title: Tumor necrosis factor alpha stimulates mycobactericidal/mycobacteriostatic activity in human macrophages by a protein kinase C-independent pathway.
abstract: Tumor necrosis factor (TNF) is a 17-kDa protein produced by endotoxin-stimulated macrophages. We have demonstrated that recombinant human TNF activates human macrophages to kill intracellular bacteria of the Mycobacterium avium complex (MAC) in a dose-related manner. TNF also primed macrophages to produce superoxide anion (O2-) following treatment with phorbol esther PMA (0.1 micrograms/ml). To investigate the intracellular pathway involved in the TNF-mediated activation of mycobacteriostatic/mycobactericidal activity in macrophages, we used two different protein kinase C (PKC) inhibitors: H7 (10(-5)-10(7) M) and staurosporine (10(-7)-10(-9) M). Mellitin (1 and 100 mM) was used as a calmodulin inhibitor. Human peripheral blood-derived macrophages cultured for 7 days were treated with H7, mellitin, or staurosporine for 1 hr prior to incubation with TNF (10(3) U/ml). Twenty-four hours after treatment with TNF the O2- release was measured spectrophotometrically following exposure to PMA. Macrophages were infected with MAC and the viable intracellular bacilli were quantitated following 4 days of treatment with TNF. All PKC inhibitors suppressed O2- production after incubation with PMA. However, treatment with either PKC or calmodulin inhibitors did not influence the intracellular killing of M. avium by TNF-stimulated macrophages. Exposure of the macrophages to cGMP inhibitor but not to cAMP inhibitor significantly impaired the response to the stimulation with TNF. In contrast, incubation of macrophages with protein kinase A (PKA) had no effect on TNF-mediated mycobacteriostatic/mycobactericidal activity. These results suggest that the TNF-mediated mycobactericidal activity in cultured macrophages probably occurs by a PKC-independent mechanism.
authors.affiliation: Kuzell Institute for Arthritis and Infectious Diseases, California Pacific Medical Center, San Francisco 94115.
authors.forename: L E
authors.initials: LE
authors.lastname: Bermudez
authors.forename: L S
authors.initials: LS
authors.lastname: Young
mesh_terms: D002147:Calmodulin / Q000037:antagonists & inhibitors; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000502:physiology; D006152:Cyclic GMP / Q000502:physiology; D006801:Humans; D007213:Indomethacin / Q000494:pharmacology; D008262:Macrophage Activation / Q000187:drug effects*; D009637:Masoprocol / Q000494:pharmacology; D009161:Mycobacterium / Q000276:immunology*; D011493:Protein Kinase C / Q000502:physiology*; D011994:Recombinant Proteins / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D002147:Calmodulin; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D009637:Masoprocol; D000242:Cyclic AMP; D011493:Protein Kinase C; D006152:Cyclic GMP; D007213:Indomethacin