Mechanism of lethal effect of human serum upon Leishmania donovani

In order to gain greater understanding of potential host defense mechanisms against Leishmania donovani, we examined the effect of nonimmune, human serum upon promastigotes and amastigotes. Fresh sera were found to be lethal for promastigotes, but had no detectable effect on amastigotes. Serum exposed promastigotes became immotile, did not take up neutral red dye, appeared to be disrupted, and failed to recover after further incubation in fresh media. Heat labile components were required for promastigote killing since heat-inactivated serum (56 degrees C, 30 min) agglutinated but did not kill them. Sera that lacked either the 5th or 6th complement (C) component had no effect when used alone, but when used together, were lethal, indicating that activation of the membrane attack complex (C5b-C9) ws necessary for the lethal effect. The mode of C activation was determined by using serum with complete, selective, deficiency of C2, and normal serum chelated with Mg-EGTA. The C2-deficient serum killed promastigotes only after the addition of purified C2, and Mg-EGTA chelated serum had no detectable lethal effect. Thus, promastigotes appeared to activate C through the classical pathway. Human IgG and IgM, detected with 125I-anti-human antibody, bound to promastigotes. Removal of antibody from serum by absorption with promastigotes eliminated the lethal effect. The effect was restored by addition of heat-inactivated serum to absorbed serum. We conclude that promastigotes bind antibody and are killed by activation of the membrane attack complex of C through the classical pathway.