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.