Eosinophil Peroxidase-derived Reactive Brominating Species
Target the Vinyl Ether Bond of Plasmalogens Generating a Novel
Chemoattractant,
-Bromo Fatty Aldehyde*
Carolyn J.
Albert
,
Arun K.
Thukkani
§,
Rita M.
Heuertz¶,
Arne
Slungaard
,
Stanley L.
Hazen**, and
David A.
Ford

From the Departments of
Biochemistry and Molecular
Biology, ¶ Internal Medicine, and Molecular Microbiology and
Immunology, St. Louis University Health Sciences Center, St.
Louis, Missouri 63104, the ** Center for Cardiovascular
Diagnostics and Prevention, Departments of Cell Biology and
Cardiovascular Medicine, Cleveland Clinic Foundation, Cleveland,
Ohio 44195, and the
Department of Medicine, Section of
Hematology, Oncology, and Transplantation, University of Minnesota,
Minneapolis, Minnesota 55455
Received for publication, November 14, 2002
 |
ABSTRACT |
Plasmalogens are a subclass of
glycerophospholipids that are enriched in the plasma membrane of many
mammalian cells. The vinyl ether bond of plasmalogens renders them
susceptible to oxidation. Accordingly, it was hypothesized that
reactive brominating species, a unique oxidant formed at the sites of
eosinophil activation, such as in asthma, might selectively target
plasmalogens for oxidation. Here we show that reactive brominating
species produced by the eosinophil peroxidase system of activated
eosinophils attack the vinyl ether bond of plasmalogens. Reactive
brominating species produced by eosinophil peroxidase target the vinyl
ether bond of plasmalogens resulting in the production of a neutral
lipid and lysophosphatidylcholine. Chromatographic and mass
spectrometric analyses of this neutral lipid demonstrated that it was
2-bromohexadecanal (2-BrHDA). Reactive brominating species produced by
eosinophil peroxidase attacked the plasmalogen vinyl ether bond at
acidic pH. Bromide was the preferred substrate for eosinophil
peroxidase, and chloride was not appreciably used even at a
1000-fold molar excess. Furthermore, 2-BrHDA production elicited by
eosinophil peroxidase-derived reactive brominating species in the
presence of 100 µM NaBr doubled with the addition
of 100 mM NaCl. The potential physiological significance of
this pathway was suggested by the demonstration that 2-BrHDA was
produced by phorbol myristate acetate-stimulated eosinophils and
by the demonstration that 2-BrHDA is a phagocyte chemoattractant.
Taken together, the present studies demonstrate the targeting of
the vinyl ether bond of plasmalogens by the reactive brominating
species produced by eosinophil peroxidase and by activated eosinophils,
resulting in the production of brominated fatty aldehydes.
 |
INTRODUCTION |
Activated phagocytes generate a variety of reactive
halogenating species, which attack adjacent cells as a part of the
normal physiological defense function of phagocytes (1, 2). Eosinophil peroxidase amplifies the oxidizing potential of hydrogen peroxide by
using it as a substrate in the presence of the micromolar
concentrations of bromide to produce hypobromous acid and its conjugate
base, which are the predominant oxygen-derived free radicals produced by activated eosinophils (3). Through the generation of reactive brominating species and their attack of viral and microbial proteins and membrane structures, eosinophil peroxidase plays a crucial role in
the antiviral and antimicrobial defense mechanisms of eosinophils (3).
Additionally, eosinophil peroxidase likely plays an important role
through the production of reactive brominating species in asthma (4,
5). In fact the involvement of eosinophil peroxidase in asthma has been
suggested by the presence of bromotyrosine in the airways and sputum of
asthmatics (4-6).
Lipids are major targets of the reactive halogenating species produced
by activated phagocytes. Reactive chlorinating and brominating species
released by activated phagocytes attack unsaturated -C=C- bonds within
the aliphatic chains of phospholipids and within the steroid nucleus of
sterols forming chlorohydrins and bromohydrins, which may disrupt
normal membrane fluid molecular dynamics (7-11). This mechanism may
represent a major cytotoxic effect of activated phagocytes. Reactive
brominating species have been shown to target unsaturated fatty acids
resulting in the production of bromohydrins that may serve as markers
not only of phagocyte-mediated inflammation but also specific
inflammatory processes mediated by brominating oxidants (9).
Plasmalogens are a subclass of glycerophospholipids found both in the
plasma membrane phospholipid pools of many mammalian tissues and in
lung surfactant (12-18). Plasmalogens possess a masked aldehyde, vinyl
ether linkage between the sn-1 aliphatic chain and the
glycerol backbone and may have an important role in both the solvation
of transmembrane ion channels and transport proteins as well as the
storage of arachidonic acid (19-22). We have recently demonstrated
that plasmalogens represent accessible molecular targets of the
membrane-permeable, reactive chlorinating species generated by
activated phagocytes resulting in the production of
-chloro fatty
aldehydes (23, 24).
Because eosinophil peroxidase has a marked selectivity for the
production of hypobromous acid at physiological halide concentrations (5, 25, 26), the present study was designed to determine whether the
vinyl ether bond of the sn-1 aliphatic chain of plasmalogens is susceptible to bromination by reactive brominating species produced
by eosinophil peroxidase and to compare this putative halogenation with
that mediated by reactive chlorinating species in eosinophils. The
results herein demonstrate for the first time that eosinophil
peroxidase utilizes micromolar concentrations of bromide at acidic pH
resulting in the production of reactive brominating species that
attack the vinyl ether bond of plasmalogens leading to the production
of lysophospholipids and
-bromo fatty aldehydes. Additionally, these
-bromo fatty aldehydes are produced by PMA-stimulated eosinophils
and thus represent a previously uncharacterized product of activated
eosinophils. The biological role of
-bromo fatty aldehydes is also
suggested by our results showing that they are a phagocyte
chemoattractant. Taken together, the present study demonstrates that
plasmalogens are attacked by eosinophil peroxidase-derived reactive
brominating species and suggests that this may represent a novel
mechanism for plasmalogen degradation during either
antiviral/antimicrobial roles of eosinophils or, alternatively, during
inflammatory tissue injury in eosinophil inflammatory states such as asthma.
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EXPERIMENTAL PROCEDURES |
Materials--
Porcine eosinophil peroxidase
(EPO)1 was isolated according
to the method of Jorg (27) employing guaiacol oxidation as the assay
(28). The purity of EPO preparations was assured before use by
demonstrating an RZ of 0.9 (A415/A280), SDS-polyacrylamide gel electrophoresis analysis with Coomassie Blue staining, and in-gel
tetramethylbenzidine peroxidase staining to confirm no contaminating
myeloperoxidase activity (29). Pentafluorobenzyl hydroxylamine was
purchased from Aldrich. Hexadecanoyl chloride was purchased from
Nu-Chek Prep. All other reagents and chemicals were purchased from
either Aldrich, Sigma, or Fisher.
Both 2-Br-[7,7,8,8-d4]-hexadecanal
(2-Br-[d4]-HDA) and
2-Cl-[7,7,8,8-d4]-hexadecanal
(2-Cl-[d4]-HDA) were synthesized from commercially available
[7,7,8,8-d4]-hexadecanoic acid (Medical Isotopes, Inc.) and purified by HPLC; their purity was confirmed by
TLC, as well as by GC-MS for its PFB oxime derivative as described previously (23, 30).
Preparation of Lysoplasmenylcholine and
Plasmenylcholine--
The lysoplasmenylcholine molecular species,
1-O-hexadec-1'-enyl-GPC, was prepared from bovine heart
lecithin and purified as described previously (31). Plasmenylcholine,
synthesized by an anhydrous reaction utilizing
1-O-hexadec-1'-enyl-GPC and hexadecanoyl chloride as
precursors with dimethylaminopyridine as a catalyst, was purified as
described previously (31). Synthetically prepared plasmenylcholine and
lysoplasmenylcholine were determined to be greater than 95% pure by
thin-layer chromatography, straight-phase HPLC, reversed-phase HPLC,
and capillary gas chromatography of the aliphatic constituents.
Lysoplasmenylcholine and 16:0-16:0 plasmenylcholine were quantified by
capillary gas chromatography.
Plasmalogen Treatment with Eosinophil Peroxidase-derived Reactive
Halogenating Species: Analysis of Reaction Products by Thin-layer
Chromatography and Capillary Gas Chromatography--
In a typical
assay, 50-200 nmol of either lysoplasmenylcholine or plasmenylcholine
was incubated in 500 µl of phosphate buffer (20 mM
NaPO4) supplemented with the indicated concentrations of NaBr and/or selected concentrations NaCl, 0.1 mM
diethylenetriaminepentaacetic acid (pH 4.0-7.0) in the presence or
absence of indicated amounts of eosinophil peroxidase and
H2O2 for indicated intervals at 37 °C.
Reactions were terminated by the addition of methanol, and reaction
products were extracted into chloroform by the method of Bligh and Dyer
(32). Reaction products were separated by TLC utilizing silica gel
60-Å plates (Whatman) and a mobile phase comprising petroleum
ether/ethyl ether/acetic acid (90/10/1, v/v/v) for the separation of
neutral lipids. Alternatively, polar lipid reaction products were
separated by TLC utilizing silica gel 60-Å plates (Whatman) and a
mobile phase comprising chloroform/acetone/methanol/acetic acid/water
(6/8/2/2/1, v/v/v/v/v). Reaction products on TLC plates containing an
aldehyde or a masked aldehyde were visualized by charring concentrated
sulfuric acid-treated plates and phosphate-containing lipids
were detected with molybdate-containing spray. In some cases,
reaction products or TLC-purified reaction products were extracted from
silica by a modified Bligh and Dyer technique (32). TLC-purified
reaction products were then subjected to capillary gas chromatography
following their derivatization with pentafluorobenzyl hydroxylamine
(see below) or methanolic HCl (70 °C for 30 min). In some cases acid
methanolysis-derivatized products were subjected to capillary gas
chromatography utilizing a Supelco SP-2330 column and detected by FID
under conditions described previously (15). Alternatively, acid
methanolysis-derivatized products were subjected to GC-MS with electron
impact ionization.
Gas Chromatography-Mass Spectrometric Analyses of
Pentafluorobenzyloxime Derivative Products--
GC-MS was performed on
reaction products or TLC-purified reaction products either directly or
following derivatization with acidic methanol or pentafluorobenzyl
hydroxylamine. In brief, derivatization with pentafluorobenzyl
hydroxylamine was performed by resuspending the reaction products in
300 µl of ethanol followed by the addition of 300 µl of 6 mg/ml
pentafluorobenzyl hydroxylamine in water. The ethanol-water
mixture was vortexed for 5 min at room temperature and allowed to
further incubate at room temperature for an additional 25 min. Reaction
products were diluted with 1.2 ml of water, extracted into
cyclohexane/diethyl ether (4/1, v/v), and resuspended in 30-100 µl
of petroleum ether prior to GC-MS analysis. GC-MS analysis of PFB
oximes of
-halo fatty aldehydes was performed on a Hewlett Packard
(Palo Alto, CA) 5973 mass spectrometer coupled to a Hewlett Packard
6890 gas chromatograph using the negative ion chemical ionization mode
with methane as the reagent gas. The source temperature was set at
150 °C. The electron energy was 240 eV, and the emission current was
300 µA. The PFB derivatives were separated on a J&W Scientific
(Folsom, CA) DB-1 column (12.5 m, 0.2 mm inner diameter, 0.33-µm film
thickness). The injector and the transfer line temperatures were
maintained at 250 °C. The GC oven was maintained at 150 °C for
3.5 min, increased at a rate of 25 °C/min to 310 °C, and held at
310 °C for an additional 5 min.
Eosinophil Activation--
Whole blood (180 ml) was taken from
hypersensitive volunteers, and eosinophils were isolated as described
previously using a Percoll gradient were subsequently purified using a
CD16 immunomagnetic bead sorting system on a Miltenyi MACS column as
described previously (33). Purified eosinophils were resuspended in
Hanks' balanced salt solution (pH 7.3) supplemented with 100 µM NaBr and immediately subjected to experimental
protocols. Eosinophils (1 × 106 cells/ml) were
treated with or without (control) 200 nM PMA for 1 h
at 37 °C. Reactions were terminated by snap-freezing in liquid nitrogen, and subsequently lipids were extracted in the presence of
internal standard (45 and 15 pmol of
2-Br-[d4]-HDA and
2-Cl-[d4]-HDA, respectively, for a total of
2.5 × 106 cells extracted). Following Bligh and Dyer
(32) extraction of eosinophil lipids, 2-BrHDA and 2-ClHDA were
quantified following derivatization to its PFB oxime by GC-MS utilizing
selected ion monitoring GC-MS (SIM-GC-MS).
Neutrophil Chemotaxis--
Whole blood (50 ml) was taken from
healthy volunteers and anticoagulated with EDTA (final concentration
5.4 mM) prior to the isolation of neutrophils using a
Ficoll-Hypaque gradient as described previously (23). Pelleted
neutrophils were resuspended in chemotaxis buffer comprised of Hanks'
balanced salt solution (pH 7.3), 1% bovine serum albumin (w/v), and 10 mM HEPES at a concentration of 4 × 106
neutrophils/ml. Neutrophil chemotaxis was assayed as described previously (34). fMLP, 2-BrHDA in Me2SO, or
Me2SO controls were diluted in modified Hanks' balanced
salt solution and loaded into the lower compartments of a Boyden
chemotaxis chamber, which was separated from the top compartment
containing 2 × 105 neutrophils (50 µl) by cellulose
nitrate filters (3 µm pore size). The Boyden chamber was incubated
for 35 min at 37 °C, the filters were then stained and dehydrated,
and chemotaxis was assessed by the leading front method as described
previously (34). Net migration through the filter was reported
in µm.
 |
RESULTS |
The present studies were directed at determining the role of
eosinophil peroxidase in the targeting of plasmalogens by reactive brominating species. As a first step, lysoplasmenylcholine
(1-O-hexadec-1'-enyl-GPC) was incubated with eosinophil
peroxidase in phosphate buffer containing hydrogen peroxide and 100 mM sodium bromide. TLC analysis of the reaction
products revealed that a neutral lipid was produced, which
migrated with an Rf
0.58 in a solvent system
that resolves neutral lipids (Fig. 1)
when lysoplasmenylcholine is treated with eosinophil peroxidase,
hydrogen peroxide, and sodium bromide at pH 4 (Fig. 1, EPO/HOBr
system). This neutral lipid did not comigrate with either
2-chlorohexadecanal (2-ClHDA) (Rf = 0.46) or
fatty acid (Rf range for palmitic, palmitoleic,
and arachidonic acid = 0.18-0.20) in this TLC system. The
production of this neutral lipid was dependent on the presence of a
complete reaction mixture comprising active eosinophil peroxidase
(thermal treatment of eosinophil peroxidase ablated
lysoplasmenylcholine loss), hydrogen peroxide, and sodium
bromide (Fig. 1).

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Fig. 1.
TLC analysis of eosinophil peroxidase-treated
lysoplasmenylcholine. 200 nmol of lysoplasmenylcholine was
incubated in the presence or absence of each of the EPO/HOBr-generating
reagents including EPO (22.6 ng), H2O2 (1 mM), and NaBr (100 mM) in 500 µl of 20 mM phosphate buffer (pH 4.0 or 7.0) as indicated at
37 °C for 5 min. Incubations were terminated by the addition of
methanol, and reaction products were sequentially extracted into
chloroform and subjected to TLC with a solvent system composed of
petroleum ether/diethyl ether/acetic acid (90/10/1, v/v/v). Reaction
products on developed TLC plates were visualized by sulfuric acid
treatment and charring.
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Similar studies were performed utilizing the plasmenylcholine molecular
species, 1-O-hexadec-1'-enyl-2-hexadecanoyl-GPC, as the
target phospholipid of the reactive brominating species generated by
eosinophil peroxidase. Reaction products were subjected to TLC analysis
with molybdate staining of inorganic phosphate, which demonstrated
plasmenylcholine loss in the presence of the complete eosinophil
peroxidase reactive brominating species system at pH 4 (Fig.
2A) concomitant with the
production of a polar lipid that comigrated with authentic LPC
(Fig. 2A). Additionally, under these conditions a neutral
lipid that migrated with the solvent front was observed when TLC plates
were charred with sulfuric acid (data not shown). Silica corresponding
to regions 1 and 2 (indicated in Fig. 2) (taken from TLC plates that
were developed concomitantly with those shown in Fig. 2A)
was scraped from TLC plates, and purified lipids were extracted from
the silica and subjected to GC following derivatization by acid
methanolysis (Fig. 2B). This GC analysis confirmed the
production of LPC in region 1 with plasmenylcholine as the target of
the reactive brominating species produced by eosinophil peroxidase
(+EPO in Fig. 2B) because only the fatty acid
methyl ester of palmitic acid was present (peak b) and the vinyl ether derivative, the dimethylacetal of palmitaldehyde, was
absent. Examination of region 2 (Fig. 2B), which stained
positively in the +EPO sample using sulfuric acid charring, by GC
analysis of acid methanolysis products revealed the presence of a peak that had an identical retention time to that of the dimethylacetal of
2-BrHDA (peak c). An additional analysis by GC of acid
methanolysis derivatives of the neutral lipid produced by eosinophil
peroxidase-derived reactive brominating species attack of
lysoplasmenylcholine (see Fig. 1) also demonstrated that the neutral
lipid was most likely 2-BrHDA, as it yielded a peak with an
identical retention time as that of peak c in Fig.
2B.

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Fig. 2.
TLC and GC analysis of eosinophil
peroxidase-treated plasmenylcholine. A, 200 nmol of
plasmenylcholine was incubated in the presence or absence of each of
the EPO/HOBr-generating reagents including EPO (45 ng),
H2O2 (1 mM), and NaBr (100 mM) in 500 µl of 20 mM phosphate buffer (pH
4) as indicated at 37 °C for 5 min. Incubations were terminated by
the addition of methanol, and reaction products were sequentially
extracted into chloroform and subjected to TLC with
chloroform/acetone/methanol/acetic acid/water (6/8/2/2/1, v/v/v/v/v) as
the mobile phase. Reaction products on developed TLC plates were
visualized by molybdate treatment. B, parallel plates were
developed identically, but silica was scraped from the
regions denoted as 1 and 2, and purified lipids
were extracted. These lipids were then subjected to acid methanolysis
and analyzed by GC with FID detection. Peaks a,
b, and c correspond to the solvent peak, the
methyl ester of hexadecanoic acid, and the putative dimethylacetal of
2-bromohexadecanal, respectively.
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To confirm that the neutral lipid reaction product from the eosinophil
peroxidase-mediated degradation of plasmalogens was 2-BrHDA, the
TLC-purified neutral lipid product that was derivatized by acid
methanolysis as well as the underivatized product were subjected to
GC-MS utilizing electron impact ionization. The mass spectrum of the
acid methanolysis product of the neutral lipid showed a base ion at
m/z 75, which is a signature ion of
dimethylacetals, acid methanolysis products of masked aldehydes
(i.e. the vinyl ether bond of plasmalogens) and free
aldehydes (Fig. 3A). The mass
spectrum of the underivatized compound included the anticipated isotopomers of the brominated parent ions of 2-bromohexadecanal (2-BrHDA) (Fig. 3B). These ions at m/z
318 and 320 are present at a 1/1 ratio characteristic of monobrominated
molecules because of the ratio of the natural isotopic abundance
of 79Br and 81Br at 1/1. Two peaks consistent
with the production of the syn- and anti-isomers
of the PFB oxime derivative of 2-BrHDA were identified by GC-MS
analyses using negative chemical ionization detection. The
fragmentation pattern of the second peak is shown in Fig. 4. One of the major fragments at
m/z 332 is monobrominated, having an ion of equal
intensity observed at m/z 334. It should be
recognized that the remaining fragmentation pattern is consistent with
the structure of the PFB oxime of 2-BrHDA (Fig. 4,
inset).

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Fig. 3.
Gas chromatography-mass spectrometry of the
lysoplasmenylcholine/eosinophil peroxidase reaction product.
Lysoplasmenylcholine (200 nmol) was incubated in the presence of the
EPO/HOBr-generating system, which includes EPO (22.6 ng),
H2O2 (1 mM), and NaBr (100 mM) in 500 µl of 20 mM phosphate buffer (pH
4.0) at 37 °C for 5 min. Incubations were terminated by the addition
of methanol, and reaction products were sequentially extracted into
chloroform and subjected to TLC as described under "Experimental
Procedures." The TLC-purified material having an
Rf = 0.58 was either subjected to acid
methanolysis prior to GC-MS (A) with electron impact
ionization detection or subjected directly to GC-MS (B) with
electron impact ionization detection as described under "Experimental
Procedures." Ions and ion fragments (the mass spectrum) of the major
peaks are shown from the two samples. The insets in
A and B illustrate the structure and putative
fragmentation patterns of the dimethylacetal of 2-bromohexadecanal and
2-bromohexadecanal, respectively.
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Fig. 4.
Gas chromatography-mass spectrometry of the
pentafluorobenzyl oxime derivative of the
lysoplasmenylcholine/eosinophil peroxidase reaction product.
Lysoplasmenylcholine (200 nmol) was incubated in the presence of the
EPO/HOBr-generating system, which includes EPO (22.6 ng),
H2O2 (1 mM), and NaBr (100 mM) in 500 µl of phosphate buffer (pH 4.0) at 37 °C
for 5 min. Incubations were terminated by the addition of methanol, and
reaction products were sequentially extracted into chloroform and
subjected to TLC. The TLC-purified neutral lipid
(Rf = 0.58) was converted to its
pentafluorobenzyl oxime derivatives as described under "Experimental
Procedures." The derivatization products were subjected to capillary
gas chromatography utilizing a DB-1 column and negative chemical
ionization mass spectrometry for detection. The mass spectrum of peak 2 of the total ion current chromatogram is shown. The inset
illustrates the structure and putative fragmentation patterns of the
pentafluorobenzyl oxime of 2-bromohexadecanal, the product of the
lysoplasmenylcholine/eosinophil peroxidase reaction.
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Further studies were designed to compare the production of 2-BrHDA in
the presence and absence of physiological NaCl levels (100 mM). The data shown in Fig. 5
show that eosinophil peroxidase mediates the attack of plasmalogens in
the presence of bromide more efficiently when also in the
presence of physiological concentrations of NaCl in comparison with in
the absence of NaCl. With only bromide present, 2-BrHDA production was
maximal at 500 µM NaBr (Fig. 5). In contrast, the bromide
requirement for maximal 2-BrHDA production was decreased to ~100-250
µM in the presence of physiological concentrations of
NaCl (100 mM) (Fig. 5). Furthermore, at physiological concentrations of NaBr (50 µM), the presence of 100 mM NaCl increased 2-BrHDA production ~2-fold (Fig. 5).
The augmentation of 2-BrHDA production by the addition of sodium
chloride is only minimally due to bromide contamination in the
chloride, because 2-BrHDA production in the presence of 100 mM NaCl with no addition of bromide was only half of that
found with only 25 µM sodium bromide added (Fig. 5).
Because of the augmentation of 2-BrHDA production by NaCl
supplementation, the pH dependence was characterized with 100 µM NaBr in the presence of 100 mM NaCl. The
pH optima for 2-BrHDA production by reactive halogenating species
produced by eosinophil peroxidase in the presence of physiological
chloride and bromide concentrations were acidic, with maximal
production observed at pH 4.5 (Fig. 6).
Additionally, 2-ClHDA production was not detected under these
conditions (data not shown).

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Fig. 5.
Augmentation of 2-BrHDA
production by eosinophil peroxidase by NaCl. 50 nmol of
lysoplasmenylcholine was incubated in the presence of the eosinophil
peroxidase reactive halogenating species-generating system, which
includes eosinophil peroxidase (22.6 ng), H2O2
(1 mM), and selected concentrations of NaBr (0-100
mM), in 2 ml of phosphate buffer at pH 4 and at 37 °C
for 5 min. In parallel incubations, treatments were supplemented with
100 mM NaCl as indicated. Lipid reaction products were
extracted and subjected to acid methanolysis in the presence of
1-hexadecanoyl-GPC (internal standard), and derivatives were analyzed
by capillary gas chromatography with FID detection as described under
"Experimental Procedures." Values are the means ± S.E. of at
least three independent experiments. 2-BrHDA was not detected
(N.D.) in the absence of both NaCl and NaBr.
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Fig. 6.
pH dependence of plasmalogen degradation by
the eosinophil peroxidase/reactive brominating species-generating
system in the presence of physiological concentrations of NaCl. 50 nmol of lysoplasmenylcholine was incubated in the presence of the
eosinophil peroxidase/reactive brominating species-generating system,
which includes eosinophil peroxidase (22.6 ng),
H2O2 (1 mM), NaCl (100 mM), and NaBr (0.1 mM) in 2 ml of phosphate
buffer at the indicated pH at 37 °C for 5 min. Lipid reaction
products were extracted and subjected to acid methanolysis in the
presence of 1-hexadecanoyl-GPC (internal standard), and derivatives
were analyzed by capillary gas chromatography with FID detection as
described under "Experimental Procedures." Values are the
means ± S.E. of at least three independent experiments.
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The specificity of the degradation of plasmalogens by eosinophil
peroxidase-generated reactive brominating species was further assessed
in reactions that included the peroxidase inhibitors sodium azide,
catalase, and 3-aminotriazole using 500 µM NaBr in the
presence of 100 mM NaCl as substrate for the peroxidase. Sodium azide, catalase, and 3-aminotriazole inhibited eosinophil peroxidase-mediated degradation of the vinyl ether bond (Fig. 7). Additionally, the specificity of the
reaction of the eosinophil peroxidase-generated reactive bromine
species toward the vinyl ether bond of plasmalogens was demonstrated
because destruction of the vinyl ether bond of plasmalogens by
treatment with hydrochloric acid fumes for 10 min ablated the
production of the eosinophil peroxidase-mediated neutral lipid product,
2-BrHDA (Fig. 7).

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Fig. 7.
Inhibition of eosinophil peroxidase-mediated
plasmalogen degradation by eosinophil peroxidase pathway inhibitors and
pretreatment of plasmalogens with acid vapors. 200 nmol of
lysoplasmenylcholine was incubated with 22.6 ng of eosinophil
peroxidase (or thermal denatured eosinophil peroxidase (Denat.
EPO), 15 min at 90 °C) in the HOBr-generating buffer system
(100 mM NaCl and 0.5 mM NaBr) at pH 4.0 at
37 °C for 5 min in the presence or absence of 3-aminotriazole
(ATZ, 50 mM; preincubated with eosinophil
peroxidase at 37 °C for 5 min prior to initiation of reaction), 10 µg/ml catalase (preincubated with reaction mixture at 37 °C for 5 min prior to the addition of eosinophil peroxidase), or 100 µM sodium azide (preincubated with eosinophil peroxidase
at 37 °C for 5 min prior to initiation of reaction) as indicated.
Alternatively, 200 nmol of lysoplasmenylcholine was pretreated with HCl
vapors (as indicated) and then incubated with 22.6 ng of eosinophil
peroxidase in the HOBr-generating buffer system at pH 4.0 at 37 °C
for 5 min. Lipid reaction products were extracted and subjected to acid
methanolysis in the presence of 1-hexadecanoyl-GPC (internal standard),
and derivatives were analyzed by capillary gas chromatography with FID
detection as described under "Experimental Procedures." The mass of
product formed was calculated by comparing the integrated areas of the
peak derived from plasmalogen degradation to that of the
internal standard. Values are the means ± S.E. of at least three
independent experiments.
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Because both eosinophil peroxidase and myeloperoxidase use both
chloride and bromide, comparisons were made between these two
peroxidases to determine their selectivity in using halides to produce
-halo fatty aldehydes. At pH 4, myeloperoxidase used either chloride
or bromide to produce reactive halogenating species, resulting in the
production of either 2-ClHDA or 2-BrHDA, respectively (Fig.
8). Additionally, in the presence of both
100 µM bromide and 100 mM chloride,
myeloperoxidase-derived reactive halogenating species attacked
plasmalogens, resulting in the production of both 2-ClHDA and 2-BrHDA
(Fig. 8). In contrast, eosinophil peroxidase was very specific for
bromide as substrate, with only small amounts of 2-ClHDA
produced in the presence of 100 mM chloride in the absence of bromide (Fig. 8). Furthermore, 2-BrHDA was selectively produced by eosinophil peroxidase in the presence of 100 µM bromide and 100 mM chloride (Fig. 8).

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Fig. 8.
Comparisons of -halo
fatty aldehyde production by eosinophil peroxidase- and
myeloperoxidase-derived reactive halogenating species. 100 nmol of
lysoplasmenylcholine was incubated in the presence of either the
myeloperoxidase- or eosinophil peroxidase-derived reactive halogenating
species-generating systems, which includes either myeloperoxidase (0.6 units) or eosinophil peroxidase (22.6 ng), H2O2
(1 mM), and the indicated concentrations of NaBr and/or
NaCl in 2 ml of phosphate buffer at pH 4 and at 37 °C for 5 min.
Lipid reaction products were extracted and subjected to acid
methanolysis in the presence of 1-hexadecanoyl-GPC (internal standard),
and derivatives were analyzed by capillary gas chromatography with FID
detection as described under "Experimental Procedures." Values are
the means ± S.E. of at least three independent experiments.
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The physiological significance of eosinophil peroxidase-mediated
production of 2-BrHDA from plasmalogens was assessed by determining that this biochemical mechanism is present in intact activated eosinophils as well as by determining that 2-BrHDA is a phagocyte chemoattractant. To determine that halogenated aldehydes are produced in activated eosinophils, GC-MS analyses of their PFB
derivatives were measured using a deuterated internal standard as
described previously (23). For these experiments, unstimulated
(control) and PMA-stimulated human eosinophils were incubated in
Hanks' balanced salt solution supplemented with 100 µM
NaBr, and the production of 2-BrHDA and 2-ClHDA was determined by GC-MS
of their respective PFB oximes. PMA activation of the eosinophils led
to the production of 2-BrHDA and a small increase in 2-ClHDA compared with nonactivated eosinophils (Fig. 9,
PMA). Additionally, PMA-stimulated 2-BrHDA and 2-ClHDA
production were blocked by the peroxidase inhibitor, sodium azide (Fig.
9). The potential physiological role of 2-BrHDA as a phagocyte
chemoattractant was tested. Both 90 nM and 90 µM 2-BrHDA induced neutrophil chemotaxis to a
significantly greater extent than their Me2SO controls
(Fig. 10). For comparison, the known
potent neutrophil chemoattractant, fMLP, is shown as a positive control
for chemoattraction (Fig. 10).

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Fig. 9.
2-BrHDA and 2-ClHDA accumulation in
PMA-stimulated eosinophils. Eosinophils isolated from
allergen-hypersensitive individuals were suspended in Hanks' balanced
salt solution at a concentration of 1 × 106 cells/ml
for 1 h under the indicated conditions. The complete system
contained 100 µM NaBr and 200 nM PMA.
Following incubation, cell suspensions were snap frozen, and
subsequently lipids were extracted in the presence of deuterated
2-BrHDA and 2-ClHDA. Halogenated aldehydes were then converted to their
PFB oximes and quantitated by selected ion monitoring using
GC-MS with chemical ionization. Experiments were performed in duplicate
as shown.
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Fig. 10.
2-Bromohexadecanal induces neutrophil
chemotaxis. 2 × 105 neutrophils were loaded into
the upper compartments of a Boyden chamber, and neutrophil chemotaxis
was elicited by 100 nM fMLP, 90 nM and 90 µM 2-BrHDA, or chemotaxis buffer (as indicated),
measured as described under "Experimental Procedures." *
and ** indicate treatments with p < 0.05 and
p < 0.005, respectively, compared with the appropriate
controls. Each value indicates the distance of neutrophil migration for
at least four independent measurements.
|
|
 |
DISCUSSION |
Plasmalogens are a predominant phospholipid molecular subclass
found in many mammalian tissues including endothelial cells and
vascular smooth muscle (16, 35). Vascular tissue is one of the major
pathophysiological host targets of activated leukocytes leading to
inflammation. The present study now demonstrates that eosinophils also
produce reactive halogenating species that attack plasmalogens. For
these studies several lines of evidence have demonstrated that 2-BrHDA
is produced from the attack on the vinyl ether bond of the plasmalogen
1-O-hexadec-1'-enyl-GPC by reactive brominating species
produced by eosinophil peroxidase. First, utilizing GC-MS analyses of
the acid methanolysis product of the reaction of reactive brominating
species with plasmalogen indicated that a dimethylacetal
derivative of an aldehyde was present. Second, the characteristic
isotopic cluster of a monobrominated molecule was observed at the
predicted mass of 2-BrHDA utilizing GC-MS analyses of the underivatized
compound. These ions at m/z 318 and
m/z 320 were present at a 1:1 ratio, which is a
signature for a monobrominated molecule because of the 1:1 natural
abundance of 79Br and 81Br, respectively.
Finally, derivatization of the neutral lipid with pentafluorobenzyl
hydroxylamine followed by GC-MS analysis with negative chemical
ionization was consistent with the derivatization of 2-BrHDA to its
pentafluorobenzyl oxime. Collectively these analyses have demonstrated
that eosinophil peroxidase-derived reactive brominating species attack
the vinyl ether bond of plasmalogens resulting in 2-BrHDA production.
Furthermore, this is the first demonstration that lipid halogenation
occurs through the activation of eosinophils.
The present studies also suggest that
-bromo fatty aldehydes are
produced by intact activated eosinophils through a mechanism that is
mediated by eosinophil peroxidase-derived reactive brominating species
that target the plasmalogen vinyl ether bond. Eosinophil peroxidase-derived reactive brominating species are produced during eosinophil activation (4, 5), and the present results show that
inhibition of their production by the inclusion of the peroxidase inhibitor azide drastically attenuates the production of 2-BrHDA by
PMA-stimulated eosinophils. It should be appreciated that in both
intact eosinophil and eosinophil peroxidase studies, physiological levels of bromide were used because normal plasma concentrations of
bromide range from 20-150 µM (36). Interestingly, in
experiments with intact eosinophils, some 2-ClHDA was produced in
response to PMA. Yet experiments with purified eosinophil peroxidase
demonstrated that it is relatively selective for the production of
reactive brominating species and the production of 2-BrHDA. Some
2-ClHDA production may be attributed to the 2% neutrophil (and hence
myeloperoxidase) contamination of the eosinophil preparation.
Eosinophil peroxidase-mediated attack of plasmalogens by reactive
brominating species occurs only at acidic pH and does not occur at
neutral pH. It is interesting to note that the airway lining fluid of
asthmatics subjects is acidic (37), and reactive brominating species
are generated following allergen challenge (5) and in severe asthma
(38). Eosinophil peroxidase-derived reactive halogenating species
selectively brominated plasmalogens with only minimal chlorination
detected. Additionally, the increase in 2-BrHDA by reactive brominating
species derived from eosinophil peroxidase with the addition of NaCl to
physiological levels of NaBr suggests that interhalogen species may be
formed (e.g. Br-Cl) that may preferentially brominate the
plasmalogen vinyl ether. This mechanism differs from that of
myeloperoxidase-mediated bromination, which appears to be mediated by
hypochlorous acid oxidation of bromide, resulting in the production of
reactive brominating species, which brominate plasmalogens at neutral
pH (30). Taken together, these studies demonstrate major differences in
the halogenation of plasmalogens by myeloperoxidase and eosinophil
peroxidase-derived reactive halogenating species. Our findings
suggest that in complex physiological systems (in vivo)
2-BrHDA would be produced via myeloperoxidase at neutral pH, whereas at
acidic pH, 2-BrHDA in the absence of 2-ClHDA production would
imply that the halogenation was mediated by eosinophil peroxidase.
Further studies with murine models of eosinophilic and neutrophilic
inflammation using eosinophil peroxidase and myeloperoxidase knockout
mice are needed to determine the relative contributions of these
distinct leukocyte heme peroxidases.
Many different aldehydes, such as 4-hydroxynonenal and
4-hydroxytetradecenal, thought to be produced by lipid peroxidation in
the course of inflammatory reactions (39, 40) have been shown to induce
significant neutrophil chemotaxis in vitro (41). Additionally, 2-ClHDA has been shown to be a chemoattractant in human
neutrophils (23). The present studies now show that 2-BrHDA also is a
neutrophil chemoattractant. The finding that 2-BrHDA is a
chemoattractant supports a role for the generation of 2-BrHDA by
eosinophils as a mechanism that might mediate the recruitment of
phagocytes to sites of active inflammation, while also lending support
to the hypothesis that aldehydic lipid oxidation or peroxidation products play important roles in such mechanisms.
The present studies suggest that the attack of plasmalogens by reactive
brominating species produced by eosinophil peroxidase might promote the
cytotoxic potential of eosinophils as a defense mechanism against
bacterial, parasitic, and viral pathogens. The cytotoxicity of this
mechanism would be through both the loss of plasma membrane
plasmalogens as well as the production of bioactive lysophospholipids
(24, 42, 43) and potentially reactive
-bromo fatty aldehydes. It is
likely that
-bromo fatty aldehydes form Schiff bases with primary
amines, which would contribute to the cytotoxicity of this mechanism
initiated by the attack of the vinyl ether-masked aldehyde of
plasmalogens. Although others have demonstrated that plasmalogens can
terminate the cytotoxicity of reactive oxygen species (44, 45), it is
possible that plasmalogens may serve as targets for the
cytotoxicity elicited by reactive halogenating species. On the other
hand, it is possible that plasmalogens may serve as protective agents
to the host cell if the products of reactive brominating species attack
of plasmalogens are relatively less cytotoxic compared with the attack
of other targets of reactive brominating species, such as proteins and
nucleic acids, which could potentially lead to greater long-term damage
to the host cell. Taken together, the present results demonstrate the
targeting of vinyl ether bonds of plasmalogens by reactive brominating
species produced by eosinophil peroxidase. This may represent a
potentially important and as yet unrecognized mechanism mediating the
generation of
-bromo fatty aldehydes and lysophospholipids, which
may both play important roles in the antimicrobial role of eosinophils as well as in inflammatory host cell injury in diseases such as asthma.
 |
FOOTNOTES |
*
This research was supported jointly by National Institutes
of Health Grants R01 HL 42665 (to D. A. F.), HL51199 (to R. M. H.),
HL61878 (to S. L. H.), and HL70937 (to A. S.) and Grant-in-aid 0151438Z (to D. A. F.) from the American Heart Association, Heartland Affiliate.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Recipient of a predoctoral fellowship award from the American Heart
Association, Heartland Affiliate.

To whom correspondence should be addressed: Dept. of
Biochemistry and Molecular Biology, St. Louis University School of
Medicine, 1402 South Grand Blvd., St. Louis, MO 63104. Tel.:
314-577-8123; Fax: 314-577-8156; E-mail: fordda@slu.edu.
Published, JBC Papers in Press, January 6, 2003, DOI 10.1074/jbc.M211634200
 |
ABBREVIATIONS |
The abbreviations used are:
EPO, eosinophil
peroxidase;
FID, flame ionization detector;
GPC, sn-glycero-3-phosphorylcholine;
GC-MS, gas
chromatography-mass spectrometry;
HPLC, high pressure liquid
chromatography;
TLC, thin-layer chromatography;
PFB, pentafluorobenzyl;
2-BrHDA, 2-bromohexadecanal;
2-ClHDA, 2-chlorohexadecanal;
PMA, phorbol
myristate acetate;
Me2SO, dimethyl sulfoxide;
fMLP, formyl-methionyl-leucyl-phenylalanine;
LPC, lysophosphatidylcholine.
 |
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Copyright © 2003 by the American Society for Biochemistry and Molecular Biology.