Haemocytes are the primary defence cells of molluscs. In the present study, extracellular-signal regulated kinase (ERK) 1/2-like proteins were identified within Lymnaea stagnalis haemocytes, with apparent molecular weights of 44 and 43 kDa, respectively. Mitogen-activated protein kinase (MAPK) activity assays have confirmed that the L. stagnalis ERK possesses kinase activity towards Elk-1. Challenge of haemocytes with bacterial lipopolysaccharide (LPS) resulted in a transient activation of ERK, and immunocytochemistry revealed that phospho-ERK was present in both the perinuclear region and the nucleus following challenge. MAPK/ERK kinase (MEK) inhibitors blocked ERK activation confirming that MEK lies upstream of ERK in haemocytes. Moreover, phagocytosis assays, using various inhibitors, showed that ERK activity was vital for efficient phagocytosis and that ERK may be activated by both Ras-dependent and Ras-independent mechanisms. Overall, this study has furthered knowledge of ERK signalling in molluscan immunity and has shown that the ERK pathway regulates the phagocytic activity of molluscan haemocytes.

Integrins play a key role in cellular immune responses in a variety of organisms; however, knowledge of integrins and their effects on cell signalling and functional responses in molluscan defence reactions is poor. Using integrin-mediated cell adhesion kits, alphaVbeta3 and beta1 integrin-like subunits were identified on the surface of Lymnaea stagnalis haemocytes. Haemocyte binding via these integrins was found to be dependent on Ca2+/Mg2+. Western blotting with an anti-phospho (anti-active) focal adhesion kinase (FAK) antibody revealed a 120-125 kDa FAK-like protein in these cells; this protein was transiently phosphorylated upon haemocyte adhesion over 90 min, with maximal phosphorylation occurring after 30 min binding. Also, integrin engagement with the tetrapeptide Arg-Gly-Asp-Ser (RGDS) resulted in a rapid increase in phosphorylation of the FAK-like protein; however, RGDS did not affect the phosphorylation of extracellular signal-regulated kinase. Treatment of haemocytes with RGDS (2 mM) inhibited phagocytosis of E. coli bioparticles by 88%. Moreover, at this concentration, RGDS reduced cell spreading by 61%; stress fiber formation was also impaired. Taken together, these results demonstrate a role for integrins in L. stagnalis haemocyte adhesion and defence reactions and, for the first time, link integrin engagement to FAK activation in molluscs.

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Our knowledge of cell signalling pathways in the molluscan immune system and their response to immunological challenge is currently poor. The present study focused on the Protein Kinase C (PKC) pathway in the immune cells (haemocytes) of Lymnaea stagnalis and its response following exposure to bacterial lipopolysaccharide (LPS). Western blotting of haemocyte proteins with either anti-PKC (pan) or anti-phospho-PKC (Ser 660) antibodies revealed the presence of two PKC-like immuno-reactive proteins of approximately 76 and 85 kDa. Challenge of haemocytes with LPS transiently increased the phosphorylation of the 85 kDa isoform, with a 2.2-fold increase in phosphorylation levels at 5 min and a return to basal levels after 20 min. This LPS-mediated response was blocked following treatment of haemocytes with GF109203X. PKC activities measured in anti-phospho-PKC immunocomplexes following haemocyte treatment with LPS and GF109203X correlated well with the observed PKC phosphorylation levels. These data show for the first time that the activity of the PKC pathway in molluscan immune cells is modulated by LPS, as it is in mammals, and suggest that cell signalling in the innate immune response may have been conserved through evolution.

Haemocytes are the primary defence cells of molluscs. In the present study, extracellular-signal regulated kinase (ERK) 1/2-like proteins were identified within Lymnaea stagnalis haemocytes, with apparent molecular weights of 44 and 43 kDa, respectively. Mitogen-activated protein kinase (MAPK) activity assays have confirmed that the L. stagnalis ERK possesses kinase activity towards Elk-1. Challenge of haemocytes with bacterial lipopolysaccharide (LPS) resulted in a transient activation of ERK, and immunocytochemistry revealed that phospho-ERK was present in both the perinuclear region and the nucleus following challenge. MAPK/ERK kinase (MEK) inhibitors blocked ERK activation confirming that MEK lies upstream of ERK in haemocytes. Moreover, phagocytosis assays, using various inhibitors, showed that ERK activity was vital for efficient phagocytosis and that ERK may be activated by both Ras-dependent and Ras-independent mechanisms. Overall, this study has furthered knowledge of ERK signalling in molluscan immunity and has shown that the ERK pathway regulates the phagocytic activity of molluscan haemocytes.