From the Department of Internal Medicine, Pulmonary and Critical Care Medicine, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27154
Received for publication, July 21, 2000, and in revised form, September 26, 2000
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ABSTRACT |
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The goal of this study was to examine arachidonic
acid (AA) metabolism by murine bone marrow-derived mast cells (BMMC)
during apoptosis induced by cytokine depletion. BMMC deprived of
cytokines for 12-48 h displayed apoptotic characteristics. During
apoptosis, levels of AA, but not other unsaturated fatty acids,
correlated with the percentage of apoptotic cells. A decrease in both
cytosolic phospholipase A2 expression and
activity indicated that cytosolic phospholipase A2 did not
account for AA mobilization during apoptosis. Free AA accumulation is
also unlikely to be due to decreases in 5-lipoxygenase and/or
cyclooxygenase activities, since BMMC undergoing apoptosis produced
similar amounts of leukotriene B4 and significantly greater
amounts of PGD2 than control cells. Arachidonoyl-CoA
synthetase and CoA-dependent transferase activities
responsible for incorporating AA into phospholipids were not altered
during apoptosis. However, there was an increase in arachidonate in
phosphatidylcholine (PC) and neutral lipids concomitant with a
40.7 ± 8.1% decrease in arachidonate content in
phosphatidylethanolamine (PE), suggesting a diminished capacity of mast
cells to remodel arachidonate from PC to PE pools. Further evidence of
a decrease in AA remodeling was shown by a significant decrease in
microsomal CoA-independent transacylase activity. Levels of lyso-PC and
lyso-PE were not altered in cells undergoing apoptosis, suggesting that
the accumulation of lysophospholipids did not account for the decrease
in CoA-independent transacylase activity or the induction of apoptosis.
Together, these data suggest that the mole quantities of free AA
closely correlated with apoptosis and that the accumulation of AA in
BMMC during apoptosis was mediated by a decreased capacity of these cells to remodel AA from PC to PE.
Mast cells play a vital role in acute and chronic allergic
inflammation by virtue of their ability to release inflammatory mediators and cytokines upon antigen activation (1-5). Arachidonic acid (AA)1 located at the
sn-2-position of membrane phospholipids is the precursor of
one important class of inflammatory mediators, collectively known as
eicosanoids (e.g. PGD2, TXB2,
LTC4, and LTB4) (6). During mast cell
activation, AA is rapidly released and utilized for the formation of
eicosanoids (7). This can be accomplished by one or more of several
PLA2 enzymes with different molecular characteristics that
have been described in mast cells (8-10). These enzymes show different
selectivity toward the hydrolysis of fatty acids at the
sn-2-position or phosphobase moieties at the
sn-3-position of phospholipids and include a relatively high molecular mass cytosolic PLA2 (cPLA2;
70-110 kDa) and several low molecular mass or secretory
PLA2s (~14 kDa) (11-15).
In mast cells and other inflammatory cells, low levels of free AA are
maintained during resting conditions by both CoA-dependent and CoA-independent mechanisms (16-19). Free AA is initially converted to arachidonoyl CoA by arachidonoyl-CoA synthetase and is then rapidly
incorporated into 1-acyl-2-lysophospholipids by
CoA-dependent acyltransferase (20, 21). AA in 1-acyl-linked
phospholipids is then gradually remodeled into 1-ether-linked
phospholipids by CoA-independent transacylase (CoA-IT), leading to
large amounts of AA in ether lipid pools (22-25).
Recent studies suggest that mast cell numbers are primarily controlled
by IL-3 and SCF as mast cells have been shown to undergo apoptosis in
the absence of these cytokines (26-29). Until now, neither the
inflammatory capacity nor the ability to regulate AA levels of mast
cells that are undergoing apoptosis has been determined. To begin to
understand how mast cells metabolize AA during apoptosis, we have
utilized an in vitro model of mast cell apoptosis induced by
cytokine depletion. Our data suggest that there is an increase in free
AA levels outside mast cells undergoing apoptosis. Importantly, the
mole quantities of AA, but not other unsaturated fatty acids,
correlated with the number of apoptotic cells and were inversely
proportional to the number of live cells. This accumulation of AA can
be explained by a decrease in CoA-IT activity, which results in a
diminished capacity of mast cells to remodel AA from
phosphatidylcholine (PC) to phosphatidylethanolamine (PE).
Materials
Octadeuterated [5,6,8,9,11,12,14,15-2H]AA
([2H8]AA) and trideuterated stearic acid
([2H3]SA) were purchased from Biomol Research
Laboratory (Plymouth Meeting, PA). LTB4,
[2H4]LTB4, PGD2,
[2H4]PGD2, TXB2, and
[2H4]TXB2 were purchased from
Cayman (Ann Arbor, MI). Essentially fatty acid-free human serum albumin
(HSA), essential and nonessential amino acids, mouse IgE
anti-dinitrophenol (IgE anti-DNP), heat-inactivated fetal bovine serum,
ionophore A23187, malondialdehyde (MDA), and
N-methyl-2-phenylindole were purchased from Sigma.
Methoxylamine HCl (MOX in pyridine), acetonitrile, pentafluorobenzyl
bromide (20% in acetonitrile), diisopropylethylamine (20% in
acetonitrile), and
N,O-bis(trimethylsilyl)-trifluoroacetamide were
purchased from Pierce. RPMI 1640 cell culture medium and Hanks'
balanced salt solution (HBSS) were purchased from Life Technologies,
Inc. PC, phosphatidylinositol (PI), phosphatidylserine (PS), and
PE were purchased from Serdary Research Laboratories (Englewood Cliffs, NJ). HPLC grade organic solvents were purchased from Fisher. A rabbit
antipeptide antibody for cPLA2 was kindly provided by
Hoffman-LaRoche. Dr. A. Sane (Wake Forest University School of
Medicine) kindly provided cPLA2 cDNA. Murine
recombinant stem cell factor (SCF) expressed in Escherichia
coli was a generous gift from Amgen (Seven Oaks, CA). Recombinant
IL-3 was purchased from Genezyme (Cambridge, MA). [3H]AA
(200 Ci/mmol), [Me3H]choline hydrochloride (85 Ci/mmol),
[1-3H]ethanolamine hydrochloride (30 Ci/mmol), and
palmitoyl-2-[1-14C-]arachidonoyl-sn-glycero-3-phosphocholine
(55 mCi/mmol) were purchased from American Radiolabel Chemical, Inc.
(St. Louis, MO). 1-[3H]Alkyl-2-lyso-GPC (56 Ci/mmol) was provided by Dr. R. Wykle (Wake Forest University School of Medicine).
Mast Cell Culture and Activation
BMMC were obtained from CBA/J mice (Jackson Laboratories, Bar
Harbor, ME) and grown in RPMI 1640 culture medium (Life Technologies, Inc.) supplemented with 10% (v/v) fetal calf serum, 50 µM 2-mercaptoethanol, 1% essential amino acids, 1%
nonessential amino acids, 2 mM L-glutamine, 5 µg/ml gentamycin, and 1% (v/v) penicillin/streptomycin. The culture
medium was enriched twice a week with 50% WEHI supernatant fluid as a
source of IL-3 for 3 weeks. Cell viability (>90%) was determined by
trypan blue exclusion.
BMMC were maintained in culture media with or without cytokines (0-100
ng/ml SCF or 100 ng/ml IL-3) for different periods of time as indicated
in the figure legends. Before stimulation with antigen, BMMC were
sensitized for 24 h with 0.5 µg/ml IgE anti-2,4-dinitrophenol (Sigma) in culture media. BMMC in HBSS containing calcium, 0.1 mg/ml gelatin, and 0.01 mg/ml fatty acid-free HSA were stimulated with antigen (2 µg/ml) or ionophore A23187 (1 µM) for 5 min at 37 °C. Cells were rapidly removed
from supernatant fluids by centrifugation (400 × g for
5 min), and the mole quantities of fatty acids and eicosanoids were
determined by negative ion chemical ionization gas chromatography/mass
spectroscopy (NICI-GC/MS) as described below.
Assessment of Apoptosis in BMMC
Caspase Activity--
BMMC were cultured without or with
cytokines for 24 h. Protease activity in BMMC homogenate was
determined using ApoAlertTM CPP32 Colorimetric Assay Kit
(CLONTECH) as directed by the manufacturers. Protease activity, defined as the amount of CPP32 required to produce 1 pmol of chromophore (p-nitroanilide, pNA) per min at 25 °C, was determined using a standard curve established using different concentrations of pNA at 405 nm. In order to establish that
the signal detected was attributable to protease activity, a control
assay was performed in which homogenates from cells that had been
placed in cytokine-depleted media were pretreated with a CPP32
inhibitor, Asp-Glu-Val-Asp-aldehyde, prior to the addition of CPP32
substrate (Asp-Glu-Val-Asp-p-nitroanilide).
Annexin V Binding to Mast Cells--
The reorientation of plasma
membrane phospholipids was monitored using the TACSTM
annexin V-FITC binding kit from Trevigen (Gaithersburg, MD). Briefly,
1 × 106 BMMC were cultured without cytokines or with
IL-3 or SCF for 24 h. BMMC were collected by centrifugation
(400 × g, 10 min) and then washed once in ice-cold
(4 °C) phosphate-buffered saline without Ca2+ and
Mg2+. The cells were resuspended in 100 µl of binding
buffer (10 mM HEPES, pH 7.4, containing 0.15 M
NaCl, 5 mM KCl, 1 mM MgCl2, and 1.8 mM CaCl2) containing annexin V-FITC and 0.5 µg of propidium iodide for 15 min at room temperature in the dark.
Binding buffer (400 µl) was then added to the mixture, and flow
cytometry was performed using a Coulter Epics XL-MCL flow cytometer.
The percentages of total cells that did not bind propidium iodide or
annexin-FITC (live cells), cells that bound annexin-FITC alone (early
apoptosis), cells that bound both propidium iodide and annexin-FITC
(late apoptosis), and cells that bound mainly propidium iodide
(necrotic cells) were determined, and the results are presented in the
form of dot plots.
DNA Ladder Formation--
BMMC were lysed in 10 mM
Tris/HCl, pH 7.6, containing 0.2% Triton X-100 and 15 mM
EDTA. The lysate was treated with 50 µg of proteinase K (Promega)
overnight at 50 °C. Subsequently, DNA was precipitated by
centrifugation (15,000 rpm, 10 min on a microcentrifuge) after
the addition of 1 ml of cold 2-propanol and 0.1 ml of NaCl (5 M). The DNA was resolved on 2.0% agarose gels by
electrophoresis, and DNA fragments were detected after ethidium bromide
staining by UV visualization.
[3H]Thymidine Incorporation into BMMC--
BMMC
were placed in growth medium with or without cytokines. Thymidine
incorporation was determined by incubating 1 × 106
BMMC with 1 µCi of [3H]thymidine (Amersham Pharmacia
Biotech) for 1 h at 37 °C. Unincorporated label was removed by
washing (twice) the cells with HBSS containing 0.25 mg/ml HSA. The cell
pellet was lysed using 0.2 N NaOH (0.25 ml for 1 h).
DNA was precipitated using 15% trichloroacetic acid (1 ml) overnight
at 4 °C. Cellular DNA was then trapped on glass microfiber filters
(Whatman GF/C). Free cellular [3H]thymidine was
removed from the filters by washes (4 ml, three times), and the amount
of radioactivity in DNA was determined by liquid scintillation counting.
Quantitation of Free Fatty Acids in Supernatant Fluids
After the addition of trideuterated stearic acid (100 ng of
[2H3]SA) as an internal standard for the
analysis of linoleic acid (LA) and oleic acid (OA) and the addition of
octadeuterioarachidonic acid ([2H8]AA, 100 ng) as an internal standard for the analysis of AA, eicosapentaenoic
acid (EPA), and docosahexaenoic acid (DHA) to an ethanolic extract of
the supernatant fluids, solvents were removed under a stream of
nitrogen. Fatty acids were then converted to pentafluorobenzyl esters,
and the mole quantities were determined by NICI-GC/MS using a Hewlett
Packard model 5989 instrument. Carboxylate anions
(m/z at 279, 281, 286, 303, 301, 327, and 311 for
LA, OA, [2H3]SA, AA, EPA, DHA, and
[2H8]AA, respectively) were monitored by gas
chromatography/mass spectroscopy. Mole quantities of fatty acids were
determined from standard curves obtained for each fatty acid using
[2H3]SA and [2H8]AA
as internal standards.
Determination of Lipid Peroxidation
BMMC were maintained in culture media without cytokines or with
cytokines for 24 h. BMMC were washed (three times) using ice-cold HBSS containing 0.25 mg/ml HSA. Subsequently, cells were lysed in 200 µl of HPLC grade H2O by repeated (four times) freezing using liquid N2 and thawing using a 37 °C water bath.
Lipid peroxidation was determined by measuring the mole quantities of
MDA, an end product of the peroxidation of unsaturated fatty acids
(30). Briefly, lysates (200 µl, 5 × 106 BMMC) were
added to a 10 mM solution of
N-methyl-2-phenylindole (650 µl) freshly made in
acetonitrile/methanol (3:1, v/v). After the addition of 150 µl of 12 N HCl, the mixture was incubated at 45 °C for 45 min.
The A586 was determined for samples and
0-10 µM MDA standards. The molar amounts of MDA in
samples were calculated using an extinction coefficient (gradient of
concentration versus A586) obtained
using 0-10 µM MDA standards.
Quantitation of Eicosanoids in Supernatant Fluids
After stimulation of BMMC,
[2H4]PGD2,
[2H4]TXB2, and
[2H4]LTB4 (10 ng of each) were
added to supernatant fluids as internal standards. Eicosanoids were
converted to methoxime-pentafluorobenzyl ester trimethylsilyl ether
derivatives. Derivatized eicosanoids were then extracted with hexane
and analyzed using combined NICI-GC/MS. Carboxylate anions for
LTB4 (m/z 479),
[2H4]LTB4
(m/z 483), PGD2
(m/z 524),
[2H4]PGD2
(m/z 528), TXB2
(m/z 614), and
[2H4]TXB2
(m/z 618) were analyzed in the single
ion-monitoring mode.
Determination of cPLA2 Activity
BMMC were washed (twice) with HBSS containing 0.25 HSA and 5 mM dithiothreitol. Cells were then suspended at
107/ml in sonication buffer (10 mM HEPES, pH
7.4, containing 80 mM KCl, 1 mM EDTA, 1 mM EGTA, 40 µg/ml leupeptin, 25 µg/ml pepstatin, 1 mM phenylethylsulfonyl fluoride, 10 mM NaF, 0.2 mM Na2VO3, and 5 mM
dithiothreitol). Sonication (10 s, three times) was performed using a
probe sonicator (Heat System Inc.) at a power setting of 2 and 10%
output. Protein content of fractions was determined using the Coomassie
Plus protein assay reagent (Pierce). cPLA2 activity was
determined using 400 pmol sonicated-vesicles of
1-palmitoyl-2-[1-14C]arachidonoyl-sn-glycero-3-phosphocholine
as substrate and 50 µg of protein from BMMC sonicates.
cPLA2 activity was initiated by the addition of substrate
to fractions that had been preincubated for 15 min at 37 °C in an
assay mixture that contained 5 mM dithiothreitol. This
preincubation was necessary for eliminating residual secretory PLA2 activity. The PLA2 reaction was stopped
after 15 min at 37 °C by extracting lipids by the method of Bligh
and Dyer (31). Free fatty acids were isolated from phospholipids by TLC
on silica gel G developed in hexane/diethyl ether/formic acid (90:60:6, v/v/v). The radioactivity in lipids was located using a
radiochromatogram imaging system (Bioscan Inc., Washington, D. C.).
Free AA and phospholipids were isolated using TLC zonal scraping, and
the radioactivity was determined utilizing liquid scintillation
counting. cPLA2 activity was calculated and expressed as
pmol of AA released/mg of protein/min.
SDS-Polyacrylamide Gel Electrophoresis and Western Blot Analysis
of cPLA2
Amounts of cPLA2 protein were determined in total
lysates of 1-2 million BMMC (32). Briefly, BMMC were incubated in
lysis buffer (100 mM Tris/HCl, pH 7.5, containing 0.1 M NaCl, 2 mM EDTA, 1% Nonidet P-40, 1 mM Na3VO4, 50 mM NaF,
0.1 mM N-tosyl-L-phenylalanine chloromethyl ketone, 0.1 mM quercetin, 1 mM
phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, and 1 µg/ml
leupeptin) for 10 min on ice. After removal of nuclei by
centrifugation, extracts were mixed with an equal volume of 2× loading
buffer (0.125 M Tris, pH 6.8, 4% SDS, 20% glycerol, 10%
mercaptoethanol, 0.05% bromphenol blue) and boiled for 5 min. Proteins
were separated by SDS-polyacrylamide gel electrophoresis on a 4-20%
polyacrylamide gel. Separated proteins were transferred onto
nitrocellulose membranes, and the blots were incubated overnight with
an anti-cPLA2 antibody (1 µg/ml). Detection of
cPLA2 was accomplished using horseradish peroxidase conjugate anti-rabbit IgG and enhanced chemiluminescence reagents (Pierce).
RNA Isolation Northern Analysis of cPLA2 from
BMMC
Total RNA was extracted from BMMC cultured with different
concentrations of SCF (0-100 ng/ml) using RNazol (Tel Test Inc., Friendswood, TX). RNA (10 µg) resolved by electrophoresis on
formaldehyde-containing agarose was transferred onto GeneScreen Plus
(PerkinElmer Life Sciences) membranes by capillary blotting. The
cPLA2 cDNA probe was labeled using a nick translation
labeling kit (PerkinElmer Life Sciences) following the recommendations
of the manufacturer. The membranes were then prehybridized in Quikhyb
(Strategene, La Jolla, CA) for 15 min at 68 °C before the direct
addition of the labeled cDNA probe (1.25-1.5 × 106 cpm/ml) to the hybridization solution. After
hybridization for 1 h at 68 °C, excess probe was washed (twice)
with 1× sodium citrate (0.15 M NaCl and 15 mM
Na3C6H5O7·2H2O)
containing 0.1% SDS (SSC) at 25 °C for 1 min followed by a 15-min
wash using 0.2× SSC, 0.1% SDS at 65 °C. Signals were detected by
exposing the blot to x-ray film with intensifying screens at
Determination of AA Incorporation into BMMC
BMMC were maintained in culture without or with cytokines. After
24 or 48 h, the cells were removed from culture media and placed
in HBSS containing 1 µCi of [3H]AA/107 BMMC
for 30 min at 37 °C. Unincorporated label was removed by washing
(three times) the cells in HBSS containing 0.25 mg/ml HSA.
Glycerolipids were then extracted from the cells, and individual glycerolipid classes were isolated by normal phase HPLC (33, 34).
Briefly, lipid extracts were reconstituted in loading buffer (hexane/2-propanol/water, 4:5.4:0.3, v/v/v) and then loaded onto an
Ultrasphere Silica column (Rainin Instrument Co., Woburn, MA) that had
been conditioned with hexane/2-propanol/ethanol/50 mM phosphate buffer (pH 7.4)/acetic acid (490:367:100:30:0.6, v/v/v/v/v). After 5 min, the amount of phosphate was increased from 3 to 5% over a
5-min period. This solvent composition was then maintained until all
major phospholipid classes had been eluted from the column. In
radiolabeled assays, 1-min fractions were collected, and the amount of
radioactivity in each lipid class was determined by liquid
scintillation counting.
The neutral lipid fraction was separated into monoglycerides,
diglycerides, free fatty acid, and triglycerides by TLC on silica gel G
using hexane/diethyl ether/formic acid (90:60:6, v/v/v).
Determination of Mole Quantities of AA in Phospholipid Classes
and Subclasses
BMMC glycerolipids extracted by the method of Bligh and Dyer
were separated into classes by normal phase HPLC as described above.
[2H8]AA (100 ng) was added to a fraction of
each glycerolipid that was subsequently subjected to base hydrolysis
(34). Fatty acids were then converted to pentafluorobenzyl esters, and
the mole quantities of free fatty acids were determined by NICI-GC/MS. Carboxylate anions (m/z) were monitored at 303 and 311 for AA and [2H8]AA, respectively, in
the single ion monitoring mode, and mole quantities of AA were
determined from standard curves obtained using
[2H8]AA as an internal standard.
To determine the distribution of arachidonate into phospholipid
subclasses, PC and PE fractions from normal phase HPLC were hydrolyzed
using 10 or 25 units of grade 1 Bacillus cereus
phospholipase C (Roche Molecular Biochemicals) for 2.5 and 6 h,
respectively. Diradylglycerides obtained from the phospholipase C
hydrolysis were converted to diradylglyceride acetates using acetic
anhydride (500 µl) and pyridine (35). 1-Acyl, 1-alkyl, and
1-alk-1'-enyl subclasses were separated by TLC on silica gel G
developed in benzene/hexane/diethyl ether (50:25:4, v/v/v). Each
subclass was extracted from the silica gel, and the mole quantities of
arachidonate were determined by NICI-GC/MS as described above.
Determination of CoA-IT Activity
For cell free assays, BMMC were cultured without or with
cytokines for 24 h and then washed (three times) using HBSS
containing 0.25 mg/ml HSA. The cells were then suspended in CoA-IT
sonication buffer (50 mM HEPES buffer, pH 7.4, containing 1 mM EDTA and 20% sucrose (w/v)). Subsequently, the cells
were broken by sonication using a probe sonicator (Heat System, Inc.)
as described above for cPLA2 assays. Cytosolic and membrane
fractions were obtained after ultracentrifugation (100,000 × g, 1 h, 4 °C). The membrane fraction was diluted in
phosphate-buffered saline containing 1 mM EGTA, and 10 µg
of total protein was utilized for CoA-IT activity determination. The
reaction was initiated by the addition of
1-[3H]alkyl-2-lyso-GPC (0.1 µCi) containing 1 nmol of 1-O-hexadecyl-2-lyso-GPC in a final volume of 100 µl. After 10 min at 37 °C, the reaction was stopped, and lipids
were extracted. Phospholipids were separated by TLC on silica gel G
developed in chloroform/methanol/acetic acid/water (50:25:8:4,
v/v/v/v). The product (1-[3H]alkyl-2-acyl-GPC) was
visualized by radioscaning (Bioscan), scrapped, and then quantified by
liquid scintillation spectroscopy.
For whole cell assays, BMMC were pulse-labeled with
[3H]AA for 30 min (1 µCi/107 cells).
Unincorporated label was removed by washing BMMC (three times) using
HBSS containing 0.25 mg/ml fatty acid-free HSA. The cells were then
placed in fatty acid-enriched cell culture media (10% fetal
calf serum) in the absence or presence of IL-3 or SCF (100 ng/ml).
After different periods of time, BMMC were removed from cell culture
media, and glycerolipid classes were obtained by normal phase
HPLC as described above. The amount of radiolabel in each lipid class
was then determined by liquid scintillation counting.
Determination of Lyso-PC and Lyso-PE Levels
BMMC (106/ml) were incubated with 0.5 µCi of
[Me3H]choline or 0.5 µCi of
[1-3H]ethanolamine for the determination of lyso-PC and
lyso-PE, respectively. After 24 h in culture media, unincorporated
label was removed by washing the cells (twice) using sterile HBSS
containing 0.25 mg/ml HSA. The cells were then cultured in the absence
or presence of IL-3 or SCF (100 ng/ml) for a further 24 h.
Phospholipids were extracted from cells as described above. The
phospholipid extract was resuspended in 50 µl of chloroform
containing 10 µg each of PC and lyso-PC or PE and lyso-PE. Lyso-PC
and lyso-PE were isolated by TLC on silica gel G using
chloroform/methanol/acetic acid/water (50:25:8:4, v/v/v/v). The
radioactivity in phospholipids (PC or PE) and lysophospholipids
(lyso-PC or lyso-PE) were determined by liquid scintillation counting.
Effects of Exogenous AA on Cellular AA Metabolism
BMMC (106/ml) were labeled with [3H]AA
for 24 h to obtain isotopic labeling of the major lipid classes
(24). After removal of unincorporated label, cells were maintained in
culture in the presence of increasing concentrations of exogenous AA
(0-50 µM). After 24 h, the distribution of
radiolabel into lipid classes was determined by TLC as described above.
Statistical Analysis
Data are expressed as the means ± S.E. of separate
experiments. Statistics (p values) were obtained using
Student's t test for paired samples. Notations used on
figures and legends are an asterisk for p < 0.05.
Apoptosis of BMMC in Cytokine-depleted Media--
Our previous
studies showed that BMMC cultured in IL-3/SCF maintained low
intracellular levels of AA (36). However, the regulation of AA has not
been described when these cells are undergoing apoptosis. Initial
experiments were designed to obtain a reproducible model of mast cell
apoptosis. In these experiments, caspase activity was examined in BMMC
cultured without or with IL-3/SCF for 24 h. BMMC placed in
cytokine-depleted media had higher protease activity (14.5 ± 0.9 CPP32 units, n = 3) compared with cells maintained in
cytokines (4.8 ± 1.1 CPP32 units). This protease activity was reduced to control levels (4.4 ± 0.1 CPP32 units) by a caspase inhibitor. These initial data suggested that a marker of apoptosis (caspase) was increased when BMMC were placed in cytokine-depleted media for 24 h.
To determine the percentage of apoptotic cells, the orientation of PS
on the surface of mast cells was examined by annexin V binding. When
BMMC were placed in cytokine-depleted media for 24 h, there was an
increase in the percentage of annexin-FITC binding cells (Fig.
1A). Consistent with this
observation, maintaining BMMC in cytokine-depleted media caused a
significant increase in the percentage of apoptotic cells concomitant
with a decrease in live cells (Table I).
Further evidence of apoptosis was obtained by examining DNA
fragmentation. As shown in Fig. 1B, BMMC cultured for
24 h with IL-3 or SCF maintained intact high molecular weight DNA.
By contrast, DNA extracted from cells placed in cytokine-depleted media
displayed distinct DNA fragmentation patterns. Finally, we determined
the capacity of BMMC to synthesize new DNA. As shown in Fig.
1C, the incorporation of radiolabeled thymidine into
cellular DNA decreased (>80%) when BMMC were placed in
cytokine-depleted media for as little as 12 h. Taken together,
these data suggest that BMMC placed in cytokine-depleted media rapidly
stop proliferating and then undergo apoptosis.
Mole Quantities of Arachidonic Acid Correlate with the Percentage
of Apoptotic Cells--
As described above, mast cells placed in
cytokine-depleted media undergo apoptosis. It is known that the
location of PS on the surface of cells is a marker of apoptosis (37).
However, it has not been established whether changes in the
phospholipid microenvironment are accompanied by changes in fatty acids
within or outside mast cells. Therefore, subsequent studies examined the mole quantities of various unsaturated fatty acids within mast
cells or released into the culture media. Removal of cytokines resulted
in a significant increase in AA levels within mast cells, while levels
of other unsaturated fatty acids (LA, OA) did not change (Table
II). Subsequent studies examined free
fatty acids outside mast cells and compared these levels with the
percentage of live or dead (annexin V binding) cells. As shown in Fig.
2A, resting levels of AA
outside mast cells were directly proportional to the percentage of
apoptotic cells (Fig. 2A) and inversely proportional to the
percentage of live cells (Fig. 2B). In both cases, there was
correlation between the percentages of dead or live cells and the mole
quantities of AA in cell culture media (R2 = 0.76 and 0.92, respectively). In contrast, mole quantities of other
unsaturated fatty acids (LA, OA, EPA, and DHA) were not related to the
percentage of dead (Fig. 2, C, D, E,
and F, respectively) or living cells (data not shown). These
data suggest that the selective accumulation of AA within or outside
mast cells is closely associated with apoptosis.
To further examine the effects of unsaturated fatty acids on apoptosis,
BMMC were incubated with different concentrations (0-50
µM) of LA, OA, or AA for 24 h, and apoptosis was
examined by annexin-FITC binding. The percentage of apoptotic cells
increased dose-dependently when AA was added to BMMC for
24 h (6.1 ± 5.6 and 12.6 ± 0.4% at 25 and 50 µM AA, respectively, n = 3, p < 0.05 at 50 µM AA). Likewise, 25 and
50 µM AA increased the percentage of necrotic cells by
26.7 ± 5.7 and 46.6 ± 2.6% (n = 3, p < 0.05), respectively. In contrast, two other
unsaturated fatty acids, LA and OA, did not affect the percentage of
dead cells at similar concentrations. These data suggest that induction
of apoptosis is selective for AA, and not other unsaturated fatty acids
when BMMC are maintained in cytokine-depleted media.
Mast Cells Undergoing Apoptosis Release More AA into Supernatant
Fluid--
Potential explanations for the accumulation of AA within or
outside BMMC include an increase in AA-releasing activities
(cPLA2), a decrease in AA incorporation and remodeling
activities or a decrease in the conversion of free AA to metabolites.
Thus, we examined the stimulus-coupled AA release from BMMC that were
undergoing apoptosis. BMMC placed in cytokine-depleted media released
757.2 ± 112.3 (n = 5) pmol of AA/5 × 106 BMMC upon stimulation with antigen for 5 min. This
level of AA release was significantly higher than AA released from
cells placed in IL-3 (259.3 ± 31.7 pmol/5 × 106
BMMC) or SCF (156.8 ± 50.6 pmol/5 × 106 BMMC).
Ionophore A23187 stimulation also resulted in more AA release from
cytokine-depleted cells compared with cytokine-treated cells (data not
shown). Taken together, these data suggest that cells cultured without
cytokines have the capacity to release significantly more AA than cells
cultured with cytokines.
Increase in Lipid Peroxidation when Mast Cells Are Undergoing
Apoptosis--
Several studies suggest that peroxidation of
unsaturated fatty acids is linked to apoptosis (38-40). To determine
whether AA accumulation is accompanied by lipid peroxidation in BMMC,
we examined the accumulation of MDA, the major product of lipid
peroxidation reactions, within BMMC induced to undergo apoptosis by
cytokine depletion. When BMMC were cultured in IL-3 or SCF-supplemented culture media, levels of MDA (6.70 ± 0.71 nmol/5 × 106 BMMC, n = 6 and 6.78 ± 1 nmol/5 × 106 BMMC, n = 6, respectively) were not altered within a 24-h incubation period. In
contrast, there was a significant increase in MDA levels (15.43 ± 1.89 nmol/5 × 106 BMMC, n = 6, p < 0.05) within BMMC that were induced to undergo apoptosis by cytokine withdrawal. Taken together, these data suggest that AA accumulation within BMMC undergoing apoptosis induced by
cytokine withdrawal is accompanied by an increase in lipid peroxidation.
cPLA2 Activity and Expression Does Not Correlate with
Mole Quantities of AA--
As described above, BMMC placed in
cytokine-depleted media for 24 h released more AA upon activation.
Since stimulus-coupled release of AA is associated with
cPLA2 activation, we next examined cPLA2
expression and activity as a possible explanation for the accumulation
of AA outside mast cells. As shown in Fig.
3A (upper panel), cPLA2 mRNA decreased when BMMC were
placed in cytokine-depleted media for 24 h. Western analysis also
revealed that there was a decrease in cPLA2 protein when
mast cells were placed in cytokine-depleted media (Fig. 3B).
Likewise, cPLA2 activity was lower in cells cultured without cytokines (Fig. 3C). Taken together, these data
suggest that changes in cPLA2 levels are not related to the
accumulation of AA in cell culture media or within BMMC when these
cells are undergoing apoptosis.
Assessment of Eicosanoid Formation by BMMC Undergoing
Apoptosis--
To determine whether the accumulation of AA outside
mast cells was due to a decrease in their capacity to form products,
levels of eicosanoids (PGD2 and LTB4) were
examined by NICI-GC/MS. Stimulation by either antigen increased
PGD2, while PGD2 levels remained unchanged after ionophore A23187 stimulation of BMMC undergoing apoptosis. There
was no significant difference in LTB4 biosynthesis by BMMC grown with or without cytokines (Table
III). These data suggest that impairment
of eicosanoid biosynthesis is not responsible for the accumulation of
free AA outside BMMC that are undergoing apoptosis.
Determination of Arachidonic Acid Incorporation into
Lipids--
Our previous data indicated that BMMC rapidly incorporated
trace amounts of AA predominantly into PC and PI pools (24). Under
resting conditions, AA is slowly remodeled from these early pools to PE
(24). To determine whether this incorporation process was blocked in
cells undergoing apoptosis, cells were pulse-labeled with
[3H]AA, and the incorporation of AA into lipids was
monitored. BMMC cultured without cytokines, with IL-3 or SCF for
24 h, incorporated the same amount of radiolabel (0.414 ± 0.097, 0.519 ± 0.148, and 0.512 ± 0.159 µCi/107 BMMC, respectively, n = 5). When
the incorporation of [3H]AA into lipid classes was
examined, cells cultured without cytokines accumulated more AA in
neutral lipids (Fig. 4A) than
cells cultured with IL-3 or SCF (Fig. 4, B and C,
respectively). In contrast, [3H]AA incorporation into PE
was lower in cytokine-depleted BMMC, while there was no difference in
the incorporation of [3H]AA into PI/PS or PC fractions
when cells were maintained without or with cytokines (Fig. 4). Thus,
impairment of AA incorporation into PC and PI does not account for the
accumulation of AA when BMMC are undergoing apoptosis.
Distribution of Arachidonate into Phospholipid Classes and
Subclasses of BMMC--
BMMC grown in cytokine-enriched media
contained arachidonate in PE (41.21 ± 5.40%, n = 5) > PC (30.95 ± 1.96) > PI (18.32 ± 2.94%) > neutral lipids (NL; 7.98 ± 2.56%). The mole
quantity of arachidonate was reduced in PE concomitant with an increase in PC when BMMC were maintained in cytokine-depleted media (Fig. 5A). Changes in the distribution of AA
within PE subclasses (1-acyl-2AA-GPE and 1-alk-1-enyl-2-AA-GPE) and PC
subclasses (1-acyl-2-AA-GPC and 1-alkyl-2-AA-GPC) mirrored the changes
in their respective subclasses (Fig. 5B). Compared with BMMC
grown in cytokine-supplemented culture media (IL-3 or SCF), there was a
significant decrease in arachidonate content of PE (40.7 ± 8.1%;
n = 3) in BMMC maintained in cytokine-depleted media
(Fig. 5C). Concomitantly, there was an increase in the
arachidonate content in NL (84.2 ± 12.8%, n = 3)
and PC (50.2 ± 18.1%, n = 3). These data reveal
that the cellular distribution of AA is altered when BMMC are
undergoing apoptosis, such that PC, and not PE, becomes the predominant
arachidonate-containing lipid pool and there is ~2-fold increase in
the arachidonate mass in NL.
Determination of CoA-independent Transacylase Activity--
As
described above, the major difference between cells cultured with
cytokines and cells undergoing apoptosis is the decrease in
arachidonate content of PE concomitant with an increase in arachidonate
content of PC and NL. Additionally, free AA levels are higher in BMMC
undergoing apoptosis. AA that is initially incorporated into PC is
remodeled to PE by CoA-IT activity, and the inhibition of CoA-IT has
been shown to result in AA accumulation in several cell types (41).
Thus, a decrease in CoA-IT activity could account for the decrease in
AA content of PE and the accumulation of AA within mast cells
undergoing apoptosis. We tested this hypothesis by determining AA
remodeling in whole cells and also measuring microsomal CoA-IT
activity. BMMC pulse-labeled with [3H]AA incorporated
arachidonate in rank order PC > PI/PS > PE > NL. In
the absence of cytokines, there was an increase of radiolabel in NL
when cells are placed in culture media for 24 and 48 h. By
contrast, [3H]AA was maintained at the same level in NL
cultured with IL-3 or SCF (Fig.
6A). Compared with
pulse-labeled cells, there was a slight increase in
[3H]AA in PE in cytokine-depleted cells at 24 and 48 h. However, the percentage of [3H]AA in PE in
cytokine-depleted BMMC was significantly lower than the levels found in
BMMC cultured with IL-3 or SCF (Fig. 6B). [3H]AA levels in PI/PS and PC decreased slightly with
time in all three groups at 24 and 48 h. However, there was no
significant difference between [3H]AA in PC and PI/PS
from cytokine-depleted and IL-3- or SCF- supplemented BMMC (Fig. 6,
C and D). Interestingly, the increase in the mole
quantities of arachidonate in PC (Fig. 5C) is not observed
in the pulse experiments (Fig. 4) or in the pulse/chase experiments in
Fig. 6D, probably because the mass amounts of
[3H]AA are very small compared with the bulk of unlabeled
arachidonate in PC. Moreover, most of [3H]AA that would
have resided in PC is shunted to the neutral lipid fraction in
apoptotic BMMC when these labeling strategies are utilized. However,
the decrease of [3H]AA in PE suggests that the activity
(CoA-IT) responsible for transferring/remodeling AA to PE is reduced in
BMMC undergoing apoptosis. To further show that the decrease in
arachidonate content in PE was due to a decrease in CoA-IT activity,
microsomal fractions were prepared from cells cultured without or with
cytokines. CoA-IT activity was lower in cells placed in
cytokine-depleted media (0.13 ± 0.05 pmol/mg/min) compared with
BMMC maintained in IL-3 (1.10 ± 0.09 pmol/mg/min,
n = 8, p < 0.05) or SCF (0.71 ± 0.16 pmol/mg/min) for 24 h. A similar CoA-IT activity profile was
determined after 48 h (data not shown). These data suggest that a
decrease in CoA-IT accounts for the decrease in AA remodeling and the
accumulation of free AA when BMMC undergo apoptosis following cytokine
depletion.
To determine whether the decrease in CoA-IT activity, or the induction
of apoptosis during cytokine depletion could be accounted for by an
increase in lysophospholipid levels within BMMC, phospholipid head
groups were radiolabeled, and the accumulation of radioactivity in
lyso-PC or lyso-PE was determined. When cells were placed in cytokine-depleted media, less [3H]choline was
incorporated into lyso-PC compared with cytokine-supplemented cells
(5757 ± 1781, 6763 ± 1909, and 9245 ± 2085 dpm/5 × 106 BMMC (n = 3) for no cytokine, IL-3,
and SCF, respectively). Similarly, BMMC labeled with
[1-3H]ethanolamine incorporated less radioactivity into
lyso-PE (1160 ± 181, 1233 ± 164, and 1524 ± 215 dpm/5 × 106 BMMC (n = 3) for no
cytokine, IL-3, and SCF, respectively). However, expressed as a
percentage of total radioactivity, there was no difference in the
amount of lyso-PC (16.7 ± 0.4, 13.8 ± 2.1, and 17.0 ± 2.2% of total radioactivity (n = 3) for no cytokine,
IL-3, and SCF, respectively) or lyso-PE (7.6 ± 0.7, 5.8 ± 0.2, and 5.8 ± 0.2% of total radioactivity (n = 3) for no cytokine, IL-3, and SCF, respectively) in cells cultured in
cytokine-depleted media when compared with cells grown with IL-3 or
SCF. These data suggest that the decrease in CoA-IT observed during
apoptosis is not due to an increase in the levels of endogenous
substrate (lyso-PC or lyso-PE). In addition, these data suggest that
lysophospholipids do not contribute to apoptosis of BMMC induced by
cytokine depletion.
AA Accumulation in Neutral Lipid Classes--
Our previous studies
suggested that inflammatory cells exposed to high concentrations of AA
shuttled excess AA into neutral lipids, with triglycerides being the
predominant class (42, 43). To determine whether a similar pathway
existed in BMMC, the neutral lipid fractions (Fig. 6A) were
further resolved into individual classes. As shown in Table
IV, there was a significant increase in
[3H]AA in all neutral lipid fractions obtained from cells
cultured without cytokines. However, the bulk of [3H]AA
resided in the TG pool. These data suggest that BMMC undergoing apoptosis accumulate more AA into neutral lipid classes than do live
cells.
Roles of Exogenous AA on Cellular AA Metabolism--
To determine
whether changes in AA metabolism described above were due to the
accumulation of AA in the cell culture media, BMMC were radiolabeled to
isotopic equilibrium using [3H]AA. The cells were
maintained in culture with different concentrations of exogenous AA. As
shown in Fig. 7, the bulk of the
radiolabel in BMMC resided in phospholipid classes (94.6 ± 0.4%,
n = 3). The addition of exogenous AA resulted in a
decrease in [3H]AA in phospholipid classes.
Concomitantly, there was a dose-dependent accumulation of
radiolabel in TGs and in free fatty acids. The bulk of the TG was
cell-associated while most of the free AA was released into the cell
culture media. These data suggest that adding exogenous AA to BMMC can
induce changes in cellular AA metabolism similar to those observed
during cytokine depletion.
The present study demonstrates an accumulation of AA within cells
and in the cell culture media of mast cells undergoing apoptosis induced by cytokine depletion. Of a series of polyunsaturated fatty
acids measured, only the levels of AA directly correlated with the
percentage of dead cells. Conversely, the percentage of live cells was
inversely proportional to the mole quantities of AA found within
apoptotic mast cells or in cell culture media. These initial data
suggested that AA metabolism played a role in apoptosis. There are
several mechanisms that could potentially account for the accumulation
of AA by mast cells undergoing apoptosis (Fig.
8). These include a decrease in AA uptake
and incorporation into phospholipids (Fig. 8a), a decrease
in AA remodeling (Fig. 8b), an increase in AA release from
phospholipid pools (Fig. 8c), or a decrease in the capacity
of mast cells to convert AA to bioactive lipid products (Fig.
8d).
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C.
RESULTS
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ABSTRACT
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Fig. 1.
Cytokine depletion results in apoptosis of
BMMC. A, BMMC were maintained with IL-3 (left
panel) or SCF (middle panel) or
without cytokines (NONE, right panel)
for 24 h. Annexin V binding was determined by flow
cytometry as described under "Experimental Procedures." Quadrants
1, 2, 3, and 4 represent dead cells, cells in late apoptosis, live
cells, and cells in early apoptosis, respectively. These data are
representative of eight separate experiments. B, DNA was
extracted from BMMC that had been placed in cell culture media for
24 h with IL-3, SCF, or without cytokines (NONE). DNA
was extracted from 2 × 106 BMMC and resolved by
agarose gel electrophoresis followed by ethidium bromide staining as
described under "Experimental Procedures." These data are
representative of five separate experiments. C, BMMC were placed in
growth media without ( ) or with 100 ng/ml IL-3 (
). After
different periods of time in culture, the incorporation of
[3H]AA thymidine into 1 × 106 BMMC was
determined. These data are the mean ± S.E. of triplicate
determinations and are representative of five separate
experiments.
Cytokine depletion induces apoptosis of BMMC
Free fatty acid levels within BMMC
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Fig. 2.
Apoptosis of mast cells correlate with AA
accumulation in culture media. Mole quantities of unsaturated free
fatty acids in the culture media of BMMC cultured without or with
cytokines for different periods of time (12-48 h) were determined by
NICI-GC/MS. The percentages of dead cells or living cells were
determined for each condition by flow cytometry. A and
B show the relationship between the mole quantities of AA
and the percentage of dead and live BMMC, respectively. C,
D, E, and F show the relationship
between the percentage of dead cells and the mole quantities of free
LA, OA, EPA, and DHA, respectively. These data were collected from four
separate experiments.
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Fig. 3.
Reduction in cPLA2 expression,
protein level, and activity during BMMC apoptosis. A,
total RNA was extracted from BMMC cultured with increasing
concentrations of SCF. RNA (10 µg) was resolved by electrophoresis on
formaldehyde-containing agarose and then transferred onto GeneScreen
Plus membranes by capillary blotting. GeneScreen Plus membranes were
probed using a labeled cPLA2 cDNA probe
(upper panel) or glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) (lower panel) as
described under "Experimental Procedures." Signals were detected by
exposing the blot to x-ray film with intensifying screens at
80 °C. These blots are representative of five separate
experiments. B, BMMC were placed in culture media with
increasing concentrations of SCF for 24 h. SDS-PAGE was
performed using a 4-20% gel, and proteins were blotted onto
polyvinylidene difluoride membranes. Immunodetection of
cPLA2 was accomplished using anti-cPLA2
monoclonal antibody and peroxidase-conjugated anti-rabbit IgG,
followed by enhanced chemiluminescence. These data are representative
of five separate experiments. C, BMMC were cultured with
increasing concentrations of SCF for 24 h. cPLA2
activity was determined using 50 µg of homogenate as described under
"Experimental Procedures." GAPDH,
glyceraldehyde-3-phosphate dehydrogenase. These data are the mean ± S.E. of four separate experiments performed in duplicate. *,
p < 0.05.
Formation of eicosanoids by BMMC undergoing apoptosis
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Fig. 4.
Influence of cytokine depletion on
[3H]AA incorporation into BMMC lipids. BMMC were
maintained in culture without or with cytokines (IL-3 or SCF) for
24 h. BMMC were subsequently pulse-labeled with
[3H]AA (0.1 µCi/1 × 106, 37 °C for
30 min). The cells were then washed (three times) with HBSS containing
0.25 mg/ml fatty acid-free HSA. Lipids were extracted, and individual
classes (NL, unknown fraction (UK), PE, PI/PS, and PC) were
isolated by normal phase HPLC as described under "Experimental
Procedures." Radioactivity in lipid classes was determined by liquid
scintillation counting. These data are representative of three separate
experiments.
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Fig. 5.
Mass distribution of arachidonate into lipid
classes and subclasses after cytokine depletion. A,
BMMC were placed in culture media without cytokines for different
periods of time. Lipids were extracted, and individual classes were
isolated by normal phase HPLC. The mole quantity of arachidonate in NL
( ), PE (
), PI/PS (
), and PC (
) was determined after base
hydrolysis by NICI-GC/MS (A). Data are shown as the
percentage of total cellular arachidonate that is found in each lipid
class and are the mean ± S.E. of five separate experiments.
B, PE and PC subclasses were isolated by TLC as described
under "Experimental Procedures." Mole quantities of arachidonate in
PE subclasses (1-acyl-2-AA-GPE (
) and 1-alk-1-enyl-2-AA-GPE (
))
and PC subclasses (1-acyl-2-AA-GPC (
) and 1-alkyl-2-AA-GPC (
))
were determined as described under "Experimental Procedures." These
data are shown as the percentage of arachidonate in each subclass and
are the mean ± S.E. of three separate experiments
(p < 0.05). C, lipids were extracted
from BMMC before (0 h) or after the cells had been cultured for 48 h without or with cytokines (IL-3 or SCF). Lipid classes were isolated
by normal phase HPLC. The mole quantity of arachidonate in NL, PE,
PI/PS, or PC of BMMC was determined at time 0 (
), after 48 h of
cytokine depletion (
), and after 48 h in IL-3 (
) or in SCF
(
) by NICI-GC/MS. Data are shown as the percentage of total cellular
arachidonate that is found in each lipid class and represent the
mean ± S.E. of five separate experiments.
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Fig. 6.
In situ CoA-independent
transacylase activity. BMMC were pulse-labeled with
[3H]AA, and unincorporated label was removed by washing
cells (three times) using HBSS containing 0.25 mg/ml fatty acid-free
HSA. The cells were then placed in fatty acid-enriched cell culture
media (10% fetal bovine serum) without cytokines ( ), with IL-3
(
), or with SCF(
). After 24 or 48 h in culture, the
percentage of radioactivity in NL (A), PE (B),
PI/PS (C), and PC (D) was determined as described
under "Experimental Procedures." These data are the mean ± S.E. of four separate experiments. *, p < 0.05.
Distribution of AA in neutral lipid classes
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Fig. 7.
Effects of exogenous AA on endogenous AA
metabolism. BMMC were labeled with [3H]AA to
isotopic equilibrium. After removal of unincorporated label, the cells
were then placed in whole media containing IL-3 and supplemented with
different concentrations of exogenous AA for 24 h. Levels of
cellular [3H]arachidonate were determined in
phospholipids ( ), free fatty acids (
), and triglycerides (
).
These data are the mean ± S.E. of three separate experiments. *,
p < 0.05.
DISCUSSION
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Fig. 8.
Proposed mechanism accounting for AA
accumulation in mast cells undergoing apoptosis. Processes that
increase cellular AA levels within mast cells include a decrease in the
uptake and incorporation of cellular AA into phospholipids
(a), a decrease in CoA-IT activity resulting in a decrease
in AA remodeling (b), an increase in PLA2
activity that results in the rapid hydrolysis of AA from cellular
phospholipids (c), or the inhibition of
cyclooxygenase (COX) and/or 5-lipoxygenase (5-LO)
(d). Hydrolysis of PC during BMMC activation may also lead
to AA accumulation (c, dashed line).
When AA levels are increased, the cells respond by incorporating free
AA into neutral lipids via de novo TG synthesis
(e). Cellular AA may also be autoxidized to form products,
which may induce apoptosis of mast cells (f).
Previous studies from our laboratory and others have indicated that
different isotypes of PLA2 are involved in the release of
AA from mast cells (8, 44-46). While secretory PLA2s are known to be nonspecific in hydrolyzing fatty acids in cell-free systems, it appears that secretory PLA2 also recruits
cPLA2 that accounts for the selective mobilization of AA
during cell activation (32). The importance of cPLA2 in AA
mobilization from mast cells has recently been confirmed using targeted
disruption of the cPLA2 gene in mice (46). In these
studies, neither immediate nor delayed prostanoid formation was
observed in mast cells obtained from cPLA2 knockout mice.
In another set of studies, cells that overexpressed a mutated
cPLA2 (Ser505 Ala505) exhibited
greatly diminished capacity to release AA compared with cells
overexpressing wild type cPLA2 (47). Since the activation of cPLA2 is important in AA release, its activation
represents a possible mechanism by which high levels of AA would be
found within or in the supernatant fluid around mast cells. However, our data suggest that, while there is an increase in AA levels within
mast cells that are undergoing apoptosis, there is a decrease in
cPLA2 mRNA, protein and activity. Therefore,
cPLA2 is not responsible for the accumulation of AA when
mast cells are undergoing apoptosis. Other studies have shown that
cPLA2 is decreased in cells undergoing apoptosis induced by
Fas activation or by contact inhibition (48-51). The decrease in
cPLA2 was shown to be due to a caspase-mediated breakdown
of the enzyme (48, 52). It has been implied from these studies that the
decrease in cPLA2 represents a novel mechanism by which
inflammatory processes are regulated when cells are undergoing apoptosis. The present study suggests that even when cPLA2
levels are decreased in cells, it may not be strictly correct to assume that inflammatory capacity is attenuated, since other enzymes regulate
AA levels. Our study also reveals that cPLA2 levels within mast cells are regulated by cytokines. In the presence of optimum levels of SCF or IL-3, BMMC proliferate in culture. Under these conditions, cPLA2 expression is maintained at high levels.
During cytokine depletion, there is a decrease in cPLA2
expression. These changes support the observation of Anderson et
al. (51) suggesting that cPLA2 is linked to cell proliferation.
Recent studies also suggest that inhibitors of cyclooxygenase or 5-lipoxygenase increase free AA levels within cells (41, 53). These inhibitors also induce apoptosis of cancer cells (53-61). Thus, changes in cyclooxygenase activity and AA levels are closely linked to apoptosis. In the present study, BMMC undergoing apoptosis produced more prostanoids than control cells. LTB4 levels remained the same, regardless of the apoptotic status of the cells, demonstrating that decreased capacity to form eicosanoids does not account for the accumulation of AA in mast cells undergoing apoptosis induced by cytokine withdrawal.
When AA is provided to mammalian cells, it is rapidly incorporated into various glycerolipid pools through both CoA-dependent and CoA-independent acylation reactions (17, 62, 63). Initially, AA is converted to AA-CoA by arachidonoyl-CoA synthetase. AA-CoA is then transferred to 1-acyl-2-lyso-sn-glycero-3-phospholipids by CoA-dependent acyltransferase. Various studies have shown that when this incorporation process is inhibited with Triacsin C, there is accumulation of AA products within cells and in the culture media (64, 65). Importantly, inhibition of this process has been associated with apoptosis of some cells (66). The present study suggests that this initial incorporation of AA into mast cells is not altered when these cells are undergoing apoptosis. Thus, a decrease in AA incorporation does not account for the increase in AA levels within mast cells or in culture media.
In mast cells, AA that is initially incorporated into 1-acyl-linked glycerophospholipids is slowly remodeled from these initial pools into 1-ether-linked glycerophospholipid pools. Our studies suggest that the remodeling of arachidonate from 1-acyl-linked to 1-ether-linked phospholipids is orchestrated by CoA-IT (7, 24). We recently demonstrated that treating cancer cell lines with CoA-IT inhibitors led to accumulation of intracellular AA (41, 67-69). Importantly, this inhibition of CoA-IT also resulted in the inhibition of cell cycle progression within breast cancer cells. The present study underscores the role of CoA-IT in AA metabolism. When mast cells were placed in cytokine-depleted media, the mole quantity of AA in PE was significantly reduced, concomitant with a decrease in CoA-IT activity in microsomal pellets. Therefore, a decrease in CoA-IT is responsible for the accumulation of AA in mast cells undergoing apoptosis. Since a decrease in CoA-IT activity results in arachidonate accumulation in PC, hydrolysis of PC by PLA2 could also potentially account for the increase in free AA levels (Fig. 8c). Because no changes were observed in lysophospholipids levels during apoptosis, and since specific activity measurements suggest that most free AA is generated from PE (7), it is likely that hydrolysis of PC plays only a minor role in free AA accumulation. However, during BMMC activation, when there is an increase in lysophospholipid levels and release of AA from all major phospholipid pools, hydrolysis of PC by PLA2 may contribute to free AA release (7, 63, 70).
Various pathways have been described for the incorporation of AA into glycerophospholipids, depending upon the concentration of AA to which cells are exposed (71). Exposure of cells to low AA concentrations results in AA incorporation into primarily 1-acyl-linked phospholipid molecular species through a high affinity, low capacity enzymatic pathway. In contrast, exposure of cells to high levels of AA will result in the incorporation of AA into neutral lipid fractions (mainly TG) and the formation of 1,2-diarachidonoyl-sn-glycero-3-phosphocholine by a low affinity, high capacity pathway (42, 71). Our data show that there is an increase in AA mass in the neutral lipid fractions of mast cells undergoing apoptosis. In addition, the AA mass in 1-acyl-linked PC species also increased in mast cells undergoing apoptosis, such that this becomes the single largest phospholipid subclass instead of 1-alk-1-enyl-2-AA-GPE. These data suggest that there is induction of the low affinity, high capacity pathway of AA metabolism in mast cells undergoing apoptosis. The addition of exogenous AA increased cellular [3H]AA incorporation into TG in a dose-dependent manner. Exogenous AA also induced free [3H]AA accumulation in the cell culture media. However, much higher levels of exogenous AA were needed to induce these changes, because exogenous AA is bound to serum albumin and is distributed over a larger volume than cell-associated AA. Overall, the increase of AA in neutral lipid fractions via de novo synthesis (Fig. 8e) and in PC subclasses (Fig. 8a) suggests that BMMC undergoing apoptosis have been exposed to high concentrations of free AA.
In addition to the accumulation of AA in some pools of lipids, another consequence of the build up of high levels of AA within and outside mast cells is the formation of oxidized products. High levels of AA may induce the formation of proapoptotic molecules such as ceramides, while fatty acids may affect various apoptotic proteins (53, 69, 72, 73). An important role for lipid peroxidation in apoptosis is further shown by our data showing a significant increase in lipid peroxidation only in mast cells that are undergoing apoptosis (Fig. 8f). Since lipid peroxidation has been linked to apoptosis (38), our present study suggests that mast cells may be undergoing apoptosis as a result of the formation of reactive lipid peroxides.
Overall, this study implies that perturbation of cellular arachidonate
metabolism is a critical process in BMMC that are undergoing apoptosis.
Importantly, AA accumulation is a predictive indicator of apoptosis in
BMMC after cytokine removal. The major enzyme activities that change
when mast cells are undergoing apoptosis are cPLA2 and
CoA-IT. It is likely that there is a link between these two activities,
since they exhibit similar biological properties. First, our data
suggest that a decrease in cPLA2 is accompanied by a
decrease in CoA-IT activity during apoptosis of mast cells. Second,
both cPLA2 and CoA-IT are selective for arachidonate (19, 74). Third, inhibitors of cPLA2 induce apoptosis of cells,
as do inhibitors of CoA-IT (51, 68). Fourth, inhibitors of CoA-IT prevent AA release and eicosanoid biosynthesis, as do inhibitors of
cPLA2 (75). Fifth, cytokine treatment of BMMC (present
study) and neutrophils results in an increase in both CoA-IT and
cPLA2 activity (76). Sixth, both activities are heat labile
and are not influenced by sulfhydryl reducing agents (77). Finally, Reynolds et al. (78) reported that cPLA2 has
weak transacylase activity. However, even with these similarities,
there are some striking differences between CoA-IT and
cPLA2. Whereas CoA-IT does not require calcium for
activation and is mainly membrane-bound, the major cPLA2
isotypes require calcium for activation and reside mainly in the
cytosol. However, cPLA2 translocates to perinuclear membranes during cell activation, and a recently cloned
cPLA2 isotype (cPLA2) is reported to be
calcium-independent and to be mainly membrane-bound (79). Since CoA-IT
has not been cloned, further studies will be required to show whether
these two activities cooperate within cells in regulating AA
metabolism. In addition to the possible link between cPLA2
and CoA-IT, the present studies also raise important implications
concerning the role of AA in apoptosis. Further investigations will be
required to determine the signal transduction processes that link AA
accumulation, lipid peroxidation, and changes in arachidonate pools to
apoptosis of BMMC.
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ACKNOWLEDGEMENTS |
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We are grateful for expert technical assistance from Chad Marion, Brooke Barham, and Michelle Edens. We thank Drs. A. Trimboli and F. Chilton for helpful suggestions.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant AI 24985 SI (to F. A. N.).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.
To whom all correspondence should be addressed: Dept. of Internal
Medicine, Section on Pulmonary and Critical Care Medicine, Wake Forest
University School of Medicine, Medical Center Blvd., Winston-Salem, NC
27157-1054. Tel.: 336-716-9923; Fax: 336-716-7277; E-mail:
afonteh@wfubmc.edu.
§ Present address: US EPA, 86 TW Alexander Dr., MD 82, Research Triangle Park, NC 27111.
Published, JBC Papers in Press, October 5, 2000, DOI 10.1074/jbc.M006551200
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ABBREVIATIONS |
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The abbreviations used are: AA, arachidonic acid; PLA2, phospholipase A2; cPLA2, cytosolic phospholipase A2; LTB4 and LTC4, leukotriene B4 and C4, respectively; PGD2, prostaglandin D2; HSA, human serum albumin; NICI-GC/MS, negative ion chemical ionization gas chromatography/mass spectrometry; GPC, glycero-3-phosphocholine; GPE, glycero-3-phosphoethanolamine; [2H8]AA, octadeuterated AA; [2H3]AA, tritradeuterated AA; LA, linoleic acid; OA, oleic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; TG, triglyceride; BMMC, murine bone marrow-derived mast cell(s); PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; HPLC, high pressure liquid chromatography; SCF, stem cell factor; IL, interleukin; FITC, fluorescein isothiocyanate; MDA, malondialdehyde; NL, neutral lipids; CoA-IT, CoA-independent transacylase; TXB2, thromboxane B2.
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