On radical production by PMA-stimulated neutrophils as monitored by luminol-amplified chemiluminescence

The means by which neutrophils within the body ward off infectious and neoplastic processes by the activation of molecular oxygen, as well as how such mechanisms dysfunction, is the subject of extensive ongoing research. Most previous studies of neutrophil activation indicate that there is a transient production of reactive oxygen species. Luminol-amplified chemiluminescence surveillance of O₂ ^{{dot minus}} and H₂O₂ supported these generalfindings. Yet, recent studies showed that production of reactive oxygen species by PMA-stimulated neutrophils is not transient by persistent; however, luminol-dependent methods do not corroborate such findings. The kinetics of O₂ ^{{dot minus}} production by human neutrophils were studied using luminol-amplified chemiluminescence (CL), spin trapping combined with electron spin resonance detection, and ferricytochrome c reduction. The effects of pH and O₂ level on luminol-amplified CL were determined using hypoxanthine/xanthine oxidase to produce O₂ ^{{dot minus}} and H₂O₂ in cell-free systems. As we have found by electron spin resonance and ferricytochrome c reduction, stimulated neutrophils continued to generate O₂ ^{{dot minus}} for several hours, yet when luminol-amplified CL was used to continuously follow radical production, CL was shortly lost. Similar loss of CL was observed with continuous enzymatic formation of O₂ ^{{dot minus}} and H₂O₂. The failure of the CL assay to report O₂ ^{{dot minus}} and H₂O₂ formation results from some luminol reaction product which interferes with the light reaction. Our results show that the cells are operative for long periods indicating that cell exposure to prolonged O₂ ^{{dot minus}} fluxes does not terminate radical production, and even when pH, [O₂], and reagents are optimized, the use of luminol-amplified CL is not a valid assay for continuous monitoring of O₂ ^{{dot minus}} and H₂O₂ generated by either stimulated neutrophils or in cell-free systems.