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.     

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.     

Cutting edge: IL-4 receptor expression by non-bone marrow-derived cells is required to expel gastrointestinal nematode parasites.

Expulsion of two gastrointestinal nematode parasites, Nippostrongylus brasiliensis and Trichinella spiralis, is similar in that both require IL-4Ralpha expression, but different in that T cells and mast cells are required for IL-4-induced expulsion of T. spiralis but not N. brasiliensis. To examine the role of IL-4Ralpha signaling in immunity to these parasites, we studied worm expulsion in chimeric mice that selectively expressed IL-4Ralpha on bone marrow-derived or non-bone marrow-derived cells. N. brasiliensis was expelled by mice that expressed IL-4Ralpha only on non-bone marrow-derived cells, but not by mice that expressed IL-4Ralpha only on bone marrow-derived cells. Although T. spiralis expulsion required IL-4Ralpha expression by both bone marrow- and non-bone marrow-derived cells, IL-4 stimulation eliminated the requirement for IL-4Ralpha expression by bone marrow-derived cells. Thus, direct IL-4Ralpha signaling of nonimmune gastrointestinal cells may be generally required to induce worm expulsion, even when mast cell and T cell responses are also required.     

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.     

Reconstitution of a functional human type II IL-4/IL-13 receptor in mouse B cells: demonstration of species specificity

IL-13 is a Th2-derived pleiotropic cytokine that recently was shown to be a key mediator of allergic asthma. IL-13 mediates its effects via a complex receptor system, which includes the IL-4R alpha-chain, IL-4Ralpha, and at least two other cell surface proteins, IL-13Ralpha1 and IL-13Ralpha2, which specifically bind IL-13. IL-13 has been reported to have very limited effects on mouse B cells. It was unclear whether this was due to a lack of receptor expression, a disproportionate relative expression of the receptor components, or an additional subunit requirement in B cells. To determine the requirements for IL-13 signaling in murine B cells, we examined IL-13-dependent Stat6 activation and CD23 induction in the murine B cell line, A201.1. A201.1 cells responded to murine IL-4 via the type I IL-4R, but were unresponsive to IL-13, and did not express IL-13 receptor. B220(+) splenocytes also failed to signal in response to IL-13 and did not express IL-13 receptor. We transfected A201.1 cells with human IL-4Ralpha, IL-13Ralpha1, or both. Transfectants expressing either human IL-4Ralpha or human IL-13Ralpha1 alone were unable to respond or signal to IL-13. Thus, human IL-13Ralpha1 could not combine with the endogenous murine IL-4Ralpha to generate a functional IL-13R. However, cells transfected with both human IL-4Ralpha and IL-13Ralpha1 responded to IL-13. Thus, the relative lack of IL-13 responsiveness in murine B cells is due to a lack of receptor expression. Furthermore, the heterodimeric interaction between IL-4Ralpha and IL-13Ralpha1 is species specific.     

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.     

IL-4 exacerbates anaphylaxis

We evaluated whether IL-4, a cytokine critical for inducing allergic responses, also contributes to the effector phase of allergy. Pretreatment of mice with IL-4 or the related cytokine, IL-13, rapidly and dramatically increased the severity of anaphylaxis induced by cross-linking Fc(epsilon)RI or FcgammaRIII. This effect was inhibited by endogenously produced IFN-gamma, was T cell-, B cell-, and common gamma-chain-independent, and required IL-4Ralpha and Stat6. IL-4Ralpha signaling also enhanced anaphylaxis in mice infected with a nematode parasite that stimulates IL-4/IL-13 production. IL-4 exacerbated anaphylaxis by acting synergistically with vasoactive mediators to increase vascular permeability. Synergy between IL-4 and vasoactive mediators during the effector phase of allergic inflammation may both contribute to allergic immunopathology and enhance protective immunity against gastrointestinal worms.     

Cutting edge: murine dendritic cells require IL-15R alpha to prime NK cells

NK cells protect hosts against viral pathogens and transformed cells, and dendritic cells (DCs) play important roles in activating NK cells. We now find that murine IL-15Ralpha-deficient DCs fail to support NK cell cytolytic activity and elaboration of IFN-gamma, despite the fact that these DCs express normal levels of costimulatory molecules and IL-12. By contrast, IL-15Ralpha expression on NK cells is entirely dispensable for their activation by DCs. In addition, blockade with anti-IL-15Ralpha and anti-IL-2Rbeta but not anti-IL-2Ralpha-specific Abs prevents NK cell activation by wild-type DCs. Finally, presentation of IL-15 by purified IL-15Ralpha/Fc in trans synergizes with IL-12 to support NK cell priming. These findings suggest that murine DCs require IL-15Ralpha to present IL-15 in trans to NK cells during NK cell priming.     

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.     

Inflammasome-mediated production of IL-1beta is required for neutrophil recruitment against Staphylococcus aureus in vivo

IL-1R activation is required for neutrophil recruitment in an effective innate immune response against Staphylococcus aureus infection. In this study, we investigated the mechanism of IL-1R activation in vivo in a model of S. aureus infection. In response to a S. aureus cutaneous challenge, mice deficient in IL-1beta, IL-1alpha/IL-1beta, but not IL-1alpha, developed larger lesions with higher bacterial counts and had decreased neutrophil recruitment compared with wild-type mice. Neutrophil recruitment and bacterial clearance required IL-1beta expression by bone marrow (BM)-derived cells and not by non-BM-derived resident cells. In addition, mice deficient in the inflammasome component apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) had the same defects in neutrophil recruitment and host defense as IL-1beta-deficient mice, demonstrating an essential role for the inflammasome in mediating the production of active IL-1beta to promote neutrophil recruitment in host defense against S. aureus. This finding was further supported by the ability of recombinant active IL-1beta to control the infection and promote bacterial clearance in IL-1beta-deficient mice. These studies define a key host defense circuit where inflammasome-mediated IL-1beta production by BM-derived cells signals IL-1R on non-BM-derived resident cells to activate neutrophil recruitment in the innate immune response against S. aureus in vivo.     

The surface expression of CD25 at different stages of proliferative response in human lymphocytes. I. The role of JAK and Src tyrosine kinases as revealed by inhibitory analysis

The long-lasting expression of an alpha-subunit of interleukin-3 receptor (IL-2Ralpha) was found to accompany the PHA-induced proliferation of human blood lymphocytes (HBL), so that to the end of the second day of mitogenic stimulation only, the large blasts may express the high affinity alphabetagamma(c) receptor for IL-2. With the selective pharmacological drugs to JAK (WHI-P131) and Src (PP2) it is shown that the non-receptor tyrosine kinases are involved in the surface CD25 expression. It is revealed that the PP-2-inhibitable expression of CD25 is timely associated with the initial stage of T cell activation, whereas WHI-P131-inhibitable expression was present during the whole G0/G1/S transition. These data indicate that at the early, antigen-dependent stage the expression of IL-2Ralpha is induced via Src-dependent signaling pathway, and prolonged increase in IL-2Ralpha expression is regulated by IL-2/IL-2 receptor interaction via JAK-dependent signaling pathway.     

Down-regulation of IL-2 receptor α (CD25) characterizes human γδ-T cells rendered resistant to apoptosis after CD2 engagement in the presence of IL-12

We recently identified a CD2-mediated, IL-12-dependent signaling pathway that inhibits apoptosis in mitogen-stimulated human gammadelta-T cells. Here we show that gammadelta-T cells which acquire resistance to mitogen-induced apoptosis upregulate IL-12 receptor beta 1 subunit (IL-12Rbeta1); in contrast, gammadelta-T cells which remain sensitive to mitogen-induced apoptosis fail to express IL-12Rbeta1. Next we show that gammadelta-T cells which are rendered resistant to mitogen-induced apoptosis attenuate their expression of the IL-2 receptor alpha chain (IL-2Ralpha/CD25), this in part accounting for their acquired resistance to IL-2-induced death. In contrast, apoptosis-sensitive gammadelta-T cells are shown to persist in their expression of IL-2Ralpha/CD25, thus remaining sensitive to IL-2-induced death. Moreover, we show that apoptosis-resistant, but not apoptosis-sensitive, gammadelta-T cells display an enhanced responsiveness to IL-15, a finding in keeping with the known function of IL-15 as a growth and survival factor. Finally, we present evidence to suggest that this differential responsiveness to IL-15 occurs in part by the increased expression of the IL-15Ralpha chain on apoptosis-resistant gammadelta-T cells, compared to apoptosis-sensitive gammadelta-T cells. The biological and clinical implications of these findings are discussed.     

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.     

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.     

IL-27 promotes IL-10 production by effector Th1 CD4+ T cells: a critical mechanism for protection from severe immunopathology during malaria infection

Infection with the malaria parasite, Plasmodium, is characterized by excessive inflammation. The establishment of a precise balance between the pro- and anti-inflammatory responses is critical to guarantee control of the parasite and survival of the host. IL-10, a key regulatory cytokine produced by many cells of the immune system, has been shown to protect mice against pathology during acute Plasmodium0 chabaudi chabaudi AS model of malaria. However, the critical cellular source of IL-10 is still unknown. In this article, we demonstrate that T cell-derived IL-10 is necessary for the control of pathology during acute malaria, as mice bearing specific deletion of Il10 in T cells fully reproduce the phenotype observed in Il10(-)(/)(-) mice, with significant weight loss, decline in temperature, and increased mortality. Furthermore, we show that IFN-γ(+) Th1 cells are the main producers of IL-10 throughout acute infection, expressing high levels of CD44 and ICOS, and low levels of CD127. Although Foxp3(+) regulatory CD4(+) T cells produce IL-10 during infection, highly activated IFN-γ(+) Th1 cells were shown to be the essential and sufficient source of IL-10 to guarantee protection against severe immune-mediated pathology. Finally, in this model of malaria, we demonstrate that the generation of protective IL10(+)IFN-γ(+) Th1 cells is dependent on IL-27 signaling and independent of IL-21.