Informace o publikaci

Silkworm (Bombyx mori) hemocytes do not produce reactive oxygen metabolites as a part of defence mechanisms.


HYRŠL Pavel LOJEK Antonín ČÍŽ Milan KUBALA Lukáš

Rok publikování 2003
Druh Článek ve sborníku
Konference 1st European workshop on the analysis of phagocyte functions.
Fakulta / Pracoviště MU

Přírodovědecká fakulta

Popis Hemocytes are basic to the invertebrate innate immune system, which is divided into cellular and humoral defence responses. Four basic types of hemocyte immune reactions have been described: phagocytosis, encapsulation, nodulation and coagulation. The generation of reactive oxygen metabolites (ROM) by hemocytes has been previously considered an important microbicidal defence mechanism of some invertebrates similar to those in vertebrate phagocytes. However, reports about similar mechanisms in insects are controversial. The aim of this study was to investigate whether hemocytes of Bombyx mori (Lepidoptera) larvae can produce ROM as a tool for the oxidative killing of invading pathogens. Understanding of defence mechanisms of Bombyx mori has high significance not only for a better insight into the evolution of animal immune systems but also has a great impact on silk production sericulture. The production of ROM measured as a luminol- and lucigenin-enhanced chemiluminescence of unstimulated or stimulated (zymosan particles, phorbol myristate acetate, calcium ionophore, rice starch or bacterium Xenorhabdus nematophila) hemocytes in whole hemolymph was not detectable. There were no changes in background CL activity when different volumes of hemolymph at various temperatures (20, 25, 37XC) were analysed. It was observed that the total radical-trapping antioxidative potential (TRAP) of the hemolymph was significantly higher in comparison with human blood plasma (5.97 ĄÓ 2.26 Ýmol/ml and 1.00 ĄÓ 0.13 Ýmol/ml, resp.) when measured by the chemiluminescence method. To eliminate the influence of high antioxidative potential of hemolymph on the analyses of ROM production, isolated hemocytes in several concentrations (0.1, 0.5, 1.0 or 1.5*106/measuring cuvette) were used in the subsequent experiments. However, spontaneous as well as the activated generation of ROM by isolated hemocytes remained at the background level. Spectrophotometry methods confirmed results obtained by the chemiluminescence technique. No superoxide anion production by hemocytes activated by PMA, ZP or Xenorhabdus nematophila was observed using an NBT test or the test based on the reduction of cytochrome c (the values were under the detection limit). Similar results were obtained by fluorometry: DHR-123 and HE assays (specific for hydrogen peroxide and superoxide anion, resp.) proved no ROM production in unstimulated as well as in activated (PMA, ZP, Ca-I, rice starch) hemocytes of Bombyx mori. To validate all the methods used, leukocytes isolated from human peripheral whole blood were applied as a positive control. In this case, significant ROM production was observed for an activators used. To conclude, none of the several experimental approaches used in this study indicated the production of ROM by hemocytes of Bombyx mori larvae as a part of their immune response.

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