JOURNAL OF CELLULAR PHYSIOLOGY 143512-523 (1990)

Lipoxin A, and Lipoxin B, Stimulate the Release but Not the Oxygenation of Arachidonic Acid in Human Neutrophils: Dissociation Between Lipid Remodeling and Adhesion SANTOSH NICAM, STEFAN0 FIORE, FRANCIS W. LUSCINSKAS, AND CHARLES N. SERHAN* Hematology Division, Department of Medicine (S.N., 5.F., C.N.S.), and the Vascular Research Division, Department of Pathology ( F . W.L.), Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02 J 15 The profiles of actions of lipoxin A, (LXA,) and lipoxin B, (LXB,), two Iipoxygenase-derived eicosanoids, were examined with human neutrophils. At nanomolar concentrations, LXA, and LXB, each stimulated the release of 11-l4C1arachidonic acid from esterified sources in neutrophils. Lipoxin-induced release of [ l I4C]arachidonic acid was both dose- and time-dependent and was comparable to that induced by the chemotactic peptide f-met-leu-phe. Time-course studies revealed that lipoxin A, and lipoxin B, each induced a biphasic release of [ l ''C]arachidonic acid, which was evident within seconds (5-1 5 sec) in its initial phase and minutes (>30 sec) in the second phase. In contrast, the all-trans isomers of LXA, and LXB, did not provoke [1-'4C]AA release. Lipoxin-induced release of arachidonic acid was inhibited by prior treatment of the cells with pertussis toxin but not by its P-oligomers, suggesting the involvement of guaninine nucleotide-binding regulatory proteins in this event. Dual radiolabeling of neutrophil phospholipid classes with [l-'4C]arachidonic acid and ['Hlpalmitic acid showed that phosphatidylcholine was a major source of lipoxin-induced release of [I -'4C]arachidonic acid. They also demonstrated that lipoxins rapidly stimulate both formation of phosphatidic acid as well as phospholipid remodeling. Although both LXA, and LXB, (1OP8-10-"M) stimulated the release of [ I I4C]arachidonic acid, neither compound evoked its oxygenation by either the 5or 1 5-lipoxygenase pathways (including the formation of LTB,, 20-COOH-LTB4, 5-HETE, or 15-HETE). LXA, and LXB, (10 'MI each stimulated the elevation of cytosolic Ca2+ as monitored with Fura 2-loaded cells, albeit to a lesser extent than equimolar concentrations of FMLP. Neither lipoxin altered the binding of [-'H]LTB, to its receptor on neutrophils. In addition, they did not stimulate aggregation or induce adhesion of neutrophils to human endothelial cells. Results indicate that both LXA, and LXB, stimulate the rapid remodeling of neutrophil phospholipids to release arachidonic acid without provoking either aggregation or the formation of lipoxygenase-derived products within a similar temporal and dose range. Together they indicate that LXA, and LXB, display selective actions with human neutrophils and suggest that these eicosanoids possess unique profiles of action which may regulate neutrophil function during inflammation.

Received August 28, 1989; accepted February 13, 1990. *To whom reprint requestsicorrespondence should be addressed. S.N. is on sabbatical leave from the Dept. of Gynecological Endocrinology, Klinikum Steglitz, Free University of Berlin. Abbreviations used: BSA, bovine serum albumin; ED, electrochemical detection; 8-trans-LXB4, 5S,14R,15S-trihydroxy-6,8, 10,12-trans-eicosatetraenoic acid; 11-trans-LXA,, 5S,6R,15Strihydroxy-7,9,11,13-trans-eicosatetraenoic acid; 15-HETE, 15shydroxy-5,8,11-cis-13-trans-eicosatetraenoicacid; 5-HETE, 5S-hydroxy-8,11,14-cis-6-trans-eicosatetraenoicacid; f-met-leuphe (FMLP), formylmethionyl-leucine-phenylalanine;HBSS-, CQ 1990 WILEY-LISS, INC

Hank's balanced salt solution without Ca' and Mg ' ; leukotriene B, (LTB,), 55,12R-dihydroxy-6,14-cis-8,10-trans-dihydroxyeicosatetraenoic acid; lipoxin A, (LXA,), 5S,GR,l5S-trihydroxy7,9,13-trans-ll-cis-eicosatetraenoicacid; lipoxin B, (LXB,,), 5S,14R,15S-trihydroxy-6,10,12-trans-8-cis-eicosatetraenoic acid; NL, neutral lipids; PA, phosphatidic acid; PBS, phosphate buffered saline; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PMN, polymorphonuclear neutrophils; PTX, pertussis toxin; RP-HPLC, reverse phase high pressure liquid chromatography; TLC, thin layer chromatography; 12-HETE, 12S-hydroxy-5,8,14-cis-l0-trans-eicosatetraenoic acid. +

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513

LIPOXINS AND HUMAN PMN

Human neutrophils can play a central role in host purchased from NEN, Du Pont Company (Boston, MA); defense by virtue of their microbiocidal activities (re- TLC silica plates linear K6D and G 6 0 were from viewed in Weissmann et al., 1980; Snyderman and Whatman (Hillbow, OR). HPLC grade solvents were Goetzl, 1981). When exposed to chemoattractants (in obtained from J.T. Baker Inc. (Phillisburg, NJ). LXA,, vitro), neutrophils may respond by aggregating, gener- LXB,, FMLP, and cytochalasin B were each stored in ating active oxygen species, remodeling phospholipids, EtOH a t -40°C and diluted just prior to use in PBS, pH and releasing arachidonic acid (Bokoch and Reed, 7.45. Lipoxin stocks in PBS were discarded following 1980; Serhan et al., 1982a, 1983; Wynkoop et al., 1986; each experiment. Pertussis toxin and pertussis toxin p Sellmayer e t al., 1987). In these cells, unesterified oligomers were from List Biological Laboratories, Inc. arachidonic acid can be oxygenated by either the 5- or (Campell, CAI. 15-lipoxygenase (Samuelsson et al., 1987). Oxygenation of arachidonic acid by the 5-LO leads to the forPreparation of human neutrophil suspensions mation of leukotriene B, (Borgeat and Samuelsson, and labeling conditions 1979), which is a potent stimulus for human neutroFor each experiment, peripheral blood was obtained phils (Ford-Hutchinson et al., 1980; Goetzl and Pickett, from healthy donors by venipuncture using heparin as 1981; Serhan et al., 1982b), while interactions between the 5- and 15-LO in these cells can lead to the forma- anticoagulant. PMNs were isolated by Ficoll-Hypaque centrifugation (LSM, Organon Teknica Co., tion of lipoxins (Serhan et al., 1984, 1986; Samuelsson gradient et al., 1987; Serhan, 1989a). Unlike leukotrienes, li- Durham, NC) followed by dextran sedimentation. Red cells were removed by hypotonic lysis followed by cenpoxins possess a conjugated tetraene structure as well trifugation (Boyum, 1968). Isolated cells were susas a unique profile of activities when compared to those pended in Dulbecco's buffered saline (PBS) evoked by other eicosanoids (reviewed in Samuelsson containing both CaC1,phosphate (0.6 mM) and MgCl, (1.0 mM), et al., 1987; Serhan and Samuelsson, 1988). For exam- pH 7.45. These suspensions contained 98 ? 1% PMNs ple, LXA, (5S,6R,15S-trihydroxy-7,9,13-trans-ll-cisdetermined by light microscopy. eicosatetraenoic acid) stimulates vasodilation (Dahlen asAfter cells were adjusted to 30 x lo6 et al., 1987,1988; Busija et al., 1989), contracts smooth PMNs/mlisolation, and incubated (20 min, 37°C) with 0.25 pCi muscle (Serhan et al., 1986; Dahlen et al., 1987, 1988; of [l-14C]arachidonic acid mciimmole). Next, Spur et al., 1988), antagonizes the actions of peptido- cells were washed twice (800(52.0 rpm for 10 min) and susleukotrienes in certain tissues (Dahlen et al., 1988; pended in PBS. Aliquots of cell suspensions were reBadr et al., 19891, and can activate subspecies of iso- moved prior to incubation and the percent of label lated protein kinase C in vitro (Hansson et al., 1986; incorporation was determined. A Beta Trac 6895 scinShearman e t al., 1989). Its positional isomer lipoxin tillation counter (Tracor Analytic Inc., Elk Grove, IL) B4(5S,14R,15S-trihydroxy-6,10,12-trans-8-cis-eicosatewas used throughout. The percent of [l-14Clarachidonic traenoic acid) has a selective radioprotective action acid incorporation was 74.5 5 11.0% (mean ? S.D., with hematopoietic stem cells (Walden, 1988). Both li= 9). Following labeling, the integrity of the I1-14C1 poxins A, and B, share the ability to stimulate endo- nlabeled cells was determined by their ability to exclude thelial cells (Brezinski et al., 1989; Lefer et al., 19881, trypan blue. 98 ? 1%of the PMN excluded trypan blue and can block and cytotoxic activities of natural killer (mean t S.D., n = 9). cells (Ramstedt et al., 1987). Previous results have shown th at LXA, displays seTreatment with pertussis toxin lective actions with human neutrophils by stimulating migration without causing aggregation (Serhan et al., Following incorporation of [l-14Cl-arachidonate the 1984; Palmblad et al., 1987). Here, we report that both labeled PMN were washed twice in Hank's balanced LXA, and LXB, stimulate arachidonate release, lipid salt solution containing bovine serum albumin 0.25% remodeling, and increments in intracellular Ca2+ without CaC1, or MgC1, (HBSS-) and adjusted to 20 x without initiating the oxygenation of free arachidonate lo6 cells/ml. For these experiments, a portion of the or aggregation within the same temporal and concen- labeled cell suspensions were treated with pertussis toxin (PTX) (2 pg/ml) and DNase type I(5 0 U/ml) for 90 tration range. min a t 37°C. The remaining portion of labeled PMN MATERIALS AND METHODS was also incubated (90 min, 37°C) in parallel with treated cells. Next, both groups, PTX-treated and unMaterials treated, were washed first in HBSS- followed by PBS Synthetic LXA,, LXB,, 5-HETE, 12-HETE, and 15- (twice). PMN were resuspended (5 x lo6 cellsiml PBS) HETE were purchased from Biomol Research Labora- and their ability to exclude trypan blue and their ratories (Plymouth Meeting, PA) and AA from Nu Check diolabel content were examined. Following these proPrep Inc. (Elysian, MN). FMLP, BSA, cytochalasin B, cedures, 97.0 2.0%of the cells excluded trypan blue ionophore A23,187,PC, PI, PE, PA, and cholesteryl (mean c S.D., n = 3) and loss of radiolabel was not dearachidonate (used for TLC standards) were from tected. In a second series of experiments, PMN labeled Sigma Chemical Co. (St. Louis, MO); Dulbecco's phos- with [ l-14C]-arachidonate were incubated with the pphate buffered saline was from Whittaker M.A. Bio- oligomers of pertussis toxin, which are responsible for products (Walkersville, MD). Hank's balanced salt so- the binding of the holotoxin to eukaryotic cell surfaces lution without Ca' and MgC* (HBSS-) was from (vide supra), followed by addition of agonists (10 sec, GIBCO Laboratories (Grand Island, NY). L3H16-keto- 37°C). In these experiments the percent of [lPGF [3HlC16:0 (palmitate), [l-14ClC20:4, L3H1LTB,, ''Cl-arachidonate incorporation was 75.8 ? 16.8 (mean and 1?4C-(U)]C20:4 (arachidonate) radiolabels were ? SD, n = 3 ) .

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NIGAM ET AL

Arachidonic acid release [l4CIC2O:4labeled PMN (5 x lo6 cells/ml PBS) were incubated at 37°C for 5 min before addition of either LXA,, LXB,, FMLP, or vehicle. Incubations performed in triplicate were terminated at the denoted intervals by addition of 3.5 ml of chloroform/methanol (2:5 v/v) and extracted as in Walsh et al. (1981). [sHl6keto-PGF,, was added to each incubation as internal standard. The extraction recovery of [3H16-keto-PGF,, was 75.9 ? 13.8% (mean S.D., n = 9, d = 135). Next, samples were acidified (pH 3.5) by addition of HC1 and the organic and aqueous phases were separated by adding chloroform (1ml) and distilled water (1 ml). Following centrifugation (1,200 rpm for 3 m i d , the organic phases were washed with 1ml of distilled water and centrifuged again (1,200 rpm for 3 min) before concentrating the organic phases by vacuum evaporation (Speed Vac, Savant Instrument Inc., Farmingdale, NY). The materials were resuspended in chloroform/ methanol (50 pl; 2:5, v/v) and spotted onto silica linear K6D plates (heat activated) and developed by using petroleum etheridiethyl ethedacetic acid (50:50:1, vl v/v). Lipids were visualized by iodine staining, scraped, and suspended in Liquiscint (National Diagnostic, Manville, N J) and the content of radiolabel was determined (Reibman et al., 1988). Unesterified arachidonic acid was identified by its Rf value and by comigration with authentic [l-14C]arachidonic acid, which was chromatographed on each TLC plate.

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Phospholipid an aly ses

Phospholipid analyses were performed by using dual radiolabel incorporation (Walsh et al., 1981) and twodimensional TLC as previously described by Serhan et al. (1982a). Briefly, PMN (30 x lo7 cells) were incubated with I3H1- palmitate (30 Ci/mmole) and l1-l4C1arachidonic acid (0.25 FCi) for 40 min at 37°C. Cells were washed twice in PBS (800 rpm, 10 min) prior to incubations with lipoxins. The percent of [ 3H]palmitate incorporation was 73.6 15.595, and Il-14C]-arachidonate incorporation was 61.5 k 6.8% (mean k S.D., n = 4 ) . Incubations with either FMLP, LXA,, LXB,, or vehicle (0.01% EtOH; v/v) were performed in duplicate, terminated by rapid addition of cold chloroform/methanol ( 2 5 v/v; 3.5 ml), and extracted (vide supra) without acidification. Samples were spotted on silica G-60 plates and two-dimensional TLC was performed by utilizing chloroform/methanol/ammonium hydroxide (65:25:6, v/v) as solvent for the first dimension and, after drying plates under NB,the second dimension was developed with chloroform/acetone/methanol/acetic acid/H,O (3:4:1:1:0.5, v/v). Phospholipids were identified and scraped, and the amounts of radiolabel in each were determined by using a double label 3H/14Ccounting program.

*

Analysis of eicosanoids Freshly isolated PMN were labeled with [14C(U)] C20:4 by incubating cells with 0.25 ~ C i / 3 0x lo6PMNI ml PBS for 20 min at 37°C. The suspensions were washed twice to remove excess [14C(U)larachidonic

acid. Percent of [ 14C(U)]arachidonic acid incorporated in these experiments was 75.0 2 9.7; n = 3. Following labeling, the cells were incubated with various stimuli or vehicle alone (1 ml PBS, pH 7.45, 37°C) and the incubations were terminated, a t the indicated intervals, by addition of cold methanol (2 vol). PGB, and 9-hydroxy-linolenic acid (300 ng) were added as internal standards and eicosanoids were extracted and analyzed as described in Serhan (1989b). Briefly, samples were placed at 4°C for 30 min and centrifuged (2,500 rpm for 15 min), and supernatants were removed. The resulting pellets were resuspended in ethanol (2 ml) and the precipitation was repeated. The resulting ethanol-methanol containing fractions were each pooled and taken to dryness by rotoevaporation. The samples were suspended in methanol/water (1:45, v/v>,acidified to pH 3.5, and rapidly loaded into a cartridge (Cl,-Sep Pack). Materials were eluted with hexane (10 ml) and methyl formate (10 ml). This latter fraction was concentrated under nitrogen, suspended in methanol (50 pl), and injected into a RP-HPLC system equipped with a Beckman Ultrasphere-ODS (4.6 mm x 25 cm) and solvent controller (LKB, Bromma, Sweden). The column was eluted with a gradient consisting of MeOH: H,O:acetic acid (65:35:0.01, vol/vol) as phase one (To20 min) and a linear gradient with Me0H:acetic acid (99.99:O.Ol v/v) as phase two (30-40 min). The flow rate was 1.0 ml/min with a pressure of 110 bar. The eluate collected in fractions every 20 sec was counted for radioactivity determination. The HPLC system was equipped with a photodiode array rapid spectral detector linked to a n AT&T PC 6300, and post-HPLC run analysis was performed by using a Wavescan EG 2146002 program (Bromma, Sweden). Products were identified by coelution with synthetic standards and by their UV spectra recorded on-line (Serhan, 1989a,b). In additional experiments, extracted materials were chromatographed on silica C, columns eluted with hexanel ether (9O:lO) and hexanelether (60:40, v/v). Materials eluting in the hexanelether fractions (60:40, v/v) were concentrated and injected into a second RP-HPLC system (Waters Associates, Millipore Col, Milford, MA). In this system, the column (Altex, Ultrasphere-ODS, 4.6 mm x 25 cm) was eluted with methanoliwaterlacetic acid (75:25:0.01, v/v) a t 1 ml/min, and the UV detector (model 481) was set a t 230 nm to monitor E-HETE, 12-HETE, and 5-HETE. A third RP-HPLC system was employed to detect picogram levels of 20-OH-LTB4and 20-COOH-LTB4 as described by Herrmann et al. (1987,1988).The column (Altex, Ultrasphere-ODs, 4.6 mm x 25 cm) was eluted with methanol/water (65:35, v/v) with TFA (1mM) and the UV detector (Waters Assoc. model 481) was set a t 270 nm (to monitor the conjugated trienes) and coupled to an electrochemical detector (Waters Assoc. model 460) to permit dual recording of the UV and ED outputs. The ED was equipped with a thin-layer glassy carbon electrode and potentials were measured against a n Ag/AgCl reference electrode filled with 3M LiCl in 65% MeOH. The electrochemical detector was set at +1.35 V with a 2 sec response time. The minimum detection of o-oxidized products of LTB, (20-OH-LTB4 and 20-COOH-LTB4) was approximately 50 pg, which is consistent with the values of 50-60 pg reported by Herrmann et al. (1987).

515

LIPOXINS AND HUMAN PMN

PMN aggregation, measurement of intracellular Ca2+, and PMN-endothelial cell adhesion a s s a y s The aggregation of human neutrophils was studied by monitoring changes in light transmittance utilizing a four channel aggregation profiler (model PAP-4, Biodata Co., Hatbow, PA). The instrument permitted simultaneous monitoring of changes in light transmittance with four separate samples (three exposed to putative stimuli and the fourth incubated in the presence of vehicle alone. PMNs, obtained from the same cell preparations used for the dose response and time course studies, were adjusted to 5 x lo6 celldm1 in PBS, pH 7.45, suspended in siliconized cuvettes (Biodata Co.), and warmed a t 37°C. In some determinations, cytochalasin B (5 pg/ml) was added 3 min before challenging cells with various agents. Magnetic stirring (-600 rpm) was employed in each of the four cuvettes. Mobilization of intracellular Ca2+ was determined as described (Luscinskas et al., 1990). Briefly, PMN were washed with HBSS- and resuspended in HBSScontaining 0.5% BSA and 0.1% glucose and incubated a t 37°C for 10 min with 1 pM Fura-2/AM (Molecular Probes, Eugene, OR). Excess Fura-2iAM was removed by centrifugation and the Fura-2 loaded PMN were suspended in HBSS' for 5 min at 37°C before addition of either FMLP, LXA,, LXB,, or the all-trans-LX isomers. The changes in fluorescence were measured by using a SPEX (Edison, NJ) Fluorolog I1 (model CM1) spectrofluorimeter equipped with continuous stirring, a beam splitter, two excitation monochrometers, and a dual mirror chopping mechanism to permit rapid alternating (30 Hz) excitation of Fura-2 a t two wavelengths, 340 nm and 380 nm. The excitation bandwidths were set at 6.6 nm. The ratio of emitted fluorescence a t 505 nm (7.2 nm bandwidth) allowed calculation of the intracellular levels of free Ca2+ ([Ca"'li) as in Grynkiewicz et al. (1985). Fluorescence signals were calibrated by using 80 FM digitonin to permit equilibration of intracellular and extracellular followed by addition of 1.0 M Tris, 300 mM Ca2' (Fmax) EGTA, pH > 10.0 (Fmi,,). Lipoxin-induced neutrophil adhesion to cultured vascular endothelial cell (HEC) monolayers was determined as described by Gimbrone et al. (1984). Briefly, human endothelial cells were isolated from the umbilical cord veins of 2-5 normal term cord segments. Primary cultures were prepared with medium 199 (M.A. Bioproducts, Bethesda, MD) with 20% fetal bovine serum. Cultures were serially passaged and maintained in M199/20% FCS supplemented with endothelial cell growth factor (50 p,g/ml; Biomedical Technologies, Inc., Stoughton, MA) and porcine intestinal heparin (100 pglrnl; Sigma, St. Louis, MO) in Costar tissue flasks. For adhesion assays, endothelial cell strains were replicate plated (passage levels 2-3) in gelatin-coat (0.1%) microtiter wells. Isolated neutrophils were labeled with "'In-oxine and coincubated (2 x lo5 PMNlwell) for 10 min a t 37°C with HEC monolayers. After addition of stimuli, non-adherent PMN were removed by gentle centrifugation (250 x g) as in Bevilacqua et al. (1985). Each incubation was performed in quadruplicate in the presence or absence of agonists. +

Binding of l3H1LTB, with neutrophils LTB, binding experiments were performed as in Lin et al. (1984). Briefly, isolated neutrophils (10 x 10' cells/ml) were incubated for 10 min a t 4°C with l3H]LTB, (1 nM) in the presence or absence of either cold LTB,, LXB,, LXA,, 5-HETE, or 15-HETE a t various concentrations. All incubations were performed in triplicate in polypro ylene tubes (12 x 75 mm) and specific binding of [BH-LTB,] was determined (Lin et al., 1984).

RESULTS U p t a k e and agonist-induced release of [l-'4Clarachidonic acid Isolated PMN incorporated 74 k 11%(n = 9; mean S.D.) of [l-14C]-labeled arachidonic acid into lipid stores following 20 min incubation a t 37°C; 27.1 -t 1.9 (n = 4; mean ? S.D.) percent of the labeled arachidonic acid was esterified into phospholipid classes. These values are in accordance with those previously obtained with human neutrophils (Walsh et al., 1981; Chilton and Murphy, 1986). Upon addition to labeled neutrophils, both LXA, and LXB, stimulated the release of [1-'4C]-arachidonic acid in a dose- and time-dependent fashion (Figs. 1 , 2 ) .In each experiment, PMN obtained from individual donors were exposed in parallel to the chemotactic peptide FMLP a s well a s LXA, and LXB, for purposes of comparison. Previous results have shown that f-met-leu-phe is a potent stimulant for lipid remodeling (Serhan et al., 1982a; Wynkoop et al., 1986).The present findings with f-met-leu-phe (Figs. 1, 2) are in agreement with those values previously reported both with respect to time course and concentration. When compared a t equal molar concentrations (10-7M), time course experiments indicated that LXA,, LXB,, and f-met-leu-phe each stimulated a rapid release of [l-14C]arachidonic acid that was evident within 5-30 sec followin addition of stimuli. Maximal levels of unesterified 11-F4C]-arachidonic acid were observed at 15 sec following exposure of the cells to either LXA,, LXB,, or f-met-leu-phe (10p7M)(Fig. 2). Here, unesterified [l-14C]-arachidonic acid was identified, following extraction, by TLC and comigration with synthetic radiolabel-containing standard. A second phase of [l-14C]arachidonicacid release was noted with each compound. Dose-response studies showed that both LXA, and LXB, at 10-7M were essentially equipotent, while a t concentrations below 10-7M LXA, was ineffective in stimulating the release of [ l-l4C1-arachidonic acid (Fig. 2A). In addition, LXB, stimulated release of arachidonic acid at 10-8M, which was comparable in magnitude to that induced by FMLP (lOpsM). The shapes of these dose-response curves are consistent with those reported for chemotactants such a s FMLP (Sellmayer et al., 1987; Palmblad et al., 1987). Next, we examined the extent of lipid remodeling and the site of [l-14C]-arachidonicacid release utilizing dual radiolabeled neutrophils. The sn-2 positions of neutrophil phospholipids were labeled with [ 1-l4C1arachidonic acid, and their sn-1 positions were labeled with ['HI-palmitic acid as described by Walsh et al. (1981). Following addition of optimal concentrations of either LXA,, LXB,, or FMLP (10p7M),the changes in radiolabel content of phospholipid classes were deter-

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516

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NIGAM ET AL.

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LXB4 Fig. 1. Lipoxin-induced release of I1 -'4Clarachidonic acid from labeled human PMN: Dose-response comparison with FMLP. Human neutrophils labeled with [l-'4Clarachidonic acid (5 x 10' cellsiml) were warmed 5 min a t 37°C followed by addition of either LXA,, LXB,, (A) or FMLP (B) (added in 20 kl aliquots). Incubations were terminated at 10 sec by addition of cold chlorofordmethanol (3.5 ml; 2:5 vlv). Unesterified [ l-14Clarachidonic acid was identified by TLC following extraction as described under "Materials and Methods." Results represent the mean -t S.E. of three separate experiments with triplicate determinations. In each experiment, the actions of the three compounds were compared with PMNs from the same individual. All values P < 0.05 vs. vehicle-control. * denotes non-statistically significant values.

mined after extraction and separation by two-dimensional TLC. LXA,, LXB,, and FMLP each induced extensive changes in the phospholipid classes of human PMN (Fig. 3). Comparisons between the [1-l4C1 and ['HI label content of PL classes following addition of stimuli revealed that the major site of [l-l4C1arachidonic acid release induced by either LXA,, LXB,, or FMLP was from phosphatidylcholine (Fig 3). This is supported by the observed decrement in 11- C I content in the PC pool, which was not accompanied by changes in the [3H] content in PC (Fig. 3B). This finding suggests t h a t lipoxins induce arachidonate release via activation of PLA,, which has been documented with other stimuli, including FMLP, A,, and zymogen (Serhan et al., 1982a; Wynkoop et a)., 1986; Walsh et al., 1981; Chilton and Murphy, 1986). Following addition of stimuli, parallel changes in both labels ([1-14C]and [3H]) were found in PA, indicating that LXA, and LXB, each stimulate phosphatidic acid formation (Fig. 3). In this respect, LXB, was more effective than LXA,, and the extent of PA formation evoked by LXB, was similar to that obtained with FMLP. LXB, also gave parallel decrements in both labels in PI as did FMLP. In contrast, LXA, did not induce statistically significant change in labeled PI. Together these results suggest that LXB, can also activate PI metabolism, possibly by a phospholipase C mechanism, and that both LXA, and LXB, promote rapid phospholipid remodeling in human PMN.

FMLP

cine fsecl Fig. 2. Time course of lpoxin-induced release of 11'*C]C20:4. Human neutrophils were labeled with [1-'4ClC20:4 as described in "Materials and Methods." Labeled PMN (5 x lo6 celldml) were incubated (1 ml) 5 min a t 37°C followed by addition of 20 p1 of either LX.4, (upper), LXB, (middle), or FMLP (lower) (final concentration 10-7M).Incubations were terminated a t the denoted intervals by addition of 3.5 ml of cold chloroform/methanol ( 2 5 , viv). Unesterified ll-'4C1C204 was extracted and analyzed as described in Figure 1. Values represent the mean I S.E. of three separate experiments with triplicate determinations. The levels of significance, P , were determined by a one tailed, paired t-test. The asterisks denote: *,P< 0.025; **, P < 0.005; ***, P < 0.0005 when compared to incubations exposed to vehicle alone at each time interval.

neutrophils via a pertussis toxin-sensitive step (Bokoch and Gilman, 1984; Okamura et al., 1985; Feltner e t al., 1986; Mong et al., 1986; Becker et al., 1986).Therefore, we examined next whether lipoxin-induced release of L1-14C]arachidonic acid was pertussis toxin-sensitive. Results in Table 1 indicate that prior treatment of [l14Cl-arachidonate-labeledPMN with pertussis toxin completely inhibited the actions of LXA,, LXB,, and Inhibition of LXA,, LXB4, and FMLP-stimulated FMLP. The results obtained here with FMLP are conarachidonic acid release by pertussis toxin sistent with those previously reported with guinea pig treatment of neutrophils neutrophils (Bokoch and Gilman, 1984). In a second A number of eicosanoids mediate their actions by group of experiments the actions of the P-oligomers of interacting with receptors which couple to G proteins pertussis toxin, extracellular C a 2 + ,and the isomers of in the plasma membrane (reviewed in Smith, 1989), LXA, and LXB, were assessed. Unlike pertussis toxin, and several chemoattractants mediate their actions on treatment of labeled PMN with the P-oligomers of the

517

LIPOXINS AND HUMAN PMN

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-20 -

RtSE, n = 4 ,d = 0 Fig. 3. Changes in ll-’*C/C20:4and IJHlC16:0 content of PMN phospholipids following addition of either LXA,, LXB,, or FMLP. Following dual labeling of PMN with [1-14ClC20:4 (A) and (B) I’HlC16:O as described under “Materials and Methods,” neutrophils (30 x 10“ cells/ml) were warmed 5 min at 37°C and incubated with 20 ~1 of either vehicle, LXA, (dotted bars), LXB, (striped bars), or FMLP (full bars) (10 7M). Incubations were terminated at 10 sec by addition of 3.5 ml of chloroformimethanol, extracted, and analyzed as described

under “Materials and Methods.” Results are the mean 2 S.E. of four separate experiment with duplicate determinations. Changes in the [1-’4ClC20:4(panel A) and I3H1C16:0(panel B) content in each phospholipid class are expressed as the percent difference obtained with PMN incubated with vehicle alone (EtOH final concentration less than 0.01%by volume). Radiolabel content within each PMN’s phospholipid class determined before addition of agonist are expressed in cpm (mean ? S.E.).

toxin did not significantly inhibit either FMLP or li- obtained following incubation of [14C(U)]-labeledPMN poxin-induced release of [l-’4Clarachidonate (Table 2). with LXA, (lOP7M).The UV tracing a t 230 nm shows The trans-isomers, e.g., 11-trans-LXA, and 8-trans- the separation of unlabeled standards (15-HETE, 12LXB,, were ineffective in stimulating the release of HETE, and 5-HETE). Neither LXA, nor LXB, stimu[1-’4C]arachidonate at equimolar concentrations to ei- lated the formation of mono-HETE’s (n = 3), suggesting ther of their native forms (10-7M). In addition, prior that lipoxins do not activate either the 5- or 15-lipoxyexposure of the labeled cells to EGTA (5 mM) resulted genases of human neutrophils at submicromolar conin approx. 40.5%inhibition of FMLP-induced response centrations. To examine if lipoxins stimulate the formation of while LXA,-induced release of [l-’4Clarachidonate LTB, or its o-oxidation products 20-OH-LTB, and 20was unaffected by chelation of extracellular Ca2+. COOH-LTB, by neutrophils, a second series of experiDo lipoxins stimulate eicosanoid formation by ments was performed utilizing the same incubation human neutrophils? conditions, and the products were analyzed following Results from several recent studies indicate that li- extraction by a gradient HPLC (see Methods). The elupoxins stimulate the formation of cyclooxygenase prod- ants were collected a t 30 sec intervals and the fractions ucts by endothelial cells (Brezinski et al., 19891, guinea were assessed for I4C content. The limit of detection for pig aortas, isolated arteris, and lung tissues (Wikstrom [ 14C(U)]-labeled eicosanoids following HPLC and scinet al., 1989; Dahlen, 1989). To determine whether li- tillation counting in this system was approximately 13 poxins stimulate the transformation of arachidonic pg. In three separate experiments, neither LXA, nor acid by lipoxygenase pathways in human PMN, we in- LXB, stimulated the formation of either leukotriene B, cubated [14C(U)] labeled cells with either LXA, or or its w-oxidation products by human PMN (data not LXB, and analyzed the extracts of these incubations shown). Since receptor-mediated formation of LTB, by for radiolabeled eicosanoids by RP-HPLC. Figure 4 isolated PMN can be both difficult to detect and vary in shows a representative HPLC profile of mono-HETE’s extent because of rapid w-oxidation of LTB,, we moni-

518

NIGAM ET AL.

TABLE 1. Pertussis toxin sensitive release of L1-'4ClC20:4 from labeled human neutrophils following addition of either LXA,, LXB.. or FMLP'

I

15-HETE

5-HETE

I

I 1-'4cIc20:4 Pertussis toxin treated cells %,

%,

Incubations Acpm increase Acpm inhibition 24 O.O? 0.0 100 PMNs + LXA, 10 7M 200 f 62 416 t_ 52 49 0.0 t 0.0 100 PMNs + LXB, IO-'M 60 0.0 ? 0.0 100 PMNs + FMLP 10 7M 513 t 90 'After labeling human neutrophils with ll-"CIC20:4, cells (20 x lofi PMNlml) were transferred in H B S S containing 0.25% of fatty acid-free BSA incubated at 37°C for 90 min in the presence ofpertussis toxin ( 2 pglml) and DNase type l(50 Uiml). Next, cells were washed to remove the pertussis toxin and adjusted to 5 x lo6 PMNiml in PBS, pH 7.45 without BSA. Aliquots of labeled PMNs from the same cell preparations were taken and incubated in parallel in the absence of PTX.After treatment, cells were incubated for 5 min a t 37°C followed by addition of either vehicle, LXA,, LXB,, or FMLP a t 10-,M (EtOH final concentration less than 0.01'2 vivi. All incubatlons were terminated a t 10 see and unesterified ll-'4C1C20:4was extracted and quantified a s described in Figure 1 i n = 3 ) . Results are the mean c S.E. of a representative experiment with triplicate determinations for each experimental point.

TABLE 2. Release of [1-14C]C20:4from labeled human neutrophils: effects of pertussis toxin p oligomers, extracellular Ca2 ' , and stereoisomers' hcum. ll-14C1C20:4 Incubations lO-,M hcpm EGTA" p-oligomers3 343.6 2 29.1 204.5 -t 37.5* 299.0 2 43.9 PMNs + FMLP PMNs + LXA, 169.0 2 7.0 171.5 2 33.0 166.2 2 17.2 7.3 2 20.0 PMNs + 11-trans-LXA, PMNs + 8-trans-LXB, -72.8 2 69.0 'PMN were labeled as in Table 1. 'EGTA, (5 mM) + Mi'+ (1 mM) was added 20 sec before the addition of either FMLP or M A , . 10-7M. 'Labeled PMN'S (20 x lo6 cellslml) were treated with p oligomers of the pertussis toxin (5 pgiml equimolar to the 2 pgiml used for the holotoxin) under the same experimental conditions described in Table 1. Results are expressed a s the mean % SE of three separate experiments with 8 determinations. *P .C 0.05.

I

I

I

1

1

0

10

20

30

40

I

Time f m h / Fig. 4. RP-HPLC profile of material obtamed following incubation of [I-'4Clarachidonic acid labeled PMN with LXA, ( I W 7 M ) . Column, Altex Ultrasphere-ODS (4.6 mm x 25cm); solvent methano1:water: acetic acid (75:25:0.01, viv); flow rate 1 mlimin. The ultraviolet detector was set at 230 nm. Upper tracing shows the elution profile of unlabeled internal standards (15-HETE, 12-HETE, 5-HETE). Lower plot gives the T'4C(U)I content of eluted fractions. The profile is representative of three separate experiments obtained with labeled PMN exposed to either LXA, or LXB, (10-7M). TABLE 3. Formation of 20-OH-LTB, and 2?-COOH-LTB4 by activated PMN: detection by ED-UV HPLC Incubation PMN + A,,,,,

PMN PMN

(5 FM)

+ FMLP ( ~ o - ~ M ) +

LXA, ( 1 0 - 7 ~ )

20-OH-LTB, 722.9 f 22.7 55.3 t 18.1 0.0 2 0.0

20-COOH-LTB4 186.2 2 5.9 21.0 2 9.3 0.0 ? 0.0

'PMN (30 x lo6 cells) in PBS (1 ml) were incubated for 20 min at 37°C with either A23,187(2.5 pM), FMLP (10-7M), LXA, (10-7M), or vehicle alone (0.01% v/v EtOH). Incubations wore stopped by addition of MeOH (2V). extracted, and chromatographed as described under Methods. The o-oxidation products of LTB, were quantitated by ED-UV RP-HPU: as in Herrmann et al. (1987).Results are expressed in nglincubation; mean -t SD of three individual experiments where the amounts detected in incubations with vehicle and PMN were subtracted from values obtained with agonists.

evident a t < l o 0 nM of either LXA, or LXB, (Table 4). tored the oxidation products of LTB, by using a selec- Both LXA, and LXB, were essentially equipotent in tive method employing ED-UV RP-HPLC (Table 3). stimulating [Ca2 li, while neither of their all-transBoth A,,,,,, and FMLP stimulated the production of isomers stimulated detectable increments in [Ca2+1,. w-oxidation products of LTB,. PMN incubated with However, neither compound ( 10-'-1OP6M) stimulated FMLP generate far lower levels of o-products than PMN aggregation in the same concentration or tempocells incubated with A23,187,as previously observed by ral range found to activate arachidonate release. RepHaurand and Flohe (1989) and Herrmann et al. (1987). resentative aggregation tracings are shown in Figure In contrast, LXA, a t 10-7M did not stimulate detect- 7. Although PMN did not aggregate in response to eiable levels of w oxidation products of LTB,. These find- ther LXA, or LXB,, the same cells did release [lings with ED-UV-RP-HPLC are consistent with those 14C120:4 in response to either compound ( n = 2). obtained with 14C-label detection after RP-HPLC. Since aggregation reflects homotypic adhesion beTaken together, these results indicate that lipoxins tween neutrophils, we also evaluated whether lipoxins stimulate arachidonic acid release without activating stimulate heterotypic adhesion by monitoring the efthe enzymes required for its further transformation by fects of these compounds in adhesion of neutrophils to neutrophils. cultured human endothelial cell monolayers. LTB,, ionophore, and PMA each stimulated the adhesion of Ca2+ mobilization, aggregation, and adhesion labeled neutrophils to monolayers of endothelial cells with v a s c u l a r endothelial cells (Table 5). In contrast, neither LXA, nor LXB, (lop6Since Ca2+ mobilization is held to play a critical role 10-8M) stimulated neutrophil adhesion to endothelial in neutrophil activation and in the activation of the cells. Together, these findings suggest that lipoxins do 5-lipoxygenase (Borgeat and Samuelsson, 19791, we not stimulate adhesion of neutrophils. next determined if lipoxins stimulate increments in Do lipoxins interact w i t h LTB, receptors on [Ca"], with Fura-2-loadedPMN. LXA, and LXB, each h u m a n PMN? stimulated a rapid yet relatively small increase in PMN possess high affinity receptors for LTB, that [Ca2+Ji which reached maximal levels within 10 sec following addition (Fig. 5). Ca2 mobilization was not may mediate its chemotactic activities (Lin et al., 1984; +

+

LIPOXINS AND HUMAN PMN

I *. c

-

I t t

fMLP ( 3 0 0 n M )

2 0 sec

B.

+COOH

Y

r c.

LXB4 ( 3 0 0 n M ) &COOH

Fig. 5. FMLP, LXA,, and LXB,-induced changes in Fura-2 loaded human neutrophils. Neutrophils were loaded with Fura-2 as described under Methods. A: FMLP (300 nM). B: LXA, (300 nM). C: LXB, (300 nM). Digitonin (80 mM) and EGTA (12.5 mM) were added to determine F,,,, and F,,,. [Ca"' 1, values were calculated by using 340/380 ratio as reported by Grynkiewicz et al. (1985). The tracings are representative of three separate experiments.

Mong et al., 1986; Goldman and Goetzl, 1984). Since lipoxins stimulated lipid remodeling without provoking other neutrophil responses (Figs. 1-5), it is possible that lipoxins may act as partial agonists on the PMN LTB, receptor. To address this question, the effects of LXA,, LXB,, 15-HETE, and 5-HETE on the binding of L3H]-LTB, were examined (Fig. 6). Increasing concentrations of unlabeled LTB, blocked the specific binding of ['HI-LTB, (1 nM) as previously reported (Lin et al., 1984). In contrast, neither LXA, nor LXB, effectively altered (10-10-10-6M) the binding of ['HI-LTB,; 15HETE, which shares C15-C20 identity with lipoxins, did partially block [3H]-LTB, binding, albeit at high concentrations ( 10P6M).These observations therefore suggest t h a t LXAl and LXB, do not act on the LTB, receptor in human neutrophils.

DISCUSSION LXA, is a highly stereospecific inducer of neutrophil chemotaxis t h a t is active in the nanomolar range, but does not elicit aggregation within the same concentration range (Serhan et al., 1984; Palmblad et al., 1987). LXA, has also been shown to be a chemokinetic agent

519

(Lee et al., 1989; Spur et al., 1988) and can stimulate oxygen radical generation by PMN when they are exposed to concentrations in excess of 1 p.M (Palmblad et al., 1988). Although lipoxins possess biological activities in several systems that are distinct from those of other eicosanoids (Samuelsson et al., 1987; Serhan and Samuelsson, 1988), little information is available regarding the cellular events upon which they act. In the present paper, we report that both LXA, and LXB, stimulate rapid lipid remodelin release arachidonic acid, and mobilize cytosolic Cak without triggering the oxygenation of arachidonic acid or neutrophil adherence within the same temporal and concentration range. Lipoxins A, and B, each stimulated the release of [l-14Clarachidonic acid from labeled PMN in a doseand time-dependent fashion. The magnitude and time course of arachidonic acid release induced by lipoxins were similar to those evoked by the chemotactic peptide f-met-leu-phe when the compounds were compared with PMN's from the same donors (Figs. 1 , 2 ) .The time course of release of [l-'4C]arachidonic acid proved to be a biphasic response with all three stimuli (Fig. 2). The levels of unesterified [ l-'4C]arachidonic acid were maximal within 15 sec and declined by 30 sec after addition of either LXA,, LXB,, or FMLP. Results obtained here with FMLP are consistent with the rapid and extensive lipid remodeling previously reported with neutrophils exposed to this chemoattractant (Agwu et al., 1989; Serhan et al., 1982a; Wynkoop et al., 1986). The actions of LXA, and LXB, were stereoselective since their all-trans-isomers were ineffective (Table 2). These results with LXA, and LXB, provide the first documentation indicating that lipoxins are potent stimulants for arachidonic acid release and lipid remodeling in human neutrophils (Figs. 1-3). Upon release, however, arachidonic acid was not transformed by either the 5- or 15-LO of human neutrophils (Fig. 4). In addition, lipoxins did not stimulate the formation of either LTB, or its w-oxidation products (Table 3). Thus, although lipoxins stimulated the release of arachidonic acid, it was not further transformed to other bioactive eicosanoids. These results with PMN are in sharp contrast to the actions of lipoxins with other cell types and tissues in which lipoxins can stimulate the release as well as transformation of arachidonic acid. For example, LXA, and LXB, stimulate endothelial cells to generate their primary arachidonic derived product, namely PGIz (Brezinski et al., 1989), while guinea pig lung strips exposed to LXA, generate thromboxane A, (Wikstrom e t al., 1989; Dahlen et al., 1989). These results are consistent with the notion that lipoxins stimulate the release of arachidonic acid but do not trigger the generation of appropriate signals a t the levels required t o activate the lipoxygenase pathways of human neutrophils. Activation of the 5-lipoxygenase in human leukocytes is regulated by a complex multicomponent system involving C a 2 + ,ATP, and several nondialyzable factors (Rouzer and Samuelsson, 1987; Puustinen et al., 1988). Therefore, our findings that LXA, and LXB, stimulate ra id but small increments in the levels of cytosolic Ca!' a s monitored with Fura-2 loaded cells (Table 1) are consistent with the low levels of Ca2 ' required to activate phospholipases, which may not be

520

NIGAM ET AL

TABLE 4. LXA, and LXB, mobilize intracellular Ca" ' in Fura-2 loaded human PMN: comparison with FMLP' lea2+I, LXB,

LXA, Concentration (nM) 300 100

30

Net increase (nM) 89 2 47* 15 2 2* 0

Net increase (nM) 86 ? 12" 15 -t 9" 1 2 2

Fold increase 2.2 2 0.5 1.3 2 0.1 0

-

FMLP Net increase (nM) 595 t 99"" 623 t 37"" 547 f 43""

Fold increase 2.8 -+ 0.2 1.3 -+ 0.1 0

Fold increase 13.6 f 2.1 12.2 +. 2.5 12.2 ? 1.4

'Agonist-induced changes in [Ca"'l, were determined by using Fura-2 loaded PMN (Luscinskas et al., 1990). Changes in Pura-2 fluorescence were monitored by using a SPEX-Fluorolog I1 spectrofluorimeter equipped with continuous stirring. Excitation of Fura-2 was monitored at two wavelen hs, 340 and 380 nm. the ratio of which (505 nm) permits calculation of [Ca2 ' I, (Grynkiewicz e t al., 1985; Luscinskas e t al., 1990). Individual values for basal [Ca 1, were substrated from peak increases in [Ca' ' 1, after addition of each compound. Values represent the mean 5 SE of three separate experiments. Statistically significant from basal ICa' 11: *iP 5 0.05); **(Pc- 0.01).

.P +

+

TABLE 5. Neutrophil adhesion to endothelial cell monolayers: effect of LXA, and LXB,' Addition

10' 'M IO-'M 10-"M LXA, 10-6M 10-'M 10 "'M LXB, 10-'M 10-'M lO-'"M Vehicle alone

Neutrophils bound/mm" HEC monolayer 2,637 2 189 1,636 t 128

1

1201

1,182 t 50 509 t 160 502 t 99 268 302 208

f f

2

60 86 46

207 ? 25 149 2 44

24i 44 254

?

48

'Standard monolayer adhesion assays were performed in the presence of the indicated concentrations of agonists (Bevilacqua et al., 1985; Gimbrone et al., 1984). Isolated PMN were labled with "'In-oxine and 100 ~1 ( 2 x lo5 cell per well) were added to endothelial cell monolayers in the presence or absence of agonists for 10 min at 37°C. Each point represents the Mean 2 SD of four determinations from a representative experiment with n = 2 .

tL *.--j(

2o 0

\ L

15-HETE

+-----+5-HETE 1 o-'O

10-*

1 o-6

above the threshold required to trigger the oxygenSD, n.3, d =9 ation of arachidonic acid in these cells (Figs. 4, 5, and Table 4). Unlike lipoxins, both leukotriene B, and 5-HETE Fig. 6. Effect of Liporins on LTB, binding. Human neutrophils were incubated 10 min at 4°C with ['HILTB, (1 nM) in the presence or are potent stimulants for elevating cytosolic Ca2 absence of LXA,, LXB,, 5-HETE, 15-HETE, or unlabeled LTB, at (Serhan et al., 1982b; O'Flaherty and Nishihira, 19871, various concentrations. Percent ["HILTB, bound was determined as and leukotriene B, promotes neutrophil adherence (re- in Lin et al. (1984). Results represent the mean t S.E. of n = 3 experviewed in Snyderman and Goetzl, 1981). Neither LXA, iments with triplicate determinations. nor LXB, stimulated aggregation, which is a n index of homotypic adhesion (Fig. 7). In addition, the lipoxins did not stimulate adhesion of neutrophils to cultured 41,000 daltons) in guinea pig neutrophils that inhibits monolayers of human endothelial cells (Table 51, indi- FMLP-induced release of [ l-14C]arachidonic acid cating that these compounds do not stimulate hetero- (Bokoch and Gilman, 1984). In addition, studies with typic adhesion. Recently, Lee e t al. (1989) reported that rabbit neutrophils suggest t h a t the receptors for neither LXA, or LXB, stimulated neutrophil adhesion FMLP, C5a, and leukotriene B, are each coupled to to endothelial cells. The present observations are con- Ni-like proteins, which are important in mediating sistent with those of Lee et al. (1989) with respect to the functional responses of these chemoattractants adhesion. In addition, they provide further evidence (Feltner et al., 1986). In a wide range of tissues, the that lipoxins display a profile of activities distinct from eicosanoids (including prostaglandins, leukotrienes, those of either LTB, or 5-HETE with human neutro- and thromboxane) generally appear to exert their acphils. tions by interacting with specific receptors that are Results from several laboratories have provided ev- thought to be linked to G proteins (reviewed in Smith, idence for the role of G proteins in receptor-mediated 1989). Prior treatment of [ l-'4Clarachidonate-labeled signal transduction in neutrophils (Bokoch and Gil- PMN with PTX completely inhibited FMLP as well as man, 1984; Okamura et al., 1985; Feltner et al., 1986; lipoxin-induced release of arachidonate (Table 1). In Mong et al., 1986; Becker et al., 1986). PTX catalyzes contrast, the P-oligomers of pertussis toxin did not inthe ADP-ribosylation of a membrane protein (approx. hibit LX-induced release (Table 2).These findings with

x?

+

52 1

LIPOXINS AND HUMAN PMN

FMLP- and PTX-treated human neutrophils are consistent with those reported for both guinea pig and rabbit neutrophils (Bokoch and Gilman, 1984; Feltner et al., 1986) and they suggest a role for G proteins in lipoxin-induced release of arachidonic acid. Since PMN possess specific receptors for leukotriene B, (Lin e t al., 1984; Feltner et al., 1986; Mong et al., 1986; Goldman and Goetzl, 1984), it was possible that the lipoxins might act through these receptors. Along these lines, LXA, has been shown to exert its actions in certain tissues by interacting with the same or a similar receptor site occupied by the cysteinyl-containing leukotrienes, namely LTC, and LTD, (Dahlen et al., 1988; Badr et al., 1989). Unlike LTC,, LXA, does not contract the guinea pig ileum. Instead, it causes a dosedependent inhibition of LTC,-induced contractions in this tissue (Dahlen et al., 1988). Recently, LXA, and LTD, were shown to interact with a common site on mesangial cells, and LXA, can competitively antagonize both the cellular and hemodynamic actions of LTD, (Badr et al., 1989). In the present study, however, neither LXA, nor LXB, altered the specific binding of LTB, to human PMN (Fig. 6). Thus, although LXA, can interact with cysteinyl-containing leukotriene receptors in certain tissues (DahlBn et al., 1988; Badr et al., 1989), lipoxins do not appear to exert their actions on human PMN via binding with LTB, receptors (Fig. 6). Whether lipoxins exert their actions on PMN by specific receptors remains to be demonstrated. A number of soluble and insoluble PMN stimuli have been shown to initiate lipid remodeling and the release of arachidonic acid (Serhan et al., 1982a, 1983; Wynkoop et al., 1986; Walsh et al., 1981). Using a doublelabel technique with [3H]arachidonic acid esterified into the sn-2 position and ['4C]palmitate esterified into the sn-1 position of distinct phospholipid classes, Walsh et al. (1981) have found that oposonized zymosan and the ionophore AZ3187 stimulate arachidonic acid release predominantly via a phospholipase A, mechanism. Utilizing a similar strategy with 13Hlpalmitate and [l-'4C]arachidonic acid labeling of neutrophils (Walsh et al., 1981)followed by separation of phospholipids by two-dimensional TLC, we find that lipoxins stimulate the release of [l-14C]arachidonic acid from predominantly phosphatid lcholine without provoking significant losses in the [2HI content of phosphatidylcholine (Fig. 3). Similar results were obtained with FMLP (Fig. 3), which are consistent with those of earlier reports (Wynkoop et al., 1986; Sellmayer et al., 1987). The results obtained with lipoxins are consistent with the role of a phospholipase A, in lipoxin-induced release of arachidonic acid. In addition, the finding that PA carried both labels following addition of either LXA,, LXB,, or FMLP suggests that, like FMLP (Serhan et al., 1982a, 1983; Agwu et al., 1989), lipoxins stimulate the formation of PA (Fig. 3). Several recent reports have provided evidence suggesting that, in addition to the well-established phospholipase C mechanism and PI cycle, neutrophils may also generate PA by means of a phospholipase D (Agwu et al., 1989; Balsinde et al., 1988). The complete mechanism of lipoxin-induced PA formation was not determined in the present study and thus remains the subject of further experiments. Nevertheless, since the levels of PA were elevated following exposure to li-

/ LXA4 \

Fig. 7. Human neutrophil aggregation: comparison between LXA,, WCB,, and FMLP 10-7M. A four channel aggregation profiler (model PAP-4, Biodata Co.) was used to study neutrophil aggregation in response to various agonists. This instrument was able to monitor changes in light transmittance in four sample cuvettes (three containing agonist and a fourth with vehicle control). PMN (5 x lo6 cells/ml) were suspended in PBS, pH 7.45 at 37"C, in siliconized cuvettes. Cytochalasin B ( 5 Kgiml) was added before addition of either stimuli or vehicle (final concentration of EtOH less than 0.018 by vol .1

The downward-pointing arrows indicate the points of addition of either FMLP, 10 7M (upper tracing), LXA,, 10 7M (middle tracing), or LXB,, 10-7M (lower tracing). Tracings are representative of 4 separate experiments where the P M N studied in aggregation were from the same cell preparation used for time course or dose response experiments.

poxins and neither compound stimulates adhesion (Figs. 3, 7, and Table 51, it appears that these two events, namely lipid remodeling and adhesion, can be dissociated in cells exposed to lipoxins. Recent results by Lee et al. (1989) have shown that preincubation of human neutrophils with LXA, inhibits (ICs0 10-'M) their subsequent chemotactic responses to either LTB, (10-7M) or FMLP (lO-'M). Preincubation with LXA, also inhibited agonist-induced Ca2 mobilization and hydrolysis of phosphoinositides (Lee et al., 1989). Lipoxin A, has recently been found to inhibit LTB,-induced plasma leakage and leukocyte migration in vivo in the hamster cheek pouch model of inflammation (Hedqvist et al., 1989).In view of these findings, our results that lipoxins stimulate rapid remodeling of neutrophil lipids and small increases in [Ca2+Iiwhich return to baseline within seconds suggest that these events may be related to the inhibitory responses of neutrophils observed following exposure of the cells to a second challenge. In summary, the present results demonstrate that lipoxins exert a selective profile of actions with human neutrophils (in vitro) and that lipid remodeling events induced by lipoxins are dissociated from both heterotypic and homotypic adhesion of these cells. Moreover, the present results suggest that lipoxins may be helpful in identifying critical steps in signal transduction pathways of human neutrophils. +

ACKNOWLEDGMENTS The authors thank M. Halm Small for skilled assistance in preparation of this manuscript, Dr. M. Elyse Wheeler for performing the neutrophil-endothelial cell

522

NlGAM ET AL.

Gimbrone, M.A., Jr., Brock, A.F., and Schafer, A.I. (1984) Leukotriene B, stimulates polymorphonuclear leukocyte adhesion to cultured vascular endothelial cells. J . Clin. Invest., 74t1552-1555. Goetzl, E.J., and Pickett, W.C. (1981) Novel structural determinants of the human neutrophil chemotactic activity of leukotriene B. J. Exp. Med., 153:482-487. Goldman, D.W., and Goetzl, E.J. (1984) Selective transduction of human polymorphonuclear leukocyte functions by subsets of receptors for leukotriene B,. J . Allergy Clin. Immunol., 74:373-377. Grynkiewicz, G., Poenie, M., and Tsien, R.Y. (1985) A new generation of CaZ indicators with greatly improved fluorescence properties. J. Biol. Chem., 260.3440-3450. Hansson, A., Serhan, C.N., Haeggstrom, J., Ingelman-Sundberg, M., Samuelsson, B., and Morris, J . (1986) Activation of protein kinase C LITERATURE CITED by lipoxin A and other eicosanoids. Intracellular action of oxygenation products of arachidonic acid. Biochem. Biophys. Res. 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adhesion assays, and Kay Case for isolation of human endothelial cells. These studies were supported by National Institute of Health grants GM38765 and A126714 (to C.N.S.), F32-HL07672 (F.W.L.), and Pol-HL36028. C.N.S. is a 1988 Pew Scholar in the Biomedical Sciences and a recipient of the J.V. Satterfield Arthritis Investigator Award from the National Arthritis Foundation. S.N. was suppored by a grant (N1 24213-1) from the “Deutsche Forschungsgemeinschaft,” Bonn, F.R.G.

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