EXTRACELLULAR PRODUCTION OF SINGLET OXYGEN BY STIMULATED MACROPHAGES QUANTIFIED USING 9,10-DIPHENYLANTHRACENE AND PERYLENE IN A POLYSTYRENEFILM

The extracellular production of singlet oxygen (O2(1DELTA(g)) by stimu lated macrophages was measured using a modification of our quantitativ e method initially developed to measure the intracellular production o f O2(1DELTA(g)) by neutrophils (Steinbeck, M. J., Khan, A. U., and Kar novsky, M. J. (1992) J. Biol. Chem. 267, 13425-13433). Glass coverslip s were coated with the specific chemical trap for O2(1DELTA(g)), 9,10- diphenylanthracene (DPA) and perylene, which is an internal standard, in a methylene chloride solution containing 0.3 mg/ml polystyrene. On evaporation, the polystyrene formed an even coating of DPA and perylen e over the surface of a glass coverslip (PDP film). Unstimulated macro phages or macrophages stimulated with 4beta-phorbol 12-myristate 13-ac etate (PMA) or formyl-methionyl-leucyl-phenylalanine (fMLP) were then added to the PDP film in a darkened room and incubated at 37-degrees-C for 30 min in a humidified 5% CO2 atmosphere. Both unstimulated and s timulated cells adhered to the PDP film in approximately equivalent nu mbers. Only stimulated cells produced measurable amounts of O2(1DELTA( g)) in a dose-dependent response to either PMA or fMLP. The production of O2(1DELTA(g)) by macrophages stimulated with PMA was maximal in re sponse to 25 ng, 17.8 +/- 1.3 nmol Of O2(1DELTA(g))/approximately 1.00 x 10(6) cells. The maximal response for fMLP was at a concentration o f 1 muM, 18.4 +/- 1.0 nmol Of O2(1DELTA(g))/approximately 1.00 x 10(6) cells. The specific detection of O2(1DELTA(g)) by this method was con firmed by thermally releasing O2(1DELTA(g)) from the DPA-O2(1DELTA(g)) reaction product, DPA-endoperoxide, regenerating the original DPA com pound. Production of O2(1DELTA(g)) by the stimulated cells was inhibit ed 80-89% by the addition of 60-120 mug of superoxide dismutase, an en zyme that converts superoxide to hydrogen peroxide and ground state mo lecular oxygen or 79-84% with the addition of 2 mM histidine, an avid quencher Of O2(1DELTA(g)). Neither of these additions interfered with adhesion of the cells to the PDP film. The ability of superoxide dismu tase to inhibit the production of O2(1DELTA(g)) suggested that O2(1DEL TA(g)) was produced via a superoxide-dependent route. The ability of a n oxidase to produce O2(1DELTA(g)) secondary to superoxide production was substantiated further using a xanthine oxidase-acetaldehyde system . Purified xanthine oxidase produced both superoxide and O2(1DELTA(g)) , and their production was inhibited by the addition of superoxide dis mutase. The production Of O2(1DELTA(g)) by this system was also verifi ed by thermally reversing the reaction. With the use of the PDP film, we have modified our original method for the intracellular detection O f O2(1DELTA(g)) and have demonstrated that extracellular production of O2(1DELTA(g)) by stimulated macrophages can be quantified and appears to be generated via a superoxide-dependent pathway.