Measurement of IL-6 inhibitory activity in cultured cell supernatants and lysates.
Interleukin-6 (IL-6) participates in a variety of cellular activities including regulation of immune and inflammatory responses. We have previously reported a discrepancy between bioactive and antigenic IL-6 secretion by lipopolysaccharide-stimulated alveolar macrophages (AMs) from smokers and have speculated that this may be due to cosecretion of an IL-6 inhibitor. In this study we further define our methods for measuring IL-6 inhibitory activity by testing the ability of serially diluted, cultured cell supernatants and lysates to suppress proliferation of an IL-6-dependent cell line, B9, to optimal concentrations of rIL-6. AM secretion of the inhibitory factor was optimal when AMs were stimulated with 1 micrograms/ml lipopolysaccharide (LPS). AMs from smokers secreted significantly greater amounts of this factor than AMs from nonsmokers. It was crucial to remove IL-6 from test samples on an IL-6 immunoaffinity column before analyzing for IL-6 inhibitory activity because (1) B9 cell proliferation could be suppressed by excess amounts of IL-6 in test supernatants and (2) excess rIL-6 added to the inhibitor assay reduced inhibitory activity. The latter finding suggested that IL-6 inhibitory activity was due to a competitive inhibitor of IL-6. This factor was shown to be specific for IL-6, because no inhibitory activity was seen on IL-2- or IL-4-dependent cell lines. Finally, we demonstrated that monocytes could also secrete an inhibitor of IL-6 bioactivity. However, secretion appeared to be less than that observed by AMs, suggesting that differentiation of monocytes into macrophages upregulated production of this factor.
Twigg
H L
HL
Department of Medicine, Indiana University Medical Center, Indianapolis 46202.
Wilkes
D S
DS
Soliman
D M
DM
D000328:Adult; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D002846:Chromatography, Affinity; D017077:Culture Media, Conditioned / Q000032:analysis* / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007376:Interleukin-2 / Q000502:physiology; D015847:Interleukin-4 / Q000502:physiology; D015850:Interleukin-6 / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D016676:Macrophages, Alveolar / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D009000:Monocytes / Q000166:cytology / Q000378:metabolism / Q000502:physiology; D011994:Recombinant Proteins / Q000494:pharmacology; D000093242:Interleukin-6 Inhibitors
D017077:Culture Media, Conditioned; D007376:Interleukin-2; D015847:Interleukin-4; D015850:Interleukin-6; D008070:Lipopolysaccharides; D011994:Recombinant Proteins; D000093242:Interleukin-6 Inhibitors