A study of the differential respiratory burst activity elicited by promastigotes and amastigotes of Leishmania donovani in murine resident peritoneal macrophages

Abstract

Acridine orange and ethidium bromide and a combination of fluorescent and transmitted light microscopy used in conjunction with the qualitative nitroblue tetrazolium assay for superoxide anion (O2-) release demonstrated dramatic differences in the binding of and respiratory burst (RB) activity elicited by promastigotes and amastigotes of Leishmania donovani in resident peritoneal macrophages (M phi) from C57BL/10ScSn mice. When amastigotes were incubated with M phi for 30 min the number of parasites per 100 M phi was 2-4-fold higher, a higher proportion of M phi became infected and the mean number of parasites per infected M phi was higher than in promastigote infections. RB activity was higher for promastigotes than amastigotes both in terms of the percentage of infected M phi containing formazan positive parasites and the percentage of individual formazan positive parasites. In an attempt to explain the differential response to promastigotes and amastigotes, RB activity was examined for sodium azide-treated, glutaraldehyde-fixed and heat-killed parasites and for various transformation intermediates between amastigotes and promastigotes. Binding and RB activity were also examined in conjunction with competitive binding assays designed to determine the specific receptors involved in ligand binding of both forms of the parasite to the M phi. The results indicate that, while amastigotes may possess an azide-sensitive mechanism which either competes for O2- produced or causes localized inactivation of RB activity, this cannot account for the full magnitude of the difference between the two forms of the parasite. The transformation and competitive binding studies suggest that the more likely explanation lies in both qualitative and quantitative differences in the distribution of surface ligands involved in binding the parasite to the M phi plasma membrane and that the well characterized mannose/fucose receptor may be important in promastigote, but not amastigote, binding and RB activity.