Original article| Volume 124, ISSUE 2, P283-292, August 1994

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Measurement of IL-6 inhibitory activity in cultured cell supernatants and lysates

  • Homer L Twigg III
    Reprint requests: Homer L. Twigg III, Indiana University Medical Center, 1001 West 10th St., OPW 425, Indianapolis, IN 46202.
    From the Department of Medicine, Division of Pulmonary and Critical Care, Indiana University Medical Center Indianapolis, Indiana USA
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  • David S. Wilkes
    From the Department of Medicine, Division of Pulmonary and Critical Care, Indiana University Medical Center Indianapolis, Indiana USA
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  • Diaa M. Soliman
    From the Department of Medicine, Division of Pulmonary and Critical Care, Indiana University Medical Center Indianapolis, Indiana USA
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  • Author Footnotes
    1 Dr. Twigg is a recipient of the Edward Livingston Trudeau Scholar Award from the American Lung Association.
    2 Dr. Wilkes is supported by a grant from the Robert Wood Johnson Foundation.
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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.


      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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