Induction of human neutrophil apoptosis by nitric oxide donors: evidence for a caspase-dependent, cyclic-GMP-independent, mechanism

Abstract

This study investigated the regulatory effects of the major inflammatory mediator, nitric oxide (NO), on human neutrophil apoptosis in vitro. Co-culture of human neutrophils with the NO donors GEA 3162 (1,2,3,4-oxatriazolium,5-amino-3-(3,4-dichlorophenyl)-chloride) (10–100 μM) and 3-morpholino-sydnonimine (SIN-1) (0.3–3 mM) caused a dramatic and concentration-dependent induction of apoptosis. However, N-formyl-methionyl-leucyl-phenylalanine (FMLP)-induced neutrophil activation (actin reorganization and chemotaxis) was inhibited by GEA 3162 treatment. The pro-apoptotic effects of the NO donors were (i) unaffected by the soluble guanylate cyclase inhibitor LY-83583 (6-anilino-5,8-quinolinedione; 100 μM), (ii) antagonized by superoxide dismutase (6 μg/mL), (iii) mimicked by exogenous peroxynitrite (at concentrations >100 μM), and (iv) inhibited by the caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone (100 μM). The pro-apoptotic effect of the NO donors was not mimicked by the cell-permeable cyclic nucleotide analogue, N 6,2- O-dibutyrylguanosine-3′,5′-cyclic monophosphate (dibutyryl-cGMP) at concentrations ≤0.2 mM. Indeed, at high concentrations (≥2 mM), dibutyryl-cGMP caused an inhibition of apoptosis. These results suggest that NO-mediated apoptosis, although caspase-dependent, is mediated by a cGMP-independent mechanism and involves the concurrent generation of oxygen free radicals and, potentially, peroxynitrite. Our data reveal a unique role for NO in inflammatory responses with differential effects upon neutrophil activation and survival, with important implications for the successful resolution of inflammation.