Actinomycin D upregulates lipopolysaccharide induction of macrophage procoagulant expression and tumour necrosis factor-alpha production
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H R Wheeler1, E J Rockett, I Clark
1Department of Clinical Oncology, Royal North Shore Hospital, Sydney, Australia.
Abstract:
The antitumour antibiotic actinomycin D (Act D) and the aminosugar D-galactosamine both enhance the sensitivity of animals to bacterial lipopolysaccharide (LPS). Lipopolysaccharide stimulates macrophage membrane-bound procoagulant activity (MPCA) and tumour necrosis factor-alpha (TNF-alpha) production in vitro. We investigated the effects of LPS combined with either Act D or D-galactosamine on procoagulant and TNF-alpha production in vitro. Actinomycin D directly induced procoagulant on the malignant monocytoid cell line WEHI 265, and synergized with LPS to enhance MPCA on both WEHI 265 cells and thioglycollate-induced peritoneal exudate macrophages. In the presence of Act D, exudate macrophages expressed procoagulant in response to concentrations of LPS 100,000-fold lower than normally required. Pulsing experiments demonstrated that LPS primed these cells within 4 h to respond to Act D, whereas 4 h priming with Act D inhibited subsequent procoagulant induction by LPS. Although its effects on TNF-alpha production were less intense, low levels of Act D more than doubled TNF-alpha produced by LPS-stimulated exudate macrophages. Procoagulant expression and TNF-alpha production were not always co-ordinately expressed; interferon-gamma (IFN-gamma) synergized with LPS to enhance both responses but when IFN-gamma was combined with Act D only procoagulant was upregulated. D-galactosamine failed to affect these macrophage responses. Results indicate different in vivo mechanisms of enhancement of LPS toxicity by these two agents.
Insights
The antibiotic actinomycin D (Act D) enhances macrophage sensitivity to bacterial lipopolysaccharide (LPS), boosting procoagulant and TNF-alpha production. D-galactosamine did not affect these responses, suggesting distinct mechanisms for LPS toxicity enhancement.
Area of Science:
- Immunology
- Pharmacology
- Cell Biology
Background:
- Bacterial lipopolysaccharide (LPS) stimulates macrophage procoagulant activity (MPCA) and TNF-alpha production.
- Actinomycin D (Act D) and D-galactosamine are known to increase animal sensitivity to LPS.
- Understanding the in vitro mechanisms of Act D and D-galactosamine on LPS-induced macrophage responses is crucial.
Purpose of the Study:
- To investigate the combined effects of LPS with Act D or D-galactosamine on macrophage procoagulant and TNF-alpha production in vitro.
- To elucidate the distinct mechanisms by which Act D and D-galactosamine modulate LPS toxicity.
Main Methods:
- Utilized the malignant monocytoid cell line WEHI 265 and thioglycollate-induced peritoneal exudate macrophages.
- Assessed macrophage membrane-bound procoagulant activity (MPCA) and tumor necrosis factor-alpha (TNF-alpha) production.
- Conducted pulsing experiments to determine the temporal effects of LPS and Act D priming.
Main Results:
- Actinomycin D directly induced procoagulant activity and synergized with LPS to significantly enhance MPCA in both cell types.
- Act D enabled macrophages to respond to LPS concentrations 100,000-fold lower than normal, with priming effects being time-dependent.
- Low-dose Act D more than doubled LPS-induced TNF-alpha production, though procoagulant and TNF-alpha responses were not always coordinated.
- Interferon-gamma (IFN-gamma) synergized with LPS for both responses, but with Act D, only procoagulant activity was upregulated.
- D-galactosamine showed no significant effect on LPS-induced MPCA or TNF-alpha production.
Conclusions:
- Actinomycin D significantly enhances LPS-induced procoagulant activity and TNF-alpha production in macrophages through distinct mechanisms compared to D-galactosamine.
- The findings suggest that Act D's enhancement of LPS toxicity involves direct procoagulant induction and potentiation of LPS signaling.
- D-galactosamine likely enhances LPS sensitivity via pathways independent of direct macrophage activation of procoagulant or TNF-alpha production.