Analysis of Human lnterleukin-5 Gene Transcription by T Cell Clones and Hybridomas

Mori, Asako; Inoue, Satomi; Mikami, Tadashi; Kaminuma, Osamu; Ohmura, Takafumi; Nishizaki, Yoko; Asakura, Yumiko; Suko, Matsunobu; Ito, Kyoko; Okudaira, H

doi:10.1159/000237036

Cited by 8 publications

(6 citation statements)

References 6 publications

(6 reference statements)

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“…pIL–5(–511)Luc was transcribed upon activation by T cell hybridomas derived from the fusion between IL–5–producing T cell clones and the IL–5–gene–nonexpressing T cell line, but not by the hybridomas derived from IL–5–nonproducing T cell clones [12, 16]. The capacity to transcribe IL–2 and IL–4 genes, and the induction of NF–AT, NF–ĸB, and AP–1 binding activities were quite comparable among IL–5–gene–expressing and nonexpressing T cell hybridomas.…”

Section: Resultsmentioning

confidence: 99%

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Regulatory Mechanisms of Human T Cell IL–5 Synthesis: Differential Roles of the Proximal Promoter–Binding Proteins in IL–5 Gene Transcription

Mori

Kaminuma²,

Ogawa³

et al. 1999

Int Arch Allergy Immunol

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Background: IL–5 is crucially involved in eosinophilic inflammation. We have previously reported that IL–5 synthesis by atopic and nonatopic asthmatics is significantly enhanced compared to control subjects. T cell IL–5 synthesis is regulated at the transcriptional level. The proximal human IL–5 promoter (–62 to –46) homologous to the conserved lymphokine element 0 (CLE0) is essential for activation–induced gene transcription by Th cells. Methods: Luciferase reporter analysis and gel shift analysis were performed. Results: The CLE0 homologous element is the overlapping binding site for a constitutive and an inducible binding factor. Site–directed mutagenesis successfully differentiated the two bindings, and revealed that the transcriptional induction was ascribed to the inducible binding, while the constitutive binding was rather inhibitory. A mutant element which lost the constitutive binding, but retained the inducible binding, exerted 3 times more transcriptional activity compared to the wild–type element. In contrast, another mutant element which lost the inducible binding and retained the constitutive binding exhibited no transcriptional induction. Gel shift analysis was performed to clarify that the inducible binding was more prominent and the constitutive binding was less in IL–5 producing Th clones compared to IL–5–nonproducing clones. Conclusion: The ratio of the inducible/constitutive binding to the CLE0 homologous element may determine the capacity of human Th cells to transcribe the IL–5 gene.

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Section: Resultsmentioning

confidence: 99%

“…Human T cell hybridomas that express or do not express IL–5 mRNA upon activation were established and cultured as described previously [12]. Transfection, preparation of protein extracts, luciferase, and β–galactosidase assay were performed as described previously [11].…”

Section: Methodsmentioning

confidence: 99%

Regulatory Mechanisms of Human T Cell IL–5 Synthesis: Differential Roles of the Proximal Promoter–Binding Proteins in IL–5 Gene Transcription

Mori

Kaminuma²,

Ogawa³

et al. 1999

Int Arch Allergy Immunol

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“…To date, the information on human IL-5 gene transcription is still limited, while quite a few studies have been performed to analyze murine IL-5 gene (23-26, 45, 46). We have employed allergen-specific Th clones and hybridomas to study cytokine synthesis involved in allergic inflammation (47), and found that human T cell IL-5 synthesis is regulated at the level of gene transcription through the 5 15 bp gene segment 5' upstream of the coding region, and unique transcriptional mechanisms distinct from those regulating IL-2 or IL-4 gene seemed to control IL-5 gene (37, 38, 48, 49). The present finding that transcriptional activity of IL-5 promoter by Con A blast lym- phocytes is correlated with the protein production further support the notion.…”

Section: Discussionmentioning

confidence: 99%

Transcriptional Regulation of IL-5 Gene by Nontransformed Human T Cells Through the Proximal Promoter Element.

et al. 2000

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Objective IL-5 is strongly involved in the eosinophilic inflammation in bronchial asthma and atopic dermatitis. Wehave previously reported that IL-5 synthesis in atopic and nonatopic asthmatics is significantly enhanced compared to control subjects. T cell IL-5 synthesis is regulated through several transcriptional elements, one of which is the proximal human IL-5 promoter (-62 to -46). The present study was undertaken to delineate the transcriptional regulation through this element using nontransformed human T cells. Methods Con A blast lymphocytes which tolerate electroporation were derived from peripheral blood lymphocytes. Luciferase reporter analysis and gel shift analysis were performed. Results The proximal promoter element is the overlapping binding site for the constitutive binding factor, Oct-1, and the inducible one, AP-1. The transcriptional induction was ascribed to the inducible binding, while the constitutive binding was rather inhibitory. A mutant element which lost the constitutive binding but retained the inducible binding exerted 3 times more transcriptional activity compared to the wild type element. In contrast, another mutant element which lost the inducible binding and retained the constitutive binding exhibited no transcriptional induction. Gel shift analysis clarified that the inducible binding was more prominent and the constitutive binding was less in IL-5-producing T cells derived from asthma patients compared to IL-5-nonproducing cells derived from control subjects. Conclusions The ratio of the inducible/constitutive binding to the proximal promoter element maydetermine the capacity of human Th cells to transcribe IL-5 gene, and its regulation may control eosinophilic inflammation. (Internal Medicine 39: 618-625, 2000)

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“…We have previously reported that human IL-5 synthesis is regulated at the transcriptional level, and a unique tran scription factor (NF-IL-5) may regulate the transcription of IL-5 gene independently from IL-2 or IL-4 gene [5], The 515-bp human IL-5 promoter/enhancer-luciferase gene construct, piL-5(-51 l)Luc, was effectively transcribed up on activation when transfected into IL-5-producing T cells. Moreover, IL-5-nonproducing T cells were unable to tran scribe pIL-5(-511 )Luc.…”

Section: Il-5 Gene Transcription Is Regulated By Multiple Promoter Elmentioning

confidence: 99%

Dissection of Human IL-5 Promoter – Essential Role of CLEO Element in Human IL-5 Gene Transcription

Mori

Kaminuma²,

Mikami³

et al. 1997

Int Arch Allergy Immunol

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Analysis of Human lnterleukin-5 Gene Transcription by T Cell Clones and Hybridomas

Cited by 8 publications

References 6 publications

Regulatory Mechanisms of Human T Cell IL–5 Synthesis: Differential Roles of the Proximal Promoter–Binding Proteins in IL–5 Gene Transcription

Regulatory Mechanisms of Human T Cell IL–5 Synthesis: Differential Roles of the Proximal Promoter–Binding Proteins in IL–5 Gene Transcription

Transcriptional Regulation of IL-5 Gene by Nontransformed Human T Cells Through the Proximal Promoter Element.

Dissection of Human IL-5 Promoter – Essential Role of CLEO Element in Human IL-5 Gene Transcription

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