YOSHINO, T.P.*; HUMPHRIES, J.E.; COPELAND, C.A.: Putative Signal Transduction Pathway Regulating Molluscan Hemocyte Behavior: Studies on In Vitro Cell Spreading and Phagocytosis

Encapsulation responses by hemocytes of the freshwater snail Biomphalaria glabrata represent the primary effector mechanism against larval stages of the blood fluke Schistosoma mansoni. Cellular adhesion and spreading are critical components of this defense reaction, but at present little is known of the receptors involved in parasite recognition, or the cell signaling pathways used to communicate hemocyte behaviors upon receiving appropriate stimulation. Using a panel of drugs known to specifically inhibit the action of various molecules involved in signal transduction, the role of protein kinase C (PKC), Ras, and mitogen-activated protein kinase (MAPK) in hemocyte spreading and phagocytosis (vs. E. coli) was studied. Calphostin C and chelerythrine chloride (PKC inhibitors) and Ras inhibitory peptide (Ras inhibitor) strongly suppressed both cell spreading and phagocytosis, although the phagocytic response was 10-fold and 4-fold more sensitive to Calphostin C and Ras inhibitory peptide, respectively. Western blot analysis revealed the presence of an immunoreactive MAPK-like protein (approx. 42 kDa) in hemocytes using an anti-ERK polyclonal antibody. Moreover, the MAPK kinase (mek) inhibitor, PD 98059, significantly reduced hemocyte spreading, implying the possible presence and involvement of a MAPK-like protein in this behavior. Overall, these results suggest that immune-related hemocyte behaviors, such as cell spreading and phagocytosis, may be regulated through receptor-stimulated upstream activation of PKC- and Ras-like molecules, with a MAPK-like protein(s) being the eventual target of downstream activation. Supported by NIAID #AI15503.