A suggested role for phospholipase C in the immune response of Galleria mellonella

Abstract

The immune response of Galleria mellonella is characterized by a variety of biological activities, including the production of antibacterial products (J. S. Chadwick and G. B. Dunphy, 1986, in “Hemocytic and Humoral Immunity in Arthropods,” A. G. Gupta, Ed., Wiley, New York). Recently, a novel inducible hemolytic activity (D. J. Phipps, R. G. Leeder, J. S. Chadwick, and W. P. Aston, Dev. Comp. Immunol. 13, 103-l 11, 1989) and a novel antitumor activity (J. K. C. Chiu, W. P. Aston, and J. S. Chadwick, Abstr. 116, “XXII Ann. Mtg. Sot. Invertebr. Pathol.,” College Park, Maryland, 1989) have been demonstrated. In addition, phospholipase C (PLC) activity has been found to increase in the cell-free hemolymph (CFH) of G. mellonella following injection of an immunizing dose (2.5 pg/larva) (P. J. De Verno, M.Sc. Thesis, Queen’s University, Kingston, Ontario, Canada, 1983) of lipopolysaccharide (LPS) derived from Salmonella typhimurium. The PLC activity was determined by a calorimetric assay using p-nitrophenylphosphorylcholine (NPPC) as the specific substrate (S. Kurioka, J. Biochem. 63, 678-680,1968; S. Kurioka and M. Matsuda, Anal. Biochem. 75, 281-289, 1976; R. G. Sheikhnejad and P. N. Srivastava, J. Biol. Chem. 261, 7544-7549, 1986; T. P. Yoshino, J. Invertebr. Pathol. 51, 32-40, 1988). The injection of endotoxin-free water did not increase the PLC activity over a 72-h period. However, LPS caused a significant increase in PLC activity, apparently reaching the maximum at 24 h postinjection. The increase and subsequent decline of PLC activity in the CFH of G. mellonella following LPS injection was found to parallel the profile of the protective immune response (Fig. 1). The isolated hemolytically active material (HAM), which can be induced by either formalized Pseudomonas aeruginosa or LPS, did not possess any PLC activity (D. J. Phipps, R. G. Leeder, J. S. Chadwick, and W. P. Aston, Dev. Comp. Zmmunol. 13, 103-111, 1989). This suggests that the PLC enzyme is a different entity from the HAM. It also differs from an LPS-induced hemolymph-derived cytotoxic activity (for human tumor cell lines), which peaks at 2 h postinjection of LPS, as described previously (J. K. C. Chiu, W. P. Aston, and J. S. Chadwick, Abstr. 116, “XXII Ann. Mtg. Sot. Invertbr. Pathol.,” College Park, Maryland, 1989). The mechanism for the LPS-induced increase in PLC activity in the hemoiymph of G. mellonella is not clearly understood. The presence of a high level of such an enzyme in the hemolymph during the height of the protective immune response suggests a role for PLC in this process. It has been shown that total hemocyte counts in G. mellonella declined rapidly upon injection of LPS (G. B. Dunphy, D. B. Morton, and J. M. Chadwick, J. Invertebr. Pathol. 47, 56-64, 1986; G. B. Dunphy and J. M. Webster, J. Gen. Microbial. 134, 1017-1028, 1988). This may be due to the damage and eventual lysis of hemocytes caused by LPS or autolysis. The membraneand/or cytosol-associated PLC enzyme might then be released into the hemolymph via cell lysis or secretion. Several investigators have reported earlier that the granular contents may be released from isolated hemocytes via exocytosis following LPS stimulation (M. W. Johansson and K. Soderhall, J. Comp. Physiol. B, 156, 175-181, 1985; K. Soderhall, V. J. Smith, and M. W. Johansson, Cell Tissue Res. 245, 43-49) 1986. It has also been shown that treatment of mammalian tissues and cells with exogenous PLC (external to cells) led to the sub-