RESUMO
Chemical screening identified three small compounds that selectively inhibited activation of the respiratory burst (RB) of human neutrophils in response to tumor necrosis factor (TNF) and formylated peptide but not phorbol ester and spared the ability of neutrophils to kill bacteria. These compounds partially inhibited TNF-triggered cytoskeletal rearrangements without blocking adhesion or transmigation of polymorphonuclear neutrophils through TNF-activated monolayers of endothelial cells. The compounds were nontoxic to neutrophils and endothelial cells. They had no direct inhibitory effect on the tyrosine kinases Src, Syk, or Pyk2. However, their differential effects on cell spreading, bacteria-induced RB, TNF-induced degranulation, TNF-induced protein tyrosine phosphorylation, and TNF-induced Syk activation suggested that each may act on different elements of neutrophil signaling pathways.
Assuntos
Movimento Celular/efeitos dos fármacos , Compostos Heterocíclicos/farmacologia , Neutrófilos/imunologia , Explosão Respiratória/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Bactérias/crescimento & desenvolvimento , Bactérias/imunologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/imunologia , Degranulação Celular/efeitos dos fármacos , Degranulação Celular/imunologia , Movimento Celular/imunologia , Células Cultivadas , Contagem de Colônia Microbiana , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/imunologia , Células Endoteliais/citologia , Células Endoteliais/imunologia , Humanos , Neutrófilos/citologia , Peptídeos/farmacologia , Proteínas Tirosina Quinases/imunologia , Explosão Respiratória/imunologia , Transdução de Sinais/imunologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Through chemical screening, we identified a pyrazolone that reversibly blocked the activation of phagocyte oxidase (phox) in human neutrophils in response to tumor necrosis factor (TNF) or formylated peptide. The pyrazolone spared activation of phox by phorbol ester or bacteria, bacterial killing, TNF-induced granule exocytosis and phox assembly, and endothelial transmigration. We traced the pyrazolone's mechanism of action to inhibition of TNF-induced intracellular Ca2+ elevations, and identified a nontransmembrane ("soluble") adenylyl cyclase (sAC) in neutrophils as a Ca2+-sensing source of cAMP. A sAC inhibitor mimicked the pyrazolone's effect on phox. Both compounds blocked TNF-induced activation of Rap1A, a phox-associated guanosine triphosphatase that is regulated by cAMP. Thus, TNF turns on phox through a Ca2+-triggered, sAC-dependent process that may involve activation of Rap1A. This pathway may offer opportunities to suppress oxidative damage during inflammation without blocking antimicrobial function.
