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Detection and Analysis of an Alternatively Spliced Isoform of Interleukin-6 mRNA in Peripheral Blood Mononuclear Cells

Oligonucleotide primers for human interleukin-6 (IL-6) that bracketed the entire coding region of the gene were used in reverse transcriptase-polymerase chain reaction (RT-PCR) studies to examine IL-6 expression in peripheral blood mononuclear cells (PBMC). In addition to the predicted 0.64-kb RT-PC...

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Bibliographic Details
Published in:Blood 1995-12, Vol.86 (12), p.4559-4567
Main Authors: Kestler, Daniel P., Agarwal, Sujata, Cobb, John, Goldstein, Keith M., Hall, Robert E.
Format: Article
Language:English
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Summary:Oligonucleotide primers for human interleukin-6 (IL-6) that bracketed the entire coding region of the gene were used in reverse transcriptase-polymerase chain reaction (RT-PCR) studies to examine IL-6 expression in peripheral blood mononuclear cells (PBMC). In addition to the predicted 0.64-kb RT-PCR product, a second 0.45-kb product was observed. Cloning and dideoxy sequence analysis of this product revealed evidence for an alternatively spliced IL-6 transcript lacking exon II. Further RT-PCR analysis using forward primers ending at or one base before the exon I donor splice site again yielded both products. Additional primers were designed and successfully used to selectively distinguish the two forms of IL-6 transcript. Both transcripts were prominent in peripheral blood monocytes and lymphocytes, whereas only the 0.64-kb, full-length transcript was prominent in the IL-6-producing 5637 (human bladder carcinoma) cell line. Northern analysis revealed, in addition to the predominant 1.3-kb transcript, several minor transcripts at 1.9 to 4.8 kb that hybridized with the alternatively spliced cDNA probe but not with an exon II probe. Western analysis revealed IL-6 polypeptides of predicted size (26 to 29 kD) in culture medium from PBMC, while showing an immunoreactive band at 17 kD in cell lysates. These findings suggest the existence of an alternatively spliced form of IL-6 mRNA, which would encode for a polypeptide missing the gp130 interactive (signal-transducing) domain contained in exon II while retaining the IL-6 receptor (p80) domain. Such a molecule could in theory function as a natural antagonist of IL-6, as it would be expected to bind to the IL-6 receptor but not lead to signal transduction.
ISSN:0006-4971
1528-0020
DOI:10.1182/blood.V86.12.4559.bloodjournal86124559