Ivermectin-induced killing of microfilariae in vitro by neutrophils mediated by NO.

Rat neutrophil granulocytes isolated after intraperitoneal casein injection of the donors exhibit high cytotoxic efficacy in vitro against microfilariae of Litomosoides carinii in the presence of ivermectin. Optimum effects of 80-90% killing of microfilariae were obtained with 100 ng ivermectin per milliliter and a microfilariae: cell ratio of 1:100. Spleen cells killed approximately 30% of the microfilariae under these conditions. Cytotoxic effects were independent of any adherence of the cell to the larvae. In contrast to the effects of spleen cells, cytotoxicity of neutrophils completely abrogated when cells and targets were separated by a membrane impermeable for the cells, suggesting a very short-living mediator in the latter case. Correspondingly, cytotoxic effects of neutrophils were completely inhibited by the addition of the arginine analogues NG-monomethyl-L-arginine and L-canavanine, indicating the involvement of reactive nitrogen intermediates. The nitric oxide scavenger hemoglobin also protected the microfilariae. Several compounds which are known to interfere with reactive oxygen intermediates were ineffective. An excess of ferrous ions in the medium in the presence of a reducing agent significantly reduced the cytotoxic efficacy of neutrophils.

Ivermectin-induced cell-dependent lethal effects on litomosoides carinii microfilariae in vitro.

Ivermectin affected the motility of Litomosoides carinii microfilariae in vitro in a dose dependent manner but did not completely immobilize the larvae and had no lethal effects when tested up to a concentration of 1000 ng/ml. However, killing of microfilariae was induced by ivermectin in vitro in the presence of spleen cells of Mastomys coucha or rats within 14 h. Optimum effects occurred at drug levels of 10-100 ng ivermectin/ml. Addition of infection serum led to increased cytotoxicity when compared with normal serum. Pretreatment in vitro of L. carinii microfilariae with ivermectin in cell-free medium and subsequent exposure to spleen cells caused also cytotoxic effects which appeared to be accelerated in comparison with simultaneous exposure of microfilariae to ivermectin and cells. Pretreated microfilariae, injected intravenously into naive M. coucha were rapidly eliminated from the blood of the recipients. These results suggest that the microfilariae become altered by the drug and thus susceptible to cell-mediated cytotoxic effects. Cytotoxicity did not depend on the attachment of cells to L. carinii microfilariae and was also induced when targets and effector cells were separated by membranes impermeable for cells. Thus ivermectin-induced cellular cytotoxicity to L. carinii microfilariae is at least partly mediated by soluble factors released by effective cells.