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During inflammatory responses, a major posttranscriptional regulation of early response and inflammatory gene expression occurs through modulation of mRNA turnover. We report that two potent inducers of the CC chemokine eotaxin, TNF-alpha and IL-4, regulate its production in airway epithelial cells by increasing eotaxin mRNA stability. In experiments using the transcriptional inhibitor actinomycin D, eotaxin mRNA half-life was significantly prolonged by cell stimulation with TNF-alpha or IL-4, with the combination of the two cytokines being the most effective in extending the mRNA half-life. Involvement of the eotaxin 3' untranslated region in the mRNA-stabilizing effect was tested by transient transfection of a construct expressing a chimeric transcript carrying a serum-inducible beta-globin reporter linked to the eotaxin 3' untranslated region. The half-life of the chimeric mRNA was markedly increased in cells stimulated with TNF-alpha and IL-4. Evidence that the mRNA-stabilizing protein HuR participated in the cytokine effect was obtained: first, HuR presence in the cytoplasm, believed to be required for HuR-mediated mRNA stabilization, increased in both transformed (BEAS-2B cell line) and primary bronchial epithelial cells following treatment with TNF-alpha and IL-4. Second, endogenous eotaxin mRNA was found to bind to HuR in vivo, as detected by immunoprecipitation of HuR-containing messenger ribonucleoprotein complexes followed by real-time RT-PCR analysis; such association increased after cell treatment with TNF-alpha and IL-4. Third, overexpression of HuR in BEAS-2B cells significantly increased the expression of eotaxin mRNA and protein. Our findings implicate mRNA stabilization in the cytokine-mediated increase in eotaxin expression and strongly suggest a role for HuR in this effect.

Background: The nuclear factor of activated T cells (NFAT) family of transcription factors plays a key role in rapidly inducing IL4 gene expression in effector T cells., Objective: Because human basophils secrete high levels of IL-4, we have examined whether specific NFAT species are expressed in these cells and whether Fc(epsilon)RI-mediated activation affects their subcellular localization and transcriptional function., Methods: Intracellular NFAT protein was identified by using 2-color flow cytometry; gene expression was done with RT-PCR. Subcellular localization of NFAT was assessed by means of Western blotting. Electrophoretic mobility shift assays assessed NFAT involvement in IL-4 transcription., Results: Basophils constitutively expressed high levels of NFAT2. In contrast, NFAT1 (NFATp), which is found in most leukocytes, was not seen in basophils. Low-level staining for NFAT4 was detected but was variably expressed among donor cells. Likewise, NFAT2 mRNA was constitutively expressed in basophils, and message for NFAT4 was seen in 3 of 5 preparations, whereas that for NFAT1 was found in only 1 of 5 preparations. NFAT2 protein accumulated in the nuclei of basophils activated for 1 hour with anti-IgE, and this was inhibited with the addition of FK506. A protein-DNA complex was formed with nuclear lysates from basophils and an IL-4 promoter NFAT consensus probe, with greater binding intensities detected in lysates of activated cells. An antibody to NFAT2 reduced the formation of the complex, whereas no effects were seen with antibodies to NFAT1, NFAT4, or unrelated transcription factors., Conclusions: The selective and specific expression of NFAT2 in basophils is unique among leukocytes. This transcription factor also appears to play a critical role in the Fc(epsilon)RI-mediated production of IL-4 in these cells.

Background: Dysregulated expression of IL-4 has been linked with allergic diseases. IL-4 expression is controlled at the level of gene transcription by the coordinated action of multiple factors that bind regulatory promoter elements. In addition, alterations in chromatin structure are thought to play a role in regulating the expression of cytokines in the T(H)2 gene cluster, although the biochemical basis for these alterations in human T cells is not well understood., Objective: We sought to define the role of histone acetylation in the regulation of IL4 gene expression in human T cells., Methods: IL-4 protein production was measured by means of ELISA. IL-4 promoter activity was measured with luciferase-based reporter constructs transiently transfected into Jurkat T cells. The acetylation status of histones associated with the IL4 gene was analyzed with chromatin immunoprecipitation assays., Results: IL-4 production from activated peripheral blood T cells was enhanced by the histone deacetylase inhibitor trichostatin A. Overexpression of the type 1 histone deacetylases 1, 2, and 3 inhibited transcription driven by the IL-4 promoter in Jurkat T cells, whereas cotransfection of the histone acetyltransferase CREB-binding protein potentiated IL-4 promoter activity. Using chromatin immunoprecipitation assays, we show that nucleosomes in the proximal IL-4 promoter are acetylated on T-cell activation., Conclusion: Our results demonstrate that the acetylation state of histones associated with the IL-4 promoter is a key regulator of IL4 gene expression.

Interleukin-4 (IL-4) is a multifunctional cytokine that plays an important role in immune and inflammatory responses. Expression of the IL-4 gene is tightly controlled at the level of gene transcription by both positive and negative regulatory elements in the IL-4 promoter. Several constitutive nuclear factors have been identified that can interact with IL-4 promoter elements in DNA binding assays. Here we report that the zinc-finger protein YY-1 (Yin-Yang 1) can bind to multiple elements within the human IL-4 promoter. Cotransfection of Jurkat T cells with different IL-4 promoter/reporter constructs together with expression vectors encoding antisense, wild-type, or zinc finger-deleted mutant YY-1 suggested that YY-1 enhanced IL-4 promoter activity in a DNA-binding domain-dependent manner. Site-directed mutagenesis revealed that a proximal YY-1-binding site, termed Y0 ((-59)TCATTTT(-53)), was essential for YY-1-driven IL-4 promoter activity. In addition, cotransfected YY-1 enhanced both IL-4 promoter activity and endogenous IL-4 gene expression in nontransformed peripheral blood T cells. Thus, YY-1 positively regulates IL-4 gene expression in lymphocytes.

Previous studies indicated that aspirin (acetylsalicylic acid [ASA]) can have profound immunomodulatory effects by regulating cytokine gene expression in several types of cells. This study is the first in which concentrations of ASA in the therapeutic range were found to significantly reduce interleukin (IL)-4 secretion and RNA expression in freshly isolated and mitogen-primed human CD4+ T cells. In contrast, ASA did not affect IL-13, interferon-gamma, and IL-2 expression. ASA inhibited IL-4, but not IL-2, promoter-driven chloramphenicol acetyltransferase expression in transiently transfected Jurkat T cells. The structurally unrelated nonsteroidal anti-inflammatory drugs indomethacin and flurbiprofen did not affect cytokine gene expression in T cells, whereas the weak cyclo-oxygenase inhibitor salicylic acid was at least as effective as ASA in inhibiting IL-4 expression and promoter activity. The inhibitory effect of ASA on IL-4 transcription was not mediated by decreased nuclear expression of the known salicylate target nuclear factor (NF)-kappaB and was accompanied by reduced binding of an inducible factor to an IL-4 promoter region upstream of, but not overlapping, the NF of activated T cells- and NF-kappaB-binding P1 element. It is concluded that anti-inflammatory salicylates, by means of a previously unrecognized mechanism of action, can influence the nature of adaptive immune responses by selectively inhibiting the expression of IL-4, a critical effector of these responses, in CD4+ T cells.

Expression of cytokine genes in T cells is thought to result from a complex network of antigen- and mitogen-activated transcriptional regulators. CP2, a factor homologous to Drosophila Elf-1 and previously found to be a critical regulator of several viral and cellular genes in response to developmental signals, is rapidly activated in T helper (Th) cells in response to mitogenic stimulation. Here we show that overexpression of CP2 enhances interleukin (IL)-4 promoter-driven chloramphenicol acetyltransferase expression, while repressing IL-2 promoter activity, in transiently transfected Jurkat cells. A CP2-protected element, partially overlapping the nuclear factor of activated T cell-binding P2 sequence, was required for IL-4 promoter activation in CP2-overexpressing Jurkat cells. This CP2-response element is the site of a cooperative interaction between CP2 and an inducible heteromeric co-factor(s). Mutation of conserved nucleotide contacts within the CP2-response element prevented CP2 binding and significantly reduced constitutive and induced IL-4 promoter activity. Expression of a CP2 mutant lacking the Elf-1-homology region of the DNA-binding domain inhibited IL-4 promoter activity in a dominant negative fashion in transiently transfected Jurkat cells. Moreover, overexpressed CP2 markedly enhanced, while its dominant negative mutant consistently suppressed, expression of the endogenous IL-4 gene in the murine Th2 cell line D10. Taken together, these findings point to CP2 as a critical IL-4 transactivator in Th cells.

Interleukin 4 (IL-4) gene expression is controlled at the level of transcription by the complex interactions of multiple factors that bind to a proximal promoter region. Nuclear factor of activated T cells (NFAT) can bind up to five purine-rich sequences in the IL-4 promoter termed the P elements (P0-P4). In this paper, we characterize a novel P element in the upstream region of the human IL-4 promoter that we term P5. P5 shares a core NFAT motif ((-353)GGAAA(-357)) and additional sequence similarity with the other P elements and supported strong interactions between the NFATp DNA-binding domain (DBD) and the AP-1 proteins cFos and cJun in DNA-binding assays. Inducibility of the IL-4 promoter was significantly impaired in a reporter construct in which the P5 element was mutated in the context of the full-length promoter. We conclude that P5 represents a novel IL-4 promoter P element that contributes to IL-4 promoter inducibility., (Copyright 2000 Academic Press.)

Interleukin 4 (IL-4) is a multifunctional cytokine that plays an important role in hematopoiesis, tumor cell growth, and cellular immune responses. Expression of the IL-4 gene is tightly controlled at the level of gene transcription, and many positive regulatory cis-elements have been identified in the proximal IL-4 promoter region. Relatively little is known about factors that downregulate IL-4 transcription. We performed a detailed deletional analysis of the proximal human IL-4 promoter and studied reporter gene activity in transiently transfected Jurkat T lymphoblasts. In this report, we characterize a novel negative regulatory element (termed P2 NRE) that is adjacent to a binding site for nuclear factor of activated T cells. Mutation of P2 NRE significantly enhanced the activity of a 175 base pair IL-4 promoter construct in transiently transfected Jurkat T lymphoblasts. Using nuclear extracts from Jurkat cells, we identify a candidate factor (termed Rep-1) that binds uniquely to the P2 NRE in DNA-binding assays. Rep-1 is not related to other factors previously shown to interact with the IL-4 promoter, and by UV cross-linking and SDS-PAGE analysis, we found that it migrates with a molecular mass of approximately 150 kDa. Characterizing the molecular mechanisms responsible for downregulating the IL-4 promoter should enhance our understanding of IL-4-gene dysregulation in disease states.

The mechanism by which glucocorticoids (GC) inhibit IL-4 gene expression is currently unknown. In T lymphocytes, IL-4 gene expression is regulated at the level of transcription by increases in intracellular calcium concentration and by the calcium-activated phosphatase calcineurin. In this paper we report that dexamethasone (Dex) inhibits calcium ionophore-induced activation of the human IL-4 promoter in transiently transfected Jurkat T cells. Inhibition of the promoter by Dex is dependent on expression of the GC receptor (GR), because it does not occur in GR-deficient cells. Dex also represses activation of the promoter induced by cotransfecting cells with a constitutively active mutant of calcineurin. Using a series of deletion constructs, we show that the proximal 95 bp of the IL-4 promoter contain a Dex-sensitive regulatory element. This region contains the P1 sequence, a proximal binding site for NF-AT. A calcium-induced but Dex-inhibited nuclear complex containing NF-AT binds to the P1 element in EMSA. Using immunoprecipitation under nondenaturing conditions, we found that the GRalpha isoform coprecipitates with NF-ATc in nuclear extracts of calcium ionophore- and Dex-treated cells. Taken together, our results show that GC inhibit IL-4 gene expression by interfering with NF-AT-dependent transactivation of the proximal human IL-4 promoter.

The differentiation of naive T-helper (Th) cells into cytokine-secreting effector Th cells requires exposure to multiple signals, including exogenous cytokines. Interleukin-4 (IL-4) plays a major role in this process by promoting the differentiation of IL-4-secreting Th2 cells. In Th2 cells, IL-4 gene expression is tightly controlled at the level of transcription by the coordinated binding of multiple transcription factors to regulatory elements in the proximal promoter region. Nuclear factor of activated T cell (NFAT) family members play a critical role in regulating IL-4 transcription and interact with up to five sequences (termed P0 through P4) in the IL-4 promoter. The molecular mechanisms by which IL-4 induces expression of the IL-4 gene are not known, although the IL-4-activated transcription factor signal transducer and activator of transcription 6 (Stat6) is required for this effect. We report here that Stat6 interacts with three binding sites in the human IL-4 promoter by electrophoretic mobility shift assays. These sites overlap the P1, P2, and P4 NFAT elements. To investigate the role of Stat6 in regulating IL-4 transcription, we used Stat6-deficient Jurkat T cells with different intact IL-4 promoter constructs in cotransfection assays. We show that, whereas a multimerized response element from the germline IgE promoter was highly induced by IL-4 in Stat6-expressing Jurkat cells, the intact human IL-4 promoter was repressed under similar conditions. We conclude that the function of Stat6 is highly dependent on promoter context and that this factor promotes IL-4 gene expression in an indirect manner.

Atopy, which predisposes individuals to develop asthma, severe systemic anaphylaxis, and atopic dermatitis, is usually associated with dramatically elevated total serum IgE levels and is thought to be controlled by a major susceptibility gene and multiple minor susceptibility genes. A recent sib-pair analysis revealed a tight linkage between markers on 5q31.1 and a major susceptibility gene controlling total serum IgE levels. Due to its location within this cluster and its biologic role in Ig class switching and Th2 cell differentiation, the IL-4 gene has emerged as one major candidate for the atopy gene. In one model, polymorphisms within IL-4 regulatory elements might result in overexpression of the gene, amplifying Th2 cell differentiation and class switching to IgE. In support of this model, we report that the human IL-4 promoter exists in multiple allelic forms that exhibit distinct transcriptional activities in IL-4-positive T cells. A particular allele has an unusually high transcriptional activity. A nucleotide substitution within a recently described OAP40 element located just upstream of an NF-AT site (P sequence) appears to be largely responsible for the increased promotor strength of this particular allelic form of the IL-4 promoter. In EMSAs, this substitution results in a markedly enhanced affinity for sequence-specific complexes exhibiting an AP-1 specificity. The identification of allelic nucleotides, which results in overexpression of the IL-4 gene, provides specific targets for a comprehensive screening of atopic and nonatopic individuals and may provide a clue for genetic predisposition for atopy.