Studies on the extracellular-signal regulated kinase pathway in 'Lymnaea stagnalis' haemocytes and its role in molluscan defence
Haemocytes, the main type of circulating defence cell in invertebrates, functionally resemble mammalian macrophages, and are responsible for phagocytosis, endocytosis and other innate immune functions. In mammalian macrophages, the extracellular-signal regulated kinase (ERK) pathway has been shown to modulate immune responses. It was therefore hypothesised that this pathway may play a similar role in haemocytes of the gastropod mollusc, 'Lymnaea stagnalis', and intermediate host of the schistosome, 'Trichobilharzia ocellata'. Two ERK-like proteins were detected by Western blotting usmg antibodies that recognise non-phosphorylated and phosphorylated forms of mammalian ERK 1/2; these proteins had approximate molecular weights of 44 and 43kDa respectively. In addition, the upstream regulator of ERK, MEK, was detected in haemocytes as well as the transcription factor downstream of activated ERK, Elk-1. By employing a mitogen-activated protein (MAP) kinase assay kit, the identified ERK-like proteins were found to possess kinase activity. When haemocytes were challenged with 'E. coli' lipopolysaccharide (LPS), the activity of the ERK pathway was found to be significantly upregulated after 5 min challenge and perinuclear migration of phosphorylated ERK was observed by immunocytochemistry. Other compounds, including Zymosan A, adrenocorticotropin hormone, noradrenaline and challenge with heat-killed 'E. coli' did not modulate haemocyte ERK pathway activity. Pharmacological inhibitors used to block signalling to the ERK pathway demonstrated that MEK, PKC, PI-3K and Ras activity are vital for phagocytosis by haemocytes. In addition, the integrin subunits [alpha]V[beta]3 and [beta]l were found to be present on the haemocyte surface and integrin engagement was found to be necessary for phagocytosis. Monosaccharides found on the surface coat of 'T. ocellata', fucose and galactose, were applied to haemocytes in the presence and absence of haemolymph. These sugars proved to have important immunomodulatory effects, since ERK, PKC and phagocytic activity were all affected by these sugars, and differences in experiments with and without haemolymph suggest an important role for serum proteins in the molluscan immune response. In conclusion, this study provided an insight into the signalling machinery involved in the molluscan defence response, and the results should stimulate further research in snail defence responses, particularly following challenge by parasites.