The effects of intracellular signalling pathway inhibitors on phagocytosis by haemocytes of Manduca sexta

The intracellular signalling pathways activated during phagocytosis by larval haemocytes of a lepidopteran, Manduca sexta, were investigated. Using fluorescein-labelled Escherichia coli as bioparticles, a fluorescence-based assay was used to quantify phagocytosis by haemocytes in monolayers in vitro, and the intracellular signalling pathways involved in phagocytosis were examined using inhibitors. Pathways known to be involved in phagocytosis by mammalian cells were selected for the study in haemocytes, and the amino acid sequences of human isoforms of the selected protein targets were used to conduct searches of two completed databases of insect proteins, those of Drosophila melanogaster and Anopheles gambiae and EST databases of moths Bombyx mori and M. sexta. Decreases in phagocytosis produced by pathway inhibitors indicated that tyrosine phosphorlylation phosphatidylinositol 3-kinase (PI3-kinase) and mitogen-activated protein kinase/extracellular-regulated kinase (ERK/MAPK) were required for internalisation of bacteria. Inhibition of Syk, a mammalian kinase, also decreased phagocytosis. JNK/SAPK did not seem to be involved in phagocytosis. The presence of protein phosphatases probably regulates phagocytosis at the intracellular level by dephosphorlyation of serine/threonine residues.