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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 bioactlve 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 μg/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.
Abbreviations:AM (alveolar macrophage), BAL (bronchoalveolar lavage), ELISA (enzyme-linked immunosorbent assay), FBS (fetal bovine serum), HEPES (N-2-hydroxyethylpiperazine-N-2-ethane-sulfonic acid), IL-6 (interleukin 6), LPS (lipopolysaccharide), PBMC (peripheral blood mono-nuclear cells), PBS (phosphate-buffered saline solution), TGF-β (transforming growth factor-β), TNF-α (tumor necrosis factor-α)
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Accepted: February 19, 1994
Received in revised form: February 15, 1994
Received: September 2, 1993
☆Supported in part by Public Health Service Grant MO1 RR750.
© 1994 Published by Elsevier Inc.