Earthworm leukocytes kill HeLa, HEp-2, PC-12 and PA317 cells in vitro

Earthworm coelomic fluid contains biologically active molecules and leukocytes that participate in phagocytosis, encapsulation. Presumably they synthesize and secrete several effector modulators of innate immune responses such as antibacterial molecules, cytotoxic proteins and cytokines. Several lytic molecules have been detected in coelomic fluid previously but it is not yet clear which are actually released from the coelomocytes. Our aim was to analyze the cytotoxic effects of coelomocytes on mammalian target cells and to provide evidence that the lytic factors originate from coelomocytes. Cell-free coelomic fluid, supernatants of short-term cultured coelomocytes, and lysates from coelomocytes--derived by mechanical and detergent extraction--were used in cytotoxicity assays performed on different mammalian standard tumor cell lines and mouse fibroblasts. We used native and denaturized (using proteinase K, and trypsin digestions, or heat-inactivation) coelomocyte lysates (CCL). The viability controls of targeted cells were made by measuring photometrically and analyzing by inverted microscopy. According to our results the coelomic fluid, the supernatant of cultured coelomocytes, and the CCL significantly decreased ratios of living cells compared to controls in a dose-dependent manner. Our experiments performed with CCLs suggest that coelomocytes are responsible for the productions of cytotoxic components presumably proteins.