Chemoattractant N-formylmethionylleucylphenylalanine (fMet-Leu-Phe) in the presence of cytochalasin B stimulates the release of leukotriene B4 (LTB4), superoxide (02) The influx and activation of neutrophils [PMN (polymorphonuclear leukocytes)] are essential for induction of inflammatory processes (1, 2). The chemoattractant N-formylmethionylleucylphenylalanine (fMet-Leu-Phe) and products of complement activation such as Ca recruit and activate the PMN to release active oxygen species and undergo degranulation (2,3). Such actions in concert with increased levels of prostaglandin E2 (PGE2) and prostacyclin (PGI2) lead to the cardinal symptoms of acute inflammation (3)(4)(5). Leukotriene B4 (LTB4), a product of the 5-lipoxygenase pathway, is itself a potent chemoattractant for PMN (6) and promotes their accumulation and adherence to the vascular endothelium proximal to the area of inflammation (3,7). Whether these actions of LTB4 supplement or mediate the effects of other chemoattractants remains unknown.Pharmacological levels of PGs inhibit superoxide (02) production induced by fMet-Leu-Phe in the presence of cytochalasin B (2) by an action purported to depend on increased cyclic AMP production (8). A similar cyclic AMP-dependent inhibition of lysosomal enzyme release has been observed by using large amounts of PGs (2, 8). Pharmacological levels of PGI2 (2.8 X 10-6 M) have also been reported to cause a partial inhibition (45%) of LTB4 release from human PMN activated with serum-treated zymosan (9).The studies in this report indicate that fMet- was obtained from J. Rokach (Merck Frosst) (10); MMM-I-135 (10,10-difluoro-13-dehydroprostacyclin) was a gift from J. Fried (University of Chicago) (11).The incubation buffer contained 130 mM NaCl, 5.5 mM KCl, 0.6 mM Na2HPO4, 0.6 mM NaH2PO4, 10 mM glucose, 1.0 mM CaC12, and 25 mM Tris HCl adjusted to pH 7.4 (12). Preparation of Cells. Rat PMN were obtained by peritoneal lavage with 40 ml of 0.9% NaCl/10 mM EDTA, pH 7.0, 16-24 hr after aseptic induction (13). PMN were recovered by centrifugation and the pellet was washed once in calcium-free incubation buffer, subjected to lysis buffer (14) for 5 min, rewashed in incubation buffer, and resuspended at a concentration of 5 x 106 cells per ml.Human peripheral PMN were isolated from fresh citrated human blood as described (15) and lysed as were the rat cells.Both preparations were >95% PMN.Incubation Conditions. Two-milliliter aliquots of PMN suspension were preincubated with calcium for 3 min at 37°C and then PGs or solvent was added for 5 min. Cytochalasin B (5 ,ug/ ml) was next added for 5 min, followed by addition of 10 ,uM fMet-Leu-Phe for 5 min. Dimethyl sulfoxide was at a final concentration of 0.7%. The incubations were terminated by centrifugation at 260 X g for 5 min at 0°C. The supernatants were analyzed for (5S)-5-hydroxy-6,8,11,14-icosatetraenoic acid (5-HETE), LTB4, and enzymes. Cell pellets were assayed for cyclic AMP.
