CHEMILUMINESCENCE IN ACTIVATED HUMAN NEUTROPHILS - ROLE OF BUFFERS AND SCAVENGERS

Human neutrophils (PMNs) suspended in Hanks' balanced salt solution (H BSS), which are stimulated either by polycation-opsonized streptococci or by phorbol myristate acetate (PMA), generate nonamplified (CL), lu minol-dependent (LDCL), and lucigenin-dependent chemiluminescence (LUC DCL). Treatment of activated PMNs with azide yielded a very intense CL response, but only a small LDCL or LUCDCL responses, when horse radis h peroxidase (HRP) was added. Both CL and LDCL depend on the generatio n of superoxide and on myeloperoxidase (MPO). Treatment of PMNs with a zide followed either by dimethylthiourea (DMTU), deferoxamine, EDTA, o r detapac generated very little CL upon addition of HRP, suggesting th at CL is the result of the interaction among H2O2, a peroxidase, and t race metals. In a cell-free system practically no CL was generated whe n H2O2 was mixed with HRP in distilled water (DW). On the other hand s ignificant CL was generated when either HBSS or RPMI media was employe d. In both cases CL was markedly depressed either by deferoxamine or b y EDTA, suggesting that these media might be contaminated by trace met als, which catalyzed a Fenton-driven reaction. Both HEPES and Tris buf fers, when added to DW, failed to support significant HRP-induced CL. Nitrilotriacetate (NTA) chelates of Mn2+, Fe2+, Cu2+, and CO2+ very ma rkedly enhanced CL induced by mixtures of H2O2 and HRP when distilled water was the supporting medium. Both HEPES and Tris buffer when added to DW strongly quenced NTA-metal-catalyzed CL. None of the NTA-metal chelates could boost CL generation by activated PMNs, because the salt s in HBSS and RPMI interfered with the activity of the added metals. C L and LDCL of activated PMNs was enhanced by aminotriazole, but strong ly inhibited by diphenylene iodonium (an inhibitor of NADPH oxidase) b y azide, sodium cyanide (CN), cimetidine, histidine, benzoate, DMTU an d moderately by superoxide dismutase (SOD) and by deferoxamine. LUCDCL was markedly inhibited only by SOD but was boosted by CN. Taken toget her, it is suggested that CL generated by stimulated PMNs might be the result of the interactions among, NADPH oxidase, (inhibitable by diph enylene iodonium), MPO (inhibitable by sodium azide), H2O2 probably of intracellular origin (inhibitable by DMTU but not by catalase), and t race metals that contaminate salt solutions. The nature of the salt so lutions employed to measure CL in activated PMNs is critical.