The precise molecular mechanisms by which phagocytic cells effect their microbicidal function is unknown. Potential mechanisms that have been identified include low intracellular pH, lysozyme, cationic proteins, lysosomal hydrolases, and oxygen metabolites (1-4). Largely from the study of leukocytes of patients with rare genetic deficiency diseases--which include chronic granulomatous disease (CGD),t gutathione peroxidase, and the reductase deficiencies, glucose-6-phosphate dehydrogenase deficiency and myeloperoxidase deficiency--it has emerged that there exists a close correlation between the respiratory burst in normal phagocytic cells and their cytocidal activity, both of which are impaired in CGD (4-7). It has long been established that one of the early sequelae of the phagocytic event in macrophages is a stimulation of the hexose monophosphate (HMP) shunt, and recent studies indicate that the ability of macrophages to kill parasites intracellularly and possibly tumor cells extracellularly is dependent upon the oxidative burst and production of oxygen radicals including superoxide anion (O2-), hydrogen peroxide (H202), hydroxyl radical, and, possibly, singlet oxygen; 102 (1-4, 8-10). The nature of the cellular enzyme or molecule(s) responsible for the initial reduction of oxygen, the biochemical nature of the electron transferring molecule, and the intracellular localization of the oxidative bactericidal mechanism and the mechanisms of its regulation in the macrophage remain important problems.In previous studies (1 I, 12) we have endeavored to design selective genetic strategies for producing variants in defined functions of cloned macrophage-like cell lines, in the hope that they may yield tractable models for the study of the molecular basis for various macrophage functions. In the present report we describe a cloned, macrophage-like cell line derived from a murine reticulum cell sarcoma that can be stimulated to oxidize glucose via the HMP shunt, and produce 02-and H202, which we believe represents a useful model of activated primary macrophages. A simple strategy was employed to select for variants lacking the ability to reduce nitroblue tetrazolium (NBT), presumably by O2-, which has led to development of a series of clones defective in oxidative metabolism. These macrophage-variant clones lack the * Supported by U. S.

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

The present study investigated the role of antigen-presenting cells (APC) in the activation of noncytolytic T cells against tumor antigens. The noncytolytic-type T cells exerted their antitumor effect by producing gamma-interferon (IFN-gamma) and by activating macrophages as the ultimate effectors. The production of IFN-gamma by these noncytolytic T cells following the stimulation with tumor cells required the participation of Ia+ APC, since the depletion of APC from cultures of tumor-immunized spleen cells resulted in almost complete inhibition of the IFN-gamma production. Both L3T4+ and Lyt-2+ subsets of T cells were capable of producing IFN-gamma, and the requirement of APC for the production of IFN-gamma was the case irrespective of whether noncytolytic T cells were of L3T4+ or Lyt-2+ phenotype. More importantly, it was demonstrated that the production of IFN-gamma by L3T4+ and Lyt-2+ T cells was inhibited by addition of the respective anti-class II and anti-class I H-2 antibody to cultures. These results indicate that antitumor L3T4+ or Lyt-2+ noncytolytic T cells are activated for the IFN-gamma production by recognizing tumor antigens in the context of self-class II or -class I H-2 molecules on APC.

No abstract

No abstract