THE DAB-MN- VALIDATION OF SPECIFICITY FOR SUPEROXIDE(+ CYTOCHEMICAL METHOD REVISITED )

We wished to assess whether the previously developed 3,3'-diaminobenzi dine (DAB)Mn++ cytochemical method, purportedly specific for superoxid e localization, is detecting superoxide O2.- and/or the superoxide pro duct, O2(1DELTAg). We show here that polymorphonuclear leukocytes (PMN s) produce O2(1DELTAg) extracellularly in response to non-phagocytic s timuli and that this production is inhibited by addition of superoxide dismutase, an enzyme typically used to demonstrate that a reaction is mediated by O2.-. Because O2(1DELTAg) is highly reactive and can be g enerated from O2.-, the reactivity of a pure chemical source of O2(1DE LTAg) with the cytochemical probe DAB was examined in the presence and absence of Mn++. Reactions between DAB and O2(1DELTAg), thermally rel eased from 1,4-dimethyl-napthalene-1,4-endoperoxide (DNE), indicated t hat O2(1DELTAg) directly reacted with DAB, forming an insoluble DAB po lymer, and that this reaction was increased by the presence of Mn++. T he direct reaction of O2(1DELTAg) with DAB was confirmed using near-IR emission spectroscopy. The near-IR emission spectrum of DNE as it was warmed showed the characteristic energy emission peak of O2(1DELTAg) and the intensity of this peak was reduced by the addition of DAB; k(q ) = 1.7 x 10(8) M-1 sec-1. The requirement of Mn++ for oxidation of DA B by O2.- was reconfirmed using potassium superoxide as a pure chemica l source of O2.-. In cell studies, however, DAB deposits were not obse rved in PMNs stimulated under conditions that lead to O2(1DELTAg) prod uction [e.g., 0.040 or 0.162 muM 4B-phorbol-12-myristate-13-acetate (P MA)], regardless of whether Mn++ was present in the cytochemical mediu m. Nor were DAB deposits found in cells stimulated with PMA in the abs ence of Mn++ of in unstimulated PMNs. Only cells incubated in cytochem ical medium containing Mn++ and stimulated to produce large amounts of O2.- (e.g., 3.24 muM PMA) contained DAB deposits. In summary, the DAB -Mn++ cytochemical method remains an excellent method for localizing t he production sites of O2.-, since the concentration of O2(1DELTAg) wi thin vesicles of stimulated cells is too low to directly oxidize DAB t o an electron-dense deposit.