Action of intracellular IL-1Ra (Type 1) is independent of the IL-1 intracellular signalling pathway.
The balance between IL-1 and its naturally occurring inhibitor IL-1 receptor antagonist (IL-1ra) is critical in determining the inflammatory response. Four splice variants of the IL-1ra gene have been identified; one secreted (sIL-1ra) and three intracellular (icIL-1ra1-3). The biological roles of the intracellular isoforms remain largely unclear. We wished to determine whether icIL-1ra1 had intracellular functions regulating IL-1 signalling. Signalling was determined using an NF-kappaB reporter assay measuring induction of the IL-8 promoter in transfected cells. Over-expression of icIL-1ra1 in HeLa cells had no effect on IL-1 stimulated IL-8 activity. In contrast over-expression of sIL-ra significantly attenuated IL-1 activity. In addition, transfection of icIL-1ra1 in HeLa cells did not cause inhibition of IL-8 promoter activity following over-expression of the IL-1 signalling components MyD88, IRAK-1, TRAF-6, Ikappakappabeta or RelA. This implies that icIL-1ra1 does not act to alter IL-1 mediated intracellular signalling in this system. We investigated whether ATP and/or over-expression of the P2X7 receptor caused icIL-1ra1 inhibition of IL-1beta mediated IL-8 reporter activation, by permitting its release. In HeLa cells, no effect of icIL-1ra1 was observed in ATP stimulated and/or P2X7 transfected cells, compared to a significant inhibition in sIL-1ra transfected cells. However, in endothelial cells stimulated with ATP, the released fraction was effective in attenuating IL-1beta activation of the IL-8 reporter. These results suggest that icIL-1ra1 does not act at an intracellular level to alter IL-1 mediated signalling, and is effective in inhibiting IL-1 responses only when released in an ATP-dependent and cell type specific manner.
Section of Functional Genomics, Division of Genomic Medicine, Royal Hallamshire Hospital, Glossop Road, Sheffield, S10 2JF, UK.
Iona
I
Evans
Steven K
SK
Dower
Sheila E
SE
Francis
David C
DC
Crossman
Heather L
HL
Wilson
D000255:Adenosine Triphosphate / Q000737:chemistry; D017398:Alternative Splicing; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology; D006367:HeLa Cells; D006801:Humans; D007249:Inflammation; D053590:Interleukin 1 Receptor Antagonist Protein; D007375:Interleukin-1 / Q000378:metabolism*; D016209:Interleukin-8 / Q000378:metabolism; D011401:Promoter Regions, Genetic; D018048:Receptors, Purinergic P2 / Q000378:metabolism; D058486:Receptors, Purinergic P2X7; D012795:Sialoglycoproteins / Q000737:chemistry* / Q000378:metabolism; D015398:Signal Transduction
C507763:IL1RN protein, human; D053590:Interleukin 1 Receptor Antagonist Protein; D007375:Interleukin-1; D016209:Interleukin-8; C546468:P2RX7 protein, human; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D012795:Sialoglycoproteins; D000255:Adenosine Triphosphate