Competition between IL-1, IL-1ra and TGF-beta 1 modulates the response of the ELA4.NOB-1/CTLL bioassay: implications for clinical investigations.
OBJECTIVE
The use of ELISA techniques to measure cytokine levels in clinical samples has chiefly replaced more labour intensive bioassays. ELISA measurements, however, do not reflect the functional activity of a cytokine within a sample; interleukin-1 (IL-1), for example, has two agonist isoforms (IL-1 alpha and IL-1 beta) and a competitive receptor antagonist (IL-1ra), and can be regulated by transforming growth factor beta1 (TGF-beta 1). The net effect of these cytokines, rather than IL-1 levels, are frequently suggested to regulate tissue inflammation, but confirming this has been difficult.


METHODS
We used the ELA4.NOB-1/CTLL co-culture IL-1 bioassay to investigate whether IL-1 activity was inhibited by IL-1ra and TGF-beta 1 in a predictable manner.


RESULTS
Thymidine incorporation into CTLL cells, induced by IL-1, was reduced dose dependently by IL-1ra and TGF-beta 1. With optimal levels of IL-1 CTLL responsiveness was reduced by 90% by 1 ng/ml TGF-beta 1 and completely abolished by 100 ng/ml IL-1ra. As expected, TGF-beta 1 and IL-1ra had independent mechanisms of action on the bioassay cell lines, and, in combination, they caused an additive, but not synergistic, effect. Importantly, the effect of these cytokines could be completely abolished in the presence of neutralising antibodies.


CONCLUSIONS
Bioassay should provide specific functional information on the net IL-1 activity of clinical samples, while the use of specific antibodies could ascertain the contribution of individual cytokines within such samples.
Ashwood
P
P
Gastrointestinal Laboratory, The Rayne Institute, St Thomas' Hospital, London SE1 7EH, United Kingdom.
Harvey
R
R
Verjee
T
T
Wolstencroft
R
R
Thompson
R P H
RP
Powell
J J
JJ
D000818:Animals; D001667:Binding, Competitive; D001681:Biological Assay; D002455:Cell Division / Q000187:drug effects; D018920:Coculture Techniques; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D053590:Interleukin 1 Receptor Antagonist Protein; D007375:Interleukin-1 / Q000494:pharmacology*; D008223:Lymphoma; D051379:Mice; D011994:Recombinant Proteins / Q000494:pharmacology; D012795:Sialoglycoproteins / Q000494:pharmacology*; D013602:T-Lymphocytes, Cytotoxic / Q000187:drug effects / Q000502:physiology*; D013936:Thymidine / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1; D014407:Tumor Cells, Cultured
C507763:IL1RN protein, human; C507764:Il1rn protein, mouse; D053590:Interleukin 1 Receptor Antagonist Protein; D007375:Interleukin-1; D011994:Recombinant Proteins; D012795:Sialoglycoproteins; C508960:TGFB1 protein, human; C508961:Tgfb1 protein, mouse; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D013936:Thymidine