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* Division of Rheumatology and Immunology, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, and
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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) respond to Mycobacterium
tuberculosis (Mtb) infection by undergoing apoptosis, a
cornerstone of effective antimycobacterial host defense. Virulent
mycobacteria override this reaction by inducing necrosis leading to
uncontrolled Mtb replication. Accordingly, M death
induced by inoculation with Mtb had the characteristics
of apoptosis and necrosis and correlated with moderate increase of
mitochondrial permeability transition (MPT), mitochondrial cytochrome
c release, and caspase-9 and -3 activation. We
hypothesized that changes in intramitochondrial Ca2+
concentration ([Ca2+]m) determine whether
M undergo either apoptosis or necrosis. Therefore, we induced
mechanism(s) leading to predominant apoptosis or necrosis by modulating
[Ca2+]m and examined their physiological
consequences. Adding calcium ionophore A23187 to M inoculated with
Mtb further increased calcium flux into the cells which
is thought to lead to increased [Ca2+]m,
blocked necrosis, stabilized MPT, decreased mitochondrial cytochrome
c release, lowered caspase activation, and accompanied
effective antimycobacterial activity. In contrast, M infected with
Mtb in presence of the mitochondrial calcium uniporter
inhibitor ruthenium red showed increased mitochondrial swelling and
cytochrome c release and decreased MPT and
antimycobacterial activity. Thus, in Mtb-infected M,
high levels of mitochondrial membrane integrity, low levels of caspase
activation, and diminished mitochondrial cytochrome
c release are hallmarks of apoptosis and effective
antimycobacterial activity. In contrast, breakdown of mitochondrial
membrane integrity and increased caspase activation are characteristic
of necrosis and uncontrolled Mtb
replication. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In broad terms, we know that defense mechanisms of the innate immune
system are of critical importance in the early suppression of the
Mtb infection. It is known that macrophages (M), the
first line of defense against tuberculosis, undergo apoptosis when
inoculated with Mtb and develop significant
antimycobacterial activity (3, 4, 5). Mtb-induced
M apoptosis requires a dual signal, the action of TNF-
(6), and the activation of cytosolic phospholipase
A2 (7), and is thought to result in
activation of caspases, including caspase-9 and caspase-3. Caspase-9 is
an initiator caspase of apoptosis and its activation, which leads
to activation of the effector caspase caspase-3, requires formation of
the "apoptosome", a complex formed by cytochrome c
released from the mitochondria into the cytoplasm, the cytosolic factor
Apaf-1, ATP, and procaspase-9 (8, 9). Apoptosis is
generally characterized by membrane blebbing, cellular condensation,
and DNA fragmentation (10), and leads to removal of the
sealed apoptotic bodies by phagocytosis predominantly through the
phosphatidylserine receptor on the M (11).
In addition to apoptosis, there exists also an alternative cell death
program, necrosis, which typically involves swelling of the cell and
organelles, resulting in lysis of the plasma membrane, release of
intracellular constituents, and induction of a strong inflammatory
response (12). Necrosis often coexists with apoptosis and
both types of cell death are observed simultaneously in many systems
including tissue injury due to ischemia-reperfusion, toxic chemicals,
and viral infections (13, 14). Although apoptosis of
Mtb-infected M is associated with diminution of the
infection, preponderance of necrosis has been associated with increased
bacterial growth (3, 4).
There was some indication that the condition of the mitochondria is the branch point leading either to necrosis or to apoptosis. Therefore, we set up an experimental approach that favors either apoptosis or necrosis. Ca2+ is an intracellular messenger involved in cell death (15, 16). In HepG2 cells, increases in [Ca2+]m lead to mitochondrial permeability transition (MPT) and to apoptosis (17). Therefore, we performed experiments which either favored apoptosis or necrosis by manipulation of the [Ca2+]m.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ca2+ ionophore A23187, cyclosporin A, ruthenium red (RR), propidium iodide (PI), and cycloheximide (CHX) were from Sigma-Aldrich (St. Louis, MO), and streptolysin O was from Cogenix (Peterborough, U.K.). 3,3'-dihexyloxycarbocyanine iodide (DiOC6(3)), pluronic-F27, and Fluo-3 were from Molecular Probes (Eugene, OR). Murine monoclonal anti-caspase-3 Abs were purchased from Transduction Laboratories (Lexington, KY), and goat anti-caspase-9 Ab from Oncogene Research Products (San Diego, CA). Ab against cytochrome c were a kind gift of Dr. R. Jermmerson (University of Minnesota Medical School, Minneapolis, MN). Mouse anti-human poly(ADP-ribose) polymerase (PARP) was 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) with 10% BSA-glucose-catalase supplement (BD Biosciences, Mountain View, CA) and 0.05% Tween 80 (Difco) and resuspended in 7H9 broth at 5 x 107 CFU/ml.
Cells and culture
Mononuclear cells from peripheral blood of healthy donors after
obtained informed consent were isolated as previously described
(7). M for the in situ TUNEL assay were cultured on
plastic cover slips (Nunc, Rochester, NY) and were plated at 2.5
x 106 mononuclear cells/ml/well in 24-well
cluster plates (Costar, Cambridge, MA). M used for Western blot
analysis were cultured on Costar 100-mm diameter tissue culture dishes
(Costar) at 2 x 107 mononuclear cells/10
ml/dish. The resulting M population (2.5 x
105 cells/coverslip or 2.0 x
106 cell/dish) were 97–99% pure as determined
by nonspecific esterase staining. The M were cultured in IMDM with
10% pooled human serum for 7 days to allow for M differentiation
before Mtb infection. M were infected with five bacteria
per cell.
Quantitation of mycobacteria
Counting of mycobacteria using the Bactec model 460TB system (BD Biosciences) was performed as described (7).
In situ analysis of programmed cell death
Apoptosis of adherent M was measured using a fluorescent in
situ TUNEL assay (In Situ Cell Death Detection kit,
tetramethylrhodamine-dUTP, catalog no. 2156792; Roche
Biosciences, Indianapolis, IN) according to the specifications of the
manufacturer. M necrosis was determined by PI staining of nuclei
(18). In brief, following the experimental procedures,
glass-adherent M were incubated with 10 µM PI at room temperature
for 10 min. The cells were then washed two times with PBS, the
coverslip dried, and PI-positive cells evaluated by fluorescence
microscopy. Of note, PI stains at the early time points tested in these
experiments only nuclei of cells whose membranes are permeable to this
dye which is a sign for necrosis. The cell membranes of apoptotic M
are impermeable to PI, unless the M undergo secondary necrosis. The
assays were performed not later than 72 h after initiation of the
cell culture, because at later time points cell membranes of apoptotic
M become permeable to PI, which indicates the onset of secondary
necrosis. In cultures harvested at and before 72 h, <10% of the
total adherent cells present at time 0 are dislodged, which guarantees
that only a minimum of total cells had been lost.
Cytochrome c release from mitochondria
M cultured in 24-well culture plates were washed twice with
cold PBS and 1 ml of 1 µg/ml streptolysin O in PBS was added to
permeabilize the cells and the plates were incubated at 4°C for 5
min. The streptolysin O solution was replaced with 1 ml of transport
buffer (78 mM KCl, 4 mM Ca Cl2, 50 mM HEPES
buffer (pH 7.2), 2 mM DTT, and 1 µg/ml of protease inhibitors). After
incubation at 37°C for 5 min and on ice for 20 min the cells were
dislodged with a rubber policeman and pelleted at 500 x
g. Supernatants were analyzed by Western blotting for
cytochrome c released from the mitochondria. The cells were
washed twice with PBS, dissolved in 1 ml of lysis buffer
containing 250 mM NaCl, 50 mM HEPES buffer (pH 7.0), 0.1% Nonidet
P-40, 50 mM NaF, 5 mM EDTA, 1 mM DTT, 0.3 mM PMSF, incubated on ice for
10 min and centrifuged at 10,000 x g for 10 min.
Protein concentrations were measured using the Bradford assay. Fifty
micrograms of cell extract were processed to determine cytochrome
c remaining in the mitochondria.
Determination of caspase activation
M were preincubated for 15 min and for the remaining time of
the experiment with CHX (10 µg/ml) which blocks de novo protein
synthesis to increase the sensitivity of the assay. Following the
experimental procedures, M were treated at 4°C with lysis buffer
for 1 h at 4 x 106 cells/ml and
centrifuged at 15,000 x g for 10 min. Fifty micrograms
of cell lysates were heated in 2x sample buffer at 95°C for 5 min
and resolved in 7.5 or 12% SDS-polyacrylamide gels, transferred to
polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford,
MA), and blocked with 10 mM Tris buffer (pH 7.5) containing 150 mM
NaCl, 0.05% Tween 20, and 5% dry milk for 2 h. The membranes
were incubated with Ab (1 µg/ml) against caspase-3, caspase-9,
cytochrome c, or PARP. Isotype-matched irrelevant Abs were
used as controls. Membranes were then washed and blotted with
HRP-protein A (Zymed Laboratories, San Francisco, CA). After extensive
washing with 10 mM Tris-HCl buffer (pH 8.0) containing 150 mM NaCl and
1% Tween 20, the membranes were developed in CL reagent (NEN, Boston,
MA) and exposed to x-ray film.
Assessment of MPT induction in M (19)
MPT induction was assessed in M to evaluate mitochondrial
membrane potential (
m) by measuring the
retention of the lipophilic cationic dye DiOC6(3)
within mitochondria as a consequence of the
m (20). MPT results in the
loss of m which causes decreased retention
of DiOC6(3) in the mitochondria. Following the
experimental procedures, CHX pretreated M cultured in 6-well plates
were washed twice with PBS. The cells were then incubated for 30 min
with 1 nM DiOC6(3) at 37°C. Digitonin was then
added to a final concentration of 7.5 µM for 20 min, the cells were
washed three times with PBS, fixed with 1% formaldehyde for 20 min,
and washed twice with PBS. Thereafter, the cells were dislodged from
the plates using a rubber policeman and analyzed for fluorochrome
incorporation in a BD FACSort flow cytometer (BD Biosciences). The M
population with reduced DiOC6(3) was determined
by setting the gate to allow separation of the cell population with
reduced staining from the M population with intact mitochondria
showing high staining with DiOC67(3).
Percentage of M with reduced DiOC6(3) staining
of the total gated cell population was determined.
Electron microscopy
M were cultured in IMEM containing 10% human serum in 10-cm
plastic petri dishes. Following the experimental procedures, the cells
were washed three times in 0.1 M sodium cacodylate (pH 7.3) and fixed
with 5 ml of MJK/2 solution for 2 h, removed by using a rubber
policeman, transferred to microcentrifuge tubes, and stored overnight
at 4°C. The cells were then exposed to 1% uranyl acetate in water
for 15 min at room temperature, washed twice with distilled water,
centrifuged into 3% agarose at 45°C, and cooled to form blocks. The
agarose blocks were dehydrated using graded steps of acetone and
embedded in Spurr’s low viscosity media. After polymerization at
65°C overnight, 80-nm sections were cut and picked up with
copper grids which were then stained with uranyl acetate and bismuth
subnitrate. The sections were analyzed with a Jeol electron microscope
(Jeol USA, Peabody, MA) and recorded on Kodak sheet film (Eastman
Kodak, Rochester, NY).
Determination of Ca2+ flux
M were plated on glass coverslips in 24-well plates, 1.0
x 105 cells/well, in calcium-free IMEM without
phenol red containing 10% pooled human serum. After the cells were
inoculated with Mtb in absence or presence of A23187 from 15
min to 2 h, the cells were loaded with 0.5 ml fluo-3/AM solution
(10 ml HEPES buffer (pH 7.2) containing 60 µg Fluo-3/AM, 10 µl
Pluronic F-127, and 40 µl of 1 M probenecid). The cells were then
incubated for 30 min at room temperature and washed three times with
PBS. Following experimental procedures, the cells were washed and the
coverslips containing the M transferred into Mackaness-type
chambers. The chambers were filled with IMEM containing 10% human
serum. Accidental infection with Mtb was prevented by
containment of the bacteria within the Mackaness-type chambers.
Ca2+ flux was assessed by mounting the
Mackaness-type chamber on a Biophysica microscope chamber which had
been modified for use with an ACAS 570 Ultima Interactive Laser
Cytometer (Meridian Instruments, Okemos, MI; Ref. 21).
Changes in [Ca2+]i were
assessed by fluorescence analysis of M at an excitation wavelength
of 488 nm and an emission wavelength of >515 nm, quantified as
relative fluorescence intensity and displayed on a pseudocolor
intensity scale.
Statistical analysis
Results are expressed as mean ± SEM. The data were analyzed using SigmaStat Statistical Software (Jandel, San Rafael, CA) using a t test for normally distributed data with equal variances and the Mann-Whitney U rank sum test for data populations with nonnormal distributions and/or unequal variances.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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with Mtb induces both apoptosis
and necrosis
M cultures incubated with Mtb for 0, 24, 48, and
72 h were assayed for apoptotic and necrotic cells. Apoptic
adherent M were determined by fluorescent microscopy using a TUNEL
assay and necrotic M by PI staining of the nuclei (see
Materials and Methods). At all indicated time points, <5%
of the cultured M became detached and were lost. At 48 and 72
h, 
50% of M were apoptotic and 15–50% of the M were
necrotic (Fig. 1
), demonstrating that
inoculation with Mtb induces cell death with the
characteristics of both apoptosis and necrosis (14, 15).
Inoculation of M with Mtb resulted also in moderate loss
of m as manifested by decreased
mitochondrial accumulation of DiOC6(3), by
release of cytochrome c from the mitochondria, and
activation of the caspases-9 and -3. (see Figs. 5, 6, and 8).
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We next investigated whether inoculation of M with
Mtb has an effect on calcium flux. Inoculation with
Mtb moderately increased calcium flux into the M which
was detectable at 2 h (data not shown) and maximal at 4 h
(Fig. 2, upper right panel).
Addition of A23187 further increased calcium levels (Fig. 2, lower left panel) and the L-type Ca2+
channel blocker dilthiazem completely inhibited Mtb-induced
calcium flux (Fig. 2, lower right panel).
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were infected in the presence of 0.5 µM A23187. With A23187, the
percentage of apoptotic M at 48 h was similar to that without
A23187 (44 ± 1% vs 46 ± 2% in M infected without
A2318, n = 3), while a significant decrease of necrotic
cells was seen in comparison to M cultures infected without A23187
(Fig. 3). Lower concentrations of A23187
had no effect (data not shown). Less than 5% necrotic M were seen
in noninfected cultures with or without A23187 (data not shown).
Although it is well recognized that
[Ca2+]i overload caused
by ionophore leads to cell death (22, 23), the increase of
[Ca2+]i induced by A23187
alone did not induce M mortality (data not shown).
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in presence of A23187 and measured the number of bacteria in the
cultures. Mtb replication in M cultures infected with
Mtb was significantly restricted after 48 h in presence
of increased concentrations of A23187 (Fig. 4, right panel; Ref.
24) in comparison to Mtb growth in M
cultured without additives paralleling the marked abrogation of M
necrosis in presence of A23187 (Fig. 4, left panel).
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mitochondria and diminishes caspase activation in
Mtb-infected M
Release of cytochrome c from the mitochondria is a
critical requirement for induction of programmed cell death (8, 9). Therefore, we determined whether release of cytochrome
c from mitochondria is altered by A23187. In M infected
with Mtb, 40% of total cytochrome c was
released from the mitochondria (Fig. 5
, first lane from right), indicating that Mtb
infection increases permeability of the inner mitochondrial membrane to
cytochrome c. Increasing
[Ca2+]i diminished
mitochondrial release of cytochrome c significantly (Fig. 5, fourth lane from left).
To determine whether activation of caspase-3 and caspase-9 correlates
with necrosis or apoptosis, we assessed the activation of caspase-3 and
caspase-9 in Mtb-infected M in presence of A23187 by
determining the amount of residual procaspase. In these experiments,
M were preincubated with CHX to prevent synthesis of procaspases
during culture. In CHX-pretreated M, both Mtb and TNF-
needed to be added to induce activation of caspase-3 and caspase-9 and
degradation of PARP, an important caspase-3 substrate (Ref.
25 ; 70 and 60% loss of procaspase-3 and procaspase-9 and
45% PARP degradation, respectively, Fig. 6). A23187 decreased procaspase-3
activation and procaspase-9 activation (29 and 15% loss of
procaspase-9 and procaspase-3, respectively), although the percentage
of apoptotic M in the cultures was increased (Fig. 1). The
inhibitory effect of A23187 on degradation of PARP was minimal.
The Ca2+ uniporter inhibitor RR enhances necrosis
of Mtb-infected M and mycobacterial replication
in Mtb-infected M cultures
RR, an inhibitor of the mitochondrial Ca2+
uniporter, blocks Ca2+ flux through mitochondrial
membranes and decreases
[Ca2+]m
(26, 27, 28). In presence of 5 ng/ml RR, a marked increase in
percentage of necrotic M after inoculation with Mtb was
seen at 48 and 72 h compared with M cultures infected without
RR (Fig. 3). In contrast, the percentage of apoptotic M was not
different from that in M cultures infected without RR (47 ±
9% vs 46 ± 2, n = 3). Uninfected M cultures
incubated with A23187 (data not shown), or with RR alone (data not
shown) did not show an increase of necrosis over background levels.
M necrosis augmented by RR (Fig. 7, left panel) correlated with an increase of mycobacterial
replication (Fig. 7, right panel).
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increased the amount of necrotic M
after 3 days from 37 ± 1 to 53 ± 2%
(p = 0.0001, n = 3). No
increase of necrosis was observed in the presence of dilthiazem alone
(data not shown). These results indicate that decrease of
[Ca2+]i induces
predominantly necrosis after infection with Mtb, but does
not induce M death in absence of infection.
RR increases cytochrome c release from M
mitochondria and enhances caspase activation
In presence of RR, Mtb inoculation dramatically
enhanced cytochrome c release from the mitochondria
resulting in 75% release of total cytochrome c (Fig. 5, second lane from right). Addition of RR to uninfected M
had no effect (Fig. 5, third lane from left).
Furthermore, RR strongly enhanced caspase-9 activation, caspase-3
activation, and PARP degradation in CHX-pretreated M infected for
12 h with Mtb in presence of TNF- (90, 80, and 94%
decrease of procaspase-9, procaspase-3, and PARP, respectively,
compared with M infected without RR; Fig. 6). RR alone had no effect
on caspase activation and PARP degradation (Fig. 6, upper left
panel, data not shown for caspase-9 and PARP). It should be
noted that necrosis of RR-treated cells is not caused by increased
activation of PARP leading to ATP depletion (30), because
addition of RR also markedly enhanced degradation of PARP (Fig. 6).
Infection of M in presence of RR causes irreversible MPT pore
opening and mitochondrial swelling
We further investigated whether MPT is required for induction of
necrosis or apoptosis. MPT causes irreversible breakdown of the
mitochondrial transmembrane potential m
(31) which correlates with the release of cytochrome
c from mitochondria. To assess MPT, accumulation of the
fluorescent cationic dye DiOC6(3)
(20) in the mitochondrial matrix was determined as a
consequence of the m. Release of
DiOC6(3) from the mitochondria is considered a
reliable indicator for m loss and of MPT
pore opening (20).
Optimal results were obtained when we measured the retention of
DiOC6(3) within mitochondria in M pretreated
with CHX at day 3. A significant decrease of dye retention in the
mitochondria was seen in M infected with Mtb alone as
compared with noninfected controls which was enhanced by addition of
TNF-. A substantial loss of dye retention was measured in infected
cultures incubated with TNF- and RR, indicating significant MPT
(Fig. 8, lane 1 from the
right). M incubated with Mtb, TNF-, and A23187
alone showed dye retention similar to cell cultures infected with
Mtb in absence of TNF- (Fig. 8, lane 4 from
right). These experiments clearly demonstrate that massive
mitochondrial damage is caused by Mtb in presence of RR that
leads to loss of DiOC6(3) retention as the
consequence of MPT and irreversible m loss.
A23187 prevents mitochondrial damage and loss of dye retention.
Similar results were obtained in electron microscopic studies that
assessed mitochondrial swelling. M incubated with Mtb and
RR for 4 days show significant mitochondrial swelling indicating
mitochondrial membrane damage, whereas uninfected M and M treated
with RR alone contained normally sized mitochondria (Fig. 9).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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inoculated with Mtb undergo apoptosis, which is
associated with strong antimycobacterial activity. Experiments with
virulent mycobacteria suggested that these pathogens are able to
override the detrimental effects of apoptosis by inducing necrosis,
which results in uncontrolled mycobacterial growth (4, 5).
We hypothesized that the branch point for either apoptosis or necrosis
is the mitochondrial membrane permeability and that the mitochondrial
membrane permeability depends on
[Ca2+]i. Ca2+ is known to be a universal secondary messenger indispensable in the majority of cellular signal transduction pathways including fertilization, proliferation, and development. Ca2+ signaling is important for membrane excitability, mitochondrial metabolism, vesicle secretion, mitosis, muscle contraction, NO production, and in the induction of cell death (32).
Previously, increase of
[Ca2+]i in M was found
to maintain mitochondrial integrity (17, 33) and to cause
apoptosis (15, 16, 17). The present study demonstrates that
increase of intracellular Ca2+ protects
mitochondria from irreversible damage by Mtb, promotes
apoptosis, and inhibits M necrosis and mycobacterial survival.
However, intracellular Ca2+ concentrations seem
to have a broad effect on multiple antimycobacterial mechanisms
including defense mechanisms that are not dependent on mitochondrial
function. Block of calcium influx was found to have a direct inhibitory
effect on phagosome-lysosome fusion (24, 34).
Down-regulation of calcium influx resulting in block of M maturation
clearly correlates with a significantly decreased defense capacity
against mycobacteria in M which do not undergo programmed cell death
(24). The importance of this finding is underscored by the
observation that virulent Mtb are able to inhibit
Ca2+ signaling and M phagosome-lysosome fusion
which results in increased intracellular survival of the Mtb
(24). Therefore, the action of intracellular calcium
resulting in antimycobacterial defense is not always dependent on the
status of the M with respect to programmed cell death.
It was suggested that moderate increase of
[Ca2+]i up-regulates the
cell’s energy state and thereby protects mitochondrial integrity
(33, 35). Consequently, ATP depletion (36)
was found to lead to irreversible loss of m
and to necrosis (37). In contrast, selective
reconstitution of the extramitochondrial ATP pool with glucose
prevented necrosis in ATP-depleted cells and restored the ability of
the cells to undergo apoptosis (38). In M, ATP is also
necessary for increased antimycobacterial activity
(39).
Our studies further indicate that increased caspase activation leads to
M necrosis, but the possibility that increased caspase activation
under necrotic conditions is the result of a general breakdown of
cellular functions cannot be excluded.
The effect of Ca2+ depletion on the mitochondria
themselves is of critical importance leading to disruption of the
mitochondrial inner membrane potential and to necrosis of the M
(40). Our findings support the hypothesis of Kroemer et
al. (41) that the relative quantity of apoptogenic factors
released vs the extent of mitochondrial dysfunction is responsible for
induction of either apoptosis or necrosis. In intact mitochondria, the
transmembrane potential (m) keeps the inner
portion of the inner mitochondrial membrane negatively charged, which
allows accumulation of the cationic dye DiOC6(3)
in the mitochondrial matrix. Reduction of
DiOC6(3) retention within the mitochondria is an
indicator for loss of m, which is the
consequence of MPT. Necrosis of Mtb-infected M in
presence of RR correlates with reduced DiOC6(3)
retention within the mitochondria and indicates the collapse of
m. High
[Ca2+]i induced by A23187
rescues mitochondria from irreversible damage which manifests itself in
an increase of DiOC6(3) retention within the
mitochondria and M apoptosis. In agreement with this model, HepG2
cells exposed to proapoptotic stimuli seem to undergo reversible
mitochondrial membrane pore opening. Reversible mitochondrial pore
opening was postulated on the basis of a transient increase in
[Ca2+]i that caused a
pulse of cytochrome c release from the mitochondria and
apoptosis. Apoptosis was thought to be dependent on resealing of the
pores and prompt recovery of the mitochondrial energy metabolism.
Despite ongoing caspase activation, the mitochondrial metabolism
required for apoptosis was found to be intact (17, 22).
We hypothesize that a similar mechanism is effective in
Mtb-infected M in which A23187 induces a transient
increase of [Ca2+]i. The
ensuing increase of
[Ca2+]m (23)
is thought to be due to the presence of a passive uniporter in the
mitochondrial membrane, a structure that increases ion diffusion down
the electrochemical gradient and does not couple the transport to that
of any other ion (42). In presence of A23187, no reduction
of DiOC6(3) retention in the mitochondria was
observed, suggesting that in presence of high
[Ca2+]m, mitochondrial
pore opening is reversible and does not lead to permanent breakdown of
m, resulting in apoptosis rather than in
necrosis.
Although in infected M, A23187 down-regulates cytochrome
c release, caspase activation and apoptosis were clearly
detectable. We speculate that our method used to detect cytochrome
c release from the mitochondria, Western blotting, is not
sensitive enough to measure the small amounts of cytochrome
c released in the presence of A23187 that are nevertheless
sufficient for activation of procaspase-9 and for triggering of the
apoptotic cascade. Alternatively, unrelated mechanisms may lead to
caspase-9 activation (43, 44).
The suggested transient nature of mitochondrial pore opening further
implies that release of cytochrome c into the cytosol does
not require collapse of m, an event that
leads to irreversible MPT pore opening (45, 46, 47) combined
with wholesale release of apoptogenic mediators (48).
Several studies indicating that cytochrome c is released
from mitochondria early in the onset of apoptosis in the absence of MPT
are in line with our studies (49, 50, 51, 52) and suggest that
irreversible MPT is not required for cytochrome c release.
Therefore, we propose that a delicate balance between signals is
required in infected M to induce apoptosis and Mtb
elimination, because on one hand sufficient amounts of apoptogenic
factors need to be released from the mitochondria, but in contrast
mitochondria need to remain intact to prevent necrosis. Our studies
strongly indicate a key role for calcium in the outcome of the type of
cell death.
Published evidence indicates that inhibition of
Ca2+ transport into the mitochondria by RR leads
to irreversible decrease of
[Ca2+]m
(28). We could document loss of
m, mitochondrial swelling, enhanced
necrosis, and bacterial growth in M incubated with Mtb
and RR as a possible consequence of
[Ca2+]m breakdown.
Surprisingly, both drugs, A23187 and RR, have no or only a slight
effect on the survival of uninfected M, indicating that signals
emanating from the mycobacteria are the primary inducers of cell
death.
Cumulatively, these findings lead to the conclusion that high
[Ca2+]i required for
effective maintenance of mitochondrial functions after infection of the
M with Mtb is of critical importance for the defense
against Mtb. Therefore, we hypothesize that drugs which
increase [Ca2+]i and aid
in the protection of mitochondrial integrity might be suitable
therapeutic tools to bolster innate immune defense mechanisms against
Mtb.
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Acknowledgments |
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Footnotes |
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2 L.D. and H.G. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Heinz G. Remold, Division of Rheumatology and Immunology, Department of Medicine, Brigham and Women’s Hospital, Smith Building, Room 526B, 75 Francis Street, Boston, MA 02115. E-mail address: hremold{at}rics.bwh.harvard.edu
4 Abbreviations used in this paper: Mtb, M. tuberculosis; M, macrophage; MPT, mitochondrial permeability transition; m, mitochondrial membrane potential; RR, ruthenium red; DiOC6(3), 3,3'-dihexyloxycarbocyanine iodide; CHX, cycloheximide; PARP, poly(ADP-ribose) polymerase; [Ca2+]i, cytosolic Ca2+ concentration; [Ca2+]m, intramitochondrial Ca2+ concentration; PI, propidium iodide.
Received for publication July 9, 2002. Accepted for publication September 10, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in the induction of apoptosis of human macrophages infected with Mycobacterium tuberculosis H37Ra. J. Immunol. 166:7469.
, a mammalian homolog of CED-3 is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose)polymerase. Cell 81:801.[Medline]
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M. Chen, H. Gan, and H. G. Remold A Mechanism of Virulence: Virulent Mycobacterium tuberculosis Strain H37Rv, but Not Attenuated H37Ra, Causes Significant Mitochondrial Inner Membrane Disruption in Macrophages Leading to Necrosis J. Immunol., March 15, 2006; 176(6): 3707 - 3716. [Abstract] [Full Text] [PDF]
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, 
) and for necrosis (
, •) at the
indicated time points. The difference in percentage of apoptosis
between M
), or in
presence of 5 µg/ml RR (
). The differences in percentage of
necrosis among the treatments at 48 h (Mtb vs
Mtb + A23187, p = 0.06,
n = 3, and Mtb vs Mtb
+ RR, p = 0.03, n =
3) and at 72 h (Mtb vs Mtb + A23187,
p = 0.004, n = 3, and
Mtb vs Mtb + RR, p =
0.006, n = 3) are statistically significant.
