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Detection and analysis of an alternatively spliced isoform of interleukin-6
mRNA in peripheral blood mononuclear cells
DP Kestler, S Agarwal, J Cobb, KM Goldstein and RE Hall
Department of Medicine, University of Tennessee Medical Center at Knoxville
37920, USA.
Oligonucleotide primers for human interleukin-6 (IL-6) that bracketed the
entire coding region of the gene were used in reverse transciptase-
polymerase chain reaction (RT-PCR) studies to examine lL-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 lL-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 lL-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 lL-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 lL-6 mRNA, which would encode for a polypeptide missing the
gp130 interactive (signal-transducing) domain contained in exon II while
retaining the lL-6 receptor (p80) domain. Such a molecule could in theory
function as a natural antagonist of lL-6, as it would be expected to bind
to the IL-6 receptor but not lead to signal transduction.
Volume 86,
Issue 12,
pp. 4559-4567,
12/15/1995
Copyright © 1995 by The American Society of Hematology
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