Inhibition of tumor necrosis factor-alpha induced neutrophil apoptosis by cyclic AMP: involvement of caspase cascade

Treatment of neutrophils with tumor necrosis factor-alpha (TNF-alpha) in th e presence of cycloheximide induced apoptosis within 3 h, as evaluated by t he occurrence of morphological nuclear changes characteristic of apoptosis. Pretreatment of neutrophils with dibutyryl cyclic AMP (dbcAMP) suppressed the TNF-alpha/cycloheximide-induced apoptosis in neutrophils in a concentra tion-dependent manner, while dbcAMP by itself did not induce any morphologi cal changes. Forskolin, or a phosphodiesterase inhibitor, also produced a c oncentration-dependent inhibition on apoptosis. This inhibition by dbcAMP w as completely reversed by pretreatment with the protein kinase A inhibitor, N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinoline sulphonamide (H-89). Dbc AMP also inhibited the TNF alpha/cycloheximide-induced activation of caspas e-3, but it had no effect on the activation of caspase-8 in human neutrophi ls. Furthermore, dbcAMP did not directly inhibit activated caspase-3 activi ty. inhibitor of protein kinase C, phosphatidylcholine-specific phospholipa se C, tyrosine kinase, nitric oxide synthase, or granulocyte colony-stimula ting factor or granulocyte monocyte colony-stimulating factor did not affec t apoptosis. These results indicate that the elevation of levels of endogen ous intracellular cyclic AMP and subsequent activation of protein kinase A play a crucial role in the prevention of apoptosis triggered by TNF-alpha/c ycloheximide in human neutrophils, and that the possible target of cyclic A MP is a product in the metabolic pathway between caspase-8 and caspase-3. ( C) 1999 Elsevier Science B.V. All rights reserved.