Eosinophils from Schistosome-Induced Hepatic Granulomas Produce Superoxide and Hydroxyl Radical

Human peripheral blood eosinophils generate superoxide (O₂^-.) in response to PMA stimulation. These cells are also capable of forming the highly reactive hydroxyl radical (HO^.) by a process that is dependent on the presence of active eosinophil peroxidase. To extend this work to tissue-resident cells, we chose to study a murine model of Schistosoma mansoni infection in which parasite ova induce granulomas whose cellular content is 50% eosinophils. In contrast to peritoneal lavage eosinophils, dispersed granuloma cells were unable to reduce ferricytochrome c (as an indicator of O₂^-.) in response to PMA stimulation. Furthermore, when human neutrophils were pretreated with conditioned medium from the granuloma cells, they also failed to reduce ferricytochrome c following PMA stimulation, implying the existence of an inhibitory factor. However, using a 5,5-dimethyl-1-pyrroline-N-oxide spin-trapping system, we were able to demonstrate significant generation of O₂^-. in response to PMA stimulation, not only in the granuloma cells, but also in the conditioned medium-treated neutrophils, demonstrating that the inhibitory factor was not affecting O₂^-. generation, but rather was interfering with ferricytochrome c reduction. In addition, using an α-(4-pyridyl-1-oxide)-N-terf-butyl-nitrone/ethanol spin-trapping system, we were able to detect HO^. formation by these same cells following PMA stimulation. This HO^. formation was inhibited by superoxide dismutase, azide, and thiocyanide, and NaSCN, consistent with a mechanism requiring O₂^-. and enzymatic peroxidase activity. These results demonstrate that tissue eosinophils associated with the schistosome-induced granuloma have the ability to form both O₂^-. and HO^., and point out potential problems associated with the measurement of O₂^-. in whole tissue preparations.