Protein kinase C signalling in 'Lymnaea stagnalis' haemocytes: a role in molluscan defence

The evolutionarily conserved protein kinase C (PKC) pathway plays an important role in vertebrate immunity, but its role in regulating defence processes in molluscs is poorly understood. Innate defence in the freshwater snail Lymnaea stagnalis, intermediate host of the avian schistosome Trichobilharzia ocellata, is largely assured by macrophage-like cells called haemocytes. Protein kinase C-like proteins were identified in L. stagnalis haemocytes using western blotting with phosphospecific anti¬PKC antibodies; the proteins appeared most similar to PKCa. and had an approximate molecular weight of 85 kDa. The phosphorylation (activation) of PKC increased over S¬lO minutes in response to the glucan-based compounds laminarin and zymosan, the bacterial endotoxin, lipopolysaccharide (LPS), and a PKC activator phorbol 12- myristate 13-acetate (PMA), with a 3.S-fold increase observed following challenge with laminarin. Activation of PKC was also shown by immunocytochemistry, and confocal microscopy revealed possible localisation of phosphorylated PKC to the plasma membrane following laminarin challenge. Pharmacological inhibitors of PKC reduced the phosphorylation of extra extracellular-signal regulated kinase 1/2 (ERK 1/2) and mitogen-activated proteinlERK kinase (MEK) in laminarin-challenged haemocytes demonstrating that the ERK pathway is a downstream target of PKC in these cells. Moreover, inhibitor experiments revealed phospholipase C (PLC), but not phosphoinositide-3 kinase (PI-3-K), to be upstream regulators of PKC in laminarin¬challenged haemocytes. The effects of the schistosome epitopes Lewis X and Lac-di¬Nac on haemocyte PKC phosphorylation were also investigated in presence of haemolymph; however, these compounds did not alter the phosphorylation status of PKC-like proteins over 30 minutes. Finally, two haemocyte defence functions were found to be partially PKC-dependent: the production of the reactive oxygen intermediate (ROI) hydrogen peroxide (H202), and cell spreading. Whereas challenge of haemocytes with laminarin resulted in a 9.S-fold increase in H202 output by haemocytes, inhibition of PKC reduced this response by up to 65 %; cell spreading was also reduced by up to 64 %. Moreover, the tyrosine kinase Src was found to play a role in haemocyte spreading; this effect could be mediated by a focal adhesion kinase (FAK)-dependent pathway. In conclusion, this study broadens our knowledge of the molecular mechanisms regulating innate defence in snails. The results should facilitate further research on molluscan immunity that could be of value in advancing our understanding of snail-schistosomes interactions