Robert Feulgen Lecture 1994. Cytochemistry and reactive oxygen species: a retrospective

This retrospective reviews the methodology we have developed over several decades for detecting reactive oxygen species (ROS), using the activated polymorphonuclear leukocyte (PMN) as the paradigm of a cell which vigorously generates ROS through activation of NADPH oxidase. In the seventies, the sites of ROS generation by PMN were not clear from biochemical data, and we sought to develop new methods for the cytochemical localization of O2.-, H2O2, and the H2O2-myeloperoxidase (MPO)-halide system. The H2O2-MPO-halide system in phagocytosing cells was localized at the fine structural level by our development of 3,3'-diaminobenzidine (DAB) as a cytochemical probe for detecting peroxidase activities. Using DAB and exogenous H2O2, we confirmed that azurophil granules discharged MPO into the phagosome, and using particles coated with DAB and relying on endogenous H2O2 to yield oxidized DAB, H2O2 was localized to phagolysosomes. The subcellular sites of H2O2 generation were shown using cerium ions which react with H2O2 and precipitate electron opaque cerium perhydroxides (Ce(OH)2OOH and Ce(OH)3OOH). The results suggested that NADPH oxidase is associated with the plasma lemma, and that the enzyme enters the phagosome along with the invaginating plasmalemma, accounting for the presence of H2O2 in the phagosome. As O2.- is the major product of NADPH oxidase, its detection was of some importance. Based on the concept that O2.- oxidizes Mn2+ to Mn3+, and Mn3+ oxidizes DAB, a medium containing DAB-Mn2+ was used to localize sites of O2.- production in stimulated PMN. The localizations were, as expected, similar to those for H2O2. These techniques have been of considerable usefulness and in general provide the foundation for cytochemistry of ROS in other systems.