Induction of apoptosis-like cell death by coelomocyte extracts from Eisenia andrei earthworms.

Earthworm's innate immunity is maintained by cellular and humoral components. Our objective was to characterize the cytotoxicity leading to target cell death caused by earthworm coelomocytes. Coelomocyte lysates induced strong cytotoxicity in tumor cell lines. Transmission electron microscopy revealed cell membrane and intracellular damage in cells treated with coelomocyte lysates. Using TUNEL-assay, within 5 min of incubation we detected DNA fragmentation. Moreover, we found phosphatidylserine translocation in target cell-membranes. Furthermore, we detected dose-dependent Ca(2+) influx and decrease of mitochondrial membrane potential in coelomocyte lysate-treated cells. Interestingly, caspase 3/8 activation was undetectable in exposed tumor cells. One such cytotoxic molecule, lysenin identified in earthworms binds to sphingomyelin and causes target cell lysis in vertebrates. Pretreatment with our anti-lysenin monoclonal antibody rescued the majority but not all target cells from coelomocyte induced death. These data suggest that, not only lysenin but also other factors participate in the caspase-independent apoptosis induced by coelomocytes.

Cold-stress induced formation of calcium and phosphorous rich chloragocyte granules (chloragosomes) in the earthworm Eisenia fetida.

The cytochemical and functional characteristics of chloragocytes of both 'control' and cold-stressed Eisenia fetida were examined. Flow cytometry revealed the heterogeneity of chloragocytes: the first group was characterized by low, the second one by high acid phosphatase (AcP) content. In 'control' animals the former, in cold-stressed ones the latter type were the dominant form. The elevated AcP-activity correlated with the accumulation of autophagic vacuoles (AVs) in chloragocytes. Both AVs and all small chloragosomes showed high AcP activity, while most of the large chloragosomes did not display any. Most 'control' granules (0.75-1.25 µm) contained high amounts of Ca and P, with less and variable quantities of S, Cl, K, Fe and Zn. Small chloragosomes with low Ca and P concentrations were seldom found. In cold-stressed animals the number of small granules (0.25-0.75 µm) increased up to 40% of total population. Their Ca and P contents were significantly lower; S and Fe concentrations were higher than those of large chloragosomes (1.0-1.5 µm). Our results prove that the formation and elemental composition of chloragosomes can be influenced by environmental stressors and suggest that the mature chloragosomes are tertiary lysosomes and their formation is coupled to autophagocytosis.