Tumor necrosis factor alpha priming of phospholipase D in human neutrophils. Correlation between phosphatidic acid production and superoxide generation.
Tumor necrosis factor alpha (TNF) primes human neutrophils (PMN) for enhanced superoxide (O2-) production if cells are subsequently stimulated with the chemotactic peptide, n-formyl-Met-Leu-Phe (fMLP). fMLP activates phospholipase D to form phosphatidic acid (PA), and a correlation may exist between PA production and O2- generation in PMN. Therefore, we assessed the ability of TNF to prime phospholipase D activation in PMN stimulated with fMLP. TNF (100 units/ml) pretreatment primed enhanced PA production in PMN challenged with 1 microM fMLP, in the absence of cytochalasin B, as demonstrated by increased production of tritiated PA from PMN label with 1-O-[9',10'-3H]hexadecyl-2-lyso-sn-glycero-3-phosphocholine ([3H]LPAF) and by increased PA mass. PA was formed via activation of phospholipase D and occurred with minimal production of diglycerides. Production of O2- was also enhanced in identically treated cells, and we demonstrated a direct correlation between enhanced PA formation and O2- production. Conversely, ethanol inhibition of PA formation led to a comparable reduction in O2- generation. This report of priming of phospholipase D by physiological agonists is the only natural system where enhanced PA formation has been dissociated from diglyceride formation. Our results suggest a link between PA production and NADPH oxidase activation in human PMN.
Bauldry
S A
SA
Department of Medicine, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, North Carolina 27103.
Bass
D A
DA
Cousart
S L
SL
McCall
C E
CE
D003571:Cytochalasin B / Q000494:pharmacology; D004075:Diglycerides / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D000431:Ethanol / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D010712:Phosphatidic Acids / Q000096:biosynthesis*; D010739:Phospholipase D / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D004075:Diglycerides; D010712:Phosphatidic Acids; D014409:Tumor Necrosis Factor-alpha; D013481:Superoxides; D003571:Cytochalasin B; D000431:Ethanol; D009240:N-Formylmethionine Leucyl-Phenylalanine; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D010739:Phospholipase D