A flow cytometric phagocytosis assay was established to investigate the role of anti-merozoite antibody, complement, and cytokines on the phagocytosis of Plasmodium falciparum merozoites by human neutrophils. This assay involved allowing fluorescein isothiocyanate-labeled merozoites to interact with phagocytes and analysis of the cells on a FACScan with Lysis II software. To differentiate the proportion of neutrophil surfacebound merozoites from the merozoites ingested by neutrophils, the fluorescence of bound merozoites was quenched by adding trypan blue. The data showed that sera from malaria-immune individuals in the Solomon Islands and Papua New Guinea promoted merozoite engulfment by neutrophils. The cytokines tumor necrosis factor alpha, gamma interferon, granulocyte-macrophage colony-stimulating factor, and interleukin-1 significantly increased the amount and the rate of merozoite phagocytosis by neutrophils. Optimum merozoite phagocytosis occurred when both cytokines and anti-malarial antibody were present.Immune responses to the merozoite stage are important in resistance to malaria, since this transitory stage, which emerges from schizonts, is responsible for the initiation and perpetuation of both the asexual and sexual parasite life cycles in the host's blood. Vaccine strategies aim to direct the immune response against merozoites (3, 4, 10, 11), since antibodies to merozoite surface proteins have been shown to block adhesion and invasion of host erythrocytes (RBC) (1,5,8,22,27). However, vaccine strategies need to take into consideration not only the merozoite invasion inhibition activity of the antibody but also its ability to promote phagocytosis of merozoites. Phagocytosis of Plasmodium falciparum merozoites has been observed in infected individuals (29, 31). Rapid phagocytosis not only prevents merozoite invasion of RBC but also reduces toxic effects known to be caused by the membrane anchor molecules of merozoite surface proteins (9, 25).Studies of merozoite phagocytosis have been hampered for several reasons. The studies carried out on human monocytes (2, 13, 14) and neutrophils (19) depended on light-microscopic observations. Morphology-based microscopic examination of merozoites (size, 1.0 to 1.2 m) is tedious, subjective, and time-consuming. Studies which used purified merozoites are rare, and in fact, mature schizonts have been commonly used as a source of merozoites (1,4,8,27).We have now developed a nonsubjective and versatile method for measuring phagocytosis of P. falciparum merozoites by human neutrophils by flow cytometric analysis. Purified merozoites were used to ensure that merozoite-specific immune mechanisms could be studied. Using this system, we have reexamined and extended the known immune factors which influence phagocytosis of P. falciparum merozoites by human neutrophils.
MATERIALS AND METHODSReagents. Human recombinant interleukin-1 (IL-1) (specific activity, 3.4 ϫ 10 4 U by lymphocyte-activating assay; Ͼ99% purity), recombinant tumor necrosis factor alpha (rTNF-␣) (sp...