Neutrophil superoxide release is required for spontaneous and FMLP-mediated but not for TNF alpha-mediated apoptosis.

Abstract

Polymorphonuclear leukocyte (PMN) lifespan is characterized by both rapid production and apoptotic cell death. The mechanisms triggering apoptosis in PMN are not completely understood. In this study, the relationship of neutrophil activation and apoptosis as related to released superoxide was investigated. PMN apoptosis was detected by DNA fragmentation, and ultraviolet and light microscopy, and was quantified by flow cytometry; superoxide release was measured by superoxide dismutase-inhibitable reduction of ferricytochrome C. Incubation of PMN with 20 ng/ml tumor necrosis factor (TNF)alpha induced superoxide release (8.8 +/- 7.5 nmol O2-/30 min, n = 7) in normal PMN and also resulted in apoptosis within 2 h, whereas a subactivating dose of 2 ng/ml TNF alpha, which did not trigger superoxide release (3.1 +/- 1.7 nmol O2-, n = 10), did facilitate apoptosis, although to a lesser degree. PMN cultured under nonstimulating conditions underwent apoptotic cell death after 8 h. Exogenous superoxide dismutase did not inhibit apoptosis induced by 20 ng/ml TNF alpha. No upregulation of endogenous manganese superoxide dismutase mRNA expression was observed in response to TNF alpha as measured by reverse transcription PCR. Formyl-methionyl-leucyl-phenylalanine (FMLP) stimulation (10(-7) M) resulting in superoxide release of 31.7 +/- 6.1 nmol O2-/30 min (n = 10) also significantly increased the percentage of apoptosis, but at 24 h (P < 0.05). Exogenous superoxide dismutase did inhibit FMLP-induced apoptosis, as well as apoptosis due to aging in culture. In conclusion, aging and FMLP-stimulated PMN undergo apoptosis by a superoxide release-dependent pathway, whereas TNF alpha-facilitated apoptosis appears to be unrelated to respiratory burst oxidase activity.