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Medical Research Council Centre for Immune Regulation, Birmingham Medical School, Birmingham University, Edgbaston, Birmingham, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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From HIV patients (2) and certain rare immunodeficient
individuals (3, 4), we know that human immunity to
mycobacterial infections constitutes an interplay between Ag-specific
CD4+ Th1 lymphocytes and host macrophages. The
macrophages promote Th1 activity by production of IL-12
(3), IL-18 (5), and IL-23 (6),
and the lymphocytes activate macrophages through the release of IFN-
(4) and TNF-
(7). These two sets of
effector cells are then thought to interact in a positive feedback
manner to contain the infection within T cell-activated
macrophages.
Mycobacteria, in turn, are thought to survive and replicate within nonactivated macrophages through a variety of strategies, including the avoidance of generation of reactive oxygen (8) and nitrogen intermediates (9) and the active inhibition of phagosome maturation (10, 11). The latter strategy preventing exposure to the antimicrobial peptides and enzymes presents within the acidic lysosomal granules. Infected cells are thought to become progressively unresponsive to further activation by Th1-derived cytokines as the pathogen undergoes intracellular replication (12).
Innate resistance to intracellular infections is also thought to be genetically determined, influencing the outcome of infection. The best characterized is the natural resistance-associated macrophage protein gene 1 (Nramp1)3 (13). This gene codes for a membrane transporter protein that acts to inhibit the replication of various intracellular pathogens, including bacille Calmette-Guérin (BCG), by excluding Fe2+ (14, 15) and other cations from the pathogen-containing phagosomes and promoting its acidification (16).
The macrophage acts as both the major reservoir of infection and the principal effector cell acting to regulate mycobacterial infections in vivo.
A major drawback in our understanding of the effector pathway(s)
operating within human macrophages is the fact that, unlike in murine
model cell systems (17), no mycobactericidal effect is
observed in vitro following stimulation of macrophages with
Th1-associated cytokines, including IFN- and TNF-
(18). This has been attributed to the inability of such
mediators to induce NO generation in human cells. However, we know that
in vivo macrophages are able to contain these organisms in the vast
majority of infected individuals. This has initiated a search for
alternative stimuli that may regulate mycobacteristatic or
mycobactericidal activity within infected human cells.
We have previously reported that a 15-min exposure to 1–3 mM ATP results in the apoptosis of BCG-infected monocyte-derived human macrophages within 6 h, with a concomitant 50–70% reduction in the viability of intracellular mycobacteria. ATP treatment of a macrophage-free culture of BCG had no effect on bacterial viability, demonstrating this to be a cell-mediated mechanism (19). Virulent strains of tuberculosis were also found to be equally as susceptible to this mechanism (20).
The effects of ATP were shown to be mediated by P2X7 receptors, as the cytocidal and bactericidal activity of ATP were blocked by pretreatment of cells with P2X7 antagonists, oxidized ATP, and KN62, and potentiated by the specific agonist benzoyl-benzyl ATP (19).
We have shown previously that the mycobactericidal effects of ATP are independent of both reactive oxygen and nitrogen intermediate generation. ATP was found to be equally effective on cells derived from patients with chronic granulomatous disease as against cells from healthy controls. Furthermore, BCG killing occurred even when such cells were additionally treated in vitro with NO inhibitors (19).
Electron microscopy (EM) studies have revealed that ATP-induced cell death of BCG-infected human macrophages is associated with the formation of cytoplasmic vacuoles into which the intracellular bacteria coalesce (21). This was thought to illustrate the promotion of phagosome-lysosome fusion and/or modulation of the phagosome pH within ATP-treated cells, resulting in the destruction of the mycobacteria.
In this study, we report the use of macrophages derived from various gene-disrupted (knockout) mice to further characterize the ATP effector mechanism involved and to confirm the selective involvement of the P2X7 receptor in this mycobactericidal effect. Using EM and confocal fluorescent microscopy, we have further analyzed the multibacillary vacuole formation that occurs following ATP stimulation. Our findings suggest these play a critical role in mediating the death of mycobacteria as they progressively acidify and fuse with cell lysosomes. Finally, we also demonstrate a key role for phospholipase D (PLD) in the P2X7 effector pathway, supporting similar findings reported by Kusner et al. (20).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The murine macrophage cell line J774a.1 was obtained from the American Type Culture Collection (Manassas, VA). The cells were cultured in DMEM (Life Technologies, Paisley, U.K.) supplemented with 10% FCS and 2 mM glutamine. The murine macrophage cell lines RAW 246.7, wild-type (WT) RAW 7.5R, and RAW 10S were a kind gift from J. M. Blackwell (Cambridge University, Cambridge, U.K.). The 7.5R and 10S lines are stable transfectants, the generation of which has been described previously (22). These were cultured in RPMI (Life Technologies) supplemented with 10% FCS and 2 mM glutamine. P2X7-/-, p47phox-/-, and inducible NO synthase (iNOS)-/- mice were kind gifts from I. P. Chessell (Glaxo Wellcome, Cambridge, U.K.), A. W. Segal (University College of London, London, U.K.), and F. Y. Liew (University of Glasgow, Glasgow, U.K.), respectively, and have been previously described (23, 24, 25). The genetic backgrounds of these knockout mice were, respectively, C57/129, C57, and 129. WT C57BL/6 and 129 were used as controls, as appropriate. Bone marrow-derived macrophages (BMDMs) were obtained as follows. Femurs and tibias were flushed with DMEM, and the bone marrow was washed and plated out overnight in DMEM supplemented with 10% FCS, 10% GM-CSF-containing medium, and 2 mM glutamine. Nonadherent cells were then removed, washed, and plated at 106/ml in the above medium. The cells were cultured for a further 9 days, with changes of the medium on days 3 and 6. On day 9, the cells were adherent and morphologically looked like mature macrophages.
M. bovis BCG Evans strain was obtained from Evans Medical (Speke, U.K.) and maintained in log phase growth in 7H9 broth supplemented with 10% albumin dextrose catalase (ADC) enrichment medium and 0.2% Tween 80. Green fluorescent protein expressing BCG (GFP-BCG) was a kind gift from D. Young (Imperial College of London, London, U.K.). These contained the gene encoding a FACS-optimized GFP protein (26) constitutively expressed under the control of the mycobacterial heat shock protein 60 promoter in a pSMT3 shuttle vector (27) construct. Stock aliquots, stored in glycerol at -70°C, were grown to log phase in 7H9 broth supplemented with 10% ADC enrichment medium, 0.2% Tween 80, and 50 µg/ml hygromycin (Sigma, St. Louis, MO).
BCG viability assay
Macrophages were grown to subconfluence in 200 µl/well
cultures in 96-well round-bottom microtiter plates. This approximates
to 5 x 104 cells in 200 µl/well. A total
of 5 x 105 BCG Evans per well was added. To
aid infection, plates were centrifuged at 700 x g for
10 min and incubated at 37°C for 60 min. Excess BCG were removed by
washing. As assessed by CFU, 
10% of BCG was taken up, giving a
ratio of one viable BCG per macrophage. Where appropriate, the
following metabolic inhibitors were then added for 60 min.
NG-Monomethyl-L-arginine
monoacetate (L-NMMA) (400 µM),
superoxide dismutase (15 U/ml), catalase (0.1 mg/ml), and butan-1-ol
(0.05–1%) were all purchased from Sigma. A 30 mM ATP (Boehringer
Mannheim, Indianapolis, IN) solution was prepared in culture medium,
adjusted to pH 7 with sodium bicarbonate, and passed through a
0.22-µM filter (Millipore, Bedford, MA) to sterilize. It was added to
the macrophage cultures to give a final concentration 3 mM, and
incubated at 37°C, 5% CO2. At appropriate time
points, the plates were centrifuged at 700 x g for 10 min. A
total of 150 µl supernatant was removed and replaced with 130 µl
7H9 medium, supplemented as above, plus 20 µl 2% saponin solution.
Cell lysis was promoted by vigorous pipetting and then incubation at
37°C for 120 min. Fifty-microliter aliquots from each well were
serially diluted in 7H9 supplemented with 10% ADC enrichment and 0.2%
glycerol and plated in 96-well flat-bottom plates. The plates were then
incubated at 37°C for 12 days. Microcolonies were counted using an
inverted microscope (x100 magnification). All experiments were
performed in quadruplets.
Lucifer yellow uptake
Pore formation was assessed using a method adapted from that described by Steinberg et al. (28). Briefly, macrophages adherent to coverslips were incubated with 500 µg/ml lucifer yellow dye, 250 µM sulfinpyrazone (Sigma), and 3 mM ATP in PBS for 5 min. They were washed in 250 µM sulfinpyrazone in PBS and viewed immediately on a Zeiss confocal laser-scanning microscope (Zeiss, Oberkochen, Germany). Cells with pores larger than 463 Da exhibit intracellular staining.
Chromium-release assay
Mouse BMDMs were cultured in 96-well round-bottom microtiter plates, as previously described. Aliquots of 51Cr (ICN Pharmaceuticals, Costa Mesa, CA) were added at a concentration of 2 µCi/well, and the plates were incubated for 16 h. Cells were washed and incubated with 3 mM ATP for a further 16 h. Supernatants were removed, and their activity was counted on a Wallac 1260 Multi Gamma II gamma counter (PerkinElmer Wallac, Gaithersburg, MD). The cells themselves were dissolved in 1% Triton X-100 solution and also counted. Results are expressed as the percentage of the total activity in the supernatant.
Confocal fluorescent microscopy
Macrophages were grown to subconfluence on 13-mm glass coverslips in 24-well plates. This approximated to 5 x 105 cells/coverslip. GFP-BCG were resuspended in DMEM, and a single cell culture was prepared by water bath sonication for 5 min. Any remaining clumps were removed by slow speed centrifugation (100 x g for 5 min). The bacterial concentration and confirmation of a single cell suspension were determined using a thoma chamber. The BCG were then added to the macrophages to give a final concentration of 2.5 x 107/coverslip. The plate was centrifuged at 700 x g for 10 min. The cells were incubated for 1 h at 37°C, 5% CO2, washed three times in DMEM to remove any extracellular bacteria, and incubated for an additional hour at 37°C, 5% CO2.
LysoTracker (Molecular Probes, Eugene, OR) was added to the cells (final concentration of 50 nM) for 1 h before stimulation. ATP was then added (final concentration of 3 mM), and the cells were incubated for the indicated times.
Before fixation, coverslips were washed three times in HBSS (Sigma). They were then fixed in 3% paraformaldehyde containing 10 mM lysine, 140 mM saline, and 40 mM HEPES (pH 7.3) for 15 min at room temperature. After washing in PBS plus 5% dextrose, the coverslip cultures were mounted on glass slides with Perma Fluor (Lipshaw Manufacturing, Detroit, MI), and the edges were sealed with nail varnish.
Slides were viewed with a Zeiss confocal laser-scanning microscope. Twenty fields were examined per slide, and representative pictures were taken.
Electron microscopy
The 3-(2,4-dinitroanilino)-3'-amino-N-methyl dipropylamine (DAMP; Molecular Probes) staining was adapted from Crowle et al. (29). BMDMs were cultured as described above on 13-mm plastic coverslips and infected with BCG Evans at an infection ratio of 5 BCG to 1 macrophage. ATP (3 mM final) was then added, and 30 min before the end of the experiment DAMP (50 µM final) was also added. The coverslips were washed three times in PBS and fixed in 2% gluteraldehyde in 0.1 M calcodylate and 2.5% sucrose (pH 7.35) for 60 min at 4°C. They were washed in 50 mM ammonia chloride, and then progressively dehydrated in ethanol, infiltrated with London Resin white (Agar Scientific, Stanstead, U.K.), and polymerized with UV light. Ultrathin sections were collected on nickel grids, which were incubated for 10 min with 0.1 M HCl. These were then blocked with 0.2% BSA and 0.2% goat serum in PBS for 45 min at room temperature. The primary Ab was a mouse antidinitrophenol monoclonal (NovoCastra, Newcastle, U.K.). A mouse IgG1 control Ab (BD PharMingen, San Diego, CA) was also used to exclude nonspecific staining. These were added at a concentration of 0.5 ng/ml for 16 h at 4°C. The secondary was a goat antimouse polyclonal Ab conjugated to 5 nM gold particles (British Biocell International, Cardiff U.K.). This was used at a dilution of 1/50 and incubated with the cells for 90 min at room temperature. Grids were counterstained with urinyl acetate and viewed on a Joel Temscan (Wellwyn Garden City, U.K.) electron microscope. Pictures were taken on Agfa Scientia (Agar Scientific) EM film. The number of gold particles per vacuole was counted. Counts were performed for 20 vacuoles, involving at least 10 macrophages for each experiment.
Statistics
The Student’s t test was used for all statistical analysis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ATP stimulation of BCG-infected J774 cells resulted in 50%
loss of mycobacterial CFU within 60 min posttreatment (Fig. 1
). Loss of mycobacterial viability was
detectable within 15 min and maximal at 2 h posttreatment.
Subsequently, all viability studies were performed at 2 h post-ATP
stimulation.
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The ATP/P2X7 effector mechanism was confirmed as a cell-mediated function, as ATP had no effect on BCG grown in macrophage-free cultures (results not shown).
ATP-mediated pore formation and macrophage death are P2X7 dependent
BMDMs from P2X7-/- mice
were examined for their responses following ATP stimulation. Cells from
WT mice were shown to rapidly take up the cell-impermeable dye lucifer
yellow following treatment with ATP for 5 min, indicating
P2X7 pore formation had occurred (Fig. 2, A and B),
whereas cells from P2X7-/-
mice failed to do so (Fig. 2, C and D). WT BMDMs
were also shown to die in response to ATP stimulation over a 16-h
period, as indicated by 51Cr release, whereas
macrophages from P2X7-/- mice
remained viable (Fig. 3a).
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Having demonstrated that macrophages from
P2X7-/- mice had no
P2X7-associated cytotoxic activity, they were
then assessed for their ability to kill intracellular mycobacteria
following ATP stimulation. BMDMs were infected with BCG and exposed to
ATP (3 mM) for 2 h. Compared with WT cells, macrophages from
P2X7-/- mice were shown to be
ineffective at killing BCG (Fig. 3
b). The percentage of BCG
killed was reduced from 50% in ATP-treated WT cells to <10% in
P2X7-/- cells. Although some
residual mycobactericidal activity was still occurring, the results
show that P2X7 is the primary transducer of the
intracellular mycobactericidal effector pathway activated following ATP
stimulation of infected macrophages.
PLD activity is essential for ATP/P2X7-mediated BCG killing, but not macrophage death
PLD is involved in a number of signaling cascades via the
production of phosphatidic acid from phosphorylcholine. If cells are
pretreated with butan-1-ol, PLD preferentially uses the primary alcohol
as a substrate producing the nonsignaling phosphatidyl butanol
(30). Treatment with butan-1-ol after BCG infection, but
before ATP stimulation, showed a concentration-dependent inhibition of
BCG killing post-ATP (Fig. 4a), but had no effect on
macrophage death, as measured by chromium release (Fig. 4b).
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Experiments were performed to investigate whether the ATP-induced mycobactericidal effects observed in murine macrophages were attributable to the generation of reactive oxygen or reactive nitrogen intermediates.
The high levels of NO required for killing mycobacteria within murine macrophages are generated by the enzyme iNOS after appropriate priming and stimulation of the cells. Macrophages from iNOS-/- mice are therefore unable to up-regulate nitrogen radical production (25). p47phox is a key enzyme in the NADPH reductase pathway, and mice with a targeted disruption of this gene are unable to produce reactive oxygen radicals (24).
BMDMs from iNOS-/- and
p47phox-/- mice and WT littermates were
infected with BCG and treated with ATP (3 mM) for 2 h. The results
(Fig. 5) reveal that ATP was equally
effective in inducing bacterial death within
iNOS-/- and
p47phox-/- macrophages as in WT
cells.
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). ATP killing of BCG was
shown to be equally as effective in all cell treatments. The data
reveal that oxygen and nitrogen radicals do not have a role in the ATP
effector mechanism. Killing post-P2X7 stimulation is not affected by Nramp1 genotype
The susceptibility of inbred mice strains to BCG is associated with a single point mutation of Nramp1 (31). Both the RAW and J774 macrophage cell lines are derived from BALB/c mice, which carry the susceptible mutation of Nramp1 (31).
To examine the possible influence of Nramp1 on the ATP-bacterial
killing effect, RAW cells, stably transfected with either the resistant
Nramp1 (RAW 7.5R) or the susceptible Nramp1 (RAW
10S), were used. ATP treatment of BCG-infected 7.5R and 10S cells was
shown to be equally as effective in inducing killing of BCG, as was
observed in the WT RAW cells (Fig. 6).
This indicates that expression of Nramp1 does not affect the ability of
RAW cells to kill BCG in response to ATP stimulation.
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BCG-containing phagosomes fuse with lysosomes post-P2X7 stimulation: confocal fluorescent microscopy studies
Mycobacteria survive within macrophages by inhibiting the maturation of their phagosomes, thus avoiding fusion with lysosomes (10). Lysosomes provide a potentially toxic environment for mycobacteria containing various hydrolases and cationic proteins in an acidic environment. The promotion of phagosome-lysosome fusion by ATP was therefore investigated as a potential mycobacterial-killing mechanism.
Confocal fluorescent microscopy was used to show that
P2X7 stimulation of infected BMDMs resulted in
the rapid fusion of the BCG-containing phagosomes with lysosomes (Fig. 7). The acidophilic dye LysoTracker
(Molecular Probes) was used to prelabel the cell lysosomes red, and
GFP-BCG were used to infect the BMDMs. When analyzed by confocal
fluorescent microscopy, the BCG in non-ATP-treated cells (Fig. 7, A and B) appeared green, indicating that they
were residing in nonacidic vesicles separate from the red-staining
lysosomes. However, in ATP-treated cells (Fig. 7, C and
D), an increasing percentage of the BCG appeared yellow (due
to colocalization with the red LysoTracker), indicating that they were
residing within acidic organelles that had fused with lysosomes. To
quantify the colocalization of the BCG, serial observations were
expressed as the percentage of colocalized BCG over total intracellular
BCG (Fig. 8). The results illustrate the
progressive acidification of the BCG-containing phagosomes. These
experiments show that BCG-containing phagosomes rapidly fuse with
lysosomes following ATP stimulation.
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, E and F). The data demonstrate that the
phagosome-lysosome fusion that occurs following ATP stimulation is
dependent on and is a function of P2X7
activation. BCG-containing phagosomes fuse to form acidified multibacillary vacuoles post-P2X7 stimulation: EM studies
High resolution transmission EM was used to investigate the
morphological changes accompanying the fusion of BCG-containing
phagosomes with lysosomes following ATP stimulation. ATP-induced
acidification of BCG-containing phagosomes was confirmed by the
prestaining of cells with DAMP, an acidophilic dye that has a
preferential affinity for acidic organelles such as lysosomes. DAMP
staining was then detected using a gold-labeled anti-dinitrophenol
Ab. Fig. 9, A and
B, illustrates a non-ATP-stimulated macrophage in which the
BCG-containing phagosome has little to no gold labeling, and is
therefore shown not to be acidic. This supports previous findings
(10, 11, 32) that BCG normally reside within individual
nonacidic phagosomes that do not fuse with lysosomes. Within 30 min of
ATP stimulation, marked changes could be observed within infected
macrophages (Fig. 9, C and D). At low power
(x5000), the BCG were observed to coalesce into multibacillary
vacuoles (Fig. 9C). These are heavily labeled with DAMP
(Fig. 9D), indicative of vacuole acidification, in contrast
to the non-DAMP-labeled individual BCG-containing phagosomes seen in
unstimulated cells.
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). Overall, the results confirm and
illustrate the rapid acidification of phagosomes that occurs post-ATP
stimulation. In addition, the EM studies illustrate that this involves
the fusion of individual BCG-containing phagosomes both with each other
and with lysosomes.
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, E and F, illustrates that at 2 h post-ATP
stimulation of P2X7-/- BMDMs,
the BCG-containing phagosomes have neither acidified nor coalesced. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The kinetics of ATP-induced BCG killing was more rapid than induction of cell death. However, although ATP-stimulated cell death and BCG killing were both shown to be specifically P2X7 dependent, their effector signaling pathways were found to diverge downstream of P2X7 activation. Blocking of cellular PLD activity with butan-1-ol then inhibited BCG killing, but not macrophage death in response to ATP stimulation.
The P2X7 receptor (formally known as P2Z) is highly expressed on cells of the immune system, predominately cells of monocytic lineage (33). However, macrophages are thought to express a number of additional purinergic receptors, including: P2Y1,2,4,5,6,10,11 and P2X1,4,5,7 (34), some of which can respond to ATP with varying sensitivity.
The fact that P2X7-/- macrophages are unable to effectively kill BCG in response to ATP demonstrates unequivocally that P2X7 is the major receptor involved in this specific effector pathway. However, we also observed a small, but nonsignificant level of residual BCG killing within ATP-treated P2X7-/- cells. This is in agreement with Kusner et al. (20), who noted that ATP was slightly more effective than the more specific P2X7 agonist benzoyl-benzyl ATP. Taken together, these findings indicate the possible involvement of an additional purinergic receptor that may contribute to the loss of bacterial viability seen in ATP-treated cells. We have provisionally characterized this additional purine receptor as P2Y11 (35).
Crossover studies using inhibitors to block both nitrogen radical production in p47phox-/- BMDMs and oxygen radical production in iNOS-/- BMDMs also demonstrated unequivocally that neither oxygen nor nitrogen radicals have a role in ATP killing of intracellular BCG. This confirms our previous findings, which showed that monocyte-derived macrophages from patients with chronic granulomatous disease are able to effectively kill BCG following ATP stimulation, and that the addition of the nitrogen radical inhibitor L-NMMA was also unable to inhibit ATP-mediated BCG killing in these cells (19).
Nramp1 is an important mechanism in murine defenses against various intracellular infections including BCG, and has been reported to be associated with human resistance to tuberculosis (36). Nramp1 is also known to be expressed on phagosome membranes (37), and has been proposed to play a role in both iron transport and phagosome acidification (16). In this study, Nramp1 was not found to be involved in the ATP-induced mycobactericidal effector process, as macrophages with either the susceptible or resistant form of the Nramp1 allele were shown to be equally capable of killing intracellular BCG following ATP stimulation.
The molecular probes LysoTracker and DAMP are weak bases that are used to study phagosome acidification because they are selectively taken up within cells by acidic organelles. In this study, both LysoTracker and DAMP preferentially targeted BCG-containing phagosomes following ATP stimulation of infected macrophages, indicating that phagosome-lysosome fusion had occurred. EM studies using DAMP labeling also illustrated that the BCG-containing phagosomes both acidified and fused with each other following ATP stimulation. This is in agreement with Molloy et al. (21), who also reported similar fusion of mycobacteria-containing phagosomes following ATP treatment.
Importantly, the phagosome acidification and phagosome fusion induced by ATP were not seen in BMDMs from P2X7 gene-disrupted mice. This illustrates that, like BCG killing, these fusion processes occur downstream of P2X7 activation.
There was an observed temporal relationship between ATP-induced phagosome acidification and BCG killing that supports the hypothesis that ATP-induced phagosome-lysosome fusion directly results in killing of the intracellular BCG. It is known that mycobacteria grow poorly in acidic environments (38) and the growth rate of intracellular M. tuberculosis is impaired when they enter large acidified vacuoles formed by coinfection with other intracellular organisms (39). Fusion with lysosomes would then expose the BCG to the detrimental effects of low pH and to the toxic actions of lysosomal proteases and antibacterial peptides. The rapid killing effect of ATP observed in this study would suggest that exposure to these lysosomal mediators results in swift mycobactericidal activity.
In confirmation of the findings reported by Kusner et al. (20), we also demonstrated that PLD activation is required for ATP-induced BCG killing. It is therefore of interest that PLD has been implicated in a number of organelle trafficking functions, e.g., degranulation by mast cells (40) and endosome fusion (41). PLD activation produces increased levels of phosphatidic acid, a known fusogen to endosome membranes (41). PLD may also activate additional phospholipases such as phospholipase A2 (PLA2) (42, 43, 44, 45). Interestingly, PLA2 is known to be induced within ATP-treated macrophages (46). This in turn may then promote endosome fusion via the generation of arachidonic acid (47).
We hypothesize that BCG actively inhibit the fusigenicity of their phagosomes, thereby preventing phagosome maturation, i.e., lysosome fusion and acidification. One mechanism by which they may achieve this effect is by altering the lipid composition of their phagosome membranes. We propose that ATP might act to overcome this inhibition via the generation of fusinogenic mediators such as PLD and/or its downstream mediators, e.g., PLA2 and arachidonic acid that act to promote organelle fusion.
Further investigation of this intracellular bacterial killing mechanism may help to elucidate a key macrophage-mediated antimycobacterial effector process. In addition, the signaling pathway(s) involved may prove to be amenable to modulation via targeted drug design for the treatment of multidrug-resistant tuberculosis.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ian P. Fairbairn, Medical Research Council Centre for Immune Regulation, Birmingham Medical School, Birmingham University, Edgbaston, Birmingham, B15 2TT, U.K. E-mail address: i.p.fairbairn{at}bham.ac.uk
3 Abbreviations used in this paper: Nramp1, natural resistance-associated macrophage protein 1; ADC, albumin dextrose catalase; BCG, bacille Calmette-Guérin; BMDM, bone marrow-derived macrophage; DAMP, 3-(2,4-dinitroanilino)-3'-amino-N-methyl dipropylamine; EM, electron microscopy; GFP, green fluorescent protein; iNOS, inducible NO synthase; L-NMMA, NG-monomethyl-L-arginine monoacetate; PLA2, phospholipase A2; PLD, phospholipase D; WT, wild type.
Received for publication October 10, 2000. Accepted for publication July 10, 2001.
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 S. L. Fernando, B. M. Saunders, R. Sluyter, K. K. Skarratt, H. Goldberg, G. B. Marks, J. S. Wiley, and W. J. Britton A Polymorphism in the P2X7 Gene Increases Susceptibility to Extrapulmonary Tuberculosis Am. J. Respir. Crit. Care Med., February 15, 2007; 175(4): 360 - 366. [Abstract] [Full Text] [PDF]
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S. D. Chakravarty, J. Xu, B. Lu, C. Gerard, J. Flynn, and J. Chan The Chemokine Receptor CXCR3 Attenuates the Control of Chronic Mycobacterium tuberculosis Infection in BALB/c Mice J. Immunol., February 1, 2007; 178(3): 1723 - 1735. [Abstract] [Full Text] [PDF]
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 E. A. Ivakine, S. M. Mortin-Toth, O. M. Gulban, A. Valova, A. Canty, C. Scott, and J. S. Danska The Idd4 Locus Displays Sex-Specific Epistatic Effects on Type 1 Diabetes Susceptibility in Nonobese Diabetic Mice Diabetes, December 1, 2006; 55(12): 3611 - 3619. [Abstract] [Full Text] [PDF]
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C. Loeuillet, F. Martinon, C. Perez, M. Munoz, M. Thome, and P. R. Meylan Mycobacterium tuberculosis Subverts Innate Immunity to Evade Specific Effectors J. Immunol., November 1, 2006; 177(9): 6245 - 6255. [Abstract] [Full Text] [PDF]
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 G. Burnstock Pathophysiology and therapeutic potential of purinergic signaling. Pharmacol. Rev., March 1, 2006; 58(1): 58 - 86. [Abstract] [Full Text] [PDF]
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A. N. Shemon, R. Sluyter, S. L. Fernando, A. L. Clarke, L.-P. Dao-Ung, K. K. Skarratt, B. M. Saunders, K. S. Tan, B. J. Gu, S. J. Fuller, et al. A Thr357 to Ser Polymorphism in Homozygous and Compound Heterozygous Subjects Causes Absent or Reduced P2X7 Function and Impairs ATP-induced Mycobacterial Killing by Macrophages J. Biol. Chem., January 27, 2006; 281(4): 2079 - 2086. [Abstract] [Full Text] [PDF]
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H. Tomioka, C. Sano, K. Sato, K. Ogasawara, T. Akaki, K. Sano, S. S. Cai, and T. Shimizu Combined Effects of ATP on the Therapeutic Efficacy of Antimicrobial Drug Regimens against Mycobacterium avium Complex Infection in Mice and Roles of Cytosolic Phospholipase A2-Dependent Mechanisms in the ATP-Mediated Potentiation of Antimycobacterial Host Resistance J. Immunol., November 15, 2005; 175(10): 6741 - 6749. [Abstract] [Full Text] [PDF]
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 A. J. Myers, B. Eilertson, S. A. Fulton, J. L. Flynn, and D. H. Canaday The Purinergic P2X7 Receptor Is Not Required for Control of Pulmonary Mycobacterium tuberculosis Infection Infect. Immun., May 1, 2005; 73(5): 3192 - 3195. [Abstract] [Full Text] [PDF]
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 R. Coutinho-Silva, L. Stahl, K.-K. Cheung, N. E. de Campos, C. de Oliveira Souza, D. M. Ojcius, and G. Burnstock P2X and P2Y purinergic receptors on human intestinal epithelial carcinoma cells: effects of extracellular nucleotides on apoptosis and cell proliferation Am J Physiol Gastrointest Liver Physiol, May 1, 2005; 288(5): G1024 - G1035. [Abstract] [Full Text] [PDF]
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L. C. Denlinger, G. Angelini, K. Schell, D. N. Green, A. G. Guadarrama, U. Prabhu, D. B. Coursin, P. J. Bertics, and K. Hogan Detection of Human P2X7 Nucleotide Receptor Polymorphisms by a Novel Monocyte Pore Assay Predictive of Alterations in Lipopolysaccharide-Induced Cytokine Production J. Immunol., April 1, 2005; 174(7): 4424 - 4431. [Abstract] [Full Text] [PDF]
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J. J. Endsley, J. L. Furrer, M. A. Endsley, M. A. McIntosh, A. C. Maue, W. R. Waters, D. R. Lee, and D. M. Estes Characterization of Bovine Homologues of Granulysin and NK-lysin J. Immunol., August 15, 2004; 173(4): 2607 - 2614. [Abstract] [Full Text] [PDF]
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B. J. Gu, R. Sluyter, K. K. Skarratt, A. N. Shemon, L.-P. Dao-Ung, S. J. Fuller, J. A. Barden, A. L. Clarke, S. Petrou, and J. S. Wiley An Arg307 to Gln Polymorphism within the ATP-binding Site Causes Loss of Function of the Human P2X7 Receptor J. Biol. Chem., July 23, 2004; 279(30): 31287 - 31295. [Abstract] [Full Text] [PDF]
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 P. J. Hanley, B. Musset, V. Renigunta, S. H. Limberg, A. H. Dalpke, R. Sus, K. M. Heeg, R. Preisig-Muller, and J. Daut Extracellular ATP induces oscillations of intracellular Ca2+ and membrane potential and promotes transcription of IL-6 in macrophages PNAS, June 22, 2004; 101(25): 9479 - 9484. [Abstract] [Full Text] [PDF]
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Z. A. Pfeiffer, M. Aga, U. Prabhu, J. J. Watters, D. J. Hall, and P. J. Bertics The nucleotide receptor P2X7 mediates actin reorganization and membrane blebbing in RAW 264.7 macrophages via p38 MAP kinase and Rho J. Leukoc. Biol., June 1, 2004; 75(6): 1173 - 1182. [Abstract] [Full Text] [PDF]
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 T. F. Pais and R. Appelberg Induction of Mycobacterium avium growth restriction and inhibition of phagosome-endosome interactions during macrophage activation and apoptosis induction by picolinic acid plus IFN{gamma} Microbiology, May 1, 2004; 150(5): 1507 - 1518. [Abstract] [Full Text] [PDF]
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 L. C. Denlinger, K. Schell, G. Angelini, D. Green, A. Guadarrama, U. Prabhu, D. B. Coursin, K. Hogan, and P. J. Bertics A novel assay to detect nucleotide receptor P2X7 genetic polymorphisms influencing numerous innate immune functions Innate Immunity, April 1, 2004; 10(2): 137 - 142. [Abstract] [PDF]
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B. M. Saunders, S. L. Fernando, R. Sluyter, W. J. Britton, and J. S. Wiley A Loss-of-Function Polymorphism in the Human P2X7 Receptor Abolishes ATP-Mediated Killing of Mycobacteria J. Immunol., November 15, 2003; 171(10): 5442 - 5446. [Abstract] [Full Text] [PDF]
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L. C. Denlinger, J. A. Sommer, K. Parker, L. Gudipaty, P. L. Fisette, J. W. Watters, R. A. Proctor, G. R. Dubyak, and P. J. Bertics Mutation of a Dibasic Amino Acid Motif Within the C Terminus of the P2X7 Nucleotide Receptor Results in Trafficking Defects and Impaired Function J. Immunol., August 1, 2003; 171(3): 1304 - 1311. [Abstract] [Full Text] [PDF]
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J. S. Wiley, L.-P. Dao-Ung, C. Li, A. N. Shemon, B. J. Gu, M. L. Smart, S. J. Fuller, J. A. Barden, S. Petrou, and R. Sluyter An Ile-568 to Asn Polymorphism Prevents Normal Trafficking and Function of the Human P2X7 Receptor J. Biol. Chem., May 2, 2003; 278(19): 17108 - 17113. [Abstract] [Full Text] [PDF]
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L. M. Sly, S. M. Hingley-Wilson, N. E. Reiner, and W. R. McMaster Survival of Mycobacterium tuberculosis in Host Macrophages Involves Resistance to Apoptosis Dependent upon Induction of Antiapoptotic Bcl-2 Family Member Mcl-1 J. Immunol., January 1, 2003; 170(1): 430 - 437. [Abstract] [Full Text] [PDF]
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G. Fenhalls, L. Stevens, L. Moses, J. Bezuidenhout, J. C. Betts, P. v. Helden, P. T. Lukey, and K. Duncan In Situ Detection of Mycobacterium tuberculosis Transcripts in Human Lung Granulomas Reveals Differential Gene Expression in Necrotic Lesions Infect. Immun., November 1, 2002; 70(11): 6330 - 6338. [Abstract] [Full Text] [PDF]
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D. H. Canaday, R. Beigi, R. F. Silver, C. V. Harding, W. H. Boom, and G. R. Dubyak ATP and Control of Intracellular Growth of Mycobacteria by T Cells Infect. Immun., November 1, 2002; 70(11): 6456 - 6459. [Abstract] [Full Text] [PDF]
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L. Duan, H. Gan, D. E. Golan, and H. G. Remold Critical Role of Mitochondrial Damage in Determining Outcome of Macrophage Infection with Mycobacterium tuberculosis J. Immunol., November 1, 2002; 169(9): 5181 - 5187. [Abstract] [Full Text] [PDF]
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 M. L. Smart, R. G. Panchal, D. N. Bowser, D. A. Williams, and S. Petrou Pore formation is not associated with macroscopic redistribution of P2X7 receptors Am J Physiol Cell Physiol, July 1, 2002; 283(1): C77 - C84. [Abstract] [Full Text] [PDF]
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