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Ivermectin-induced killing of microfilariae in vitro by neutrophils mediated by NO
H Zahner1, D Schmidtchen, J A Mutasa
1Institut für Parasitologie, Justus-Liebig-Universität Giessen, Federal Republic of Germany.
Abstract:
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.
Insights
Rat neutrophils and ivermectin effectively kill microfilariae in vitro. This cytotoxic effect involves reactive nitrogen intermediates, not reactive oxygen intermediates, highlighting a novel therapeutic pathway.
Area of Science:
- Immunology
- Parasitology
- Biochemistry
Background:
- Microfilariae, the larval stage of parasitic nematodes like Litomosoides carinii, are a target for antiparasitic drugs.
- Neutrophils are key immune cells involved in host defense against pathogens.
Purpose of the Study:
- To investigate the in vitro cytotoxic efficacy of rat neutrophil granulocytes against Litomosoides carinii microfilariae.
- To elucidate the mechanism of action, particularly the role of reactive nitrogen and oxygen intermediates.
Main Methods:
- Isolation of rat neutrophil granulocytes following casein injection.
- In vitro co-incubation of neutrophils and microfilariae with varying concentrations of ivermectin.
- Assessment of microfilariae killing using different cell-to-larvae ratios and separation methods (membranes).
- Inhibition studies using arginine analogues (NG-monomethyl-L-arginine, L-canavanine) and nitric oxide scavengers (hemoglobin).
Main Results:
- Neutrophils demonstrated significant in vitro cytotoxicity against microfilariae, achieving 80-90% killing with 100 ng/mL ivermectin and a 1:100 microfilariae:cell ratio.
- Cytotoxicity was mediated by a short-lived factor released by neutrophils, independent of cell adherence.
- The effect was abrogated by inhibitors of nitric oxide synthesis and nitric oxide scavengers, indicating the involvement of reactive nitrogen intermediates.
- Reactive oxygen intermediates did not appear to play a significant role, and excess ferrous ions reduced efficacy.
Conclusions:
- Rat neutrophils, in combination with ivermectin, exhibit potent in vitro anti-microfilarial activity.
- The mechanism relies on reactive nitrogen intermediates, suggesting a targeted approach for parasitic infections.
- This finding opens avenues for developing novel therapeutic strategies against filarial parasites.