BACTERIAL LIPOPOLYSACCHARIDE PRIMES HUMAN NEUTROPHILS FOR ENHANCED RELEASE OF ARACHIDONIC-ACID AND CAUSES PHOSPHORYLATION OF AN 85-KD CYTOSOLIC PHOSPHOLIPASE A(2)

Production of leukotriene B-4 (LTB(4)) by human neutrophils (PMN) in r esponse to different stimuli is increased after pretreatment with lipo polysaccharides (LPS). We have analyzed the steps in arachidonic acid (AA) metabolism affected by LPS by examining release of AA and its met abolites from [H-3]AA prelabeled PMN. Pretreatment of PMN for 60 min w ith up to 1 mu g/ml of LPS alone had no effect, but release of [H-3]AA was stimulated up to fivefold during subsequent stimulation with a se cond agent. In the absence of LPS-binding protein (LBP), priming was m aximal after pretreatment of PMN with 10 ng of LPS/ml for 60 min; in t he presence of LBP maximal priming occurred within 45 min at 0.1 ng of LPS/ml and within 15 min at 100 ng of LPS/ml. Treatment of PMN with 1 0 ng of LPS/ml also increased uptake of opsonized zymosan by up to 60% . Phospholipids are the source of released [H-3]AA. No release was obs erved from [C-14]oleic acid (OA)-labeled PMN suggesting that phospholi polysis may be specific for [H-3]AA-labeled phospholipid pools. Cytoso l from PMN primed with LPS contains two to three times the phospholipa se A(2) (PLA(2)) activity of control PMN, against almitoyl-[2-C-14]ara chidonoyl-phosphatidylcholine. This activity is Ca2+ dependent and dit hiothreitol resistant. LPS priming is accompanied by reduced migration during SDS-PAGE of an 85-kD protein, identified as a cytosolic PLA(2) . The extent and kinetics of this effect of LPS on cPLA(2) parallel th e priming of [H-3]AA release, both depending on LPS concentration eith er with or without LBP. These findings suggest that priming by LPS of AA metabolism by PMN includes phosphorylation of an AA-phospholipid-se lective cytosolic PLA(2) that is dissociated from activation until a s econd stimulus is applied.