Oxidant Generation by Single Infected Monocytes after Short-Term Fluorescence Labeling of a Protozoan Parasite

Abstract

Leishmania spp. are intracellular protozoa residing in mononuclear phagocytes. Leishmania organisms are susceptible to microbicidal responses generated in response to phagocytosis. Assuming that both phagocyte and parasite populations are heterogeneous, it is advantageous to examine the response of individual cells phagocytosing living parasites. Because Leishmania spp. lose virulence during the raising of transfectants, we developed a method to label live Leishmania chagasi short-term with fluorescent dyes. Up to six parasite divisions were detected by flow cytometry after labeling with carboxyfluorescein diacetate succinimidyl ester (CFSE), dioctadecyl-tetramethylindo carbocyanine perchlorate, or chloromethyl tetramethylrhodamine. Labeled parasites entered mononuclear phagocytes as determined by confocal and time-lapse microscopy. Dihydroethidium (DHE) was used to detect macrophage-derived oxidants generated during phagocytosis. Presumably Leishmania organisms are opsonized with host serum/tissue components such as complement prior to phagocytosis. Therefore, we investigated the effects of opsonization and found that this increased the efficiency of CFSE-labeled parasite entry into monocytes (84.6% ؎ 8.8% versus 20.2% ؎ 3.8% monocytes infected; P < 0.001). Opsonization also increased the percentage of phagocytes undergoing a respiratory burst (66.0% ؎ 6.3% versus 41.0% ؎ 8.3% of monocytes containing CFSE-labeled parasites; P < 0.001) and the magnitude of oxidant generation by each infected monocyte. Inhibitor data indicated that DHE was oxidized by products of the NADPH oxidase. These data suggest that opsonized serum components such as complement lead to more efficient entry of Leishmania into their target cells but at the same time activate the phagocyte oxidase to generate microbicidal products in infected cells. The parasite must balance these positive and negative survival effects in order to initiate a viable infection.Leishmania protozoa are intracellular parasites residing in macrophages or other mononuclear cells of their mammalian hosts (13). The parasites are inoculated into mammalian cutaneous tissue in the form of a promastigote, which must locate an appropriate phagocyte and stimulate its own internalization in order to survive. However, promastigotes are susceptible to killing by the reactive oxygen species generated by the macrophage during phagocytosis (19,20,27). Once intracellular, parasites convert to the more resistant obligate intracellular amastigote form. The Leishmania spp. have developed a number of mechanisms to protect themselves from oxidant-mediated killing while in their susceptible promastigote form. These include a surface glycolipid coat called lipophosphoglycan, antioxidant defense molecules such as iron superoxide dismutase, peroxidoxins, and a unique thiol-reducing factor, trypanothione (16,33,34). Furthermore, Leishmania spp. have the ability to suppress gamma interferon (IFN-␥)-mediated activation of macrophages through enhancing macrophage phosphatase activity (9, 30).Th...

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