Proc. Nati. Acad. Sci. USA Vol. 87, pp. 6248-6252, August 1990 Medical Sciences Selective incorporation of (15S)-hydroxyeicosatetraenoic acid in phosphatidylinositol of human neutrophils: Agonist-induced deacylation and transformation of stored hydroxyeicosanoids (inflammation/arachidonic acid/lipoxygenase products/leukotrienes/lipoxins) MARK E. BREZINSKI AND CHARLES N. SERHAN* Hematology Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115 Communicated by Eugene P. Kennedy, May 16, 1990

ABSTRACT The uptake and mobilization of (15S)- chial lavage fluids obtained from asthmatics after antigen hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid (15-HETE), challenge (6) and (ii) alterations in 15-HETE levels are a major product ofarachidonic acid metabolism, was examined associated with both psoriatic skin lesions of humans (7) and with human neutrophils (polymorphonuclear leukocytes; arthritic synovial fluid obtained from canines (8). Although PMNs). Upon exposure to labeled 15-HETE, PMNs rapidly (15 elevated levels of 15-HETE have been detected in mamma- sec to 20 min) incorporated r20% of the label into phosphati- lian tissues and 15-HETE is known to have distinct biological dylinositol, while <4% was associated with other phospholipid activities, the role of this eicosanoid in inflammation and classes and neutral lipids. This pattern was distinct from that other physiologic responses remains to be fully appreciated ofeither labeled arachidonate or labeled(5S)-hydroxy-8,11,14- (for reviews, see refs. 1, 5, and 9). cis--trans-eicosatetraenoic acid (5-HETE), which within 20 Stenson and Parker (10) first demonstrated that the 5- min were predominantly associated with triglycerides and lipoxygenase product (5S)-hydroxy-8,11,14-cis-6-trans- phosphatidylcholine. After reversed-phase HPLC, >98% of eicosatetraenoic acid (5-HETE) is incorporated into phos- the label in phosphatidylinositol, isolated from PMNs, was pholipids and triglycerides (TGs) of PMNs, a finding that has released with phospholipase A2. Upon exposure to either been extended to include a wide range of cell types (see ref. chemotactic peptide (FMLP), phorbol 12-myristate 13-acetate, 1). Recent results with glomerulosa cells show that when or an ionophore (A23187), 15-HETE-labeled PMNs released these cells contain 5-HETE, but not 15-HETE, in their mono- 15-HETE from phosphatidylinositol and displayed an impaired and diglycerides, their ability to release aldosterone is inhib- ability to generate leukotriene B4 (LTB4), 20-OH.LTB4, and ited (11). In view of recent observations with 15-HETE and 20-COOH-LTB4. Deacylated [3H]15-HETE was converted to its transformation products (5), the present studies were (5S,15S)-dihydroxy-6,13-trans-8,11-cis-eicosatetraenoic acid undertaken to examine three questions. First, is 15-HETE (5,15-DHETE), lipoxin A4, and lipoxin B4, each carying 3H incorporated into human PMN lipids in a profile similar to label. PMNs labeled with 5-HETE also released and trans- that of either AA or 5-HETE? Second, can either esterified formed this HETE when stimulated. However, the profre of 15-HETE or 5-HETE be mobilized upon activation of PMNs labeled products differed between PMNs with either esterified and, if so, are they subject to oxygenation? Third, are 15-HETE or 5-HETE. When activated, 5-HETE-labeled PMNs functional responses of PMNs [i.e., leukotriene B4 (LTB4) generated both 5,20-DHETE and 5,15-DHETE but not labeled formation, aggregation] altered by these events? lipoxins. Threshold aggregation induced by FMLP with 15- HETE-labeled PMNs was inhibited (z2 orders of magnitude), MATERIALS AND METHODS while the threshold response was relatively unimpaired with either A23187 or phorbol 12-myristate 13-acetate-induced ag- Materials. Radiolabeled eicosanoids were purchased from gregation. Results indicate that 15-HETE is rapidly esterified NEN Research Products, Boston, MA. 15-HETE and 5- into phosphatidylinositol ofPMNs, which can be mobilized and HETE were isolated and prepared by SnCl2 reduction of transformed upon exposure of the cells to a second signal. 15S-HPETE generated with soybean lipoxygenase and 5S- Moreover, they suggest that eicosanoid intermediates other HPETE from potato tubers, respectively (12). The identities than arachidonic acid can be stored by cells, released via signal and concentrations of 15-HETE and 5-HETE were confirmed transduction, and oxygenated to generate alternative profiles of by GC-MS, reversed-phase HPLC, and chiral column (J. T. eicosanoids. Baker, Baker Bond, Phillipsburg, NJ) HPLC (as described in ref. 13). Other materials were as described in ref. 4. A wide range of mammalian cell types can generate (15S)- Methods. PMNs were obtained from healthy human vol- hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid (15- unteers by Ficoll/Hypaque gradient centrifugation of hep- HETE) from unesterified arachidonic acid (AA) by lipoxy- arinized venous blood (14). The cells were suspended in genase-, cyclooxygenase-, or cytochrome P-450-dependent reactions. They include human polymorphonuclear leuko- Abbreviations: AA, arachidonic acid; CE, cholesterol ester; 5- cytes (PMNs), eosinophils, macrophages, keratinocytes, and HETE, (5S)-hydroxy-8,11,14-cis-6-trans-eicosatetraenoic acid; 15- cells in ref. 15-HETE dis- HETE, (15'S)-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid; tracheal epithelial (reviewed 1). 5,15-DHETE, (5S,15S)-dihydroxy-6,13-trans-8,11-cis-eicosatetrae- plays biological properties in vitro: it can inhibit human noic acid; LXA4, lipoxin A4 [(5S,6R,15S)-trihydroxy-7,9,13-trans- vascular cyclooxygenase activity (2), block leukotriene pro- 11-cis-eicosatetraenoic acid]; LXB4, lipoxin B4 [(5S,14R,15S)- duction (3), and serve as a substrate for lipoxin biosynthesis trihydroxy-6,10,12-trans-8-cis-eicosatetraenoic acid]; LTB4, leuko- (ref. 4; reviewed in ref. 5). These findings are ofinterest since triene B4 [(5S,12R)-dihydroxy-6,14-cis-8,11-trans-eicosatetraenoic (i) elevated levels of 15-HETE are detected in human bron- acid]; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PIP, phosphatidylinositol 4-phosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; PMA, phorbol 12-myristate The publication costs of this article were defrayed in part by page charge 13-acetate; TG, triglyceride; PMN, polymorphonuclear leukocyte; payment. This article must therefore be hereby marked "advertisement" FMLP, formyl methionylleucylphenylalanine. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 6248 Downloaded by guest on September 24, 2021 Medical Sciences: Brezinski and Serhan Proc. Natl. Acad. Sci. USA 87 (1990) 6249 Dulbecco's phosphate-buffered saline (PBS) and contained 98% ± 1% PMNs by light microscopy. Less than 2% were permeable to trypan blue in reported experiments. Suspen- ,24 0 PC 1 sions (30 x 106 cells per ml) were warmed to 370C for 5 min. For time-course experiments, (15S)-hydroxy-5,8,11-cis-13- 20- Lii trans-[5,6,8,9,11,12,14,15-3H(N)]eicosatetraenoic acid I ([3H]15-HETE) (183.5 Ci/mmol; 1 Ci = 37 GBq) was added 16 to cell suspensions and the incubations were stopped by addition of MeOH/CHCl3 [5:2 (vol/vol); 3.5 ml] at the u, 12 indicated intervals. In parallel incubations, PMNs were ex- 8 T posed to either (5S)-hydroxy-8,11,14-cis-6-trans-[5,6,8,9,11,- 12,14,15-3H(N)]eicosatetraenoic acid ([3H]5-HETE) (192.0 0 4 Ci/mmol) or [1-14C]arachidonic acid ([14C]AA) (52.8 mCi/ mmol). When inositol phospholipids were examined, incu- bations were stopped with CHC13/MeOH [1:2 (vol/vol); 3.75 0 1 2 3 4 5 6 7 8 9 10 20 ml] for analysis (15). Time (min) Phospholipids were separated by a modification of de- scribed techniques (16), which permitted analysis of individ- FIG. 1. Time course of [3H]15-HETE incorporation into phos- ual phospholipid, TG, and cholesterol ester (CE) from the pholipids. PMNs (30 x 106 cells per ml) were suspended in PBS (1 same incubation. Materials from the chloroform phase were ml; 370C, pH 7.45). Samples were extracted and phospholipids were separated by two-dimensional TLC. Values represent the means ± dried with argon and suspended in MeOH/CHCl3 [5:2 (vol/ SE of four or five experiments. *, [3H]15-HETE incorporated with vol); 30 Al]. Two-dimensional TLC was utilized, consisting of P < 0.05 when compared to the amounts associated with other CHC13/MeOH/glacial acetic acid/water (60:50:1:4) for the phospholipid classes. first dimension and heptane/diethylether/glacial acetic acid (60:40:2) for the second dimension. Lipids were visualized by esterify [3H]15-HETE into the sn-2 of PI. Although the exposure to iodine vapors, and the identities of individual incorporation of 15-HETE into either phosphatidylinositol phospholipids were established by comigration with syn- 4-phosphate (PIP) or phosphatidylinositol 4,5-bisphosphate thetic standards and were confirmed by using spray reagents (PIP2) represented a relatively low percentage of added obtained from Sigma (i.e., iodoplatinate, orcinol ferric chlo- material, the relative levels of [3H]15-HETE esterified were ride, and molybdenum blue). Inositol phospholipids were comparable to those observed with [14C]AA (Fig. 2). Both extracted and separated (15). The identities of inositol lipids PIP and PIP2 are present in low amounts compared to other were confirmed with standards and with 32P-labeled products phospholipids of PMNs (19). Thus, their ability to incorpo- prepared from platelets (17). rate 15-HETE may represent sites of enrichment within the For analysis ofeicosanoids, incubations were stopped with phospholipid classes, since a relatively large percentage of MeOH containing prostaglandin B2 (100 ng) as internal stan- these inositol lipids contain radiolabel. When 15-HETE con- dard, and products were extracted with Sep-Pak C18 car- centrations were increased (range, 0.03-30 gM), uptake into tridges (Waters) (4). Materials eluting with methyl formate PI of PMNs increased in a nonlinear fashion, which appeared were injected into a reversed phase HPLC system, which to consist of two components (data not shown). consisted of a dual pump gradient (LKB; Bromma, Sweden) Comparison of the incorporation patterns for 15-HETE, equipped with a Novo Pak C18 column (3.9 x 7.5 mm). The 5-HETE, and AA with isolated PMNs revealed column was eluted with MeOH/H20/acetic acid [68:32:0.01 differences (vol/vol)] at a flow rate of 0.39 ml/min, and the system was equipped with a photodiode array detector. Post-HPLC A 'uaU) analyses were performed with a 2140-202 Wavescan program 81 (Bromma, Sweden) and a Nelson Analytical 3000 series .5- 0 * 0. chromatography data system (Paramus, NJ). Products were U) 61 a-0 quantitated from their UV spectra (220-340 nm) and molar X-C absorption coefficients were used (18). n 4 x For aggregation studies, PMNs (3 107 cells per ml) were 0 either eicosanoids (30 or vehicle alone for 0 2!Oor incubated with AM) c 0 1~~~~~~~AP 20 min at 37°C, centrifuged (3 min), and washed twice in PBS c ~o-5 (30 ml). Aggregation was monitored with a four-channel ag- -4. gregation profiler (model PAP-4, Biodata, Hatsboro, PA). 0 x PMNs (5 106 cells per ml) were challenged with either a 0 8 0 L vehicle (0.4 vol% EtOH) or various concentrations of agonist. m.5 A two-tailed Student's t test was used for statistical analysis. 6so 0 RESULTS 0L) 4*0f Upon exposure to [3H]15-HETE, PMNs rapidly incorporated 0 0O label into phospholipids (Fig. 1). Incorporation was time 0I.e 2 dependent and was associated with phosphatidylinositol (PI) (=20% of recovered label), while uptake into other phospho- 0.5 1 5 20 lipid classes, CE, or TG represented <4% (Fig. 2 and Table Time (min) 1). To determine whether labeled 15-HETE was esterified in position, labeledPI was isolated from PMNs by TLC the sn-2 FIG. 2. Incorporation of[3H115-HETE and [14C]AA into inositol (as in Fig. 1) and incubated with snake venom phospholipase phospholipids. PMNs (30 x 106 cells per ml) were incubated in PBS A2 (240C, 60 min. pH 6.5). After extraction and reversed- (1 ml; 370C) with either [3H]15-HETE (A) or [14C]AA(B). Incuba- phase HPLC, 98.9% of the radiolabel was released from tions were stopped and materials were extracted as described. PMN-derivedPI, which coeluted with authentic 15-HETE (n Values represent the mean ± SE ofthree experiments. **, P < 0.005; = 2). Together these findings indicate that PMNs rapidly *, P < 0.05. Downloaded by guest on September 24, 2021 6250 Medical Sciences: Brezinski and Serhan Proc. Natl. Acad Sci. USA 87 (1990) Table 1. Comparison of [3H]5-HETE, [3H]15-HETE, and [14C]AA incorporation into PMN lipids 15-HETE/ 15-HETE/ [3H]15-HETE [3H]5-HETE [14C]AA [3H]15-HETE [3H]15-HETE/AA PI 20.0 ± 6.0 0.6 ± 0.1 4.6 ± 0.6 3.0 ± 1.0 1.1 ± 0.5 PC 1.0 ± 0.1 1.5 ± 0.2 6.7 ± 0.9 0.5 ± 0.2 0.1 ± 0.1 PE 3.0 ± 1.0 0.8 ± 0.1 2.2 ± 0.3 1.7 ± 0.9 1.0 ± 0.5 PS 0.6 ± 0.1 0.4 ± 0.0 0.4 ± 0.1 0.7 ± 0.5 0 ± 0 SP 0.5 ± 0.1 1.0 ± 0.3 0.1 ± 0.0 0.2 ± 0.1 0 ± 0 TG 0.0 ± 0.0 8.0 ± 1.0 28.0 ± 4.0 0.2 ± 0.2 0.1 ± 0.1 CE 0 ± 0 0.1 ± 0.1 0 ± 0 0.1 ± 0.1 0 ± 0 PMNs (30 x 106 cells per ml) were incubated with either [3H]15-HETE, [3H]5-HETE, or [14C]AA for 20 min at 37TC, or PMNs (30 x 106 cells per ml) were incubated with either 15-HETE (30 ,tM) or AA (30 ,uM) + 15-HETE (30 1tM) added simultaneously with [3H]15-HETE for 20 min at 370C. Incubations were stopped, products were extracted, and the distribution of radiolabel was determined after two-dimensional TLC. Values represent means ± SEM for three to five separate experiments. PS, phosphatidylserine; SP, Sphingomyelin. between the profiles (Table 1). Unlike 15-HETE, incorpo- followed by washing and stimulation with ionophore (5 ,M; ration ofeither 5-HETE or AA was predominantly associated 20 min at 37°C) generated amounts of LTB4, 20-OH-LTB4, with TG (8=8% and -28%, respectively). Of the lipid classes, and 20-COOH-LTB4 that were not statistically different from 15-HETE was dominant in PI among the phospholipids of PMNs treated with vehicle alone (n = 3 donors with duplicate PMNs, while both 5-HETE and AA were incorporated determinations). mainly into phosphatidyicholine (PC). The values obtained We next determined whether agonist-induced release of with both 5-HETE and AA are consistent with those reported esterified HETEs also promotes their oxygenation by PMNs. earlier (10). Unlabeled 15-HETE blocked the incorporation In response to either A23187, PMA, or FMLP, PMNs deacy- of [3H]15-HETE (Table 1). In addition, when PMNs were lated 15-HETE and converted it to (5S,15S)-dihydroxy-6,13- exposed simultaneously to equal quantities of unlabeled trans-8,11-cis-eicosatetraenoic acid (5,15-DHETE), lipoxin A4 15-HETE and AA together with [3H]15-HETE, the incorpo- and lipoxin B4 (LXB4). The specific activities of ration of label was reduced but not completely blocked. (LXA4), Since PMNs stored 15-HETE in PI, we determined 5,15-DHETE and deacylated 15-HETE were essentially equal whether PMNs mobilize this source of 15-HETE upon acti- while those of the lipoxins were lower, suggesting that addi- vation. After labeling with [3H]15-HETE, PMNs were ex- tional sources of unlabeled substrate can contribute to their posed to either selected agonists or vehicle alone (Fig. 3). formation. In the absence of an agonist, neither [3H]5,15- FMLP activates PMNs via interaction with specific recep- DHETE nor [3H]lipoxins were detected (Table 2). PMNs also tors, while A23187, phorbol 12-myristate 13-acetate (PMA), released and transformed esterified 5-HETE; however, a and AA each activates PMN responses via circumventing different profile of products was obtained (Table 2). With all receptor-ligand interactions (20). PMNs exposed to either three stimuli, 5-HETE was converted to both its co-oxidation FMLP (100 nM), PMA (100 nM), or A23187 (5 ,M) each product, 5,20-DHETE, and 5,15-DHETE. In contrast to 15- showed a significant reduction in labeled PI. In contrast, AA, HETE, none of the stimuli induced the formation of at a concentration (e.g., 30 ,uM) that stimulates O- gener- [3H]lipoxins from esterified [3H]5-HETE. ation by PMNs (21), did not provoke a loss of label from PI. The impact of esterified 15-HETE on agonist-induced Both 20-COOH- and 20-OH-LTB4 were generated in re- aggregation of PMNs is given in Table 3. Threshold concen- sponse to those agonists (albeit at lower quantities than with trations required to induce aggregation were determined in ionophore). PMNs labeled with 15-HETE also displayed an each case rather than monitoring relative changes in light impaired ability to generate LTB4 and its w-oxidation prod- ucts (Fig. 4). In contrast, PMNs incubated (20 min at 37°C) 800 separately with either 5-HETE (30 ,uM) or AA (30 ,uM) 600+ 70- = PMNs with esterified 1 5-HETE a-L = PMNs a) 60- (4) (4) 0)~~~~~~~~~~~4 S._ 400 - c 50- 1 (4) co'ITQj 40 200 - 30- 2020 (3) 0 LrzL, FT1 10 A231 87 PMA FMLP .'g (5 AM) (100 ng/ml) (100 nM) 0 A23187 PMA FMLP AA FIG. 4. Agonist-induced release of LTB4 with 15-HETE- (5 AM) (100 nM) (100 nM) (30 tkM) esterified PMNs. Isolated PMNs (3 x 107 cells per ml) were first incubated with either vehicle [0.4% EtOH (vol/vol)] or 15-HETE (30 FIG. 3. Agonist-induced release of [3H]15-HETE from PI. PMNs ,uM) at 37°C for 20 min. Both groups of cells were centrifuged, were labeled with [3H]15-HETE (as described in Figs. 1 and 2) for 20 washed with PBS, and suspended in PBS (1 ml). At time zero, either min. Stimuli or vehicle were added to 30 x 106 PMNs per ml and the stimuli or vehicle (4 ,ul, EtOH) was added. Incubations (20 min at incubations continued for 20 min at 37°C. Incubations were stopped, 37°C) were stopped and products were extracted and quantitated. products were extracted, and PI was isolated from each after Values represent mean total LTB4 levels (i.e., sum of 20-OH-LTB4, two-dimensional TLC. Values represent the mean + SE of three or 20-COOH-LTB4, and LTB4) (ng per incubation ± SE) obtained in four (numbers in parentheses) separate experiments. three separate experiments. Downloaded by guest on September 24, 2021 Medical Sciences: Brezinski and Serhan Proc. Natl. Acad. Sci. USA 87 (1990) 6251

Table 2. Agonist-induced release and transformation of stored 5-HETE and 15-HETE in human PMNs [3H]15-HETE-labeled PMNs, cpm [3H]5-HETE-labeled PMNs, cpm Agonist LXA4 LXB4 5,15-DHETE 15-HETE LXA4 5,20-DHETE 5,15-DHETE 5-HETE A23187 (5 uM) 62 32 752 72 0 1225 172 1113 PMA (100 nM) 10 20 175 645 0 885 45 800 FMLP (100 nM) 15 15 45 1300 0 2150 110 2220 Vehicle 0 0 0 30 0 107 0 247 PMNs (3 x 107 cells per ml) were labeled with either [3H]15-HETE and 15-HETE (30 ,uM) or [3H]5-HETE and 5-HETE (30 tIM) for 20 min at 370C. After washing in PBS, cells were exposed to agonists or vehicle alone [0.4% EtOH (vol/vol)] and incubated for 20 min at 37TC. Products were extracted and analyzed by reversed phase-HPLC equipped with a rapid spectral detector. Values represent the average cpm associated with PMN-derived products obtained for two representative experiments. Products were identified by both coelution with synthetic standards and characteristic UV spectra. transmission, since the latter may not represent an accurate In the present paper, we report that PMNs rapidly incor- value when comparing two distinct groups of cells (i.e., porate 15-HETE into inositol-containing phospholipid with PMNs vs. 15-HETE esterified PMNs) with potentially dif- the majority esterified into the sn-2 position ofPI (Figs. 1 and ferent light scattering properties (22). For PMNs exposed to 2). The overall pattern of 15-HETE uptake in PMN lipids was FMLP, the threshold concentration required to provoke distinct from that of either AA or 5-HETE (Table 1). In aggregation was =2 orders of magnitude higher for cells with addition, we present the evidence that esterified sources of esterified 15-HETE (P < 0.05). In contrast, with either either 15-HETE or 5-HETE can be mobilized and trans- A23187 or PMA, statistically significant differences in the formed to more polar products upon subsequent activation of threshold concentration required to induce aggregation were these cells (Fig. 3 and Table 2). The profile of products not evident. Moreover, when PMNs were incubated with generated by activated PMNs with esterified HETEs differed either AA (30 or 5-HETE (30 followed by washing, and was dependent on the structure of the esterified precur- ttM) ,uM) sor. PMNs that have esterified 15-HETE also display an the concentrations of these stimuli required to induce aggre- impaired ability to both generate LTB4 and aggregate in gation were not altered. The threshold concentrations of response to receptor-mediated stimulation (Fig. 4 and Table A23187 required were 50 + 0 nM, 50 + 0 nM, and 36.7 ± 10.9 3). In contrast, aggregation was not dramatically inhibited in nM for vehicle treated, 5-HETE, and AA, respectively (n = response to either A23187 or PMA. 3). Threshold concentrations with FMLP were 6.7 ± 1.4 nM, PMNs take up both 5-HETE and 12-HETE. In the case of 3.7 ± 1.1 nM, and 3.7 ± 1.1 nM for vehicle treated, 5-HETE, 12-HETE, a product of human platelets, PMNs deposit and AA, respectively (n = 3). 23.1% in TG with 2.0%o in PC (28). PMNs also incorporate 5-HETE with >60% of the added label in TG and -20% in DISCUSSION phospholipid [7.82% in PI, 7.37% in PC, and 4.25% in phosphatidylethanolamine (PE)] (10). Uptake of their pre- The signs of inflammation are evoked in part by eicosanoids, cursor AA is also >50% into TG followed by its acylation into which can stimulate vasodilation, increase vascular perme- phospholipid stores (10, 29). In the present study, [3H]15- ability, and promote the influx of leukocytes. In particular, HETE was rapidly esterified into inositol-containing phos- leukotrienes are potent stimulants of chemotaxis and can pholipid (Fig. 2) without appreciable incorporation into TG of induce changes in vascular permeability (23). Mono-HETEs PMNs. A preference for 15-HETE incorporation into PI has (5-HETE, 12-HETE, and 15-HETE), which are also gener- also recently been demonstrated in both endothelial cells and ated at sites of inflammation (6, 24), can promote chemotaxis, neuroblastoma cells and was previously shown in a macro- albeit at higher levels than those required of LTB4 (23, 25). phage cell line, which raises the possibility of a PI-specific 15-HETE injected into plaque lesions of psoriasis vulgaris in acyltransferase (30-32). Since >75% of the 15-HETE in humans results in complete regression of psoriatic lesions PMN phospholipid was found in inositol phospholipid (Figs. (26), and intraarticular administration of 15-HETE in canines 1 and 2 and Table 1), its presence may represent a site for with arthritis significantly reduces both the clinical severity regulatory events in receptor-mediated signaling via inositol and the volume of synovial effusates (27). However, the lipid metabolism (33). biochemical bases for these actions of 15-HETE remain to be Although evidence for agonist-induced release and further elucidated. oxygenation of stored HETEs has not been previously re- ported with human PMNs, it is well established that incor- Table 3. Comparison of agonist-induced aggregation with porated AA can be released and transformed by PMNs 15-HETE-esterified PMNs exposed to appropriate stimuli (10, 19, 34). FMLP, PMA, and A23187 each led to the release of label from PI (Fig. 3) and Threshold agonist concentration transformation of the HETE to more polar products (Table required for aggregation 2). [3H]15-HETE was released and converted to labeled 15-HETE-esterified Student's 5,15-DHETE, LXA4, and LXB4. The ionophore proved to be Agonist PMNs, nM PMNs, nM t test more effective in stimulating the formation of lipoxins from A23187 37.0 ± 12.2 366.7 ± 108.9 NS [3H]15-HETE originating from esterified sources. This find- PMA 0.4 ± 0.2 0.7 ± 0.3 NS ing is consistent with its ability to activate phospholipases FMLP 4.0 ± 2.6 666.7 ± 136.1 P < 0.05 and the 5-lipoxygenase (19). FMLP as well as PMA also stimulated the formation of labeled 5,15-DHETE and li- After cells were labeled with 15-HETE (30 ,uM) as described in poxins. In both cases, however, the majority ofthe recovered Table 1, PMNs (5 x 106 cells per ml) were challenged with various These concentrations of each agonist. The concentrations were increased deacylated [3H]15-HETE remained intact. observa- by increments of 0.5 order of magnitude until the minimum concen- tions were not restricted to the deacylation and oxygenation tration required to induce aggregation (threshold) was determined. ofesterified sources of 15-HETE but were also demonstrable Values represent the mean threshold concentration ± SE required with 5-HETE (Table 2). 5-HETE is esterified into PMN lipids for aggregation with duplicate determinations for PMNs from three (10, 35, 36). After labeling, stimulated PMNs released and separate donors. NS, not significant. transformed 5-HETE to both labeled 5,15-DHETE and 5,20- Downloaded by guest on September 24, 2021 6252 Medical Sciences: Brezinski and Serhan Proc. Natl. Acad Sci. USA 87 (1990) DHETE. In contrast to PMNs labeled with [13H]15-HETE, 5. Serhan, C. N. (1990) J. Bioenerget. Biomembr., in press. neither LXA4 nor LXB4 was generated from esterified 6. Murray, J. J., Tonnel, A. B., Brash, A. R., Roberts, L. J., II, sources of [3H]5-HETE with the stimuli examined. The Gosset, P., Workman, R., Capron, A. & Oates, J. A. (1986) N. Engl. J. Med. 315, 800-804. different profiles of products generated from cellular stores of 7. Duell, E. A., Ellis, C. N. & Voorhees, J. J. (1988) J. Invest. labeled HETEs are consistent with those observed with Dermatol. 91, 446-450. exogenous substrates (4). Also, the ability of FMLP to 8. Herlin, T., Fogh, K., Ewald, H., Hansen, E. S., Knudsen, V. E., stimulate the release and transformation of 5-HETE to its Holm, I., Kragballe, K. & Bunger, C. (1988) Acta Path. Microbiol. 15-hydroxy derivative provides further evidence that the Immunol. Scand. 96, 601-604. 15-lipoxygenase activity of human PMNs can be stimulated 9. Samuelsson, B., Dahldn, S.-E., Lindgren, J. A., Rouzer, C. A. & However, since Serhan, C. N. (1987) Science 237, 1171-1176. by receptor-mediated signal transduction. 10. Stenson, W. F. & Parker, C. W. (1979) J. Clin. Invest. 64, 1457- the major oxygenated product formed with each stimulus and 1465. [3H]5-HETE-labeled PMNs proved to be 5,20-DHETE, it is 11. Richards, C. F. & Campbell, W. B. (1989) Prostaglandins 38, likely that the w-oxidation route of 5-HETE dominates the 565-580. lipoxygenation when this HETE is liberated within PMNs. 12. Sekiya, J., Aoshima, H., Kajiwara, T., Togo, T. & Hatanaka, A. 5,15-DHETE enhances degranulation while the biological (1977) Agric. Biol. Chem. 41, 827-832. 13. Hawkins, D. J., Kfuhn, H., Petty, E. H. & Brash, A. (1988) Anal. consequences of 5,20-DHETE formation remain to be deter- Biochem. 173, 456-462. mined (37). 14. Boyum, A. (1968) Scand. J. Clin. Lab. Invest. 21, Suppl. 97, 77-89. The formation of lipoxins by FMLP-stimulated labeled 15. Van Dongen, C. J., Zwiers, H. & Gispen, W. H. (1985) Anal. PMNs demonstrates receptor-mediated generation of these Biochem. 144, 104-109. eicosanoids from stores within a single cell type. Recently, 16. 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L. & Galbraith, W. (1989) Biochem. Pharmacol. 38, 4151-4154. lipoxins. This form of "priming" and/or remodeling of 25. Waldman, J. S., Marcus, A. J., Soter, N. A. & Lim, H. W. (1989) membranes to give altered profiles of eicosanoids after a J. Invest. Dermatol. 92, 112-116. 26. Fogh, K., Sogaard, H., Herlin, T. & Kragballe, K. (1988) J. Am. second challenge may be relevant in inflammation and other Acad. Dermatol. 18, 279-285. physiologic events. 27. Fogh, K., Hansen, E. S., Herlin, T., Knudsen, V., Henriksen, Remodeling of PMN phospholipid with HETEs such as T. B., Ewald, H., Bunger, C. & Kragballe, K. (1989) Prostaglandins 5-HETE and 12-HETE has been suggested to alter membrane 37, 213-228. characteristics such as fluidity (10, 28). However, evidence 28. Stenson, W. F. & Parker, C. W. (1979) Prostaglandins 18, 285-292. 29. Chilton, F. H. & Murphy, R. C. (1986) J. Biol. Chem. 261, 7771- for agonist-induced release and lipoxygenation of esterified 7777. HETEs has not been previously established. The finding that 30. Shen, X.-Y., Figard, P. H., Kaduce, T. L. & Spector, A. A. (1988) PMNs with esterified 15-HETE in PI show an impaired Biochemistry 27, 996-1004. ability to generate LTB4 and did not aggregate to usual 31. McKinney, M. (1987) J. Neurochem. 49, 331-341. threshold concentrations ofFMLP suggests that these events 32. Stenson, W. F., Nickells, M. W. & Atkinson, J. P. (1983) Prosta- be related to some of the "anti-inflammatory" glandins 26, 253-263. may causally 33. Berridge, M. J. (1989) J. Am. Med. Soc. 262, 1834-1841. actions recently observed with injections of 15-HETE (26, 34. Haurand, M. & Flohe, L. (1989) Biochem. Pharmacol. 38, 2129- 27). Moreover, our findings suggest that cells can be primed 2137. by lipid remodeling to express new profiles of eicosanoids, 35. Bonser, R. W., Siegel, M. I., Chung, S. M., McConnell, R. T. & the balance of which may regulate the actions of proinflam- Cuatrecasas, P. 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