Exogenous Leukotriene B 4 ( LTB 4 ) Inhibits Human Neutrophil Generation of LTB 4 from Endogenous Arachidonic Acid During Opsonized Zymosan Phagocytosis 1

The effect of exogenous leukotriene B4 (LTB4) on opsonized zymosan-stimulated human neutrophil formation of 5-lipoxygenase products and arachidonic acid release was directly assessed using reverse-phase HPLC/tandem mass spectrometric methods for quantitation. Stable isotopically labeled LTB4, [1,2C2]LTB4, caused a dose-dependent inhibition of LTB4 production in isolated human neutrophils with significant inhibition (60 6 7% of control levels) when 0.12 nM [C2]LTB4 was present. Production of 5-hydroxy-6,8,11,14-eicosatetraenoic acid and release of free arachidonic acid were also dosedependently inhibited by exogenous LTB4. Metabolites of LTB4, 20-hydroxy-LTB4 and 3(S)-hydroxy-LTB4, also significantly reduced LTB4 production to levels as low as 10 6 6% and 10 6 7% of control levels, respectively, when present exogenously at 10 nM. Exogenous 5-hydroxy-6,8,11,14-eicosatetraenoic acid at concentrations as high as 10 nM produced no significant reduction in LTB4 biosynthesis during zymosanstimulated human neutrophil production of LTB4. The inhibitory effect of LTB4 could be partially reversed by the LTB4 receptor antagonist U 75302. Furthermore, an alternative stimulus, Nformyl-methionyl-leucyl-phenylalanine (100 nM), did not inhibit the production of LTB4 in opsonized zymosan-stimulated human neutrophils. These results suggest that activation of the LTB4 receptor on the human neutrophil during phagocytosis limits the ultimate biosynthesis of LTB4. This autocrine effect is opposite to that observed when neutrophils have much of the signal transduction pathways bypassed when stimulated with calcium ionophore A23187 or treated with exogenous free arachidonic acid. LTB4 is a potent chemotactic agent produced from arachidonic acid in several cell types, including the human polymorphonuclear leukocyte (neutrophil), after activation of 5-lipoxygenase and intermediate formation of LTA4. The activation of 5-lipoxygenase involves a complex series of events, which include translocation of 5-lipoxygenase from the cytosol of the perinuclear envelope (Ford-Hutchinson et al., 1994). At the nuclear membrane, association of this monooxygenase takes place with an auxiliary protein, 5-lipoxygenase-activating protein (FLAP), required for 5-lipoxygenase activity in the intact cell. During the activation of 5-lipoxygenase, it is also likely that cPLA2 is translocated to a similar site in the nucleus and may be responsible for the specific release of arachidonic acid that is acted on by 5-lipoxygenase (Channon and Leslie, 1990). The neutrophil also contains the cytosolic enzyme LTA4 hydrolase, which converts the 5-lipoxygenase product LTA4 into LTB4 through the stereospecific addition of water to the triene epoxide (Radmark et al., 1994). However, LTA4 can be exported from the neutrophil in normal tissue environments and subsequently converted into LTB4 by adjacent cells rather than the cell synthesizing LTA4 (Marcus and Hajjar, 1993). Thus the formation of LTB4 entails multiple biosynthetic steps and probably occurs to a significant degree outside of the neutrophil. Many of the events of neutrophil activation are linked to these biochemical steps of LTB4 biosynthesis through complex signal transduction networks. The priming of neutrophils for LTB4 synthesis is thought to require coordination of two or more pathways, with concomitant elevation of intracellular free calcium ion. Because the neutrophil has a specific G protein-linked receptor for LTB4 (Yokomizo et al., 1997), it can respond to exogenous LTB4 by elevation of intracellular calcium ion, which suggests that LTB4 syntheReceived for publication March 5, 1998. 1 This work was supported by a grant from the National Institutes of Health (HL25785) and a predoctoral training grant (GM07635). 2 Present address: Glaxo/Wellcome, 5 Moore Drive, Research Triangle Park, NC 27709. ABBREVIATIONS: LTB4, leukotriene B4; LC/MS/MS, reverse-phase HPLC tandem mass spectrometry; 5-HETE, 5-hydroxy-6,8,11,14-eicosatetraenoic acid; 20-OH-LTB4, 20-hydroxy-LTB4; fMLP, N-formyl-methionyl-leucyl-phenylalanine; LTA4, leukotriene A4; 5-HpETE, 5-hydroperoxy6,8,11,14-eicosatetraenoic acid; cPLA2, cytosolic phospholipase A2; BSA, bovine serum albumin; HBSS, Hank’s buffered saline solution; PMN, polymorphonuclear leukocyte; PFB, pentafluorobenzyl ester; MRM, multiple reaction monitoring. 0022-3565/98/2871-0150$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 287, No. 1 Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 287:150–156, 1998 150 at A PE T Jornals on A ril 3, 2017 jpet.asjournals.org D ow nladed from sis could be regulated by LTB4 itself (McDonald et al., 1992; McDonald et al., 1994). Recent studies have suggested that free arachidonic acid as well as products of 5-lipoxygenase may act to increase the activity of phospholipase A2 in the human neutrophil. In ionophore-stimulated cells, 5(S)-HETE was found to potentiate markedly the release of arachidonic acid in human neutrophils and to increase the biosynthesis of 5(S)-HETE and LTB4 (Billah et al., 1985). Exogenous arachidonic acid and LTB4 were reported to have similar effects when added to cells before they were stimulated with calcium ionophore, which suggests that increased phospholipase A2 activity was a result of the addition of these exogenous substrates. An additional study has shown that the activity of the 85-kDa cytosolic PLA2 isolated from human neutrophils shown to have unique specificity for arachidonic acid was increased 2-fold when cells were exposed to 5-HETE, LTB4 or free arachidonic acid (Wijkander et al., 1995). It has been proposed that the increase in cPLA2 activity initiated by exogenous free arachidonic acid results from conversion of this arachidonic acid into LTB4, because the increase in activity initiated by arachidonic acid could be blocked by treatment of the neutrophils with inhibitors of 5-lipoxygenase (Lew et al., 1991). Separate studies have suggested that LTB4 activates human neutrophil 5-lipoxygenase indirectly, probably through signal transduction pathways. Incubation of cells with stable isotopically labeled LTB4 and arachidonic acid resulted in significant increases in LTB4 biosynthesis and the synthesis of 5,15-diHETE from 15-HpETE (McDonald et al., 1992). In these studies, a LTB4 receptor antagonist reduced LTB4 biosynthesis. The calcium ionophore in the presence of exogenous LTB4 was found to increase 5-lipoxygenase translocation to the nuclear fraction of neutrophils (Serio et al., 1997), adding further support to the idea that LTB4 may stimulate its own biosynthesis via the membrane LTB4 receptor. We have recently developed a mass spectrometry-based method for the simultaneous quantitation of LTB4, v-oxidized metabolites of LTB4 and 5-HETE in order to assess multiple 5-lipoxygenase products in a single analytical determination (Wheelan and Murphy, 1997). This method has detection limits in the picogram level and so makes it possible to study leukotriene biosynthesis under physiologically relevant conditions where the absolute quantity of LTB4 produced is below detection limits that typically apply when we use HPLC with UV detection. In addition, the ability to characterize exogenously added LTB4 uniquely as a carbon13-labeled analog two atomic mass units heavier than endogenously produced LTB4 made possible the direct assessment of newly synthesized LTB4 from endogenous arachidonic acid in the presence of exogenous [C2]LTB4. With this approach, exogenous LTB4 at concentrations as low as 0.1 nM was found to inhibit significantly the opsonized zymosan-stimulated production of LTB4 by human neutrophils. Furthermore, the release of free arachidonic acid was found to be inhibited by exogenous LTB4, which suggests that both responses may be under LTB4 receptor control and linked to unique signaling events initiated by phagocytosis. Materials and Methods Materials. The following drugs and chemicals were kindly provided by or obtained from the sources indicated: LTB4, [6,7,14,15d4]LTB4, [5,6,8,9,11,12,14,15-d8] 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid (d8-5-HETE), 5(S)-HETE, 20-OH-LTB4 and [5,6,8,9,11,12,14,15-d8]arachidonic acid (d8-AA) (Cayman Chemical Co., Ann Arbor, MI), U 75302 (Biomol Research Laboratories, Plymouth, PA), zymosan A and [1,2-C2]LTB4 (Sigma Chemical Co., St. Louis, MO), 3(S)-OH-LTB4 [Dr. J.R. Falck (Wheelan et al., 1994)], fMLP (Vega Biotechnologies Inc., Tucson, AZ) and the calcium ionophore A23187 (Calbiochem, La Jolla, CA). The chemical purity and structural identity of all synthetic LTB4 isotopimers were checked by UV spectroscopy, HPLC and mass spectrometry. All solvents were HPLC grade obtained from Fisher Scientific (Fair Lawn, NJ), and other commercially available reagents were the highest purity avail-