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It is well known that lipopolysaccharide (LPS) of Gram-negative bacteria triggers antibacterial responses to mammalian macrophages [Weinstein, S., Gold, M. R. & DeFranco, A. (1991) Proc. Natl Acad. Sci. USA 88, 4148-4152] and insect hemocytes [Charalambidis, N.D., Zervas, C.G., Lambropoulou, M., Katsoris, P.G. & Marmaras, V.J. (1995) Eur J. Cell Biol. 67, 32-41], via protein-tyrosine phosphorylation. In this study we show that insect hemocytes in response to LPS facilitate internalization of LPS (either cell-associated or cell-free). According to our data, the recognition and covalent association of LPS (either cell-associated or cell-free) to the hemocyte surface are essential initial steps for LPS internalization. LPS (Escherichia coli) recognizes membrane effector 47-kDa protein (p47) and then crosslinks to membrane-associated p47 (mp47) via the intermediacy of tyrosine derivatives generated by the action of phenol oxidase, as is the case for cuticular protein-chitin crosslinks during sclerotization [Shaefer, J., Kramer, K.J., Garbow, J.R., Jacob, G.S., Stejskal, E.O., Hopkins, T.L. & Speirs, R.D. (1987) Science 235, 1200-1204]. The covalent association of LPS to the hemocyte surface appears to be a prerequisite for LPS internalization as judged by the resistance of LPS binding to dissociation by proteinase K. In addition, our results show that the effector molecules participating in LPS covalent association at the cell surface and LPS internalization are not involved in LPS-induced activation of hemocytes. However, the fact that genistein, as well as the inhibitors of LPS-dependent secretion, block LPS covalent association at the cell surface and LPS internalization provides a preliminary characterization of an LPS signal-transduction-dependent process which is apparently involved.

The mechanism of recognition of foreignness and entrapment of invaders by the immune system of insects is unknown. In this report using hemocyte monolayer preparations and biochemical analysis we demonstrate the requirements for recognition of E. coli in vitro, their entrapment by hemocytes, and nodule formation. A model system consisting of an isolated hemocyte protein (47 KDa), isolated hemocyte tyrosinase, isolated hemocytes, tyrosine, and E. coli was used to obtain these results. The 47 kDa polypeptide has the ability to form adducts with tyrosine derivatives generated by the action of tyrosinase and to attach to the E. coli surface. The latter process takes place independently of tyrosinase activity. When the E. coli-47KDa protein complex was overlaid on hemocyte monolayers followed by tyrosine and tyrosinase or vice versa, the bacteria were entrapped by hemocytes. The same results were obtained when the monolayers were overlaid with 47 KDa protein, followed by E. coli-47 KDa protein complex and then tyrosine and tyrosinase. The same experimental procedure in test tubes resulted in nodule formation. These results permit us to propose that the most likely explanation for the entrapment of E. coli to hemocytes and the formation of nodules is the production of E. coli-47 KDa complexes, and their crosslinking through a quinone intermediate generated by the action of tyrosinase on hemocytes.

The results indicate that certain hemocyte proteins of the medfly, Ceratitiscapitata, are responsible for the recognition of foreignness, since they are able to bind to the surface of Escherichia coli in vitro. Furthermore, when the E. coli-hemocyte protein complex was incubated in the presence of tyrosine and phenoloxidase, the bacteria were immobilized, forming large aggregates. The formation of aggregates seems to be due to reactive tyrosine intermediate (quinone) generated by the action of phenoloxidase. o 1992 wiiey-~iss, Inc