Production of a T‐Cell Clone Which Reacts with Membrane Proteins of <i>Acholeplasma laidlawii</i>

Kuwano, Koichi; Ono, Shigeaki; Akashi, Akira; Ohishi, Masahiro; Shigematsu, Hideki; Arai, Sumio

doi:10.1111/j.1348-0421.1997.tb01198.x

Cited by 4 publications

(4 citation statements)

References 19 publications

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“…The A. laidlawii PG8 was cultured in Pleuropneumonia‐like organism (PPLO) medium (Difco, Detroit, MI) and heat‐inactivated for 30 min at 60° as described previously 16 . It was then centrifuged for 30 min at 20 000 g , followed by lyophilization.…”

Section: Methodsmentioning

confidence: 99%

Opposing roles of activator protein‐1 and CCAAT/enhancer binding protein β in the regulation of inducible granulysin gene expression in a human monocytic cell line, THP‐1

2002

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SUMMARYWe previously reported that inducible granulysin gene expression in a human monocytic cell line, THP-1 is dominantly dependent on transcription factor activator protein-1 (AP-1). Here, we further examined the precise regulatory mechanisms underlying granulysin gene expression using THP-1 cells treated with Acholeplasma laidlawii. Transfection of reporter gene constructs into THP-1 cells indicated that the presence of a positive regulatory element(s) is located from À329 to À85 base pairs, containing two distinct AP-1 binding sites and one nuclear factor-kB (NF-kB) binding site. Deletion or mutation of the NF-kB binding site failed to affect inducible promoter activity, whereas deletion or mutation of both the AP-1 binding sites abrogated the promoter activity. Interestingly, deletion of the putative CCAAT/enhancer binding protein b (C/EBPb) binding site upstream of the positive regulatory element induced the augmentation of granulysin promoter activity. Electrophoretic mobility shift assays demonstrated that nuclear extract prepared from A. laidlawii-treated THP-1 cells generated a speci®c binding to oligonucleotides, including AP-1, C/EBPb, and NF-kB element. Furthermore, over-expression of liver-enriched transcriptional activator protein, a subunit of C/EBPb, augmented A. laidlawii-induced granulysin promoter activity, whereas over-expression of liver-enriched transcriptional inhibitory protein inhibited the promoter activity. NF-kB p50 homodimer had no transactivation property, although it bound to the NF-kB site. These results indicate that AP-1 and C/EBPb, but not NF-kB participate in the regulation of inducible granulysin gene expression in THP-1 cells. Moreover, the Toll-like receptor 2-dependent signalling pathway may be involved in A. laidlawii-induced transactivation of the granulysin promoter. Thus, these results suggest that the gene expression of granulysin in macrophages would be exquisitely regulated by positive and negative transcription factors when microbial invasion occurs.

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Section: Methodsmentioning

confidence: 99%

Opposing roles of activator protein‐1 and CCAAT/enhancer binding protein β in the regulation of inducible granulysin gene expression in a human monocytic cell line, THP‐1

2002

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“…A. laidlawii PG8 was cultured in PPLO medium (Difco, Detroit, MI) and heat inactivated for 30 min at 60°, as described previously 13 . It was then centrifuged for 30 min at 20 000 g , followed by lyophilization.…”

Section: Methodsmentioning

confidence: 99%

Acholeplasma laidlawii up‐regulates granulysin gene expression via transcription factor activator protein‐1 in a human monocytic cell line, THP‐1

Kida¹,

Kuwano²,

Zhang³

et al. 2001

Immunology

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Summary An antimicrobial protein granulysin is constitutively expressed in cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. However, little is known about the precise regulatory mechanisms underlying granulysin gene expression. In this study, we examined the regulatory mechanisms underlying granulysin gene expression using a human monocytic cell line, THP‐1, treated with Acholeplasma laidlawii. The level of granulysin mRNA expression in THP‐1 cells was significantly augmented in response to stimulation with A. laidlawii. The transfection of reporter gene constructs into THP‐1 cells indicated that DNA sequences between residues −329 and −239, relative to the transcriptional start site of the granulysin gene, are responsible for mediating gene induction. In addition, mutagenesis of a putative activator protein‐1 (AP‐1)‐binding site between residues −277 and −271 in the granulysin promoter resulted in the reduction of granulysin promoter activity. Electrophoretic mobility shift assays (EMSA) demonstrated that nuclear extract prepared from A. laidlawii‐treated THP‐1 cells can generate specific binding to DNA oligonucleotides encompassing the AP‐1‐binding site, whereas unstimulated nuclear extract from the cells failed to do so. Furthermore, competition and supershift assays confirmed that A. laidlawii can induce the activation of AP‐1. These results indicate that AP‐1 dominantly participates in the regulation of inducible granulysin gene expression in THP‐1 cells. Therefore, the finding of inducible granulysin gene expression by A. laidlawii suggests that inducible granulysin in macrophages may function as a protective weapon when microbial invasion occurs.

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“…Acholeplasma laidlawii PG8 was cultured in PPLO medium (Difco, Detroit, MI) and heat-inactivated for 30 min at 60°as described previously. 32 They were then centrifuged for 30 min at 20 000 g followed by lyophilization. For stimulation, the lyophilized A. laidlawii were suspended in the supplemented RPMI-1640 described above.…”

Section: Acholeplasma Laidlawiimentioning

confidence: 99%

“…Acholeplasma laidlawii PG8 was cultured in PPLO medium (Difco, Detroit, MI) and heat‐inactivated for 30 min at 60° as described previously 32 . They were then centrifuged for 30 min at 20 000 g followed by lyophilization.…”

Section: Acholeplasma Laidlawiimentioning

confidence: 99%

Conditional expression of liver‐enriched transcriptional activator protein augments Acholeplasma laidlawii‐induced granulysin gene expression in a human monocytic cell line, THP‐1

2004

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SummaryThe antimicrobial protein granulysin is considered to play an important role in the defence mechanism against bacterial infection. We previously reported that Acholeplasma laidlawii-induced transactivation of the granulysin promoter in a human monocytic cell line, THP-1, is regulated by activator protein-1 and CCAAT ⁄ enhancer binding protein-b (C ⁄ EBPb), but not by nuclear factor-jB. Moreover, liver-enriched transcriptional inhibitory protein (LIP), a C ⁄ EBPb isoform, was strongly induced in A. laidlawii-stimulated THP-1 cells. However, the level of liver-enriched transcriptional activator protein (LAP), another C ⁄ EBPb isoform, was essentially constant. Accordingly, we speculated that LIP would downregulate A. laidlawii-induced granulysin gene expression in THP-1 cells. In the present study, we examined whether LAP augments A. laidlawiiinduced granulysin gene expression using conditional LAP-expressing THP-1 cells in a tetracycline-controlled expression system. Our results indicated that conditional expression of LAP augmented A. laidlawiiinduced expression of granulysin mRNA. In addition, the granulysin protein was observed in A. laidlawii-stimulated, LAP-expressing THP-1 cells. Our results suggest that the expression of LAP plays a critical role in the expression of the granulysin gene in macrophages.

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Production of a T‐Cell Clone Which Reacts with Membrane Proteins of Acholeplasma laidlawii

Abstract: Abstract:The role of cellular immunity in mycoplasma infection is not completely understood.

Cited by 4 publications

References 19 publications

Opposing roles of activator protein‐1 and CCAAT/enhancer binding protein β in the regulation of inducible granulysin gene expression in a human monocytic cell line, THP‐1

Opposing roles of activator protein‐1 and CCAAT/enhancer binding protein β in the regulation of inducible granulysin gene expression in a human monocytic cell line, THP‐1

Acholeplasma laidlawii up‐regulates granulysin gene expression via transcription factor activator protein‐1 in a human monocytic cell line, THP‐1

Conditional expression of liver‐enriched transcriptional activator protein augments Acholeplasma laidlawii‐induced granulysin gene expression in a human monocytic cell line, THP‐1

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