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in the Induction of Apoptosis of Human Macrophages Infected with Mycobacterium tuberculosis H37Ra1
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Department of Medicine, Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital and Harvard Medical School, Boston MA 02115; and
Partners Asthma Center, Brigham and Women’s Hospital, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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) apoptosis, an important innate microbial
defense mechanism induced by Mycobacterium tuberculosis
(Mtb) H37Ra, depends on the induction of TNF-
synthesis. When protein synthesis is blocked, both infection with
Mtb and addition of TNF- are required to induce
caspase 9 activation, caspase 3 activation and apoptosis. In this
study, we show that the second protein synthesis-independent signal
involves activation of group IV cytosolic phospholipase A2
(cPLA2). Apoptosis of Mtb-infected M and
concomitant arachidonic acid release are abrogated by group IV
cPLA2 inhibitors (methyl arachidonyl fluorophosphate and
methyl trifluoromethyl ketone), but not by inhibitors of group VI
Ca2+-independent (iPLA2 ; bromoenol
lactone) or of secretory low molecular mass PLA2. In
M homogenates, the predominant PLA2 activity showed the
same inhibitor sensitivity pattern and preferred arachidonic acid over
palmitic acid in substrates, also indicating the presence of one or
more group IV cPLA2 enzymes. In concordance with these
findings, M lysates contained transcripts and protein for group IV
cPLA2- and cPLA2-
. Importantly, group IV
cPLA2 inhibitors significantly reduced M
antimycobacterial activity and addition of arachidonic acid, the major
product of group IV cPLA2, to infected M treated with
cPLA2 inhibitors completely restored the antimycobacterial
activity. Importantly, addition of arachidonic acid alone to infected
M significantly reduced the mycobacterial burden. These findings
indicate that Mtb induces M apoptosis by independent
signaling through at least two pathways, TNF- and cPLA2,
which are both also critical for antimycobacterial defense of the M
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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)3 infected with
mycobacteria undergo apoptosis or programmed cell death, which is
considered an important innate defense mechanism that prevents spread
of infection by sequestering pathogens within apoptotic bodies and
protecting the surrounding tissue from their harmful effects
(1, 2, 3, 4). Abs to TNF-, a proinflammatory cytokine of
central importance in protective antimicrobacterial immunity (5, 6), abrogated M apoptosis induced by Mycobacterium
tuberculosis (Mtb) (7), indicating that
apoptosis is dependent on the action of TNF- (8). In
contrast, TNF- does not induce apoptosis in the absence of
mycobacterial infection (7), strongly suggesting that
additional signals are required for initiation of M apoptosis
by Mtb.
Induction of M apoptosis by Mtb involves transmission of
signals by cell surface receptors belonging to the TNF receptor gene
superfamily (9) including TNFR1 (10) which
triggers apoptosis on interaction with TNF- trimers leading to
recruitment and activation of caspases, cysteine proteases that cleave
after aspartic acid. Caspase 3, the executing caspase essential for
apoptosis (11), is activated by upstream caspases
including caspase 9 (12). Caspase 9 activation ensues from
complex formation of pro-caspase 9 with the adapter protein
Apaf-1 and cytochrome c following release of
cytochrome c from the mitochondrial compartment
(13).
Phospholipases A2 (PLA2),
lipolytic enzymes that release fatty acids from the sn-2
position of glycerophospholipids (14), are involved in the
induction of apoptosis of a number of cell lines (15, 16, 17, 18).
In this study. we therefore thought to characterize the second signal
that cofunctions with TNF- in Mtb-induced apoptosis of
host M concentrating specifically on PLA2.
The mammalian PLA2 enzymes fall broadly into four
groups. The low molecular mass enzymes belonging to the groups I, II,
III, V, and X are cysteine-rich, secreted proteins that require
millimolar concentrations of calcium for activity and have no
distinguishing preference for a fatty acid in the sn-2
position of the phospholipid substrate (14). The second
class of PLA2 enzymes includes specific acetyl
hydrolases of platelet-activating factor. Two forms of group VI
Ca2+-independent PLA2
(iPLA2) were described from the myocardium
(19) and from Chinese hamster ovary cells and M
(20, 21). Three group IV cytosolic phospholipase
A2 (cPLA2) are known. The
85-kDa cytosolic group IV cPLA2- requires
micromolar Ca2+ and shows preference for
arachidonic acid (22). cPLA2-
contains an amino-terminal calcium-dependent lipid-binding C-2 domain
that mediates Ca2+-dependent translocation to the
nuclear envelope. The recently described
cPLA2-
has a
Mr 110,000 and shares 30% identity
with cPLA2-, including a functional C-2 domain
(23, 24). The recently cloned
Ca2+-independent cPLA2-
lacks a C-2 domain, has a Mr 61,000,
29% sequence identity with cPLA2-, and a
preference for arachidonic acid at the sn-2 position of
phosphatidylcholine as compared with palmitic acid, in distinction to
the group VI iPLA2 (24, 25).
Several lines of evidence indicate also that antimycobacterial activity
is enhanced in M undergoing apoptosis. In tuberculosis patients in
vivo, apoptosis was commonly observed in granulomas infected with
Mtb (3, 7), suggesting an antimycobacterial
defense function for apoptosis. Apoptosis of bacillus
Calmette-Guérin-infected M induced by ATP, but not
necrosis, resulted in a significant reduction of viable intracellular
bacteria (26). Also, addition of uninfected fresh M to
apoptotic M infected with Mycobacterium avium reduced
bacterial numbers greatly, whereas no such effect was seen when
uninfected M were added to necrotic infected M (4).
These studies indicate that significantly increased antimycobacterial
activity of the M is closely associated with apoptosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant human TNF- and Abs against human TNF- were
obtained from R&D Systems (Minneapolis, MN). Bromoenol lactone (BEL),
an inhibitor of group VI iPLA2 with an
IC50 of 60 nM; arachidonyl trifluoromethyl ketone
(AACOCF3) and methyl arachidonyl fluorophosphate
(MAFP), both inhibitors of group IV cPLA2 and
group VI iPLA2 with an IC50
of 0.5 µM; and 12-episcalaradial, an inhibitor of low molecular mass
secretory PLA2 (sPLA2) with
an IC50 of 5.4 µM were obtained from Biomol
(Plymouth Meeting, PA). SB203347, an inhibitor of group IIA and group V
sPLA2 with an IC50 of 0.5
µM against group IIA sPLA2 was a gift from L.
Marshall (SmithKline Beecham, King of Prussia, PA).
Z-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl ketone (Z-DEVD-FMK),
a specific inhibitor of caspase-3, was purchased from Enzyme System
Products (Livermore, CA). Arachidonic acid was purchased from Sigma
(St. Louis, MO). Rabbit Ab to cPLA2- (batch
44282) was a gift from L.-L. Lin (Genetics Institute, Cambridge, MA). A
rabbit Ab against cPLA2- (batch K037, 200
µg/ml) and murine mAb against poly(ADP-ribose) polymerase (PARP) and
against actin were obtained from Santa Cruz Biotechnology (Santa
Cruz, CA).
Bacteria
The attenuated Mtb strain H37Ra (American Type
Culture Collection, Manassas, VA) was grown in 7H9 broth (Difco,
Detroit, MI) containing 10% BSA-glucose-catalase supplement (BD
Biosciences, Mountain View, CA) and 0.05% Tween 80 (Difco) and
resuspended in 7H9 broth at a concentration of 5 x
107 CFU/ml. In all experiments, M were
infected with five bacteria per cell for 4 h.
Cells and culture
Peripheral blood was obtained from healthy donors after
obtaining informed consent, and mononuclear cells were isolated as
previously described (4). M used in the in situ TUNEL
studies were cultured on 13-mm Thermanox plastic coverslips (Nunc,
Naperville, IL) and were plated at a concentration of 1.0 x
106 mononuclear cells/ml/well in 24-well cluster
plates (Costar, Cambridge, MA). The M used for Western blot analysis
were cultured on Falcon 100-mm diameter tissue culture dishes (BD
Labware, Franklin Lakes, NJ) at a concentration of 2 x
107 mononuclear cells/10 ml/dish. The resulting
M population (1.0 x 105 cells/coverslip
and 2.0 x 106 cells/dish) were 97- 99%
pure as determined by nonspecific esterase staining. Cells were
cultured in IMDM with 10% pooled human serum for another 7 days to
allow for M differentiation before infection with Mtb. In
the experiments using cycloheximide (CHX; 0.1 µM), CHX was present
during the 30-min preincubation, infection and subsequent culture for
24 h.
Quantitation of mycobacteria
Counting of mycobacteria using the Bactec model 460TB system (BD Biosciences) was performed as previously described (4).
In situ apoptosis analysis
M apoptosis was determined using the in situ TUNEL technique
of DNA strand breaks (27) as previously described
(4). M adherent to plastic coverslips were infected
with five H37Ra per cell in the presence or absence of CHX and
phospholipase inhibitors. In experiments in which the slow-acting
PLA2 inhibitor AACOCF3
(21) was used, the agent was added 3 h before
addition of the bacilli. For experiments using the
PLA2 inhibitors 12-episcalaradial, SB203347, BEL,
and MAFP, the agents were added 15 min before infection. In experiments
with Z-DEVD-FMK, M cultures were preincubated with 20 µM
Z-DEVD-FMK for 4 h before the addition of the mycobacterial
inoculum. The cells were then washed and Z-DEVD-FMK was reconstituted
to 20 µM. After 3 days, the cells were harvested and further
processed. In experiments using CHX-treated M, apoptosis was
measured 24 h after infection with Mtb because M are
highly sensitized to death signals when treated with CHX
(28).
Measurement of [3H]arachidonic acid release
Cells were cultured in 24-well plates as monolayers with 2
x 105 cells/well in triplicate. Cells were
labeled with 0.3 µCi/ml
[5,6,8,9,11,12,14,15-3H]arachidonic acid (NEN,
Boston, MA) and incubated at 37°C for 18 h. Unincorporated
[3H]arachidonic acid was removed by washing
three times with HBSS. Cells were then incubated in 1 ml/well fresh
medium for 3 h before stimulation. In experiments using
AACOCF3, the agent was added 3 h before
addition of H37Ra or the addition of TNF-. BEL and MAFP were added
15 min before infection. In experiments with Z-DEVD-FMK, M cultures
were preincubated with 20 µM Z-DEVD-FMK for 4 h before addition
of the mycobacteria. The Z-DEVD-FMK concentration was reconstituted
after washing to 20 µM for the remainder of the experiment.
Supernatants were collected at different times and the cells were
dissolved in 1 ml 0.2% SDS-HBSS. A total of 0.4 ml of each supernatant
and of each lysate was mixed with 3.6 ml of scintillation fluid, and
the radioactivity was evaluated by liquid scintillation counting.
Western blotting
Cells were lysed on ice in lysis buffer (250 mM NaCl, 50 mM
HEPES, 0.1% Nonidet P-40, 50 mM NaF, 5 mM EDTA, 100 mM sodium
orthovanadate, 1 mg/ml leupeptin, 1 mg/ml aprotinin, 1 M DTT, 50 mM
PMSF, and 5 µl/ml diisopropylphosphofluoridate) at 4 x
106 cells/ml and then centrifuged at 15,000
x g for 10 min to remove the nuclei. Fifty microliters of
cell lysates were heated in 2x sample buffer at 95°C for 5 min and
resolved in 12% SDS-polyacrylamide gels, transferred to polyvinylidene
difluoride membranes (Immobilon-P; Millipore, Bedford, MA), and blocked
for 2 h with 10 mM Tris buffer (pH 7.5) containing 150 mM NaCl,
0.05% Tween 20, and 5% dry milk. The membranes were incubated with
murine Ab against caspase 3 (C31720, 1 µg/ml; Transduction
Laboratories, Lexington, KY), rabbit anti-human
cPLA2- (0.1 µl of immune serum/ml), rabbit
anti-human cPLA2- (1 µg/ml), or mouse
anti-human PARP (1 µg/ml). Isotype-matched irrelevant Abs were
used as controls. Membranes were then washed, and blotted with
species-specific goat anti-murine or goat anti-rabbit
biotinylated secondary Abs (0.1 µg/ml; Vector Laboratories,
Burlingame, CA). After extensive washing with 10 mM Tris-HCl buffer (pH
8.0) containing 150 mM NaCl and 0.05% Tween 20, the membranes were
developed in chemiluminescence reagent (NEN) and exposed to x-ray
film.
RT-PCR
M mRNA was extracted in 4 M guanidinium isothiocyanate
(Fisher Scientific, Pittsburgh, PA) and 0.1 M 2-ME (Sigma)
(29), purified by CsCl gradient ultracentrifugation,
washed, and quantified by absorbance at 260 nm. RNA (2 µg in 50 µl)
was reverse transcribed into cDNA at 42°C for 60 min using Moloney
murine leukemia virus reverse transcriptase (Promega, Madison,
WI) and random hexamer primers (Roche Diagnostics, Indianapolis, IN).
Two microliters of the reaction mixture was amplified via PCR using
Taq DNA polymerase (Life Technologies, Grand Island, NY).
The PCR was conducted in 25 µl reaction mixture consisting of
reaction buffer, 1.5 µM MgCl2, 0.5 µM dNTP, 1
µM of each of the primers, and 1 U Taq DNA polymerase
using a step program for cPLA2- (94°C, 1
min; 56°C, 1 min; 72°C, 2 min), and for
iPLA2, cPLA2-, and
cPLA2- (94°C, 1 min; 58°C, 1 min; 72°C,
2 min) for 35 cycles followed by a 7-min final extension at 72°C. The
PCR products were subjected to electrophoresis in a 1.5% agarose gel
and stained with ethidium bromide. The primer pairs were composed of
the following sequences: cPLA2-, 5'-ATC TCT
ACA ACC CCT GAC AG-3', 5'-ACA CCA GAG AAT CCC ACC AT-3' which amplified
to a 436-bp product; cPLA2-, 5'-CTG TTG GAT
GCC GTC ACG TA-3', 5'-GGA AAG TCA GGT CTC TCT CAG-3' which amplified a
453-bp product; iPLA2, 5'-CTG GTG AAC TTC CAG CAG
TT-3', 5'-TGA GGC GTT CTT TCC TAG GA-3' which amplified a 351-bp
product; and cPLA2-, 5'-GCA GCT CAA GAA TGT
CAT GGA-3', 5'-ACA AGC CTC ACC ACT TGA CCA-3' which amplified a 439-bp
product. The GAPDH control amplimer set was obtained from Clontech
Laboratories (Palo Alto, CA) (5'-TGA AGG TCG GAG TCA ACG GAT TTG GT-3',
5'-CAT GTG GGC CAT GAG GTC CAC CAC-3' which amplified a 983-bp
product).
Characterization of PLA2 activity
cPLA2 activity was assessed in lysates of
M by the hydrolysis of
1-palmitoyl-2-[14C]arachidonyl-phosphatidylcholine
to liberate [14C]arachidonic acid using a
liposome-based assay (30). Adherent M were dislodged
using a rubber policeman, resuspended in RPMI 1640 containing 10% male
human serum at 107 cells/ml, and lysed by three
cycles of freeze thawing in the absence of serum in lysis buffer
consisting of 0.05 M phosphate buffer (pH 7.2) with 0.15 M NaCl, 1
µg/ml leupeptin and 1 mM DTT. To assess iPLA2
activity, 25–50-µl samples of lysate or medium alone were adjusted
to a final volume of 125 µl containing 5 mM EDTA, 1 mM DTT, 10 mM
Tris-HCl (pH 7.4), and 3.6 µM
1-palmitoyl-2-[14C]arachidonyl-phosphatidylcholine
for 1 h at 37°C. Ca2+-dependent
PLA2 activity was assessed in the presence of 4
mM CaCl2 in the absence of EDTA. The reaction was
stopped by the addition of 625 µl of Dole’s reagent. Free
[14C]arachidonic acid was extracted in
n-heptane and counted in a liquid scintillation counter
(17). In selected experiments, 1-palmitoyl-2-
[14C]palmitoyl-phosphatidylcholine was used as
a substrate. To examine the sensitivity of the
PLA2 activity to inhibitors, BEL,
12-episcalaradial, or MAFP were dissolved in DMSO and diluted so that
all dilutions of inhibitor contained 10% DMSO (v/v) in 10 mM Tris-HCl,
pH 7.4. One microliter of inhibitor or 10% DMSO (v/v) in 10 mM
Tris-HCl (pH 7.4) was added to the reaction mixture (100 µl) and
incubated for 10 min at 37°C before the addition of substrate (25
µl).
Statistical analysis
Results are expressed as mean ± SD. Data were analyzed using ANOVA and analysis of covariance, with time as the covariant. Analyses were performed using the SAS general linear models procedure (SAS Proprietary Software Release 6.12; SAS Institute, Cary, NC). Treatments, concentrations of substances used, and time were treated as fixed effects. In ANOVA models, all pairwise or nonorthogonal a priori contrasts were used to test comparisons among treatments or among levels. In analysis of covariance models, submodels were used to test comparisons among treatments. In both cases significance tests for these comparisons were controlled for multiple comparisons.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It was found before that inoculation of M with Mtb
induces apoptosis which requires TNF- synthesis (7). To
determine whether additional signals are required for
Mtb-induced apoptosis, M were preincubated for 30 min
with the protein synthesis inhibitor CHX before infection with
Mtb. Inhibition of protein synthesis of the M blocked
Mtb-induced apoptosis, but addition of as little
as 5 ng/ml TNF- restored apoptosis (Fig. 1
). On the other hand, addition of
TNF- to uninfected M in the absence or presence of CHX increased
apoptosis only minimally (Ref. 7 ; Fig. 1). These
experiments indicate that in addition to the protein
synthesis-dependent events involving TNF-, a protein
synthesis-independent signal is also required for
Mtb-dependent M apoptosis.
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apoptosis. Apoptosis of
TNF-/CHX-treated Mtb-infected M cultures was decreased
from 35.2 ± 1.4 to 7.4 ± 0.5% in the presence of
Z-DEVD-FMK (p = 0.0001, n = 3),
indicating that Mtb-induced apoptosis of M is caspase 3
dependent.
It is known that the upstream enzyme caspase 9 is activated by the
increase of mitochondrial membrane permeability and subsequent release
of cytochrome c into the cytoplasma, which leads to
activation of caspase 3 (11). We therefore examined
activation of caspase 9 and caspase 3 in Mtb-infected M.
In the presence of CHX, both Mtb infection (12 h) and the
action of exogenous TNF- were required to induce activation of
caspase 9 and caspase 3 (Fig. 2) as
assessed by Western blot analysis. Caspase 3 degrades proteins
necessary for cellular repair including PARP (31, 32).
Although infection with Mtb alone (12 h) or addition of
exogenous TNF- alone to CHX-treated Mtb-infected M
increased the concentration of the major cleavage product of PARP which
migrates on SDS-PAGE at 85 kDa, both Mtb infection and
exogenous TNF- were required for significant degradation of PARP
(116 kDa; Fig. 2, lane 4 of PARP panel). These experiments
demonstrate that caspase 9 and caspase 3 are activated in
Mtb-inoculated M and that this caspase activation, like
apoptosis, requires two independent signals.
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apoptosis
cPLA2 has been implicated in the induction
of apoptosis of cell lines in response to various stimuli
(15, 16, 17, 18). To investigate the possible role of
PLA2 in the induction of Mtb-dependent
M apoptosis, M were incubated with inhibitors of different
PLA2 species in a concentration-dependent manner.
PLA2 inhibitors included 12-episcalaradial, an
inhibitor of sPLA2, SB203347, an inhibitor of
both group IIA and group V sPLA2,
AACOCF3, and MAFP, both inhibitors of group IV
cPLA2 and group VI iPLA2,
and BEL, a specific inhibitor of group VI iPLA2.
Apoptosis of Mtb-infected M was significantly inhibited
by AACOCF3 and MAFP in a dose-dependent manner,
but not by the specific group VI iPLA2 inhibitor
BEL (Fig. 3). SB203347 and
12-episcalaradial, inhibitors of low molecular mass
sPLA2 enzymes, had no effect on
Mtb-induced M apoptosis, which excludes a role of
sPLA2 (33). This finding is
consistent with a requirement for group IV cPLA2
in the induction of M apoptosis. Addition of increasing amounts of
arachidonic acid, the predominant product of
cPLA2 (34), to
AACOCF3-treated M restored
Mtb-induced M apoptosis in a dose-dependent manner (Fig. 3). Arachidonic acid alone did not induce apoptosis of uninfected M
(data not shown).
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To provide further evidence of the involvement of group IV
cPLA2 in Mtb-induced M apoptosis,
we measured PLA activity in M extracts employing the substrate
1-palmitoyl-2-[14C]
palmitoyl-phosphatidylcholine in a liposome assay in the presence or
absence of Ca2+ and/or the
Ca2+ chelator EDTA. The assay was performed in
the presence of DTT to inactivate the cysteine-rich
sPLA2 enzymes. Most of the
PLA2 activity in M lysates was
Ca2+ independent, with an 
20% increase in
activity in the presence of Ca2+ without EDTA
(data not shown). Moreover, the iPLA2 activity in
the M lysates was inhibited by MAFP with an
IC50 of 10-7 M (Fig. 4). The IC50 for
BEL and 12-episcalaradial was >10-5 M,
suggesting that the PLA2 activity was not that of
group VI iPLA2. The specific activity of the
M-PLA2 toward 1-
palmitoyl-2-[14C]arachidonyl-phosphatidylcholine
and
1-palmitoyl-2-[14C]palmitoyl-phosphatidylcholine
was 43 ± 8 pmol/h/106 cells
(n = 4) and 3.2 ± 2.5
pmol/h/106 cells (n = 2),
respectively, suggesting that the M activity is predominantly that
of group IV cPLA2-. In contrast,
Ca2+-independent group VI
iPLA2 prefers palmitic acid to arachidonic acid
in the sn-2 position (33, 34).
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was also examined using RT-PCR with primers for group
IV and group VI PLA2 enzymes. We detected
transcripts for both group IV cPLA2- and for
group IV cPLA- (22) in M (Fig. 5, upper panel) but not
transcripts for group IV cPLA2
(23) or group VI iPLA2 (35, 36) (data not shown) after 40 cycles.
cPLA2 transcripts were detectable at 35 cycles.
The presence of cPLA2 and
cPLA2 were also demonstrated by Western blot
analysis of cytosolic M extracts which revealed a single peptide
band with an apparent Mr 60,000
consistent with the molecular weight of cPLA2-
(20) and a doublet at Mr
110,000 consistent with cPLA2- (Fig. 5, lower panel). Infection with Mtb slightly induced
cPLA2- protein production in the M. These
bands were not seen when irrelevant isotype-matched Ab was used (data
not shown).
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with Mtb
To demonstrate activation of M cPLA2 by
Mtb infection, M were prelabeled with
[3H]arachidonic acid and then infected with
Mtb. The release of [3H]arachidonic
acid into the medium was then measured (33) in a
time-dependent fashion. Arachidonic acid release from M was
significantly increased 4–10 h after infection with Mtb,
whereas there was minimal arachidonic acid release over time from
noninfected M or M treated with TNF- alone (Fig. 6). Equal amounts of arachidonic acid
were released from Mtb-infected M in the presence or
absence of CHX, indicating that de novo protein synthesis is not
required for PLA2 activation.
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cultures in the absence and presence of various
PLA2 inhibitors. Arachidonic acid release and
apoptosis induced by Mtb was blocked by
AACOCF3 and MAFP, inhibitors of group IV
cPLA2 and group VI iPLA2,
but not by BEL, the selective inhibitor of group VI
iPLA2 (Fig. 7a), suggesting participation
of group IV cPLA2 and not of group VI
iPLA2 in arachidonic acid release. Of interest,
the caspase 3 inhibitor Z-DEVD-FMK did not inhibit arachidonic acid
release induced by Mtb, indicating that
cPLA2 acts upstream from caspase 3 (Fig. 7b). These studies show also that the release of
radioactivity after infection of
[3H]arachidonic acid-labeled M with
Mtb represents free arachidonic acid and not released
membrane vesicles containing [3H]arachidonate,
because the release of radioactivity into the cell supernatants was
specifically inhibited by cPLA2 inhibitors.
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Apoptosis of Mtb-infected M is associated with
significantly increased antimycobacterial activity (4, 26). To identify a possible role for cPLA2
in the M defense against Mtb, bacterial burden was
measured in infected M cultures after treatment with
cPLA2 inhibitors, with arachidonic acid or with
both. We determined the number of Mtb in the culture at day
4 of the infection, because at this time 35% of the M are
apoptotic, <5% of the cells are necrotic, and de novo mycobacterial
growth is negligible (4). The number of viable
phagocytosed Mtb by M was initially determined at 4
h of incubation. After 4 days, the number of viable Mtb in
the M cultures was reduced by 50% (Fig. 8). In the presence of the
cPLA2 inhibitors, AACOCF3
and MAFP, Mtb numbers were not reduced, suggesting that
cPLA2 activity contributes to the
antimycobacterial activity of the M (Fig. 8a). Addition
of 10–50 µM arachidonic acid to Mtb-infected M
reversed the effect of AACOCF3, and addition of
arachidonic acid alone to infected M inhibited growth of
Mtb almost completely (Fig. 8b). Thus,
arachidonic acid, the predominant product of
cPLA2, is an efficient antimycobacterial agent in
M. There was no direct effect of arachidonic acid on the bacteria,
because addition of arachidonic acid to a final concentration of 10–50
µM to Mtb cultures did not reduce the viability of the
bacilli (data not shown). These experiments suggest that
cPLA2 action is not only required for the
induction of apoptosis, but also for the antimycobacterial activity in
the M.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. A CHX-sensitive
signal is identical to the action of TNF-, consistent with previous
studies (7) and a protein synthesis-independent signal is
transmitted by the action of cPLA2.
TNF- was described to induce the death of L929 cells and WEHI 164
cells (37), but most cell lines and primary cells are
resistant to the cytotoxic activity of TNF- (38). It
now appears that cells sensitive to TNF- can be distinguished from
resistant cells by their capacity to produce arachidonic acid. When the
properties of TNF--resistant L929 fibroblasts were compared with
those of the TNF--sensitive parent cells, the resistant cells were
unable to release arachidonic acid in response to TNF- treatment
(39), suggesting that the action of
PLA2 is required for the cell to undergo
TNF--dependent apoptosis. It was later found that group IV
cPLA2 is involved in the induction of
TNF--dependent cytotoxicity of murine 3T3-like fibroblasts, L929
cells, and human leukemic cells (15, 17, 18). The degree
of cPLA2 activity correlated well with the
susceptibility of the cells to TNF- in presence of the second
sensitizing signal, CHX or actinomycin (17), and the
susceptibility of the fibroblasts to TNF- depended on sustained
phosphorylation of cPLA2- (40).
Likewise, inhibition of cPLA2 activity by
specific inhibitors (15, 16) or by transfection with
cPLA2 antisense oligonucleotides
(17) caused resistance to cell death and expression of
cPLA2 in TNF--resistant cells increased their
sensitivity to TNF--induced cytotoxicity (18).
In Mtb-infected M, cPLA2 is
essential for induction of apoptosis because the
cPLA2 inhibitors AACOCF3
and MAFP block apoptosis of the cells (Fig. 3
) and inhibition of
apoptosis by cPLA2 inhibitors is reversed by
addition of exogenous arachidonic acid, the predominant product of
cPLA2 (34). Arachidonic acid has
been implicated in TNF--induced death of TA1 adipogenic cells
(41) in the presence of the second stimulus CHX in that it
generates reactive oxygen intermediates (ROI) by interacting with the
mitochondrial electron transport chain (42). RO1 have been
found to be required for apoptosis induction, possibly by activation of
caspase 3 (43, 44). We and others (4, 26) have found that apoptosis of M correlates with increased
antimycobacterial activity. We are now able to link apoptosis with
antimycobacterial activity by either addition of
cPLA2 inhibitors or arachidonic acid.
cPLA2 inhibitors diminished the capacity of M
to suppress Mtb growth, whereas addition of arachidonic acid
to Mtb-infected M almost completely abrogated
Mtb growth (Fig. 8b), suggesting that the
cPLA2 products are part of the antibacterial
defense system of the M (44). Although the role of
arachidonic acid, especially its potential to generate ROI
(41) in antimycobacterial activity, is not well
understood, we recognize an obvious therapeutic potential in altering
arachidonic acid levels in Mtb-infected M and think that
drugs increasing the arachidonic acid concentration in the infected
M might be candidates for therapeutic down-regulation of
intracellular Mtb growth.
As discussed above, there are several groups of
PLA2 enzymes (14). The low molecular
species of PLA2 were considered unlikely
candidates to be involved in the induction of Mtb-dependent
M apoptosis because two different inhibitors of these enzymes,
12-episcalaradial and SB203347, failed to inhibit apoptosis while
AACOCF3 and MAFP were effective at nanomolar
concentrations. We detected two group IV PLA2
enzymes, cPLA2- and
cPLA2-, in M by RT-PCR and Western
blotting. The susceptibility of group IV
cPLA2- to various inhibitors of
PLA2 has not been described. It is therefore not
possible to determine which of the cPLA2 species
present in the M is required for Mtb-induced apoptosis.
Nevertheless, PLA2 activity independent of
Ca2+ was detected in lysates of M, and this
activity was 100-fold more susceptible to MAFP than to
12-episcalaradial and BEL in vitro (Fig. 4), exhibiting the same
profile of susceptibility to inhibitors as the
PLA2 that participates in Mtb-induced
apoptosis of M (Fig. 3). This PLA2 activity in
cultured M was distinct from group VI iPLA2 by
its resistance to BEL and by its relative preference for arachidonic
acid in the sn-2 position of phosphatidylcholine. The
cumulative evidence therefore points to a crucial role for group IV
cPLA2, possibly cPLA2-,
in Mtb-induced apoptosis of human M.
Although the function of group IV cPLA2 in
inducing apoptosis is thought to be based on the generation of
arachidonic acid necessary to generate ROI (42),
arachidonic acid might have additional functions in the signaling
pathways leading to apoptosis. Three unrelated cellular compartments
are thought to interact in the induction of apoptosis
(45). First, stimuli such as oxidants or
Ca2+ influx can lead to disruption of the
mitochondrial transmembrane potential and mitochondrial permeability
transition resulting in release of cytochrome c into the
cytoplasm. Cytochrome c binds APAF-1, causing the activation
of caspase 9 which results in activation of the effector caspase 3
(46). Caspases are necessary for the induction of
TNF--dependent apoptosis (47). Second, triggering
through the TNF- receptor 55-kDa TNFR1 results in the recruitment of
docking proteins, including Fas-associated death domain protein
and TNFR-associated death domain protein to the death domain of the
receptor, which then associate with caspase 8, leading eventually also
to activation of the effector caspase 3 (48). A third
pathway, which may be important in some cases, involves stress exerted
on the endoplasmic reticulum; e.g., release of
Ca2+ from intracellular stores activates caspase
12 located within the endoplasmic reticulum, which results in
cytotoxicity by amyloid-. This pathway might not be involved in all
types of apoptosis (46). It is presently unclear how the
different pathways induced by death receptor triggering and
mitochondrial permeability transition cooperate and result in apoptosis
(48) and where signals generated by
cPLA2 fit into this scenario. Findings described
in this study indicate that caspase 3 activation, arachidonic acid
release, and apoptosis are significantly inhibited by the
cPLA2 inhibitor AACOCF3,
whereas cPLA2 activity is not blocked by the
specific caspase 3 inhibitor Z-DEVD-FMK. These findings are consistent
with a model suggesting that cPLA2 operates
upstream of caspase 3.
In conclusion, our findings indicate that infection of human M with
Mtb independently leads to activation of
cPLA2 and synthesis of TNF-. The activity of
these two components is integrated to provide activation of caspase 3,
initiation of apoptosis, and antimycobacterial activity.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Heinz G. Remold, Brigham and Women’s Hospital, Smith Building, Room 526B, 75 Francis Street, Boston MA, 02115. E-mail address: hremold{at}rics.bwh.harvard.edu
3 Abbreviations used in this paper: M, macrophage; AACOCF3, arachidonyl trifluoromethyl ketone; BEL, bromoenol lactone; CHX, cycloheximide; PLA2, phospholipase A2; cPLA2, cytosolic PLA2; iPLA2, Ca2+-independent PLA2; MAFP, methyl arachidonyl fluorophosphate; Mtb, Mycobacterium tuberculosis; PARP, poly(ADP-ribose) polymerase; sPLA2, secretory PLA2; ROI, reactive oxygen intermediates; Z-DEVD-FMK, Z-Asp (OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl ketone.
Received for publication November 20, 2000. Accepted for publication April 13, 2001.
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|---|
interferon, interleukin-4, and tumor necrosis factor. Infect. Immun. 58:2675. in mycobacteria-induced granuloma formation in tumor necrosis factor--deficient mice. Lab. Invest. 79:379.[Medline]
. J. Biol. Chem. 274:17063., that is prenylated and contains homology to cPLA2. J. Biol. Chem. 273:21926., tumor necrosis factor-, and cycloheximide. Endocrinology 139:4860., a mammalian homolog of CED-3 is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose)polymerase. Cell 81:801.[Medline]
: effects on proliferation of normal and transformed cells in vitro. Science 230:943. is mediated by lipoxygenase metabolites of arachidonic acid. Biochem. Biophys. Res. Commun. 188:538.[Medline]
. Nature 403:98.[Medline]
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), or 12-epi-scalaradial
(12-epi, 
) using a liposome assay. Activity is expressed as
percentage of baseline activity (ordinate). The differences among the
treatments are statistically significant (p =
0.01). Inhibition by MAFP compared with BEL and 12-episcalaradial is
statistically significant at 10-7 M and 10-6
M (p = 0.02, n = 3).
), with TNF-
), with Mtb alone (
), and 10 µM
BEL (