ACTIVATION OF SUPEROXIDE FORMATION AND LYSOZYME RELEASE IN HUMAN NEUTROPHILS BY THE SYNTHETIC LIPOPEPTIDE PAM3CYS-SER-(LYS)4 - INVOLVEMENT OF GUANINE-NUCLEOTIDE-BINDING PROTEINS AND SYNERGISM WITH CHEMOTACTIC PEPTIDES

Upon exposure to the bacterial chemotactic peptide fMet-Leu-Phe, human neutrophils release lysozyme and generate superoxide anions. The synthetic lipoamino acid N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteine (Pam3Cys), which is derived from the N-terminus of bacterial lipoprotein, when attached to Ser-(Lys)4 [giving Pam3Cys-Ser-(Lys)4], activated superoxide anions formation and lysozyme release in human neutrophils with an effectiveness amounting to about 15% of that of fMet-Leu-Phe. Palmitic acid, muramyl dipeptide, lipopolysaccharide and the lipopeptides Pam3Cys-Ala-Gly, Pam3Cys-Ser-Gly, Pam3Cys-Ser, Pam3-Cys-OMe and Pam3Cys-OH did not activate superoxide anions formation. Pertussis toxin, which ADP-ribosylates guanine-nucleotide-binding proteins (G-proteins) and functionally uncouples formyl peptide receptors from G-proteins, prevented activation of superoxide anions formation by fMet-Leu-Phe and inhibited Pam3Cys-Ser-(Lys)4-induced superoxide anion formation by 85%. Lipopeptide-induced exocytosis was pertussis-toxin-insensitive. Superoxide anions formation induced by Pam3Cys-Ser-(Lys)4 and fMet-Leu-Phe was enhanced by cytochalasin B, by a phorbol ester and by a diacylglycerol kinase inhibitor. Addition of activators of adenylate cyclase and removal of extracellular Ca2+ inhibited superoxide anions formation by fMet-Leu-Phe and Pam3Cys-Ser-(Lys)4 to different extents. Pam3Cys-Ser-(Lys)4 synergistically enhanced fMet-Leu-Phe-induced superoxide anions formation and primed neutrophils to respond to the chemotactic peptide at non-stimulatory concentrations. Our data suggest the following (1) Pam3Cys-Ser-(Lys)4 activated neutrophils through G-proteins, involving pertussis-toxin-sensitive and -insensitive processes. (2) The signal transduction pathways activated by fMet-Leu-Phe and Pam3Cys-Ser-(Lys)4 are similar but not identical. (3) In inflammatory processes, bacterial lipoproteins and chemotactic peptides may interact synergistically to activate superoxide anions formation, leading to enhanced bactericidal activity.