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 02 or H202 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, H202. This was accomplished by allowing the cells to phagocytize zymosan particles covalently coupled with glucose oxidase (GO-Zy particles). Approximately onethird of the H202 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 H202 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 H202 production required to kill a single parasite (8.7 x 10' nmol/min) by GO-Zy particles in suspension and to formulate a first approximation of the killing potency ofthe reconstituted cells-i.e., number of parasites expected to be killed-that correlated well with the observed growth of the parasites intracellularly.Appreciation of the importance of oxidative cytocidal mechanisms in intracellular killing of a variety of microorganisms ingested by phagocytic cells derives in part from the study of patients with a variety of genetic deficiency diseases. Polymorphonuclear leukocytes (PMN) and monocytes from patients with chronic granulomatous disease (CGD), which serves as the paradigm of such conditions, fail to kill a variety of bacteria and are known to be defective in oxygen metabolism (1-7). A number of oxygen metabolites produced by phagocytic cells could mediate this cytocidal activity, including°a, OH-, singlet 2, and hypohalide. There are very few instances in which the precise oxygen metabolite responsible for killing a particular organism within cells has been established.For the past several years we and others have developed and studied continuous cloned macrophage-like cell lines as models for understanding mononuclear phagocyte function (8-10). We have previously described a murine cloned macrophage cell line, J774.16 which, upon stimulation with phorbol 12-myristate 13-acetate or aggregated immunoglobulins, oxidizes glucose via the hexose monophosphate shunt and produces°-and

Interaction of primate alveolar macrophages and Legionella pneumophila.

“…Recent reports have implicated reactive oxygen metabolites, formed by the respiratory burst, in the antimicrobial effect of mononuclear phagocytes on intracellular protozoan pathogens (24,(30)(31)(32)(33)(34)(35)(36). Reduction of NBT to blue-black formazan, visualized microscopically as a ring of NBT-positive material around organisms within macrophage phagosomes, has been employed as a measure of the respiratory burst (24,34).…”

Induction of Apoptosis in Macrophage Cell Line, J774, by the Cell‐Free Supernatant from Pseudomonas aeruginosa

“…Bloom. The macrophages were cultured in Dulbecco's modified Eagle's medium (DMEM) (Nissui Pharmaceutical Co., Ltd., Japan) supplemented with 10% fetal bovine serum (FBS) and 50 U of penicillinstreptomycin per ml at 37 C in the presence of 5% CO 2 overnight (25). In some experiments, the cells were cultured in culture dishes containing glass slides.…”

Chagas' disease

“…Within the parasitophorous vacuole, parasites may be killed by cytocidal mechanisms such as production of hydrogen peroxide. This process may be associated with lysosomal fusion (247,256 …”