title: 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.
authors.affiliation: Xenova Ltd, Slough, Berkshire, England.
authors.forename: T
authors.initials: T
authors.lastname: Mander
authors.forename: S
authors.initials: S
authors.lastname: Hill
authors.forename: A
authors.initials: A
authors.lastname: Hughes
authors.forename: P
authors.initials: P
authors.lastname: Rawlins
authors.forename: C
authors.initials: C
authors.lastname: Clark
authors.forename: G
authors.initials: G
authors.lastname: Gammon
authors.forename: B
authors.initials: B
authors.lastname: Foxwell
authors.forename: M
authors.initials: M
authors.lastname: Moore
mesh_terms: D016227:Benzoquinones; D001665:Binding Sites; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism / Q000502:physiology; D003907:Dexamethasone / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D016753:Interleukin-10 / Q000494:pharmacology; D015847:Interleukin-4 / Q000494:pharmacology; D047029:Lactams, Macrocyclic; D008070:Lipopolysaccharides / Q000494:pharmacology; D016403:Lymphoma, Large B-Cell, Diffuse / Q000201:enzymology / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism / Q000502:physiology; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D009000:Monocytes / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism*; D011809:Quinones / Q000494:pharmacology; D017828:Rifabutin / Q000031:analogs & derivatives; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D019311:Staurosporine / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis*
chemical_list: D016227:Benzoquinones; D004791:Enzyme Inhibitors; D047029:Lactams, Macrocyclic; D008070:Lipopolysaccharides; D011809:Quinones; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10; D017828:Rifabutin; D015847:Interleukin-4; C020754:herbimycin; D003907:Dexamethasone; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate