Down-regulation of IL-7Ralpha expression in human T cells via DNA methylation

IL-7 is critical for the development and survival of T cells. Recently, we found two subsets of human CD8+ T cells expressing IL-7Ralpha(high) and IL-7Ralpha(low) with different cell survival responses to IL-7. Although these CD8+ T cell subsets have differential IL-7Ralpha gene expression, the mechanism for this is unknown. DNA methylation is an important gene regulatory mechanism and is associated with the inactivation of gene expression. Thus, we investigated a role for DNA methylation in differentially regulating IL-7Ralpha gene expression in human CD8+ T cells and Jurkat T cells. IL-7Ralpha(high)CD8+ T cells had decreased methylation in the IL-7Ralpha gene promoter compared with IL-7Ralpha(low)CD8+ T cells and Jurkat T cells with low levels of IL-7Ralpha. Treating Jurkat T cells with 5-aza-2'-deoxycytidine, which reduced DNA methylation, increased IL-7Ralpha expression. Plus, the unmethylated IL-7Ralpha gene promoter construct had higher levels of promoter activity than the methylated one as measured by a luciferase reporter assay. These findings suggest that DNA methylation is involved in regulating IL-7Ralpha expression in T cells via affecting IL-7Ralpha gene promoter activity, and that the methylation of this gene promoter could be a potential target for modifying IL-7-mediated T cell development and survival.     

Responsiveness of naive CD4 T cells to polarizing cytokine determines the ratio of Th1 and Th2 cell differentiation

The intrinsic features of naive CD4 T cells that affect their ability to respond to polarizing signals for Th cell differentiation are not well understood. In this study, we show that naive CD4 T cells from mice transgenic for the Hlx gene expressed lower levels of IL-4Ralpha. The down-regulation of IL-4Ralpha diminished IL-4 signaling and the Th2 response and enhanced the Th1 response under suboptimal polarizing conditions. In nontransgenic CD4 T cells, blocking IL-4Ralpha with Abs had the same effect in an Ab dose-dependent manner. Conversely, Hlx haploinsufficiency caused higher expression of IL-4Ralpha to favor Th2 cell differentiation. Thus, the IL-4Ralpha level on naive CD4 T cells is genetically controlled by Hlx and determines the ratio of Th1 and Th2 cell differentiation.     

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.     

Cutting edge: IL-7-independent regulation of IL-7 receptor alpha expression and memory CD8 T cell development

Expression of IL-7Ralpha on a subset of Ag-specific effector CD8 T cells is believed to identify memory cell precursors. However, whether IL-7 regulates IL-7Ralpha expression in vivo and is responsible for selective survival of IL-7Ralpha(+) effector cells is unknown. Our results show that in the absence of IL-7, IL-7Ralpha expression was extinguished on the majority of CD8 T cells responding to virus infection, sustained on a subset of effector cells transitioning to memory, and expressed at high levels by memory cells. Additionally, an IL-7-deficient environment was capable of supporting bcl-2 up-regulation and memory cell development in response to virus infection. Thus, IL-7Ralpha regulation occurs independently of IL-7 in responding CD8 T cells, indicating that CD8 memory T cell precursors are not selected by IL-7/IL-7Ralpha interactions.     

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.     

Antisense inhibition of membrane-bound human interleukin-5 receptor-alpha chain does not affect soluble receptor expression and induces apoptosis in TF-1 cells

Binding of human interleukin-5 (HuIL-5) to its membrane-anchored receptor (IL-5R) triggers multiple signaling pathways, cellular proliferation, and maturational responses, as well as protection from apoptosis. In contrast, soluble forms of the HuIL-5R have been shown to inhibit IL-5 signaling and, therefore, may represent naturally occurring negative regulators of IL-5 function. Because of the central role of IL-5 in promoting eosinophilia and airway hyperresponsiveness in animal models of asthma, antisense oligonucleotides specific either for the membrane form alone or for sequences shared between both the membrane and soluble forms of the HuIL-5Ralpha ligand binding chain were designed. The activities of these oligonucleotides were characterized in IL-5R-expressing erythroleukemic TF-1 cells. Herein we report that an antisense oligonucleotide targeted to a sequence unique to the alternatively spliced membrane-bound form of the HuIL-5Ralpha chain has been developed that selectively inhibits membrane, but not soluble, mRNA isoform expression. Both this membrane-specific oligonucleotide and an antisense oligonucleotide targeted to sequence common to both membrane and soluble isoforms were found to potently suppress cell surface IL-5Ralpha levels and IL-5-mediated cell survival by inducing apoptosis similar to IL-5 withdrawal. Thus, these oligonucleotides represent unique genetic agents with therapeutic potential for diseases with an eosinophilic component.     

Antigen controls IL-7R alpha expression levels on CD8 T cells during full activation or tolerance induction

The high-affinity chain of the IL-7 receptor, IL-7Ralpha (CD127), is expressed by effector CD8 T cells that have the capacity to become memory cells. IL-7Ralpha expression is uniformly high on naive CD8 T cells, and the majority of these cells down-regulate expression upon antigenic challenge. At the peak of expansion, the fraction of effectors expressing high IL-7Ralpha varies depending on the response examined. The signals that a CD8 T cell receives during a response to Ag that lead to altered expression of IL-7Ralpha have not been fully defined. In vitro experiments demonstrated that Ag alone is sufficient to down-regulate IL-7Ralpha on all cells and most of the cells rapidly re-express the receptor upon removal from Ag. Expression was not altered by the B7.1 costimulatory ligand or when IL-12 was present to provide the signal needed for development of effector functions, indicating that TCR engagement is sufficient to regulate IL-7Ralpha expression. Consistent with this, in vivo priming with peptide Ag resulted in IL-7Ralpha expression that inversely correlated with Ag levels, and expression levels were not changed when IL-12 or adjuvant were administered with Ag. A large fraction of the cells present at the peak of expansion had re-expressed IL-7Ralpha, but most of these cells failed to survive; those that did survive expressed high IL-7Ralpha levels. Thus, Ag-dependent signals regulate IL-7Ralpha levels on responding CD8 T cells, and this occurs whether the responding cells become fully activated or are rendered tolerant by administration of peptide Ag alone.     

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.     

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.     

IL-13 signaling through the IL-13alpha2 receptor is involved in induction of TGF-beta1 production and fibrosis

Interleukin (IL)-13 is a major inducer of fibrosis in many chronic infectious and autoimmune diseases. In studies of the mechanisms underlying such induction, we found that IL-13 induces transforming growth factor (TGF)-beta(1) in macrophages through a two-stage process involving, first, the induction of a receptor formerly considered to function only as a decoy receptor, IL-13Ralpha(2). Such induction requires IL-13 (or IL-4) and tumor necrosis factor (TNF)-alpha. Second, it involves IL-13 signaling through IL-13Ralpha(2) to activate an AP-1 variant containing c-jun and Fra-2, which then activates the TGFB1 promoter. In vivo, we found that prevention of IL-13Ralpha(2) expression reduced production of TGF-beta(1) in oxazolone-induced colitis and that prevention of IL-13Ralpha(2) expression, Il13ra2 gene silencing or blockade of IL-13Ralpha(2) signaling led to marked downregulation of TGF-beta(1) production and collagen deposition in bleomycin-induced lung fibrosis. These data suggest that IL-13Ralpha(2) signaling during prolonged inflammation is an important therapeutic target for the prevention of TGF-beta(1)-mediated fibrosis.     

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.     

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.     

Smad-dependent cooperative regulation of interleukin 2 receptor alpha chain gene expression by T cell receptor and transforming growth factor-beta

The interleukin 2 receptor alpha chain (IL-2Ralpha) is a component of high affinity IL-2 receptors and thus critically regulates T cell growth and other lymphoid functions. Five positive regulatory regions together control lineage-restricted and activation-dependent IL-2Ralpha induction in response to antigen and IL-2. We now show that TGF-beta cooperates with T cell receptor (TCR) signaling to increase IL-2Ralpha gene expression. Moreover, we identify a sixth positive regulatory region that regulates IL-2Ralpha expression in cells treated with anti-CD3 + anti-CD28 as well as TGF-beta and show that this region contains binding sites for Smad3, AP-1, and cAMP-responsive element-binding protein/ATF proteins. The importance of Smad complexes is indicated by impaired IL-2Ralpha induction by TGF-beta in CD4+ T cells from both Smad3-/- and Smad4-/- mice. Thus, we have identified a novel positive regulatory region in the IL-2Ralpha gene that mediates TGF-beta-dependent induction of the gene. These findings have implications related to IL-2Ralpha expression on activated T cells and regulatory T cells.