Abstract
Transforming growth factor (TGF)-beta and interleukin (IL)-10 inhibited lipopolysaccharide (LPS)-induced macrophage production of the inflammatory cytokines tumor necrosis factor-alpha (TNF), IL-1 alpha, and IL-1 beta by contrasting post-transcriptional mechanisms. TGF-beta acted slowly and late, as it required 12-16 h to exert a suppressive effect, and inhibited TNF production even when added 6 h after LPS. TGF-beta affected neither the level of TNF mRNA, the release of preformed TNF nor the degradation of TNF. Thus, TGF-beta appeared to inhibit translation of TNF mRNA. IL-10 not only suppressed TNF release to a 25-fold greater extent than TGF-beta, but also inhibited release of IL-1. In contrast to TGF-beta, IL-10 acted on an early step in cytokine production, its effect being maximal 3 h after addition of LPS. Unlike TGF-beta, IL-10 markedly suppressed TNF, IL-1 alpha, and IL-1 beta mRNA levels. However, this was accomplished without suppressing transcription of the corresponding genes. Moreover, cycloheximide antagonized the IL-10-dependent reduction in cytokine mRNA levels. Thus, IL-10 may induce a ribonuclease active on cytokine transcripts or may induce a protein that enhances the susceptibility of TNF, IL-1 alpha, and IL-1 beta mRNAs to ribonucleolytic action. We conclude that IL-10 and TGF-beta induce different phenotypes of macrophage deactivation, and deactivate macrophages by different mechanisms: IL-10 promotes degradation of cytokine mRNA, while TGF-beta primarily suppresses translation.
Highlights
Cytokine Production and Assay-Monolayers of macrophages were stimulated with LPS (+ IFN-r), S. aureusenterotoxin B, ora combination of phorbol myristate acetate and A23187 either in the presence or absence of IL-10 or Transforming growth factor (TGF)-@for the time periods indicated
IL-10 and TGF-P Suppress LPS-induced T N F Production to Different Extents and with Distinct Stimulation Requirements-Exposure of LPS-stimulated macrophages to IL-10 diminishes the accumulation of tumor necrosis factor-a (TNF) in their conditioned medium [15]
SEBinduces macrophage TNF production [28,29], and caombination of the phorbolester PMA and the calcium ionophore A23187 primes macrophages for tumor cell cytotoxicity, which might reflect TNF production [30]. We confirmed that these agents were not contaminated with LPS, yet induced TNF release, and demonstrated that TGF@ inhibited TNF production to a similar extent, whether TNF was induced by SEB, PMA and A23187, or LPS itself (Table I)
Summary
Activation of macrophages plays an important role in immune responses against microbes, tumors, and normal host tissues. Cytokines and OtherReagents-Supernatants from COS7 cells transfected with murine IL-10 cDNA Cytokine Production and Assay-Monolayers of macrophages were stimulated with LPS (+ IFN-r), S. aureusenterotoxin B, ora combination of phorbol myristate acetate and A23187 either in the presence or absence of IL-10 or TGF-@for the time periods indicated. 10, IFN-7,and TGF-@(18, 20): Briefly, serial 2-fold dilutions of the test supernatants as well as of recombinant murine TNF were incubated with 3-4 X lo' WEHI cells in the presence of actinomycin D (0.5 pglml). Preparation of Total Cell Lysates-Macrophage monolayers were washed and scraped as described above, but the cell pellet was lysed in 150 mM NaCl, 10 mM Tris, pH 8, 1 mM phenylmethylsulfonyl fluoride, 0.5 unit/ml aprotinin,and 1%Triton X-100 (60min, rotating a t 4 T ). Data Presentation-Unless indicated otherwise, results are means +S.E. for the number of experiments performed
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