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.     

Stat6 is necessary and sufficient for IL-4's role in Th2 differentiation and cell expansion

IL-4 plays a critical role in the differentiation of TCR-stimulated naive CD4 T cells to the Th2 phenotype. In response to IL-4, the IL-4R activates a set of phosphotyrosine binding domain-containing proteins, including insulin receptor substrate 1/2, Shc, and IL-4R interacting protein, as well as Stat6. Stat6 has been shown to be required for Th2 differentiation. To determine the roles of the phosphotyrosine binding adaptors in Th2 differentiation, we prepared a retrovirus containing a mutant of the human (h)IL-4R alpha-chain, Y497F, which is unable to recruit these adaptors. The mutant hIL-4Ralpha, as well as the wild-type (WT) hIL-4Ralpha, was introduced into naive CD4 T cells. Upon hIL-4 stimulation, Y497F worked as well as the WT hIL-4Ralpha in driving Th2 differentiation, as measured by Gata3 up-regulation and IL-4 production. Furthermore, IL-4-driven cell expansion was also normal in the cells infected with Y497F, although cells infected with Y497F were not capable of phosphorylating insulin receptor substrate 2. These results suggest that the signal pathway mediated by Y497 is dispensable for both IL-4-driven Th2 differentiation and cell expansion. Both WT and Y497F hIL-4Ralpha lose the ability to drive Th2 differentiation and cell expansion in Stat6-knockout CD4 T cells. A constitutively activated form of Stat6 introduced into CD4 T cells resulted in both Th2 differentiation and enhanced cell expansion. Thus, activated Stat6 is necessary and sufficient to mediate both IL-4-driven Th2 differentiation and cell expansion in CD4 T cells.     

Stat6 and IRS-2 Cooperate in Interleukin 4 (IL-4)-Induced Proliferation and Differentiation but Are Dispensable for IL-4-Dependent Rescue from Apoptosis

Stat6 and IRS-2 are two important signaling proteins that associate with the cytoplasmic tail of the interleukin 4 (IL-4) receptor. Data from numerous in vitro experiments have led to a model for IL-4 signal transduction in which the Stat6 signaling pathway is responsible for the IL-4 induced changes in gene expression and differentiation events, while the IRS-2 signaling pathway provides mitogenic and antiapoptotic signals. In order to determine the relative contributions of these signaling molecules in primary lymphocytes, we have examined IL-4 responses in T cells from mice deficient for either Stat6 or IRS-2 as well as from mice doubly deficient for both genes. Both IRS-2 and, especially, Stat6 are shown to be critically involved in IL-4-induced proliferation of T cells, presumably through the cooperative regulation of the Cdk inhibitor p27kip1. Like Stat6-deficient Th cells, IRS-2-deficient cells are also compromised in their ability to secrete Th2 cytokines, revealing a previously unrecognized role for IRS-2 in Th2 cell development. Although Stat6 and/or IRS-2 expression is required for IL-4-induced proliferative and differentiative responses, both signaling proteins are dispensable for the antiapoptotic effect of IL-4. However, treatment of lymphocytes with a protein tyrosine phosphatase inhibitor is able to block the antiapoptotic effect of IL-4 specifically in Stat6- or IRS-2-deficient cells and not in wild-type cells. Our results suggest that Stat6 and IRS-2 cooperate in promoting both IL-4-induced proliferative and differentiating responses, while an additional signaling mediator that depends on protein tyrosine phosphatase activity contributes to the antiapoptotic activities of IL-4 in primary T cells.     

Stat4 limits DNA methyltransferase recruitment and DNA methylation of the IL-18Ralpha gene during Th1 differentiation

Stat4 is required for Th1 development, although how a transiently activated factor generates heritable patterns of gene expression is still unclear. We examined the regulation of IL-18Ralpha expression to define a mechanism for Stat4-dependent genetic programming of a Th1-associated gene. Although Stat4 binds the Il18r1 promoter following IL-12 stimulation and transiently increases acetylated histones H3 and H4, patterns of histone acetylation alone in Th1 cells may not be sufficient to explain cell-type-specific patterns of gene expression. The level of DNA methylation and recruitment of Dnmt3a to Il18r1 inversely correlate with IL-18Ralpha expression, and blocking DNA methylation increases IL-18Ralpha expression. Moreover, there was decreased Il18r1-Dnmt3a association and DNA methylation following transient trichostatin A-induced histone hyperacetylation in Stat4-/-Th1 cultures. Increased association of Dnmt3a and the Dnmt3a cofactor Dnmt3L with the promoters of several Stat4-dependent genes was found in Stat4-/- Th1 cultures, providing a general mechanism for Stat4-dependent gene programming. These data support a mechanism wherein the transient hyperacetylation induced by Stat4 prevents the recruitment of DNA methyltransferases and the subsequent repression of the Il18r1 locus.     

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.     

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.     

An absolute requirement for STAT4 and a role for IFN-gamma as an amplifying factor in IL-12 induction of the functional IL-18 receptor complex

IL-12 and IL-18 are both proinflammatory cytokines that contribute to promoting Th1 development and IFN-gamma expression. However, neither IL-12R nor IL-18R is expressed as a functional complex on most resting T cells. This study investigated the molecular mechanisms underlying the induction of an IL-18R complex in T cells. Resting T cells expressed IL-18Ralpha chains but did not exhibit IL-18 binding sites as detected by incubation with rIL-18 followed by anti-IL-18 Ab, suggesting a lack of IL-18Rbeta expression in resting T cells. Although they also failed to express IL-12R, stimulation with anti-CD3 plus anti-CD28 generated IL-12R. Exposure of these cells to IL-12 led not only to up-regulation of IL-18Ralpha expression but also to induction of IL-18R binding sites on both CD4(+) and CD8(+) T cells concomitant with IL-18Rbeta mRNA expression. The IL-18 binding site represented a functional IL-18R complex capable of exhibiting IL-18 responsiveness. IL-12 induction of an IL-18R complex and IL-18Rbeta mRNA expression was not observed in STAT4-deficient (STAT4(-/-)) T cells and was substantially decreased in IFN-gamma(-/-) T cells. However, the failure of STAT4(-/-) T cells to induce an IL-18R complex was not corrected by IFN-gamma. These results indicate that STAT4 and IFN-gamma play an indispensable role and a role as an amplifying factor, respectively, in IL-12 induction of the functional IL-18R complex.     

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.     

Identification of novel IL-4/Stat6-regulated genes in T lymphocytes

IL-4, primarily produced by T cells, mast cells, and basophiles, is a cytokine which has pleiotropic effects on the immune system. IL-4 induces T cells to differentiate to Th2 cells and activated B lymphocytes to proliferate and to synthesize IgE and IgG1. IL-4 is particularly important for the development and perpetuation of asthma and allergy. Stat6 is the protein activated by signal transduction through the IL-4R, and studies with knockout mice demonstrate that Stat6 is critical for a number of IL-4-mediated functions including Th2 development and production of IgE. In the present study, novel IL-4- and Stat6-regulated genes were discovered by using Stat6(-/-) mice and Affymetrix oligonucleotide arrays. Genes regulated by IL-4 were identified by comparing the gene expression profile of the wild-type T cells induced to polarize to the Th2 direction (CD3/CD28 activation + IL-4) to gene expression profile of the cells induced to proliferate (CD3/CD28 activation alone). Stat6-regulated genes were identified by comparing the cells isolated from the wild-type and Stat6(-/-) mice; in this experiment the cells were induced to differentiate to the Th2 direction (CD3/CD28 activation + IL-4). Our study demonstrates that a number a novel genes are regulated by IL-4 through Stat6-dependent and -independent pathways. Moreover, elucidation of kinetics of gene expression at early stages of cell differentiation reveals several genes regulated rapidly during the process, suggesting their importance for the differentiation process.