Evidence for perforin-like activity associated with earthworm leukocytes

Earthworm (Eisenia fetida) coelomic fluid contains several leukocytes (coel omocytes): basophils, acidophils and neutrophils as well as chloragocytes. Small coelomocytes and coelomocyte lysate are cytotoxic for the tumor cell target K562. The expression of a lytic factor was investigated by immunocyt ochemistry using light and transmission electron microscopy. A rat-anti-mou se-perforin-mAb labeled mainly small coelomocytes (nearly 20%) as visualize d by light microscopy. TEM analysis using immunogold showed a homogenous la beling in the cytoplasm of small coelomocytes. The highest number of immuno gold particles was estimated in coelomocytes with many small cytoplasmic gr anules. Coelomocytes with large lysosomal granules were also labeled but le ss intensely. No antibody binding was observed for chloragocytes either in light or electron microscopy. This suggests that the perforin-like activity is associated with only one cell type and that chloragocytes are responsib le for other lytic activities. MALDI-MS revealed calreticulin usually assoc iated with perforin in mammalian cells that mediate lysis (e.g. NK, CTL). T ogether, results strongly suggest the presence of putative perforin in eart hworms. This in turn supports the hypothesis that perforin is a conserved c omponent important in immune defense during evolution.