-13-induced AHR and identify a potential pathway distal to cytokine receptor interactions for the control of IL-13-mediated bronchoconstriction in asthma.     

Comparative roles of IL-4, IL-13, and IL-4Ralpha in dendritic cell maturation and CD4+ Th2 cell function

IL-4 and IL-13 play key roles in Th2 immunity and asthma pathogenesis. Although the function of these cytokines is partially linked through their shared use of IL-4Ralpha for signaling, the interplay between these cytokines in the development of memory Th2 responses is not well delineated. In this investigation, we show that both IL-4 and IL-13 influence the maturation of dendritic cells (DC) in the lung and their ability to regulate secretion of IFN-gamma and Th2 cytokines by memory CD4(+) T cells. Cocultures of wild-type T cells with pulmonary DC from allergic, cytokine-deficient mice demonstrated that IL-4 enhanced the capacity of DC to stimulate T cell secretion of Th2 cytokines, whereas IL-13 enhanced the capacity of DC to suppress T cell secretion of IFN-gamma. Because IL-4Ralpha is critical for IL-4 and IL-13 signaling, we also determined how variants of IL-4Ralpha influenced immune cell function. T cells derived from allergic mice expressing a high-affinity IL-4Ralpha variant produced higher levels of IL-5 and IL-13 compared with T cells derived from allergic mice expressing a low-affinity IL-4Ralpha variant. Although DC expressing different IL-4Ralpha variants did not differ in their capacity to influence Th2 cytokine production, they varied in their capacity to inhibit IFN-gamma production by T cells. Thus, IL-4 and IL-13 differentially regulate DC function and the way these cells regulate T cells. The affinity of IL-4Ralpha also appears to be a determinant in the balance between Th2 and IFN-gamma responses and thus the severity of allergic disease.     

Complex role of the IL-4 receptor alpha in a murine model of airway inflammation: expression of the IL-4 receptor alpha on nonlymphoid cells of bone marrow origin contributes to severity of inflammation

Recent studies have suggested the IL-4Ralpha expressed on lung epithelium is necessary for TH2-mediated goblet cell differentiation and mucus hypersecretion in a murine model of allergic lung disease. However, the IL-4Ralpha is expressed on numerous cell types that could contribute to the overall pathology and severity of asthma. The relative role of the receptor on these cells has not yet been conclusively delineated. To dissect the contribution of IL-4Ralpha in the development of pulmonary allergic responses, we generated murine radiation bone marrow (BM) chimeras. BM from IL-4Ralpha(+) or IL-4Ralpha(-) mice was transferred into recipient mice that expressed or lacked IL-4Ralpha. In the absence of IL-4Ralpha in recipient mice, there was no goblet cell metaplasia or mucus hypersecretion in response to OVA, even in the presence of TH2 cells and substantial eosinophilic infiltration. More importantly, we found that expression of the IL-4Ralpha on a nonlymphoid, MHC class II(+), BM-derived cell type contributes to the severity of inflammation and mucus production. These results suggest that IL-4 and IL-13 contribute to the development of allergic inflammation by stimulating a complex interaction between IL-4Ralpha(+) cell types of both bone marrow and non-bone marrow origin.     

The effect of IL-5 and eotaxin expression in the lung on eosinophil trafficking and degranulation and the induction of bronchial hyperreactivity

The mechanisms regulating the selective migration and degranulation of eosinophils in the asthmatic lung and the subsequent development of airways hyperreactivity (AHR) have not been fully delineated. In this investigation, we have employed a novel transgene model to facilitate the dissection of the contributions of IL-5 and/or eotaxin to eosinophil function in the absence of complex tissue signals derived from the allergic lung. Gene transfer of IL-5 and/or eotaxin to the lungs of naive mice induced a pronounced and selective airways eosinophilia, but did not result in eosinophil degranulation or AHR. Airways eosinophilia occurred independently of the induction of a blood eosinophilia, but was markedly augmented by the coexpression of both cytokines and/or by the transient mobilization of eosinophils from the bone marrow by the administration of i.v. IL-5. However, for eosinophil degranulation and AHR to occur, the inhalation of Ag was required in association with IL-5 and eotaxin expression. Investigations in IL-5-deficient mice linked eosinophilia, and not solely IL-5 and eotaxin, with the induction of AHR. Furthermore, eosinophil degranulation and AHR were dependent on CD4+ T cells. Importantly, this investigation shows that IL-5 regulates eosinophilia within the lung as well as in the circulation and also amplifies eotaxin-induced chemotaxis in the airway compartment. Moreover, the interplay between these cytokines, CD4+ T cells, and factors generated by Ag inhalation provides fundamental signals for eosinophil degranulation and the induction of AHR.     

Distinct spatial requirement for eosinophil-induced airways hyperreactivity

T helper (Th)-2-derived cytokines and their involvement in the recruitment and activation of inflammatory cells crucially orchestrate asthma pathogenesis. A notable cellular component of this allergy-induced inflammation is the eosinophil. However, whether the eosinophil is an obligatory mediator for enhancing airways hyperreactivity (AHR) to cholinergic stimuli, a watershed of the asthmatic lung, is somewhat controversial. In this investigation we have endeavoured to define the spatial requirements for IL-4 and IL-13, and the downstream effector molecules, IL-5 and the CC chemokine eotaxin, for the recruitment of eosinophils and the development of AHR in a murine model of allergic pulmonary disease. These studies are of particular importance considering clinical trials, with either the soluble IL-4Ralpha subunit or a humanized anti-IL-5 antibody, are being conducted. Interestingly, our studies show that depletion of both IL-4 and IL-13 is necessary to ablate pulmonary eosinophilia and AHR, and that this may be attributed to the role these cytokines play in regulating the expression of the eosinophil- activating molecules, IL-5 and eotaxin. While it is clear that depletion of IL-5 diminishes pulmonary eosinophilia, we demonstrate in BALB/c mice that a deficiency in both IL-5 and eotaxin is necessary to abolish both the trafficking of eosinophils to the lung and AHR. However, in contrast to the neutrophil-rich inflammation observed in mice deficient in both IL-4 and IL-13, inflammation per se in mice deficient in both IL-5 and eotaxin is significantly attenuated. This suggests that asthma immunotherapy may be better directed towards the eosinophil- activating molecules IL-5 and eotaxin, rather than towards pleiotrophic molecules such IL-4 and IL-13, which are additionally important in modulating alternative inflammatory responses.     

A murine IL-4 receptor antagonist that inhibits IL-4- and IL-13-induced responses prevents antigen-induced airway eosinophilia and airway hyperresponsiveness

The closely related Th2 cytokines, IL-4 and IL-13, share many biological functions that are considered important in the development of allergic airway inflammation and airway hyperresponsiveness (AHR). The overlap of their functions results from the IL-4R alpha-chain forming an important functional signaling component of both the IL-4 and IL-13 receptors. Mutations in the C terminus region of the IL-4 protein produce IL-4 mutants that bind to the IL-4R alpha-chain with high affinity, but do not induce cellular responses. A murine IL-4 mutant (C118 deletion) protein (IL-4R antagonist) inhibited IL-4- and IL-13-induced STAT6 phosphorylation as well as IL-4- and IL-13-induced IgE production in vitro. Administration of murine IL-4R antagonist during allergen (OVA) challenge inhibited the development of allergic airway eosinophilia and AHR in mice previously sensitized with OVA. The inhibitory effect on airway eosinophilia and AHR was associated with reduced levels of IL-4, IL-5, and IL-13 in the bronchoalveolar lavage fluid as well as reduced serum levels of OVA-IGE: These observations demonstrate the therapeutic potential of IL-4 mutant protein receptor antagonists that inhibit both IL-4 and IL-13 in the treatment of allergic asthma.     

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.     

Cyclooxygenase inhibition augments allergic inflammation through CD4-dependent, STAT6-independent mechanisms

Nonselective cyclooxygenase (COX) inhibition during the development of allergic disease in a murine model causes an increase in type 2 cytokines and lung eosinophilia; however, the mechanisms responsible for this augmented allergen-induced inflammation have not been examined. Ab depletion of CD4 and CD8 cells revealed that the heightened allergic inflammation caused by COX inhibition was CD4, but not CD8, dependent. Allergen sensitization and airway challenge alone led to undetectable levels of IL-5 and IL-13 in the lungs of IL-4, IL-4Ralpha, and STAT6 knockout (KO) mice, but COX inhibition during the development of allergic inflammation resulted in wild-type levels of IL-5 and IL-13 and heightened airway eosinophilia in each of the three KO mice. These results indicate that the effect of COX inhibition was independent of signaling through IL-4, IL-4Ralpha, and STAT6. However, whereas COX inhibition increased IgE levels in allergic wild-type mice, IgE levels were undetectable in IL-4, IL-4Ralpha, and STAT6 KO mice, suggesting that IL-13 alone is not a switch factor for IgE synthesis in this model. These results illustrate the central role played by products derived from the COX pathway in the regulation of allergic immune responses.     

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 may mediate allergen-induced hyperresponsiveness independently of IL-5 or eotaxin by effects on airway smooth muscle

IL-13 is a mediator of allergen-induced airway hyperresponsiveness (AHR). The aim of this study was to evaluate whether eotaxin and IL-5 were implicated in the effects of IL-13 on allergen-induced AHR or whether IL-13 may exert its effects through direct actions on airway smooth muscle (ASM). To study this question airway inflammation and AHR were induced in mice by sensitization and subsequent challenge on three successive days with ovalbumin. A monoclonal anti-IL-13 antibody administered before each challenge significantly reduced AHR without affecting airway eosinophilia. No changes of mRNA in BAL and lung tissues or protein levels in BAL of IL-5 or eotaxin were found following anti-IL-13 treatment. Combined injection of monoclonal anti-IL-5 and antieotaxin antibodies before each antigen challenge blocked airway eosinophilia but failed to reduce AHR. IL-13 induced calcium transients in cultured murine ASM cells and augmented the calcium and contractile responses of these cells to leukotriene D4. These results suggest that IL-13 plays an important role in allergen-induced AHR and is important in the early phases of the inflammatory process. Its effects on AHR are mediated independently of IL-5 and eotaxin and may involve a direct effect on ASM to augment its responsiveness.     

The role of IL-13 in established allergic airway disease

The effectiveness of targeting IL-13 in models where airway hyperresponsiveness (AHR) and airway inflammation have already been established is not well-described. We investigated the effects of blocking IL-13 on the early and late phase airway responses and the development of AHR in previously sensitized and challenged mice. BALB/cByJ mice were sensitized (days 1 and 14) and challenged (days 28-30) with OVA. Six weeks later (day 72), previously sensitized/challenged mice were challenged with a single OVA aerosol and the early and late phase response and development of AHR were determined. Specific in vivo blockade of IL-13 was attained after i.p. injection of a soluble IL-13Ralpha2-IgG fusion protein (sIL-13Ralpha2Fc) on days 71-72 for the early and late responses and on days 71-73 for the development of AHR. sIL-13Ralpha2Fc administration inhibited the late, but not early, phase response and the OVA challenge-induced changes in lung resistance and dynamic compliance; as well, sIL-13Ralpha2Fc administration decreased bronchoalveolar lavage eosinophilia and mucus hypersecretion following the secondary challenge protocols. These results demonstrate that targeting IL-13 alone regulates airway responses when administrated to mice with established allergic airway disease. These data identify the importance of IL-13 in the development of allergen-induced altered airway responsiveness following airway challenge, even when administered before rechallenge of mice in which allergic disease had been previously established.     

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.     

Immune Modulatory Effects of IL-22 on Allergen-Induced Pulmonary Inflammation

IL-22 is a Th17/Th22 cytokine that is increased in asthma. However, recent animal studies showed controversial findings in the effects of IL-22 in allergic asthma. To determine the role of IL-22 in ovalbumin-induced allergic inflammation we generated inducible lung-specific IL-22 transgenic mice. Transgenic IL-22 expression and signaling activity in the lung were determined. Ovalbumin (OVA)-induced pulmonary inflammation, immune responses, and airway hyperresponsiveness (AHR) were examined and compared between IL-22 transgenic mice and wild type controls. Following doxycycline (Dox) induction, IL-22 protein was readily detected in the large (CC10 promoter) and small (SPC promoter) airway epithelial cells. IL-22 signaling was evidenced by phosphorylated STAT3. After OVA sensitization and challenge, compared to wild type littermates, IL-22 transgenic mice showed decreased eosinophils in the bronchoalveolar lavage (BAL), and in lung tissue, decreased mucus metaplasia in the airways, and reduced AHR. Among the cytokines and chemokines examined, IL-13 levels were reduced in the BAL fluid as well as in lymphocytes from local draining lymph nodes of IL-22 transgenic mice. No effect was seen on the levels of serum total or OVA-specific IgE or IgG. These findings indicate that IL-22 has immune modulatory effects on pulmonary inflammatory responses in allergen-induced asthma.     

Deletion of IL-4Ralpha on CD4 T cells renders BALB/c mice resistant to Leishmania major infection

Effector responses induced by polarized CD4+ T helper 2 (Th2) cells drive nonhealing responses in BALB/c mice infected with Leishmania major. Th2 cytokines IL-4 and IL-13 are known susceptibility factors for L. major infection in BALB/c mice and induce their biological functions through a common receptor, the IL-4 receptor alpha chain (IL-4Ralpha). IL-4Ralpha-deficient BALB/c mice, however, remain susceptible to L. major infection, indicating that IL-4/IL-13 may induce protective responses. Therefore, the roles of polarized Th2 CD4+ T cells and IL-4/IL-13 responsiveness of non-CD4+ T cells in inducing non-healer or healer responses have yet to be elucidated. CD4+ T cell-specific IL-4Ralpha (Lck(cre)IL-4Ralpha(-/lox)) deficient BALB/c mice were generated and characterized to elucidate the importance of IL-4Ralpha signaling during cutaneous leishmaniasis in the absence of IL-4-responsive CD4+ T cells. Efficient deletion was confirmed by loss of IL-4Ralpha expression on CD4+ T cells and impaired IL-4-induced CD4+ T cell proliferation and Th2 differentiation. CD8+, gammadelta+, and NK-T cells expressed residual IL-4Ralpha, and representative non-T cell populations maintained IL-4/IL-13 responsiveness. In contrast to IL-4Ralpha(-/lox) BALB/c mice, which developed ulcerating lesions following infection with L. major, Lck(cre)IL-4Ralpha(-/lox) mice were resistant and showed protection to rechallenge, similar to healer C57BL/6 mice. Resistance to L. major in Lck(cre)IL-4Ralpha(-/lox) mice correlated with reduced numbers of IL-10-secreting cells and early IL-12p35 mRNA induction, leading to increased delayed type hypersensitivity responses, interferon-gamma production, and elevated ratios of inducible nitric oxide synthase mRNA/parasite, similar to C57BL/6 mice. These data demonstrate that abrogation of IL-4 signaling in CD4+ T cells is required to transform non-healer BALB/c mice to a healer phenotype. Furthermore, a beneficial role for IL-4Ralpha signaling in L. major infection is revealed in which IL-4/IL-13-responsive non-CD4+ T cells induce protective responses.     

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.     

CD4+ T cell-specific deletion of IL-4 receptor alpha prevents ovalbumin-induced anaphylaxis by an IFN-gamma-dependent mechanism

IL-4Ralpha-mediated STAT6 activation serves an essential role in various animal models of allergy and asthma at both the sensitization and effector phases. IL-4 and IL-13 signaling via the IL-4Ralpha chain exacerbates murine anaphylaxis, but the cell-specific requirements for IL-4Ralpha expression are unclear. The purpose of this study was to elucidate the mechanisms of systemic anaphylaxis to OVA in gene-targeted mice with a deletion of the IL-4Ralpha chain in the macrophage/neutrophil or CD4+ T lymphocyte population. Results demonstrated that anaphylaxis in this model was entirely dependent upon the FcgammaRII/III and was associated with mast cell degranulation. Expression of the IL-4Ralpha on CD4+ T cells, but not macrophages or neutrophils, was critical for severe anaphylaxis, characterized by diarrhea, hypothermia, and death. Ab depletion experiments demonstrated that IFN-gamma protected against mortality and severe intestinal pathology despite the presence of Ag and specific Ab. This protection was associated with reduced levels of mast cell protease, a marker of mast cell degranulation, suggesting that IFN-gamma may inhibit mast cell degranulation in vivo. These data suggest that it may be possible to limit the severity of anaphylaxis using rational therapies designed to increase numbers of IFN-gamma-producing cells by targeting IL-4Ralpha signaling in CD4+ T lymphocytes.     

Expression of IL-4 receptor on non-bone marrow-derived cells is necessary for the timely elimination of Strongyloides venezuelensis in mice, but not for intestinal IL-4 production

In rodents and in humans, Strongyloides infection induces an immune response which is predominantly Th2 in nature. In an attempt to understand the role of the IL-4R/STAT6 signaling pathway, the pathway activated by the Th2 cytokines IL-4 and IL-13, in the induction of protection during Strongyloides venezuelensis infection, we have carried out experiments in mice lacking the IL-4Ralpha chain. Experiments were also carried out in STAT6 (STAT6(-/-)) and IL-12-deficient (IL-12(-/-)) mice for comparison. There was enhancement of IL-13 and abolition of IFN-gamma production in the small intestine of 7 day-infected IL-12(-/-) animals but worm elimination proceeded with very similar kinetics to those of wild-type mice. In IL-4Ralpha- or STAT6-deficient mice, there was a delay in parasite elimination and a large number of S. venezuelensis adult worms was still present in the small intestine 14 days after infection. Moreover, IgE production was completely abolished in IL-4Ralpha- or STAT6-deficient mice but tissue eosinophilia was normally induced by the parasite infection in deficient mice. Bone marrow transfer experiments showed that worm elimination occurred when a functional IL-4 receptor was present only in non-bone marrow-derived cells but not when IL-4R was only expressed in bone marrow cells. The induction of IL-4, but not IL-13, occurred independently of IL-4R. We believe these results are the first direct evidence that the mechanism responsible for the timely elimination of S. venezuelensis is dependent on the activation of IL-4R and STAT6. Moreover, a functional protective response is dependent on the expression of IL-4Ralpha on non-bone marrow-derived cells.