Monoclonal antibody recognized hemocyte subpopulations in juvenile and adult Lymnaea stagnalis: functional characteristics and lectin binding.

The mouse monoclonal antibody LS1 recognizes a membrane epitope present on circulating hemocytes of the gastropod mollusc Lymnaea stagnalis. In both juvenile and adult pond snails, LS1+ (LS1 positive) hemocytes have the morphology of immature cells. The percentage of LS1+ hemocytes is higher in juveniles (ca. 39%) than it is in adults (ca. 14%). Functional characteristics of LS1+ hemocytes and lectin binding to these cells were studied. In both age groups, the proliferative activity, as measured by the incorporation of deoxybromouridine, is much higher for LS1+ hemocytes than it is for LS1- (LS1 negative) cells. LS1+ hemocytes are phagocytically less active and have a lower lysosomal enzyme (peroxidase) content as compared to hemocytes that lack the epitope. Histochemical staining of the total population of circulating hemocytes shows that the lectins DBA, BS-l-A4 and BS-l-B4, PNA, SBA and ECA do not react with the hemocytes. LTA, APA, WGA, Con A and LCA bind to all hemocytes. RCA and STA recognize surface carbohydrate moieties present on subpopulations of hemocytes only. The LS1+ hemocyte population virtually lacks the carbohydrate residues recognized by STA, whereas the LS1- population never shows binding of RCA. Our results support the findings that the LS1 epitope is a membrane marker of less differentiated hemocytes in both juvenile and adult L. stagnalis. Furthermore, they suggest a correlation between the presence of the LS1 epitope and the absence of STA binding, whereas absence of the LS1 marker may correlate with the presence of a sugar recognized by RCA.

The production of toxic oxygen metabolites by hemocytes of different snail species.

The phagocytic hemocytes of four snail species were investigated for their ability to generate reactive oxygen metabolites upon stimulation by foreign material. Hemocytes of the pond snail Lymnaea stagnalis and of the garden snail Helix aspersa showed a luminol-dependent chemiluminescence (CL) when they phagocytosed zymosan particles. This CL was inhibited by superoxide dismutase (SOD) and sodium azide, indicating the involvement of oxygen intermediates. Hemocytes of the planorbid snails Planorbarius corneus and Biomphalaria glabrata did not give a detectable CL response. This is probably due to the presence of hemoglobin in the hemolymph; after isolation of the cells and subsequent stimulation, however, still no CL could be measured. Hemocytes of all four snail species showed a SOD-sensitive nitroblue tetrazolium (NBT) reduction, indicating the generation of superoxide anions. Regarding the NBT reaction, no differences were observed between strains of B. glabrata that were susceptible or resistant to PR-1 Schistosoma mansoni; neither did exposure to the parasite have an effect on the ability of the hemocytes to reduce NBT. Also, hemocytes from Trichobilharzia ocellata-infected L. stagnalis did not react differently from hemocytes of uninfected snails. It is now clear that phagocytically stimulated hemocytes of several molluscan species can generate reactive forms of oxygen; the relevance of this fact for the phylogeny of killing systems operative in leukocytes is discussed.

Possible role of reactive forms of oxygen in in vitro killing of Schistosoma mansoni sporocysts by hemocytes of Lymnaea stagnalis.

The interactions between sporocysts of Schistosoma mansoni and hemocytes of the pond snail Lymnaea stagnalis were studied in an in vitro system. All sporocysts were encapsulated and killed in the hemolymph of juvenile or adult snails within 24 h; the killing patterns of both age groups were not very different. The separation of hemolymph into hemocytes and plasma and subsequent incubation of sporocysts showed that the cells, not the plasma, were responsible for parasite killing. The possible involvement of reactive oxygen intermediates and the enzyme peroxidase in the cytotoxic mechanism was studied. Chemiluminescence (CL) could not be detected during the hemocyte-parasite encounter. Histochemical staining with nitroblue tetrazolium (indicative for superoxide) and diaminobenzidine (indicative for hydrogen peroxide) showed that oxygen intermediates were produced by snail hemocytes that encapsulated sporocysts. Histochemistry also suggested the presence of peroxidase activity at the hemocyte-sporocyst interface. The cytotoxic mechanism underlying parasite killing by hemocytes and the role of reactive forms of oxygen and peroxidase are discussed.

A comparative study on the internal defence system of juvenile and adult Lymnaea stagnalis.

The immunological immaturity of juvenile specimens of some snail species, e.g. Lymnaea stagnalis, may contribute to their greater susceptibility to infection by schistosome parasites. In a comparison between juvenile and adult specimens of the pond snail L. stagnalis, we have shown that the blood cells (amoebocytes) of juvenile snails are less efficient at phagocytosing: fewer amoebocytes are competent and the average number of particles engulfed per cell is lower. This functional immaturity seems to correlate with morphological immaturity of the amoebocytes. Opsonic and haemagglutinating activities are low in juvenile snail plasma, but much higher in adult plasma. Finally, however, the initial rate at which injected bacteria are eliminated from the circulation seems only slightly slower in juvenile snails than it is in adults.