THE JOURNAL OF BIOLOGICAL CHEMISTRY
`© 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
`
`Vol. 274, No. 40, Issue of October 1, pp. 28395–28404, 1999
`Printed in U.S.A.
`
`Inflammatory Platelet-activating Factor-like Phospholipids in
`Oxidized Low Density Lipoproteins Are Fragmented Alkyl
`Phosphatidylcholines*
`
`(Received for publication, May 12, 1999, and in revised form, July 6, 1999)
`
`Gopal K. Marathe‡, Sean S. Davies‡, Kathleen A. Harrison§, Adriana R. Silva¶,
`Robert C. Murphy§, Hugo Castro-Faria-Neto¶, Stephen M. Prescotti**, Guy A. Zimmermani, and
`Thomas M. McIntyre‡i ‡‡
`From the Departments of ‡Pathology and iInternal Medicine and the **Huntsman Cancer Institute, University of Utah,
`Salt Lake City, Utah 84112, the §Department of Pediatrics, National Jewish Medical and Research Center, Denver,
`Colorado 80206, and the ¶Deptamento de Fisiologia & Farmacodinåmica, IOC, Oswaldo Cruz, Fiocruz, Rio de Janeiro,
`Brazil 21045-900
`
`Oxidation of human low density lipoprotein (LDL)
`generates proinflammatory mediators and underlies
`early events in atherogenesis. We identified mediators
`in oxidized LDL that induced an inflammatory reaction
`in vivo, and activated polymorphonuclear leukocytes
`and cells ectopically expressing human platelet-activat-
`ing factor (PAF) receptors. Oxidation of a synthetic
`phosphatidylcholine showed that an sn-1 ether bond
`confers an 800-fold increase in potency. This suggests
`that rare ether-linked phospholipids in LDL are the
`likely source of PAF-like activity in oxidized LDL. Ac-
`cordingly, treatment of oxidized LDL with phospho-
`lipase A1 greatly reduced phospholipid mass, but did not
`decrease its PAF-like activity. Tandem mass spectrometry
`identified traces of PAF, and more abundant levels of 1-O-
`hexadecyl-2-(butanoyl or butenoyl)-sn-glycero-3-phospho-
`cholines (C4-PAF analogs) in oxidized LDL that comigrated
`with PAF-like activity. Synthesis showed that either C4-
`PAF was just 10-fold less potent than PAF as a PAF receptor
`ligand and agonist. Quantitation by gas chromatography-
`mass spectrometry of pentafluorobenzoyl derivatives
`shows the C4-PAF analogs were 100-fold more abundant in
`oxidized LDL than PAF. Oxidation of synthetic alkyl
`arachidonoyl phosphatidylcholine generated these C4-
`PAFs in abundance. These results show that quite minor
`constituents of the LDL phosphatidylcholine pool are the
`exclusive precursors for PAF-like bioactivity in oxidized
`LDL.
`
`Platelet-activating factor (PAF)1 is a phospholipid autacoid
`with a wide variety of actions, primarily on cells and events
`that comprise the inflammatory system. PAF initiates the
`
`rapid inflammatory response as it is the leukocyte activating
`molecule produced and displayed by stimulated endothelial
`cells (1). PAF does not induce the bactericidal effector functions
`of leukocytes, but rather stimulates their adhesive and migra-
`tory behavior that allows them to transit the endothelial bar-
`rier. Leukocytes (polymorphonuclear leukocytes or PMN),
`monocytes, and eosinophils, as well as platelets, express the
`PAF receptor and accordingly are activated by PAF in concen-
`trations ranging from picomolar to nanomolar levels. The po-
`tency of PAF, its broad actions, and the potentially deleterious
`events it invokes rationalize the tight regulation of PAF syn-
`thesis (2).
`PAF is recognized by a single, specific receptor that is a
`member of the family of seven-transmembrane-spanning, G-
`protein-linked receptors (3, 4). Alone among this large family of
`receptors and related orphan sequences, the PAF receptor rec-
`ognizes an intact phospholipid, and does so with a marked
`specificity. The PAF receptor shows a several hundredfold se-
`lectivity for the sn-1 ether bond of PAF, and complete specific-
`ity for the sn-2 acetyl residue compared with the long chain
`fatty acyl residue of most alkyl phosphatidylcholines (5, 6). The
`choline headgroup confers a several thousandfold advantage
`over the related phosphatidylethanolamine analog (7). Thus,
`compared with Edg-2 and Edg-4 receptors for lysophosphatidic
`acid (8), the PAF receptor has two additional, important recog-
`nition requirements; one is for a specific headgroup, and the
`second is for a specific, atypical sn-2 residue.
`The PAF receptor responds to synthetic analogs that contain
`short sn-2 fatty acyl residues, and this too is relevant to in-
`flammatory pathophysiology. PAF-like analogs with this struc-
`ture are produced by oxidation of cellular (9), low density
`lipoprotein (10–13), or foodstuff (14) phosphatidylcholines. The
`predominant biologic phosphatidylcholines are lipids of the
`diacyl subclass, and so the oxidation products are expected to
`be diacyl species. These oxidatively generated PAF analogs
`stimulate monocytes (15), leukocytes (16), and platelets (17).
`Oxidation of phosphatidylcholines to PAF-like lipids also oc-
`curs in vivo following exposure to the strong oxidant stress of
`cigarette smoke (15, 18). Additionally, oxidatively fragmented
`phosphatidylcholines are found in atherosclerotic plaques (13),
`and they circulate at detectable levels in human plasma (19).
`Oxidation of phosphatidylcholines generates a plethora of
`chemically related phosphatidylcholines and, as sn-1 alkyl or
`acyl phosphatidylcholines oxidize in a similar fashion (20),
`there is heterogeneity at both the sn-1 and sn-2 position. Only
`some of these will stimulate the PAF receptor, but identifica-
`tion of the biologically active species in the mix of similar
`28395
`
`* This work was supported by National Institutes of Health Grants
`HL 44513 (to T. M. M.), HL 44525 (to G. A. Z.), HL 50153 P50 (to
`S. M. P.), and HL 34303 (to R. C. M.) and by a grant from the Margolis
`Foundation (to G. A. Z.). The costs of publication of this article were
`defrayed in part by the payment of page charges. This article must
`therefore be hereby marked “advertisement” in accordance with 18
`U.S.C. Section 1734 solely to indicate this fact.
`‡‡ To whom correspondence should be addressed: Human Molecular
`Biology and Genetics, 15 N. 2030 E., University of Utah, Salt Lake City,
`Utah 84112-5330. Tel.: 801-585-0716; Fax: 801-585-0701; E-mail:
`tom.mcintyre@hmbg.utah.edu.
`1 The abbreviations used are: PAF, platelet-activating factor; LDL,
`low density lipoprotein; PMN, polymorphonuclear leukocyte; HAPC,
`1-O-hexadecyl-2-arachidonoyl-sn-glycero-3-phosphocholine; GC, gas
`chromatography; LC, liquid chromatography; MS, mass spectroscopy;
`HPLC, high performance liquid chromatography; BHT, butylated hy-
`droxytoluene; HBSS/A, 0.5% human serum albumin in HBSS.
`
`This paper is available on line at http://www.jbc.org
`
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`28396
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`Bioactive Alkyl Phosphatidylcholines in Oxidized LDL
`
`The recombinant human plasma form PAF acetylhydrolase and
`hPAFR293 cells expressing the human PAF receptor were from ICOS
`Corp. (Bothell, WA), while phospholipase A2 (bee venom), phospho-
`lipase C (Bacillus cereus), phospholipase D (cabbage), and butylated
`hydroxytoluene (BHT) were from Sigma. Dialysis tubing (6000–8000-
`kDa cut-off) was from Spectrum Medical Industries, Inc. (Houston, TX),
`and glass fiber filter papers were from VWR Scientific (Westchester,
`PA). FURA-2AM ester was from Molecular Probe (Eugene, OR). All the
`solvents (J.T. Baker, Inc.) were HPLC grade. Lipase from Rhizopus
`arrhizus was from Roche Molecular Biochemicals. 1-O-Hexadecyl-2-
`arachidonoyl-sn-glycero-3-phosphocholine (HAPC), PAF, 1-palmitoyl-2-
`acetyl-sn-glycero-3-phosphocholine (acyl-PAF), and lysoPAF were from
`Biomol Research Laboratories (Plymouth Meeting, PA). The long chain
`phospholipids were purified by reversed phase HPLC prior to use.
`Commercial lysoPAF was subjected to mild alkaline hydrolysis as
`described below and acetylated with acid chlorides (acetyl, butyryl, or
`crotonyl) in the presence of perchloric acid (23) to generate PAF and its
`C4 analogs. These were then purified by reversed phase HPLC and
`analyzed by GC/MS as described below. The total mass of the material
`was determined by lipid phosphorus analysis (24).
`Isolation and Oxidation of Human LDL—Human LDL was isolated
`by density flotation from normolipidic subjects (25) as described in
`detail (10), except that we employed Pefabloc (200 mM) as a non-toxic
`alternative to diisopropyl fluorophosphate to inactivate PAF acetylhy-
`drolase (26) and allow oxidized products to accumulate (10). Isolated
`LDL was oxidized with 10 mM CuSO4 for 18–24 h at 37 °C. Control LDL
`was not subjected to oxidation and was prevented from oxidation by 100
`mM BHT.
`Separation of PAF-like Lipids—Total lipids were extracted from LDL
`by the method of Bligh and Dyer (27) before neutral lipids, fatty acids,
`and phospholipids were separated by aminopropyl chromatography
`(10). The phospholipid fraction was further separated on a reversed
`phase column (ODS silica, 250 3 4.6-mm Microsorb MV; Rainin Instru-
`ment Co., Woford, MA) with a mobile phase of methanol/acetonitrile/
`H2O (840:150:10) containing 1 mM ammonium acetate and BHT (10 mM)
`at a flow rate of 1 ml/min. Fractions were collected for every minute for
`the first 10 min, and PAF-like lipids elute between minutes 5 and 8.
`Recovery of a [3H]PAF internal standard added to the LDL particle in
`the HPLC fractions was .75%. Fractions found to contain leukocyte
`agonists (as described below) were pooled, the solvent removed by a
`stream of N2, reconstituted with chloroform:methanol (2:1) containing
`BHT (10 mM), and stored at 220 °C. Authentic PAF and PAF-like lipids
`were suspended in HBSS/A and sonicated prior to use.
`PAF-like lipids isolated from LDL were further purified by straight
`phase chromatography prior to determining their specific bioactivity.
`For this, a portion of the PAF-like lipids separated on reversed phase
`HPLC were treated with lipase from R. arrhizus (28) and then injected
`onto a 5-mm silica column (2 3 150 mm, Phenomenex, Torrance, CA)
`and the column developed with an isocratic solvent system (hexane:
`isopropanol:20 mM ammonium acetate, pH 7 (3:4:0.7, v/v/v)) (29) at a
`flow rate of 0.2 ml/min. Fractions were dried under nitrogen and used
`for bioassays and mass spectrometry.
`PMN Adhesion—Human neutrophils were isolated by dextran sedi-
`mentation and centrifugation over Ficoll (30). CD18-dependent adhe-
`sion of activated neutrophils to a gelatin surface after 10 min of incu-
`bation at 37 °C was quantified using a video microscopy imaging system
`to count adherent cells. Authentic PAF was used as a positive control
`and to establish the daily sensitivity of the cells. In experiments where
`recombinant PAF acetylhydrolase was used, PAF-like lipids or PAF
`were treated with 4 mg of this enzyme in HBSS/A for 1 h at 37 °C before
`addition of the agonist to neutrophils. The enzyme itself caused no
`activation at this concentration. Alternatively, neutrophils were treated
`with 10 mM WEB 2086 for 20 min prior to the addition of agonist as a
`means to competitively block the PAF receptor.
`Pleurisy Model—Wistar rats (150–200 g) were injected (0.1 ml total
`volume) intrathoracicaly with pooled HPLC fractions 6, 7, and 8 resus-
`pended in 0.1% bovine serum albumin in sterile saline. Some animals
`were treated with the PAF receptor antagonist (20 mg/kg) 1 h before
`challenge. Some pooled HPLC aliquots were treated with recombinant
`PAF acetylhydrolase (2 mg) for 20 min at 37 °C, the lipids reextracted,
`dried, and resuspended in injection buffer before use. The animals were
`euthanized 6 h after injection in a CO2 chamber, and the thoracic cavity
`opened and washed with 3 ml of heparinized (Liquemine; Roche, Rio de
`Janeiro, Brazil) saline (10 units/ml). The pleural wash was recovered,
`and the volume measured with a graduated syringe. Pleural washes
`were diluted in Turk fluid (2% acetic acid) for total cell counts in
`Neubauer chambers. Differential analysis was performed in cytosmears
`stained by the May Grunwald-Giemsa method. The protein content of
`
`FIG. 1. Polar lipids purified from oxidized LDL are inflamma-
`tory. Lipids from native or Cu1-oxidized LDL were extracted and
`purified by reversed phase chromatography and then pooled fractions
`6–8 were injected into the pleural space of Wistar rats as described
`under “Materials and Methods.” Some rats were treated with the PAF
`receptor antagonist WEB 2086 (20 mg/kg) 1 h prior to agonist challenge,
`while others received lipids that had been treated with recombinant
`PAF acetylhydrolase (2 mg for 20 min at 37 °C, followed by re-extrac-
`tion). Pleural analysis of cell number and lavage protein content were
`performed 6 h after the intrathoracic injection. Statistically significant
`differences (p , 0.05) compared with control animals receiving BSA in
`saline are marked *, while differences compared with animals injected
`with lipids purified from oxidized LDL are marked 1. Each bar is the
`mean 1 S.E. from at least four animals. Mono, monocytes; PMN,
`neutrophils; Eo, eosinophils.
`
`oxidation products has been complicated by this heterogeneity.
`Here we show that one difficulty in identifying biologically
`active agents has been their profound dilution with related, but
`less active, diacyl homologs. We find that all of the PAF recep-
`tor agonists generated during the oxidation of LDL are derived
`from oxidation of the alkyl phosphatidylcholines found in very
`low abundance in LDL (21, 22). Removing the contaminating
`diacyl oxidation products allowed us to identify and quantitate
`fragmented alkyl phosphatidylcholines in oxidized LDL. While
`a trace amount of PAF was generated by oxidative fragmenta-
`tion, major bioactive species are butanoyl- and butenoyl-PAF,
`which are also products of hexadecyl arachidonoyl phosphati-
`dylcholine fragmentation. Thus, oxidation of rare phospholipid
`species in LDL generates bioactive, short chain PAF-analogs.
`
`MATERIALS AND METHODS
`Tissue culture grade chemicals were from Whittaker Bioproducts
`Inc., (Walkersville, MD), and tissue culture dishes were from Falcon
`Labware (Lincoln Park, NJ). Four-well multiwell dishes for PMN ad-
`hesion assays were from Nunclon (Nunc, Roskilde, Denmark). Trypsin/
`EDTA was from Life Technologies, Inc., fetal Bovine Serum was from
`Hyclone Laboratories (Logan, UT), and human albumin was from Bax-
`ter Health Care Corp. (Glendale, CA). WEB 2086 was a generous gift
`from Boehringer Ingelheim Pharmaceuticals, Inc. (Ridgefield, CT).
`[3H]WEB 2086 (13.5 Ci/mml) was purchased from NEN Life Science
`Products. Aminopropyl columns were from J.T. Baker Inc. (Phillips-
`burg, NJ), and Pefabloc was from Pentapharm AG (Basel, Switzerland).
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`Bioactive Alkyl Phosphatidylcholines in Oxidized LDL
`
`28397
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`FIG. 3. Effect of an sn-1 ether bond on PAF-like activity of
`oxidized phosphatidylcholine. 1-O-hexadecyl-2-arachidonoyl-sn-
`glycero-3-phospholcholine (HAPC) or 1-palmitoyl-2-arachidonoyl-sn-
`glycero-3-phosphocholine (PAPC) were oxidized with Cu1, the bioactive
`phospholipid separated by isocratic chromatography, and their concen-
`tration was determined by phosphorus analysis. Aliquots were added to
`FURA-2-loaded leukocytes, and the increase in intracellular Ca21
`was determined as described under “Materials and Methods.”
`
`FIG. 2. Phospholipids from oxidized LDL demonstrate PAF-
`like activity. A, reversed phase HPLC purification of leukocyte ago-
`nists in oxidized LDL. Phospholipids were extracted from native or
`oxidized LDL, and separated by aminopropyl and C18 reversed phase
`HPLC as described under “Materials and Methods.” Fractions were
`collected every minute, and an aliquot of this was dried under nitrogen
`before being reconstituted in HBSS/A. The ability of duplicate aliquots
`to stimulate PMN, as measured by their CD11/CD18-dependent adhe-
`sion to a gelatin-coated surface, was determined as a percentage of the
`maximal response to PAF by that donor’s cells. The effect of the PAF
`receptor antagonist WEB 2086 (10 mM) on PMN adhesion, or the effect
`of pretreating the fractions with recombinant human PAF acetylhydro-
`lase (4 mg/fraction) is also shown. This experiment is representative of
`two independent experiments. B, PAF-induced accumulation of intra-
`cellular Ca21 in hPAFR293 cells. hPAFR293 cells were loaded with
`FURA2-AM and then stimulated with the stated concentration of PAF.
`Emission changes as fluorescence excitation jumped from 340 nm to 380
`nm was captured as a function of time. The concentrations were as
`follows: a, HBSS/A buffer alone; b, 10212 M PAF; c, 10211 M PAF; d,
`10210 M PAF; e, 1029 M PAF. Inset, fluorescence ratio of FURA2-loaded
`untransfected 293 cells exposed to 1028 M PAF. C, activation of
`hPAFR293 cells by aliquots of purified LDL phospholipids. FURA-2-
`
`loaded hPAFR293 cells were exposed to aliquots of HPLC fractions 5–7
`from unoxidized LDL or Cu1-oxidized LDL as shown by the filled arrow
`(immediately adjacent fractions failed to alter Ca21 levels in these cells
`and are not presented). After the fluorescence ratio returned to a stable
`base line, 10210 M PAF was added (as shown by the open arrow) to
`measure receptor desensitization. In one series of measurements with
`aliquots from the same fraction, the cells were pretreated with WEB
`2086 to block PAF receptor function. In a second series with material
`from these fractions, the aliquots were pretreated with recombinant
`human PAF acetylhydrolase. Individual components of this experiment
`were performed at least twice with similar findings. D, displacement of
`[3H]WEB 2086 from hPAFR293 cell membranes. Membranes from
`hPAFR293 cells were purified, and their ability to bind [3H]WEB 2086
`was determined as described under “Materials and Methods.” Left, PAF
`displacement. Increasing concentrations of PAF displace [3H]WEB 2086
`from hPAFR293 cell membranes. Total [3H]WEB 2086 binding was
`2457 6 210 dpm, and the nonspecific binding, determined with 1025 M
`unlabeled PAF, was 116 6 20 dpm. Right, aliquots of fractions 5, 6, and
`7 were used to displace bound [3H]WEB 2086. Some aliquots of fractions
`5, 6, and 7 were treated with recombinant human PAF acetylhydrolase
`prior to addition. This experiment is representative of one other.
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`28398
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`Bioactive Alkyl Phosphatidylcholines in Oxidized LDL
`
`the pleural wash was determined by a Biuret reaction after clearing by
`centrifugation at 500 3 g for 10 min.
`Measurement of Intracellular Ca21 in hPAFR 293 Cells—Subcon-
`fluent hPAFR293 cells (ICOS Corp., Bothell, WA) that stably express
`the human PAF receptor were treated with Versene (Life Technologies,
`Inc.) and resuspended in fresh culture medium (;1.1 3 107 cells/ml).
`FURA-2 AM was loaded into cells at 1 mM from a 1 mM Me2SO stock,
`and after incubation in the dark for 45 min at 37 °C, the cells were
`washed with HBSS/A and resuspended in HBSS/A at a density of
`2.25 3 106 cells/ml. Fluorescence of 1.5 ml of cells was measured at
`24 °C, with dual excitation at 340 nm and 380 nm with the emission
`recorded at 510 nm (31). The response of each batch of cells was tested
`with 0.1 and 1 nM authentic PAF to generate the maximal PAF re-
`sponse. Control 293 cells were processed in the same way, and their
`response was tested with PAF, or with thrombin or lysophosphatidic
`acid as positive controls. For some experiments, we confirmed the
`results obtained with hPAFR293 cells by performing parallel experi-
`ments in FURA2-labeled PMN. Ligand displacement of [3H]WEB 2086
`from hPAFR293 cell membranes ectopically expressing the human PAF
`receptor was as described for Chinese hamster ovary cell membranes
`(32).
`Structural Analysis—PAF-like lipids were treated with 5 units of
`lipase from R. arrhizus in HBSS/A for 11 h at 37 °C and then tested
`directly for their ability to mobilize Ca21 in hPAFR 293 cells (28).
`Acyl-PAF (1-palmitoyl-2-acetyl-sn-glycero-3-phosphocholine) and PAF
`served as controls. In a similar fashion, PAF-like lipids were treated
`with phospholipase C (B. cereus), bee venom phospholipase A2, and
`cabbage phospholipase D before being tested for the ability to mobilize
`Ca21 in PMN and hPAFR293 cells. The presence of an sn-1 ether bond
`was investigated by subjecting PAF-like lipids, PAF, or acyl-PAF to
`saponificaion with 0.5N NaOH in methanol for 2 h at 24 °C. Saponified
`material, containing free fatty acids and either lyso-PAF (1-O-hexade-
`cyl-glycerophosphocholine) from glycerolipids with an sn-1 ether bond
`or glycerophosphocholine from diacyl phospholipids, did not induce
`Ca21 accumulation in hPAFR293 cells. This material was reacetylated
`with excess acetyl chloride in the presence of perchloric acid (23), and
`then reexamined for the ability to mobilize intracellular Ca21 in the
`receptor-transfected cells.
`Mass Spectrometric Analysis of Normal Phase HPLC Fractions—
`Direct LC/MS and LC/MS/MS analysis was carried out with a Sciex
`API-III1 triple quadrupole mass spectrometer (PE-Sciex, Thornhill,
`Ontario). For all electrospray ionization experiments, the curtain gas
`flow was 1.2 liter/min nitrogen with a nebulizer pressure at 38 p.s.i. The
`orifice potential was maintained at 75 V, and the electrospray ioniza-
`tion potential at 14200 V for detection of positive ions. For the analysis
`of negative ions, the ion spray potential was adjusted to 22800 V and
`purified air (zero air) was used to reduce any possibility for glow
`discharge at the electrospray needle. The orifice potential was main-
`tained at 295 V. Selected ion recording experiments and multiple
`reaction monitoring experiments were carried out using the tandem
`quadrupole mass settings as indicated in the text. Normal phase HPLC
`was carried out in a 2 3 150-mm normal phase silica column (Phenome-
`nex, Rancho Cordova, CA) using a mobile phase of hexane/isopropa-
`nol/20 mM ammonium acetate (3/4/0.7) at the flow rate of 200 ml/min.
`The GC/MS analysis of PAF-like lipids was carried out following hy-
`drolysis of the glycerophosphocholine lipids with phospholipase C, fol-
`lowed by derivatization of the liberated diglycerides with pentafluoro-
`benzoyl chloride as described previously (33). For the quantitative
`analysis of target molecules, [2H3]PAF was added as internal standard
`(10 ng) to each aliquot taken for GC/MS analysis prior to treatment
`with phospholipase C.
`
`RESULTS
`Oxidation of LDL Generates Inflammatory Mediators—We
`extracted and purified the polar lipids from native and oxidized
`LDL and injected this into the pleural cavity of naive rats. The
`lipids isolated from oxidized LDL, but not its unoxidized coun-
`terpart, induced acute inflammation within 6 h as marked by
`leukocyte accumulation (Fig. 1A) and proteinaceous edema
`(Fig. 1B). The leukocyte accumulation was characterized by
`
`saponified material was chemically acetylated with acetyl chloride be-
`fore addition to FURA2-loaded hPAFR293 cells. The tracings are as
`follows: a, untreated material; b, after saponification; c, after acetyla-
`tion of saponified material.
`
`FIG. 4. Alkyl phosphatidylcholines account for the PAF-like
`activity found in oxidized LDL. A, the effect of phospholipase A1
`treatment on the Ca21 flux induced in hPAFR293 cells by oxidized
`phospholipids. PAF and acyl-PAF (top panels) were treated, or not, with
`the lipase from R. arrhizus (5 units) in HBSS/A for 11 h at 37 °C and
`then added to FURA-2-loaded hPAFR293 cells. Changes in fluorescence
`as the excitation wavelength jumped between 340 and 380 nm was
`recorded as before. Synthetic phosphatidylcholines (middle panels)
`were oxidized, purified by isocratic HPLC, quantitated by phosphorus
`analysis, and treated with R. arrhizus lipase, or not, before adding to
`FURA2-loaded hPAFR293 cells. Two maximally active fractions, frac-
`tions 6 and 7, from oxidized LDL (lower panels) were treated or not with
`lipase according to the above protocols, added to FURA2-loaded
`hPAFR293 cells, and changes in the fluorescence ratio were determined
`as before. These experiments were repeated five times in different
`batches of LDL preparations. B, the effect of chemical saponification
`and reacetylation on the Ca21 flux induced in hPAFR293 cells by
`phospholipids from oxidized LDL. PAF, its acyl analog (upper panels),
`or fractions 6 and 7 from the isocratic reversed phase separation of
`oxidized LDL (lower panels) were treated with 0.5 N NaOH in methanol
`for 2 h as described under “Materials and Methods.” A portion of the
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`Bioactive Alkyl Phosphatidylcholines in Oxidized LDL
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`FIG. 5. Normal phase LC/MS/MS
`analysis of the reverse phase HPLC
`fraction 6 obtained from oxidized
`LDL. HPLC retention times are indicated
`above each peak. A, elution of glycero-
`phospholipid molecular species as indi-
`cated by the total ionization current de-
`rived from those components generating
`m/z 184 (phosphocholine cation) by elec-
`trospray ionization and collisional activa-
`tion. Measurement of biological activity
`present in each HPLC fraction (0.5 min) is
`indicated in the bar graph as elevation of
`intracellular calcium ions in human poly-
`morphonuclear leukocytes (see “Materials
`and Methods”). B, selected ion recording
`for the collisional activation of m/z 524,
`generating ions at m/z 184. This specific
`ion transition is the most abundant prod-
`uct ion following collisional activation of
`platelet-activating factor. C, selected ion
`recording for the collisional activation of
`m/z 550, generating ions at m/z 184.
`This specific ion transition is the most
`abundant product ion of collisional activa-
`tion of butenoyl-PAF (16:0e/4:1-GPC). D,
`selected ion recording for the collisional
`activation of m/z 550, generating ions at
`m/z 184. This specific ion transition is the
`most abundant product ion of collisional
`activation of butanoyl-PAF (16:0e/4:0-
`GPC). E, mass spectra of all precursor
`ions for m/z 184 which eluted from the
`HPLC from 24.5 to 25.5 min. F, mass
`spectra of all precursor ions for m/z 184
`which eluted from the HPLC from 20.0 to
`21.0 min.
`
`mononuclear cell and early eosinophil influx, but especially by
`a neutrophilic effusion. Treatment of the lipid preparation with
`recombinant human plasma acetylhydrolase (which specifi-
`cally hydrolyzes phospholipids with short sn-2 acyl residues;
`Refs. 34 and 35) prior to injection into the animals blocked
`cellular infiltration and the edema. That the inflammatory
`principle was PAF or PAF-like analogs was strengthened by
`the potent inhibition of the inflammatory response by in vivo
`blockade of the PAF receptor with the specific antagonist WEB
`2086.
`Accumulation of PAF-like Lipids after Oxidation of
`LDL—We purified the leukocyte agonist in oxidized LDL by
`quantitating neutrophil adhesion, a measure of CD11/CD18
`activation (36). The lipids derived from oxidized LDL that
`eluted between 5 and 7 min were leukocyte agonists, and these
`lipids were not present in native, unoxidized LDL (Fig. 2A).
`Like the in vivo events induced by the lipids isolated from
`oxidized LDL, ex vivo leukocyte activation was blocked by a
`specific PAF receptor antagonist WEB 2086 and by pretreating
`these fractions with purified, recombinant PAF acetylhydro-
`lase. Treatment of these fractions with phospholipase A2, phos-
`pholipase C, or phospholipase D inactivated the stimulatory
`compounds in fractions 5–7 (data not shown). This is an impor-
`tant confirmation that the biologically active species were still
`phospholipids, and were not simply fragments released from
`oxidizing polyunsaturated acyl residues. We established that
`the active agent(s) acted through the PAF receptor using 293
`
`cells stably transfected with the human PAF receptor that
`allows these cells to respond to PAF (Fig. 2B). Each fraction
`that activated neutrophils also induced a Ca21 flux in these
`cells and by doing so, desensitized the ectopic PAF receptor to
`a second stimulus with PAF (Fig. 2C). The Ca21 flux in these
`cells was blocked by co-incubation with WEB 2086 or by pre-
`treatment with PAF acetylhydrolase. Lipids from unoxidized
`LDL did not activate these cells, showing oxidation truly gen-
`erates PAF-like phospholipids. We quantitated the amount of
`PAF equivalents in the active fractions to determine whether
`this shadowed leukoctye stimulation using a competitive
`[3H]WEB 2086 displacement assay and purified membranes
`from hPAFR293 cells (Fig. 2D). We calculate that there was
`twice the amount of PAF-like material (equivalent to 20 nM
`PAF) in fraction 6 than in either fraction 5 or 7 (which con-
`tained 9 and 10 nM PAF equivalents, respectively.) Following
`the treatment of each fraction with recombinant PAF acetyl-
`hydrolase competition with [3H]WEB 2086 was lost, and sur-
`rounding fractions, or equivalent fractions from unoxidized
`LDL, also failed to displace [3H]WEB 2086.
`PAF-like Lipids in Oxidized LDL Are Alkyl Phospholipids—
`Oxidation of synthetic diacyl phosphatidylcholines generates
`PAF-like activity (11, 16, 37), suggesting that some particular
`modification of the fragmented sn-2 acyl residue can overcome
`the normally strong preference for an sn-1 ether bond. We
`tested this prediction by oxidizing 1-palmitoyl-2-arachidonoyl-
`sn-glycero-3-phospholcholine and its sn-1 ether homolog 1-O-
`
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`Bioactive Alkyl Phosphatidylcholines in Oxidized LDL
`
`FIG. 6. Normal phase LC/MS/MS
`analysis of the reverse phase HPLC
`fraction 7 obtained from oxidized
`LDL. HPLC retention times are indicated
`above each peak. A, elution of glycero-
`phospholipids as indicated by the total
`ionization current derived from those
`components generating m/z 184 (phos-
`phocholine cation) by electrospray ioniza-
`tion and collisional activation. Measure-
`ment of biological activity present in each
`HPLC fraction (0.5 min) is indicated in
`the bar graph as elevation of intracellular
`calcium ions in human polymorphonu-
`clear
`leukocytes (see “Materials and
`Methods”). B, selected ion recording for
`the collisional activation of m/z 524, gen-
`erating ions at m/z 184. This specific ion
`transition is the most abundant product
`ion following collisional activation of
`platelet-activating factor. C, selected ion
`recording for the collisional activation of
`m/z 550, generating ions at m/z 184.
`This specific ion transition is the most
`abundant product ion of collisional activa-
`tion of butenoyl-PAF (16:0e/4:1-GPC). D,
`selected ion recording for the collisional
`activation of m/z 550, generating ions at
`m/z 184. This specific ion transition is the
`most abundant product ion of collisional
`activation of butanoyl-PAF (16:0e/4:0-
`GPC). E, mass spectra of all precursor
`ions for m/z 184 which eluted from the
`HPLC from 24.0 to 25.0 min. F, mass
`spectra of all precursor ions for m/z 184
`which eluted from the HPLC from 20.0 to
`21.0 min.
`
`hexadecyl-2-arachidonoyl-sn-glycero-3-phospholcholine, puri-
`fying the oxidation products, and quantitating their mass by
`phosphorus analysis. When the concentration of the two homol-
`ogous oxidation products was adjusted to give equivalent
`amounts of Ca21 release in leukocytes, we found (Fig. 3) that
`800-fold more diacyl products were required. This suggests
`that there is no highly preferred sn-2 residue in oxidized diacyl
`phosphatidylcholines that can overcome the requirement for an
`sn-1 ether bond.
`In light of this information, we determined the nature of the
`sn-1 bond of the bioactive phospholipids in oxidized LDL. This
`was done by hydrolyzing diacyl phosphatidylcholines with
`phospholipase A1 before analysis in the hPAFR293 cell Ca21
`flux assay. Control experiments (Fig. 4A) showed the acyl an-
`alog of PAF (which is about 1% as potent as PAF in this assay)
`was destroyed by this digestion, while PAF with its sn-1 ether
`bond was unaffected. An identical result was obtained when
`the oxidation products of 1-palmitoyl-2-arachidonoyl-glycero-
`phosphocholine and 1-hexadecyl-2-arachidonoyl-glycerophos-
`pholcholine were digested. Similarly, phospholipase A1 diges-
`tion destroyed nearly all of the phospholipid mass in fractions
`5 through 7 derived from oxidized LDL as determined by phos-
`phorus staining of the lipids resolved by TLC (not shown). In
`contrast, phospholipase A1 did not detectably reduce the PAF-
`like bioactivity in these fractions (Fig. 4A). We confirmed this
`result using chemical saponification to completely hydrolyze
`diacyl compounds, which abolished PAF-like activity of both
`PAF and acyl-PAF (Fig. 4B). Chemical acetylation returned the
`PAF sample to its original level of activity (compare tracings a
`and c), but did not have a similar effect with acyl-PAF. Sapon-
`ification of fractions 6 and 7 from oxidized LDL, also completely
`inactivated the PAF-like activity. Acetylation of the hydrolysis
`
`products restored PAF-like activity, a result not possible if
`fractions 6 and 7 just contained oxidation products derived
`from diacyl phospholipids.
`Identification of PAF-like Lipids in Oxidized LDL—We took
`advantage of the above findings to obtain highly purified PAF-
`like lipids from oxidized LDL for tandem mas