Transcription factor GATA-3 is involved in repression of promoter activity of the human interleukin-4 gene.

GATA-3 was shown to bind to two sites of the IL-4 gene promoter in human T-cell lines PER-117 and Jurkat. A motif located in the region of position -860 and responsible for GATA-3 binding was detected for the first time. Mutation or deletion of this site increased the promoter activity. The findings suggest a direct involvement of GATA-3 in regulation of the human IL-4 gene transcription as a repressor of the promoter activity.

Dexamethasone suppresses human interleukin-5 gene promoter.

Synthetic glucocorticoid dexamethasone suppressed interleukin-5 gene expression in PER-117 human T cells at the level of transcription. The conserved lymphokine element 0 in the interleukin-5 gene promoter context served as a target for dexamethasone.

GATA-3 has dual regulatory functions in human interleukin-5 transcription.

Interleukin-5 (IL-5) is a T-cell cytokine involved in Type 2 diseases and is commonly described as being coordinately regulated with other Type 2 cytokines, such as IL-4 and IL-13. Considering the unique control of eosinophilia by IL-5, such coordinate regulation would be surprising. In fact, the biological specificity of eosinophilia and its control by IL-5 suggests a unique and independent control of IL-5 regulation. In this report we show the binding of GATA-3 to three sites in the human IL-5 promoter in the human T-cell line PER117. The previously identified -70 site and another site at position -152 are shown to positively regulate IL-5 transcription. More importantly, the site located at -400 acts as a powerful repressor of IL-5 transcription with mutagenesis of this site allowing a high level expression of IL-5 without the activation of other factors normally required for IL-5 expression. Whereas GATA-3 has been proposed to be involved in the regulation of the IL-4/IL-5/IL-13 locus, we show here that it has another function in controlling IL-5 transcription that supports the observed unique biological function of this cytokine.

Binding of octamer factors to the murine IL-5 CLE0 in primary T-cells and a T-cell line.

Interleukin-5 (IL-5) is an inducible T-cell derived cytokine with remarkable specificity for the eosinophil lineage. It is controlled at the level of transcription and regulation of the gene is an obvious target for therapy of eosinophil-dependent allergic disorders such as asthma, eczema and rhinitis. Using a T-cell line and primary T-cells we have shown for the first time that the Oct1 and Oct2 transcription factors combine to form a complex with the functionally critical murine IL-5 cis-regulatory element, conserved lymphokine element 0 (CLE0), and contribute to positive regulation of the gene. These results show the increasingly important role of octamer factors in regulation of the IL-5 gene.

Regulation of IL-5 expression.

Interleukin 5 (IL-5) is a cytokine primarily involved in the pathogenesis of atopic diseases. It specifically controls the production, activation and localization of eosinophils, the major cause of tissue damage in atopic diseases. IL-5 belongs to a gene family shared by IL-3, IL-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) and is predominantly regulated at the transcriptional level. A variety of stimuli and modulators have been identified as regulating production of IL-5 both in vivo and in vitro, indicating a highly complex series of control mechanisms. However, a better understanding of the biology of IL-5 and the regulation of its expression is crucial for the development of new therapeutic agents for allergic disease. This review covers the major molecular aspects of IL-5 research.

Identification of regions within the third FnIII-like domain of the IL-5Ralpha involved in IL-5 interaction.

Previously, two binding sites for interleukin 5 (IL-5) were identified on the IL-5 receptor alpha chain (IL-5Ralpha). They are located within the CD loop of the first fibronectin type III (FnIII)-like domain and the EF loop of the second FnIII-like domain. The first binding site was identified by exploiting the different abilities of human IL-5Ralpha (hIL-5Ralpha) and mouse IL-5Ralpha (mIL-5Ralpha) to bind hIL-5. Here we show that ovine IL-5 (oIL-5) has the ability to activate the hIL-5Ralpha but not the mIL-5Ralpha. By using chimeras of the mIL-5Ralpha and hIL-5Ralpha we demonstrate that residues within the first and third FnIII-like domains of mIL-5Ralpha are responsible for this lack of activity. Furthermore, mutation of residues on hIL-5Ralpha to mIL-5Ralpha within the predicted DE and FG loop regions of the third FnIII domain reduces oIL-5 activity. These results show that regions of the third FnIII domain of IL-5Ralpha are involved in binding, in addition to the regions in domains one and two of the IL-5Ralpha that were identified in an earlier study.

Binding of octamer factors to a novel 3'-positive regulatory element in the mouse interleukin-5 gene.

The development of eosinophilia is regulated by interleukin (IL)-5. The biological specificity of eosinophilia suggests a tight and independent regulation of IL-5 expression. A number of regulatory regions in the 5'-end of the IL-5 gene have been described; many of them are involved in the regulation of other genes, and it is not clear how the specific expression of IL-5 is regulated. In this study, we report the finding of a novel 3'-regulatory element. Data base analysis of a 2-kilobase fragment of the 3'-end of the mouse IL-5 gene revealed the presence of a 40-base pair-long repetitive sequence that consists of four direct repeats of ATGAATGA distributed in a symmetrical manner. This sequence, named mouse downstream regulatory element-1 (mDRE1), was shown to be protected in DNase I footprinting assays in vitro. Electrophoretic mobility shift assays using specific antibodies identified the transcription factors Oct-1 and Oct-2 as responsible for the formation of the specific complexes with mDRE1 and nuclear extracts from both EL4 and primary T-cells. Competition electrophoretic mobility shift assays with oligonucleotides containing different numbers of ATGAATGA repeats showed that Oct-1 and Oct-2 bind to different motifs in the mDRE1 sequence. Deletion of mDRE1 from a 9.5-kilobase IL-5 gene construct significantly decreased the expression of the luciferase reporter gene, suggesting that it plays a positive role in the expression of the IL-5 gene.

Interleukin-5: a drug target for allergic diseases.

There is a large body of evidence that eosinophils are a key component of the allergic response in asthma. Interleukin (IL) 5 is uniquely involved in the production of eosinophils, and with a variety of other cytokines and factors controls their activation, localization and survival. Thus, IL-5 is an important drug target for new anti-asthmatics. The routes to drug discovery are based on screens for inhibitors of IL-5 production, ligand antagonists, control of receptor expression and receptor activation. In this review, we will discuss specific targets and screening assays with examples of some of the compounds in development.

Role of nuclear factor of activated T cells (NFAT) in the expression of interleukin-5 and other cytokines involved in the regulation of hemopoetic cells.

NFAT (nuclear factor of activated T cells) is a transcription factor that plays a role in the regulation of various cytokines, including those involved in the regulation of hemopoetic cells such as granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-4 (IL4), interleukin-3 (IL3), interleukin-13 (IL13) and interleukin-5 (IL5). In this report we provide a summary of the various locations in the promoters of each of these cytokines where NFAT has been shown or suggested to bind, and at which sites NFAT has been shown to be involved in transcriptional regulation. We also provide experimental data to show that the binding of NFAT to the nucleotides GAA at positions -113 to -111 of the human IL5 promoter is associated with functional activity in human T cells.

A human T-cell line with inducible production of interleukins 5 and 4. A model for studies of gene expression.

The production of interleukin-5 (IL5) and interleukin-4 (IL4) by activated T-cells is important in the pathogenesis of helminth infections and allergy. Human Jurkat cells express IL4 but one of the main factors restricting studies of human IL5 expression has been the lack of human T-cell lines which express significant levels of IL5 in an inducible fashion. We report that the human T-cell leukemia cell line (PER-117), previously shown to produce IL2, also produces IL5 and IL4, and is a useful model for the study of the regulation of IL5 and IL4 gene expression. We show that expression of IL5 and IL4 mRNAs in PER-117 cells is stimulation dependent. IL5 and IL4 reporter constructs are also transiently expressed in these cells in an inducible fashion. IL5 production in the PER-117 cell line can be activated by phorbol 12-myristate 13-acetate alone and further enhanced by calcium ionophore A23187, cyclic adenosine 3', 5'-monophosphate or anti-CD28 antibodies. The conditions used to stimulate the PER-117 cells determined whether IL5 production was inhibited by cyclosporin A or dexamethasone. These data indicate that the PER-117 cell line is a model to study signal transduction and transcriptional activation of the human IL5 gene in human T-cells.

Identification of residues involved in binding of IL5 to betacom using betaIL3 and betacom chimeras.

In mice there are two forms of the beta chain used in the IL3 receptor system, betacom and betaIL3. betacom is used by the IL3, IL5 and GM-CSF receptors whereas betaIL3 is only used in the IL3 receptor. In this work an assay was developed to identify residues of beta1L3 that restrict IL5 activity. It was found that such residues reside within the 2nd CRM of the molecule. Furthermore, when residues in the betaIL3 B'-C' loop were replaced with betacom sequence a form of betaIL3 was produced that was able to respond to IL5. This region is also responsible for IL3 binding to betaIL3 in the absence of alpha chain. It is therefore an important structural motif of betacom and betaIL3 responsible for both ligand interaction and specificity.

Nuclear factor of activated T cells and YY1 combine to repress IL-5 expression in a human T-cell line.

BACKGROUND: IL-5 is an inducible T-cell cytokine with the unique ability to induce eosinophilia without increases in other cell compartments. Regulation of IL-5 expression is controlled primarily at the level of transcription. The role of eosinophilia in allergic disorders indicates IL-5 as a target for therapy. OBJECTIVE: This report aims to increase our understanding of IL-5 gene regulation by identifying distal control elements in the human (h) IL-5 promoter, determining the transcription factors that bind these elements and elucidating their role in control of hIL-5 gene expression. METHODS: Methods used in this study include deoxyribonuclease I footprint analysis, electrophoretic mobility shift assay, and functional analysis by transfection of PER-117 cells with site-directed mutants of the hIL-5 promoter. RESULTS: We have identified a protected region in the distal hIL-5 promoter that has sequence homology to the previously identified negative regulatory element within BR3. This protected region has not been previously reported and is shown to contain overlapping binding sites for YY1 and nuclear factor of activated cells. The binding sites exist between positions -447 and -459, and this sequence was named hPRE2-IL5. Substitution mutations that abolish binding of these proteins to hPRE2-IL5 result in a 2- to 3-fold increase in hIL-5 promoter activity in activated human T cells. CONCLUSION: We report the novel combination of YY1 and nuclear factor of activated T cells transcription factors binding to a distal hIL-5 promoter element where both factors are involved in down-regulation of hIL-5 gene expression in human T cell.

The activity of the human interleukin-5 conserved lymphokine element 0 is regulated by octamer factors in human cells.

Interleukin-5 (IL-5) controls the development of eosinophilia and contributes to a number of disease states including asthma. Expression of IL-5 is inducible under tight transcriptional regulation. This requires the contribution of several promoter elements; however, the conserved lymphokine element 0 (CLE0) in particular, is essential for expression of IL-5. In this study, we report the nuclear factors which regulate human IL-5 CLE0 activity in the human cell line PER-117. Using specific antibodies, we identified the transcriptional factors Oct-1 and Oct-2 binding to the 5' region of the CLE0 element. The involvement of Oct factors with CLE0 has not been reported previously in any of the lymphokines. In addition, the CLE0 element also appeared to complex with the transcriptional activator AP-1, consisting of the family members Jun D and Fra-2. We observed the binding of Oct-1 to be constitutive in comparison to Oct-2 and AP-1, both of which were induced in response to cell activation by PMA/A23187. Although the interaction of all three factors with CLE0 was closely linked and overlapping, residues critical to their binding were identified. We demonstrate, using site-directed mutagenesis and cotransfection experiments, that the CLE0 element is indispensable for IL-5 promoter activity and that Octamer factors contribute to the positive regulation of the hIL-5 gene.

Binding of YY1 and Oct1 to a novel element that downregulates expression of IL-5 in human T cells.

BACKGROUND: IL-5 controls development of eosinophilia and has been shown to be involved in the pathogenesis of allergic diseases. In both atopic and nonatopic asthma, elevated IL-5 has been detected in peripheral blood and the airways. IL-5 is produced mainly by activated T cells, and its expression is regulated at the transcriptional level. OBJECTIVE: This study focuses on the functional analysis of the human IL-5 (hIL-5) promoter and characterization of cis -regulatory elements and transcription factors involved in the suppression of IL-5 transcription in T cells. METHODS: Methods used in this study include DNase I footprint assays, electrophoretic mobility shift assays, and functional analysis by mammalian cell transfection involving deletion analysis and site-directed mutagenesis. RESULTS: We identified 5 protein binding regions (BRs) located within the proximal hIL-5 promoter. Functional analysis indicates that the BRs are involved in control of hIL-5 promoter activity. Two of these regions, BR3 and BR4 located at positions -102 to -73, have not previously been described as regulators of IL-5 expression in T cells. We show that the BR3 sequence contains a novel negative regulatory element located at positions -90 to -79 of the hIL-5 promoter, which binds Oct1, octamer-like, and YY1 nuclear factors. Substitution mutations, which abolished binding of these proteins to the BR3 sequence, significantly increased hIL-5 promoter activity in activated T cells. CONCLUSION: We suggest that Oct1, YY1, and octamer-like factors binding to the -90/-79 sequence within the proximal IL-5 promoter are involved in suppression of IL-5 transcription in T cells.

Analysis of the 5' and 3'UTRs in the post-transcriptional regulation of the interleukin-5 gene.

Post-transcriptional regulation is emerging as an important control point in cytokine gene expression. However, the role that it plays in IL-5 gene expression is unclear with some conflicting reports. Here we investigate the importance of post-transcriptional regulation and the role of the 5' and 3' untranslated regions (UTRs) in mIL-5 gene expression. To do this, IL-5 expression from a panel of cDNA constructs was compared. We found it essential to remove the 5' synthetic oligonucleotide tails, introduced during the cloning of the mIL-5 cDNA, when studying IL-5 expression. The presence of these oligo(G) tails acted as potent inhibitors of translation of both SV40 and SP6 transcripts. Furthermore, the length of the tails was found to be critical to the translational efficiency. Taking this into account, we found no evidence to suggest that IL-5 is regulated at the level of mRNA stability or translation efficiency by either the 5' or 3'UTR. These results suggest that post-transcriptional control is not a major factor regulating IL-5 expression.

Expressed luciferase viability assay (ELVA) for the measurement of cell growth and viability.

An expressed luciferase viability assay (ELVA) has been developed for cell viability and cell number based on detecting the expression of luciferase transfected into the cells. Stable transfectants were produced that expressed luciferase constitutively. Like many endogenous enzymes, luciferase is rapidly degraded following cell death, so that the enzyme can be used as a measure of cell viability. A modified luciferase assay was used in which the reagents were added directly to the cells in a microplate. The main advantages compared to other cell viability assays are the wide dynamic range, high sensitivity, low background, and the absence of any requirement to wash or harvest the cells. Stable transfectants of three factor-dependent cell lines (B13, Ba/F3 and CTLL) were produced and used in cytokine assays. Three strategies of selection after electroporation were tested: (1) using a plasmid containing both the genes encoding firefly luciferase and a selectable marker (neo), (2) cotransfection of a plasmid containing luciferase and a plasmid containing a selectable marker (puromycin resistance), and (3) cotransfection of a plasmid containing luciferase and a plasmid containing the human IL-5Ralpha-chain, and selecting in IL-5. This latter strategy produces an IL-5 responsive cell line expressing luciferase in a single step without the need for antibiotic selection.

Identification of two novel palindromic regulatory elements in the murine interleukin-5 promoter.

Interleukin-5 has remarkable specificity for the eosinophil lineage. This fact, combined with the biological specificity of eosinophilia suggests tight and independent regulation of IL-5 expression. Here we report two novel palindromic regulatory elements (PRE) which contain positive regulatory motifs (PRM) that control transcription of the murine IL-5 gene. The first element, mPRE1-IL5 (-79 to -90) contains the mPRM1 at positions -87 to -89 which operates as a positive regulatory element with mutation of this motif resulting in a 64% decrease in gene activity. Gene expression was reduced by 67% when a similar mutation was introduced into the mPRM2 (-467 to -469) of mPRE2-IL5 (-459 to -470). Both elements specifically bind proteins from EL4-23 cell nuclear extracts forming constitutive DNA-protein complexes. EMSA experiments utilising mutated mPRE-IL5 oligonucleotides indicate that in both elements, the mPRMs are essential for protein binding.

New directions in understanding interleukin-5 gene expression.

Specific regulation of the interleukin-5 (IL-5) gene is implied by the unique control of eosinophilia which is regulated by IL-5. In studies of IL-5 gene expression, the only control elements identified for the IL-5 gene have been transcriptional elements in the 5' untranslated region (UTR). Significant differences exist in the arrangement of the murine and human IL-5 promoters, which is surprising considering the tight regulation of the gene. Novel palindromic regulatory elements involved in transcriptional regulation have been found in the 5' UTR and new results show the presence of transcriptional elements in the 3' UTR. Post-transcriptional control mechanisms in both the 5' and 3' UTRs have also been described.

Interleukin-5 binds to heparin/heparan sulfate. A model for an interaction with extracellular matrix.

Interleukin-5 (IL-5) is the major cytokine regulating eosinophil production. In allergic disease tissue damage is primarily caused by eosinophils. Heparan sulfate proteoglycans are components of the bone marrow stroma, which supports hemopoietic cell differentiation and proliferation. We show that at low IL-5 concentrations heparan sulfate enhances the proliferation of an IL-5-dependent cell line. To investigate a mechanism for this effect we used an artificial proteoglycan to establish an enzyme-linked immunosorbent assay for the binding of heparin to proteins. Using this assay we demonstrate that IL-5 binds to heparin. The IL-5/heparin interaction is inhibited by ethylenediaminetetraacetate and enhanced by low concentrations of zinc ions. IL-5 interacts with iduronic acid containing glycosaminoglycans, and heparan sulfate preparations that have numerous N-sulfated domains per chain are especially efficient at inhibiting heparin binding. Both IL-5/heparin binding and the synergistic effect of IL-5 and heparan sulfate on cell proliferation were inhibited by an anti-IL-5 monoclonal antibody. These data suggest that the binding of IL-5 to heparan sulfate modulates IL-5 activity.

Biological and molecular characteristics of interleukin-5 and its receptor.

Interleukin-5 (IL5) is a T cell-derived cytokine involved in the pathogenesis of atopic diseases. It specifically controls the production, the activation and the localization of Eosinophils. The Eosinophils are the major cause of tissue damage resulting in the symptoms of asthma and related allergic disorders. T cells purified from bronchoalveolar lavage and peripheral blood of asthmatics secrete elevated amount of IL5. Therefore IL5 emerges to be an attractive target for the generation of new anti-allergic drugs. Agents which inhibit either the production or the activity of IL5 could be expected to ameliorate the pathological effects of the allergic response. A better understanding of the biology of IL5 and the regulation of its expression is, however, a prerequisite for the development of new therapeutic agents. This review covers the major biological, molecular and structural aspects of IL5 research since the identification of this cytokine ten years ago.