Reconstitution of a variant macrophage cell line defective in oxygen metabolism with a H2O2-generating system

Abstract

A variant clone, C3C, derived from the cloned macrophage cell line J774.16 lacks the capacity to produce O2- or H2O2 after appropriate stimulation. When the parental and variant cell lines were infected with epimastigotes of Trypanosoma cruzi, the parasites were killed or their growth was inhibited by the parental line, but they grew readily in the variant clone C3C. It was possible to reconstitute the variant cell line with an enzyme system targeted to the lysosomal compartment capable of generating a single oxygen metabolite, H2O2. This was accomplished by allowing the cells to phagocytize zymosan particles covalently coupled with glucose oxidase (GO-Zy particles). Approximately one-third of the H2O2 theoretically expected to be produced by the ingested GO-Zy particles could be detected outside the cells by the cytochrome c peroxidase assay; this fraction may represent the efficiency of extracellular assays for H2O2 production. When T. cruzi-infected clone C3C cells were reconstituted with GO-Zy particles, upon addition of glucose, intracellular killing of the parasites occurred. It was possible to estimate the level of H2O2 production required to kill a single parasite (8.7 x 10(-7) nmol/min) by GO-Zy particles in suspension and to formulate a first approximation of the killing potency of the reconstituted cells--i.e., number of parasites expected to be killed--that correlated well with the observed growth of the parasites intracellularly.