Μεταγωγή μηνυμάτων σε απόκριση βακτηρίων στα αιμοκύτταρα του εντόμου Ceratitis capitata

Abstract

The recognition and phagocytosis of microbes by macrophages is a principal aspect of innate immunity that is conserved from insects to humans. Insect hemocytes recognize a variety of foreign targets by both humoral and cell surface receptors. A doublet of medfly hemocyte proteins with a molecular mass of about 55 kDa and 50 kDa were precipitated with LPS. Antibodies raised against human CD14 recognize specifically the same doublet of proteins. These results support that mammalian CD14 and the doublet of protein bands in medfly hemocytes share common epitopes. This doublet of protein bands is released from hemocytes upon LPS triggering. The membrane-bound LPS-binding protein appears to intervene in LPS internalization and E. coli phagocytosis, but not in LPS signaling. We recently demonstrated that LPS promotes activation of the Ras/mitogen-activated protein cascade in hemocytes and that phagocytosis of E. coli by insect hemocytes is mediated by an integrin-dependent process [Foukas et al., 1998]. Data reported in this thesis concern the focal adhesion kinase (FAK) tyrosine phosphorylation status in hemocytes in response to E. coli. It was found that E. coli-triggering stimulates a significant increase in tyrosine phosphorylation of FAK in hemocytes. Furthermore, immunoblotting analysis using anti-Y397 demonstrated intense FAK activity at the Y397/SH2-binding site in hemocytes treated with E. coli. In addition, antibody-mediated inhibition of FAK and Src-kinase has been shown to abolish FAK phosphorylation and E. coli phagocytosis, indicating a specific role for the FAK/Src complex in the processes of promoting cell phagocytosis. These findings expand the known signaling functions of FAK and provide insight into signal transduction events associated with hemocyte phagocytosis in response to E. coli. Finally, in an attempt to further elucidate the effects of LPS on medfly hemocytes, we focused on ERK behaviour. It was initially observed ERK activation and its kinetic upon LPS or E. coli triggering. The medfly hemocytes respond to LPS by changing their morphology, inducing the activation of several signaling pathways, including Ras/MEK/ERK and contributing in LPS uptake. The present results with the actin reorganization inhibitors denote that LPS-induced ERK phosphorylation appears to be actin remodeling independent, as the inhibitors of actin cytoskeleton reorganization did not affect ERK activation. In support are the results with the MEK1/2 inhibitor, U0126, which blocks ERK activation and LPS uptake. These data strongly support that LPS uptake is independent of actin remodeling in insect hemocytes, indicating once again the conservation of intracellular signaling mechanisms in insect and mammals.