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Differential effects on TNF alpha production by pharmacological agents with varying molecular sites of action
Abstract:
This study describes the activation conditions for tumor necrosis factor-alpha (TNF alpha) production in myelomonocytic U937 cells and human primary peripheral blood monocytes in response to lipopolysaccharide (LPS) and/or phorbol 12-myristate 13-acetate (PMA). PMA itself induced only low levels of TNF alpha production with delayed kinetics (e.g. 0.758 +/- 0.128 ng/ml from U937 cells after 48 h) while LPS induced greater levels of TNF alpha production in less time (e.g. 2.083 +/- 0.96 ng/ml from monocytes in 24 h). Pharmacological agents with various molecular sites of action were used to validate the two systems, with the protein serine-threonine kinase inhibitors staurosporine and Ro-31-8220, the protein tyrosine kinase inhibitor herbimycin A (HBA) and dexamethasone exhibiting the greatest potency (IC50S 5-350 nM). In contrast to the effect on TNF alpha production, PMA induced strong phosphorylation/activation of p42/p44mapk in monocytes by 10 min determined in a mobility shift assay, while LPS was a weaker inducer. Additionally, staurosporine (to LPS and PMA) and HBA (to LPS only) inhibited the activation of these mitogen-activated protein kinase (MAPK) isoforms at doses 10-100 fold higher than those required to inhibit maximal TNF alpha production. These data indicate the involvement of the p42/p44mapk signalling pathway in LPS-induced pro-inflammatory cytokine production but suggest that other signalling pathways are also implicated in this phenomenon.
Insights
Lipopolysaccharide (LPS) more potently activates tumor necrosis factor-alpha (TNF alpha) production in monocytes than phorbol 12-myristate 13-acetate (PMA). Kinase inhibitors effectively block TNF alpha production, suggesting complex signaling pathways beyond p42/p44mapk activation.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Tumor necrosis factor-alpha (TNF alpha) is a key pro-inflammatory cytokine.
- Understanding TNF alpha production is crucial for inflammatory disease research.
- Lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA) are common stimuli for cytokine production.
Purpose of the Study:
- To investigate the activation conditions for TNF alpha production in human monocytes and U937 cells.
- To compare the efficacy of LPS and PMA in inducing TNF alpha.
- To identify signaling pathways involved in TNF alpha production.
Main Methods:
- Stimulation of U937 cells and human monocytes with LPS and/or PMA.
- Quantification of TNF alpha production using cell-based assays.
- Pharmacological inhibition of signaling pathways using kinase inhibitors (staurosporine, Ro-31-8220, herbimycin A) and dexamethasone.
- Assessment of mitogen-activated protein kinase (MAPK) activation via mobility shift assays.
Main Results:
- LPS induced higher levels of TNF alpha production more rapidly than PMA.
- Protein kinase inhibitors demonstrated high potency in blocking TNF alpha production (IC50s 5-350 nM).
- PMA strongly activated p42/p44mapk, while LPS was a weaker inducer of this specific pathway; however, kinase inhibitors affected TNF alpha production at lower doses than p42/p44mapk inhibition.
Conclusions:
- LPS is a more potent stimulus for TNF alpha production than PMA in monocytes.
- Multiple signaling pathways, not solely p42/p44mapk, are involved in LPS-induced TNF alpha production.
- Kinase signaling pathways are critical targets for modulating inflammatory responses.