Spin-trapping detection of superoxides in polymorphonuclear leukocytes stimulated with serum-opsonized zymosan

To clarify where oxygen radicals are generated in polymorphonuclear leukocytes (PMNs) during phagocytosis, superoxides (O2-) from opsonized symosan (OZ)--stimulated human PMNs were detected by the ESR and spin-trapping methods. PMNs were preactivated with OZ for the indicated periods of time at 37 degrees C. Then a spin-trapping agent, 5, 5-dimethyl-1-pyrroline N-oxide (DMPO), was added to them, and they were further incubated for 30 sec for ESR observations. The ESR spectra consisted of two components due to the DMPO-OOH and DMPO-OH spin adducts. To clarify where these spin-adducts were present, cells were separated from extracellular fluid by brief centrifugation and resuspended in Hanks' balanced salt solution. ESR examination of two fractions showed that the DMPO-OOH adducts was present in the cell fraction, whereas the DMPO-OH adducts were present in the extracellular fluid. When DMSO was used as a scavenger of hydroxyl radicals (.OH), DMPO-CH3 adducts were observed in the fluid fraction but not in the cell fraction. Both spin adducts were completely abolished by Cu, Zn-SOD but not catalase. These results indicated that O2- were produced inside phagosomes of OZ-stimulated PMNs and .OH were produced outside them by spontaneous decomposition of the DMPO-OOH adducts.