Earthworm leukocyte populations specifically harbor lysosomal enzymes that may respond to bacterial challenge

Abstract

Earthworm leukocytes (coelomocytes) are responsible for innate cellular immune functions such as phagocytosis and encapsulation against parasites and pathogens. Microbial killing results from the combined action of the phagocytic process with humoral immune factors such as agglutinins (e.g., lectins), lysosomal enzymes (e.g., acid phosphatase, lysozyme), and various cytotoxic and antimicrobial molecules. There is also evidence of weak adaptive immune responses against foreign transplants. This study focused on aspects of the innate immune response. First, anti-human acid phosphatase (anti-AcP) polyclonal antibody characterized different acid hydrolase patterns in coelomocytes. Second, flow cytometry identified a strongly immunoreactive coelomocyte population. Third, ultrastructural and cytochemical analyses revealed acid phosphatase in discrete granules (lysosomes) of effector hyaline and granular coelomocytes but not in mature chloragocytes. Coelomocytes were exposed to bacteria to assess how phagocytosis influences: (a) the production of acid phosphatase using Western blot, and (b) release of acid phosphatase using ELISA from cell-free coelomic fluid. Fourth, after phagocytosis, acid phosphatase levels differed between controls and experimentals. Fifth, we found a 39-kDa molecule that reacted intensely with anti-AcP. Our results suggest that effector earthworm coelomocytes may not eliminate pathogens only by phagocytosis but also by extracellular lysis.

Cathepsin L and cystatin B gene expression discriminates immune cœlomic cells in the leech Theromyzon tessulatum

“…5A). Such ultrastructural features have been described for oligochaetes chloragocytes (or eleocytes) in coelomic cavity [35]. Unlike the hirudinoïd species, T. tessulatum, which belongs to glossiphoniid leeches, does not possess botryoïdal tissue into the coelomic cavity.…”

“…The probes labelled with DIG-UTP were revealed using alkaline phosphataseconjugated antibodies as previously described [16]. For double in situ hybridization, DIGlabelled cathepsin L riboprobes (40-100 ng per slide) and 35 S-labelled cystatin B riboprobes (100 ng or 1 × 10 6 c.p.m. per slide) were mixed in the same hybridization buffer and hybridization was carried out as described before.…”

Still waiting for the toll?

“…This system has already been valuable for gene function research in vertebrates. Upcoming experiments shall prove the usefulness of the method when applied to functional research of putative genes in invertebrate immunity coupled with other previously defined procedures [59][60][61][62][63] ( Table 4). …”

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

“…According to the literature, coelomocytes can be divided into three main populations by light microscopic cytology: hyaline and granular amoebocytes, and chloragocytes; however, two cell populations (large coelomocytes and small coelomocytes) were detected by the first flow cytometric analysis [9]. Our recent flow cytometric and confocal microscopic studies showed three well-separated cell populations, expressing different cell surface and intracellular markers [8,30,31]. Further recent studies identified cellular cytotoxicity related to earthworm coelomocytes [10].…”