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*
Medical Research Council Center for Immune Regulation, Division of Immunity and Infection, University of Birmingham, Birmingham, United Kingdom; and
Division of Medicine, University of Bristol, Bristol, 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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We previously demonstrated that the addition of extracellular ATP (ATPe) to Mycobacterium bovis bacille Calmette-Guérin (BCG)-infected human macrophages resulted in macrophage death with a concomitant reduction in the viability of the intracellular mycobacteria (13), and this confirmed the earlier observations of Molloy et al. (14). Recent studies showed that virulent intracellular Mycobacterium tuberculosis was also susceptible to killing by ATPe (15, 16). One-third of the world’s population is currently estimated to harbor M. tuberculosis, the causative agent of tuberculosis, and there are 8 million new cases and 3 million deaths annually from this disease (17). An increase in the incidence of tuberculosis has been compounded by the HIV epidemic and the occurrence of multidrug-resistant strains of tuberculosis (17). Therefore, the mechanism by which ATPe promotes mycobacterial killing within infected human macrophages may provide a target for the design of novel immunotherapeutics in the treatment of multidrug resistant strains of tuberculosis.
Mycobacteria are facultative intracellular bacteria that survive within host macrophages by modulating the internal environment of the cytosolic compartment in which they reside. This compartment retains features of phagosomes and/or early endosomes, failing to acquire lysosomal markers such as lysosomal-associated membrane glycoproteins, the small GTP-binding protein, rab 7, and cathepsin D (18, 19, 20, 21, 22, 23). Furthermore, mycobacterial phagosomes are only mildly acidified and the vesicular proton pump required for vacuole acidification is excluded (21, 22, 23). The mechanism by which mycobacteria-containing organelles evade phagosome-lysosome fusion (PL-F) and acidification has not been fully elucidated. Recent evidence provided by Malik et al. (24) showed that an increase in the [Ca2+]c within macrophages infected with M. tuberculosis was associated with enhanced PL-F and reduced survival of the bacteria within host cells.
Given the likely expression of at least two types of Ca2+-mobilizing ATP receptors on macrophages, we investigated the relationship between the effects of ATP on Ca2+ mobilization and the killing of BCG within infected human macrophages. The data presented show that P2X7 receptor-mediated macrophage death is Ca2+ independent, whereas the killing of the intracellular mycobacteria is Ca2+ dependent. Although the entry of eCa2+ via the P2X7 purinoceptor subtype cannot be excluded as a mechanism sufficient to kill intracellular mycobacteria, we demonstrate that the mobilization of intracellular Ca2+ (iCa2+) via an ATP-activated P2Y purinoceptor other than P2Y2 may also be operating to mediate the bactericidal effects of ATP. Furthermore, mycobacterial phagosomes within ATP-stimulated macrophages are acidified, and this process is also similarly Ca2+ dependent.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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EGTA was obtained from Sigma-Aldrich (Poole, Dorset, U.K.). Thapsigargin was obtained from Research Biochemicals (Natick, MA). ATP, sodium salt was obtained from Boehringer Mannheim (East Sussex, U.K.). Ionomycin was obtained from Novabiochem/Calbiochem (Nottingham, U.K.). BAPTA/AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester), 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine (DAMP), and fura-2/AM were obtained from Molecular Probes (Eugene, OR).
Bacterial culture
Stock cultures of BCG (Evans strain) were maintained as described previously (13, 25). The concentration of the BCG stock was determined by direct counting using a Thoma counting chamber (Weber Scientific, Hamilton, NJ) under dark ground illumination. Macrophages were infected with BCG at a ratio of 5:1 (bacteria/cells) unless otherwise stated. Intracellular BCG viability was determined by microcolony-forming unit assessment as described previously (13, 25).
Human monocyte-derived macrophage cultures
PBMC were prepared as described previously (13, 25) and resuspended at 2.5 x 106 cells/ml in RPMI 1640 medium (Life Technologies, Paisley, U.K.) containing 5% pooled AB+ male human serum (First Link, West Midlands, U.K.) and 2 mM L-glutamine (Life Technologies) (complete medium). The PBMC were then plated onto either 1) 13-mm glass coverslips (Merck/BDH Chemicals, Leics, U.K.) contained within 24-well tissue culture plates (Sarstedt, Leics, U.K.) (1 ml/well), 2) 22-mm glass coverslips (Merck/BDH) within 12-well tissue culture plates (ICN Biomedicals, Oxfordshire, U.K.) (2 ml/well), or into 3) 96-well round-bottom microtiter plates (Sarstedt) (0.2 ml/well), and allowed to adhere overnight. The coverslips and/or microtiter plates were washed with warm medium to remove nonadherent cells and incubated at 37°C under 5% CO2. The cells were cultured for 5–7 days before infection overnight with BCG opsonized with complement-containing human serum. The extracellular bacteria were removed by washing three times with warm HBSS (Sigma-Aldrich).
Assessment of macrophage cytotoxicity (sodium [51Cr] chromate (51Cr) release)
Human macrophage monolayers cultured for 5–7 days in 96-well round-bottom plates were labeled with 51Cr (ICN Biomedicals) at 2 µCi/well for 16 h as described previously (13, 25). The labeled cells were washed three times in warm HBSS to remove excess 51Cr, resuspended in either HBSS with 1 mM CaCl2 (Ca2+-containing medium) or HBSS without CaCl2 and with 2 mM EGTA (Ca2+-free medium) to chelate residual Ca2+, and treated as described for individual experimental details. Following treatment, the macrophages were washed, replaced with an equal volume of complete medium, and incubated overnight. Supernatants (150 µl/well) were aspirated from individual wells into LP2 plastic counting tubes (Life Sciences International, Hampshire, U.K.), and aliquots of 150 µl of 1% Triton X-100 were added to each well to lyse the remaining adherent cells. The pellets were subsequently aspirated and transferred to LP2 tubes, and the activity of both supernatants and pellets assessed by measurement of 51Cr (cpm) within a gamma counter (1260 Multigamma II; LKB Wallac, Turku, Finland). The specific release of radioactive chromium was then calculated as previously described (25).
Dynamic video imaging of cytosolic calcium
Video imaging of fura-2-loaded human monocyte-derived
macrophages was performed as described previously for 
T3-1 cells
(26). Macrophages cultured on 22-mm glass coverslips for
5–7 days were washed and incubated in 1 ml of physiological saline
solution (PSS; 127 mM NaCl, 1.8 mM CaCl2, 5 mM
KCl, 2 mM MgCl2, 0.5 mM
NaH2PO4, 5 mM
NaHCO3, 10 mM glucose, 0.1% BSA, and 10 mM
HEPES, pH 7.4) containing 2 µM fura-2/AM (final concentration) for 30
min at 37°C. Cells were washed several times in PSS, and the
coverslips loaded into a holder that was fitted into a heating chamber
at 37°C. Cells were suspended in either 500 µl PSS with 1.8 mM
CaCl2 (Ca2+-containing
medium) or in PSS with 100 µM EGTA and no CaCl2
(Ca2+-free medium), and image capture was
performed using Magical hardware (Joyce-Loebl, Gateshead, U.K.), Tardis
software (Joyce-Loebl), and a Nikon Diaphot microscope (Nikon U.K.,
Kingston-upon-Thames, U.K.). The macrophages were excited alternately
at 340 and 380 nm, and emitted light was collected at 510 nm averaging
the data from 16 video frames and subtracting background values before
ratioing. The ratio of fluorescence at 340 and 380 nm was calculated at
a pixel-by-pixel basis and used to determine the
Ca2+ concentration assuming a dissociation
constant of 225 nM for fura-2 and Ca2+ at 37°C.
Calibration was performed as previously described
(26).
DAMP uptake and electron microscopy
Human macrophage monolayers grown for 5–7 days on 13-mm glass coverslips were infected with opsonized BCG for 1–2 h and then washed three times in warm HBSS to remove extracellular bacteria. Cells were resuspended in HBSS without CaCl2 and with 2 mM EGTA (Ca2+-free medium), and were prestimulated for 30 min with 5 µM thapsigargin. ATP (3 mM) was then added to the cultures for 30 min at 37°C, and the macrophages were washed and replaced in complete medium for 3.5 or 5.5 h post-ATP treatment. Thirty minutes before fixation, the cultures were incubated with 50 µM DAMP (final concentration), washed twice in complete medium and twice in PBS, and then fixed in 1 ml per well of 2% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.35, containing 2.5% sucrose, for 60 min at room temperature. The fixed cells were dehydrated in ethanol and embedded in LR White (Agar Scientific, Essex, U.K.). Ultrathin sections were cut and mounted on Formvar (Agar Scientific, Stansted, U.K.)-coated nickel grids, and these mounted sections were processed for immunogold labeling essentially as described elsewhere (23). Briefly, the grids were 1) incubated with 0.1 M HCl for 10 min, 2) washed with water, 3) blocked with 5% normal goat serum in 0.5 M NaCl, 0.01 M sodium phosphate (pH 7.0), 0.1% Tween 20 (solution A) for 20 min, 4) incubated for 1 h at room temperature or 4°C overnight with 5 µg/ml mouse mAb against 2,4-dinitrophenyl (Clone PAK; Vector Laboratories, Peterborough, U.K.) diluted in solution A, 5) washed with solution A, 6) incubated for 1 h at room temperature or 4°C overnight with 1:50 dilutions of goat anti-mouse IgG conjugated with 8 nm colloidal gold (Jackson ImmunoResearch Laboratories, West Grove, PA), 7) washed with solution A, and 8) washed with water. Finally, the grids were stained with 2% aqueous uranyl acetate and the labeled samples viewed in a Jeol 1200EX electron microscope. Control studies revealed that colloidal gold staining was dependent upon the exposure of the macrophages to DAMP and the primary mouse mAb against 2,4-dinitrophenyl (data not shown).
The relative delivery of DAMP to acidic compartments was enumerated by counting vesicles that contained electron-dense gold particles. These vesicles consisted of those that contained BCG and electron-dense compartments that did not (presumably lysosomes), and comparisons were made between treatments. Forty vesicles were counted for gold particle labeling per treatment, and values were plotted as a percentage of compartments containing <2 (0–1), <5 (2, 3, 4), <10 (5, 6, 7, 8, 9), <20 (10, 11, 12, 13, 14, 15, 16, 17, 18, 19), or <40 (20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39) gold particles. The localization of gold particles within vesicles lacking BCG (i.e., lysosomes) was not influenced by the treatments herein; therefore, the data are reported solely for BCG-containing compartments.
RT-PCR for P2Y11
Total RNA was isolated from 7-day-cultured human
monocyte-derived macrophages or human THP-1 monocytic cells
preincubated with or without 100 U/ml rhIFN-
(Genzyme, Cambridge,
MA) for 24 h, using an RNeasy kit (Qiagen, Crawley, U.K.). The RNA
was digested using RNase-free DNase I (Qiagen), and 1 µg of RNA was
incubated with 6 µg of random primers (Life Technologies) at 70°C
for 10 min. This mixture was then reverse transcribed to cDNA in a
total reaction volume of 20 µl containing 4 µl of 5x first strand
buffer (Life Technologies), 2 µl of 0.8 mM dNTPs (Sigma-Aldrich), 2
µl of 10 mM DTT (Life Technologies), 2 µl of 400 U monkey murine
leukemia virus reverse transcriptase (Life Technologies), and 0.5 µl
of 1 U of RNAguard (Amersham Pharmacia Biotech, Little Chalfont, U.K.)
at 42°C for 1 h. The reaction was terminated by heating for 5
min at 95°C. To check for possible DNA contamination, the reactions
were also performed substituting distilled water for reverse
transcriptase. The cDNA was then diluted 1 in 5 in water and 1 µl was
added to 19 µl of a PCR buffer mix containing 2 µl of PCR buffer
(10 mM Tris-HCl, pH 8.3, 5 mM KCl) (Perkin-Elmer, Cambridge, U.K.), 2
µl of 2.5 mM MgCl2 (Perkin-Elmer), 2 µl of
0.8 mM dNTPs (Sigma-Aldrich), 1 µM of each forward and reverse
primer, 1.25 U of Amplitaq (Perkin-Elmer), and 8.75 µl of water. The
P2Y11 primer sequences used were as follows:
P2Y11 forward, 5'-CTG GTG GTT GAG TTC CTG GT-3'
and P2Y11 reverse, 5'-GTT GCA GGT GAA GAG GAA
GC-3' (27). The PCR was performed on a Hybaid thermal
cycler using the following conditions: 95°C for 5 min (denaturation
step), 35 cycles of 95°C for 30 s, 57°C for 1 min, and 72°C
for 1 min, and a final extension step at 72°C for 10 min to generate
a product of 234 bp. A 
-actin PCR was also performed using
macrophage cDNA to assess template quality as described above for
P2Y11. The -actin primer sequences used were
as follows: -actin forward, 5'-TTC AAC TCC ATC ATG AAG TGT GAC
GTG-3', and -actin reverse, 5'-CTA AGT CAT AGT CCG CCT AGA AGC
ATT-3'. The -actin PCR was performed on a Hybaid thermal cycler
using the following conditions: 95°C for 5 min (denaturation step),
35 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for
1 min, and a final extension step at 72°C for 4 min to generate a
product of 309 bp.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ATPe interacts with ligand-gated ionotropic P2X purinoceptors and G protein-coupled metabotropic P2Y purinoceptors on macrophages. The activation of P2X purinoceptors promotes the influx of eCa2+ via ligand-gated cation channels while having no effect on the mobilization of iCa2+ stores. Hence, in Ca2+-free medium, the [Ca2+]c would presumably be unaffected in ATP-stimulated cells expressing only P2X purinoceptors. Conversely, the stimulation of P2Y receptors mobilizes Ca2+ from intracellular stores due to the activation of Ins (1, 4, 5)P3 receptors located on the surface of the endoplasmic reticulum. Therefore, the stimulation of P2Y purinoceptors with ATP would increase the [Ca2+]c both in the absence or presence of eCa2+. Following the depletion of the iCa2+ store, the influx of eCa2+ within cells expressing P2Y purinoceptors results from the capacitative entry of eCa2+ via Ca2+ release-activated Ca2+ channels located within the plasma membrane (28, 29). Macrophages have been demonstrated to express P2Y2 purinoceptors (3, 4, 5, 6), which are equally activated by ATP and UTP (7, 8, 9), and P2X7 purinoceptors, which are ATP-activated and UTP-insensitive (4, 5, 6, 10, 11). Based on the physiological and pharmacological properties of these receptor subtypes, the Ca2+-mobilizing effects of ATP and UTP were examined in fura-2-loaded, human monocyte-derived macrophages in the presence or absence of eCa2+.
In Ca2+-containing medium, UTP (Fig. 1
a) and ATP (Fig. 1b) both caused rapid and biphasic (spike-plateau type)
increases in [Ca2+]c,
although the responses to 1 mM ATP were consistently larger and more
sustained than those to 1 mM UTP. On comparing responses to ATP in the
presence and absence of eCa2+ (Fig. 1b), it was apparent that a large proportion of the
Ca2+ increase in
Ca2+-containing medium (as determined by the
plateau phase of the Ca2+ response) was due to
the entry of eCa2+, presumably via the
P2X7 receptor ligand-gated ion channel. The
plateau phase of the UTP-mediated response in
Ca2+-containing medium (Fig. 1a) was
much less pronounced than that to ATP, most probably due to such entry
being exclusively capacitative rather than occurring due to the opening
of a ligand-gated ion channel. Preliminary dose-response studies (data
not shown) had established that the concentrations of ATP and UTP used
were maximally effective at increasing
[Ca2+]c, and pore
formation was not apparent within this time scale as leakage of fura-2
did not occur (data not shown).
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, a and b). Because ATP and UTP are
reported to be equipotent at P2Y2 purinoceptors
(7, 8, 9) and these receptors have been shown to rapidly
desensitize (30, 31), we next assessed whether such
desensitization could be exploited to further delineate responses
mediated by distinct P2Y purinoceptor subtypes. On examining
nucleotide-mediated single cell responses to verify that human
macrophages were both UTP and ATP sensitive (Fig. 2, a and b), it was
shown that 100 µM UTP promoted an increase in
[Ca2+]c (in
Ca2+-containing medium), but this had returned to
near baseline levels within 1–2 min of stimulation. The addition of 1
mM UTP after 2.5 min (Fig. 2b) did not further increase
[Ca2+]c measurably,
whereas stimulation with 1 mM ATP (in Ca2+-free
medium) (Fig. 2a) caused a pronounced increase in
[Ca2+]c. In repeat
experiments, the initial stimulation of macrophages with UTP (in
Ca2+-containing medium) increased
iCa2+ from 31 ± 6 to 160 ± 35 nM
(n = 6). UTP failed to further increase the
Ca2+ response in UTP prestimulated macrophages
(34 ± 10 to 40 ± 15 nM; n = 3), whereas ATP
stimulation of UTP-desensitized cells (in
Ca2+-free medium) did mobilize
iCa2+ (51 ± 17 to 194 ± 6 nM;
n = 3) reflecting the single cell data presented within
Fig. 2. The ATP-mediated Ca2+ response in
UTP-desensitized macrophages was lower than that mediated by ATP alone
(445 ± 19 nM; n = 4) (data not shown), suggesting
that UTP desensitizes only a subset of ATP-activated P2 purinoceptors
in human macrophages. Furthermore, all UTP-prestimulated macrophages
that subsequently responded to ATP were also UTP-activated; therefore,
this ATP response was not a result of cells being UTP-insensitive.
Thus, it appeared that UTP-activated P2Y2
receptors expressed on human macrophages underwent rapid homologous
desensitization. Such findings were recently demonstrated by Humphreys
and Dubyak (32) with regard to UTP effects on the human
THP-1 monocytic cell line. As complete cross-desensitization of
P2Y2 purinoceptors was not apparent (i.e., ATP
was still able to mobilize
[Ca2+]c in
UTP-desensitized cells in Ca2+-free medium; Fig. 2a), these preliminary observations provided evidence for
the existence of other Ca2+-mobilizing P2Y
purinoceptors on human macrophages in addition to the
P2Y2 receptor subtype.
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a), 1 mM ATP (Fig. 3b), or 1 mM UTP (Fig. 3c) in
Ca2+-free medium, followed by stimulation with 5
µM ionomycin (also in Ca2+-free medium). As
previously demonstrated in Fig. 1, the preliminary
Ca2+ increase promoted by ATP (Fig. 3b) was higher than that generated by UTP (Fig. 3c) (in Ca2+-free medium) within
macrophages derived from the same human donors. The
[Ca2+]c increase
elicited by ionomycin within control macrophages (Fig. 3a)
was similar to that mediated in cells prestimulated with UTP (Fig. 3c) (p > 0.05). In contrast, the
response to ionomycin in ATP-prestimulated macrophages (Fig. 3b) was significantly lower (p <
0.05) than the Ca2+ increase promoted by
ionomycin alone (Fig. 3a). These data further differentiated
the iCa2+-mobilizing responses mediated by ATP
and UTP, revealing that although ATP effectively depleted the
iCa2+ store, UTP failed to do so.
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), thapsigargin
completely prevented the ATP-stimulated increase in
[Ca2+]c (in
Ca2+-free medium), thus confirming that
thapsigargin and ATP mobilize Ca2+ from a common
intracellular pool. The ability of thapsigargin to deplete the
ATP-mobilizable iCa2+ store was used in
subsequent experiments to delineate Ca2+
mobilization and entry in ATP-stimulated macrophages.
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Having demonstrated that ATP not only could simulate Ca2+ entry via ionotropic P2X purinoceptors, but also could mobilize iCa2+ by activating metabotropic P2Y receptors, we sought to establish the relative contribution of these Ca2+ sources to the cytotoxic and bactericidal effects of ATP by 1) examining ATP responses in Ca2+-containing and Ca2+-free medium and 2) comparing ATP-mediated effects to responses promoted by the Ca2+ ionophore, ionomycin, in the presence or absence of eCa2+.
In Ca2+-containing medium, both ATP and ionomycin
stimulated macrophage death (Fig. 5a) with an apparent
concomitant reduction in the viability of the intracellular BCG (Fig. 5b). ATP and ionomycin were ineffective against
extracellular mycobacteria (data not shown), revealing that the
bactericidal effects of these agonists were macrophage-mediated. In
Ca2+-free medium, ionomycin-mediated macrophage
death was completely blocked (Fig. 5a); therefore, the
cytotoxic effect of the Ca2+ ionophore on
macrophages occurred following the influx of
eCa2+ rather than due to the mobilization of
iCa2+. However, the ability of ionomycin to
mediate mycobacterial killing was reduced but not blocked in
Ca2+-free medium, demonstrating that the
mobilization of iCa2+ was sufficient to cause
this bactericidal effect. In contrast, ATP-mediated cytotoxic and
bactericidal effects were not reduced in
Ca2+-free medium (Fig. 5, a and
b, respectively), showing that the influx of
eCa2+ was not responsible for either of these
responses.
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The data above suggested that the elevation of [Ca2+]c was a sufficient stimulus for macrophage death and BCG killing, but the retention of the cytotoxic and bactericidal effects of ATP in Ca2+-free medium implied that these responses were either Ca2+-independent or were dependent upon the mobilization of Ca2+ from intracellular stores (rather than due to the entry of eCa2+ via P2X-activated ion channels). To establish whether ATP-mediated responses were dependent on the mobilization of Ca2+ from intracellular stores, the cytotoxic and bactericidal effects of ATP were examined (in Ca2+-free medium) in macrophages prestimulated with 1) thapsigargin, which depletes Ins (1, 4, 5)P3-sensitive iCa2+ stores, or 2) BAPTA/AM, a cell-permeable chelator of iCa2+.
As shown (Fig. 6), ATP promoted
macrophage death and BCG killing in Ca2+-free
medium, as previously revealed in Fig. 5. Neither thapsigargin
nor BAPTA/AM were themselves cytotoxic (Fig. 6a)
or bactericidal (Fig. 6b), and neither of the inhibitors
prevented ATP-mediated macrophage death (Fig. 6a). Indeed,
the chelation of iCa2+ stores by BAPTA/AM
actually enhanced the cytotoxic effect of ATP, further
demonstrating that ATP-mediated macrophage death was
Ca2+-independent. Conversely, BAPTA/AM alone
enhanced the survival of the intracellular mycobacteria within control
macrophages, whereas both BAPTA/AM and thapsigargin inhibited the
mycobactericidal effects of ATP (Fig. 6b)
(p < 0.05). These data showed that the
mobilization of iCa2+ was linked to both the
survival of intracellular mycobacteria within control macrophages and
the death of mycobacteria within ATP-stimulated macrophages.
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b), whereas the cytotoxic effect of ATP was similar to that
for ATP and BAPTA/AM in Ca2+-free medium (Fig. 6a). These data implied that the influx of
eCa2+ via the P2X7
purinoceptor would probably be sufficient to promote the killing of
intracellular mycobacteria in the absence of an effect contributed by
other Ca2+-mobilizing P2Y purinoceptor
subtypes. Effect of ATP and thapsigargin on the acidification of BCG-containing phagosomes
Mycobacterium species reside within intracellular
compartments in macrophages that do not acquire lysosomal markers
(18, 19, 20, 21) and fail to acidify below pH 6.3–6.5
(21, 22, 23). Based on these observations, we examined whether
ATP-mediated BCG killing coincided with the acidification of
mycobacterial phagosomes. For this study, we used the cell-permeable
probe, DAMP, which accumulates selectively and can be detected within
acidic organelles in intact cells (23). Vacuole
acidification was determined by estimating the number of gold particles
colocalizing with BCG-containing compartments at 4 (Fig. 7a) and 6 h (Fig. 7b) post-ATP stimulation, respectively.
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), the vast majority of BCG-containing compartments
within control, nonstimulated macrophages contained fewer than two gold
particles, suggesting that BCG normally resided within an intracellular
compartment that was not acidic, in agreement with the previous
findings of others (21, 22, 23). Stimulation of infected
macrophages with 3 mM ATP resulted in a pronounced increase in the
acquisition of gold particles to BCG-containing phagosomes, indicative
of vacuole acidification. BCG-containing compartments within
macrophages stimulated with ATP contained proportionally more gold
particles than control macrophages (p < 0.0001
at 4 h and p < 0.005 at 6 h post-ATP
stimulation, respectively). To determine the role of Ins
(1, 4, 5)P3-sensitive Ca2+
stores in the acidification of mycobacterial phagosomes within
ATP-stimulated macrophages, the effect of thapsigargin was then
examined. Thapsigargin treatment of BCG-infected macrophages did not measurably influence the accumulation of gold particles to BCG-containing compartments. However, thapsigargin pretreatment significantly reduced (p < 0.01) the acquisition of gold particles to BCG-containing phagosomes within ATP-stimulated macrophages, such that the number of gold particles colocalized with BCG-containing phagosomes within ATP-stimulated, thapsigargin-pretreated cells was not significantly different (p > 0.05) from the number of gold particles contained within control, nonstimulated macrophages at both time points examined. The data showed that the mobilization of Ins (1, 4, 5)P3-sensitive iCa2+ stores was essential for the ATP-stimulated acidification of mycobacteria-containing phagosomes in Ca2+-free medium.
Electron microscopic analysis of macrophages labeled with the
acidotropic probe, DAMP, revealed electron-dense vesicles resembling
lysosomes colocalizing with gold particles (Fig. 8a). The acquisition of gold
particles to these electron-dense vesicles was not influenced by the
stimulation of macrophages with ATP, suggesting that ATP treatment of
macrophages alone did not enhance organelle fusion (data not shown).
Mycobacteria-containing phagosomes within ATP-stimulated macrophages
showed enhanced colocalization with gold particles at 4 h post-ATP
stimulation in the absence of obvious morphological damage to the
bacteria (Fig. 8b). At 6 h post-ATP stimulation,
morphological damage to the mycobacteria that colocalized with gold
particles was apparent, as revealed in Fig. 8c. Therefore,
the data provide preliminary evidence demonstrating that the
ATP-mediated acidification of mycobacterial phagosomes is accompanied
by morphological damage and/or degradation of the BCG, correlating with
a pronounced decrease in bacterial viability, as determined by
microcolony-forming unit assessment in Figs. 5 and 6.
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It was demonstrated that BCG-infected human macrophages killed intracellular mycobacteria when stimulated with ATP by a mechanism that was dependent on the mobilization of iCa2+ but was not P2Y2-mediated. The latter was shown by the inability of UTP to promote mycobacterial killing (13). Studies by Jin et al. (34) revealed that in addition to the P2Y2 receptor subtype, human monocytes expressed mRNA for P2Y1, P2Y4, and P2Y6 purinoceptors. Due to the pharmacological properties associated with these purinoceptor subtypes (34), it was unlikely that such receptors were operating to mediate the mycobactericidal effects of ATP demonstrated here. Communi et al. (35, 36) recently described a P2Y receptor expressed on human HL-60 cells, termed P2Y11, that was 1) more potently activated by 3'-O-(4-benzoylbenzoyl)-ATP (BzATP) rather than ATP, 2) was UTP-insensitive, and 3) coupled to PLC to activate Ins (1, 4, 5)P3-sensitive iCa2+ stores. Therefore, we examined whether human macrophages expressed mRNA for this purinoceptor subtype.
Fig. 9 shows that human THP-1 monocytic
cells pretreated with IFN- expressed mRNA for
P2Y11. Furthermore, primary human macrophages
derived from two donors previously determined to be sensitive ("high
responder") or resistant ("low responder") to the cytotoxic and
mycobactericidal effects of ATP (13) were shown to exhibit
equivalent levels of mRNA expression for the
P2Y11 purinoceptor subtype (Fig. 9a).
IFN- pretreatment of human macrophages derived from both donors
failed to promote a demonstrable increase in the levels of
P2Y11 mRNA (Fig. 9a), as previously
has been revealed for the P2X7 purinoceptor subtype
(32), if the levels of -actin were comparable between
samples (Fig. 9b). Thus the P2Y11
purinoceptor subtype provides an additional candidate
Ca2+-mobilizing receptor for further
investigation in the ATP-mediated mycobactericidal events described in
this study.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, the cytotoxic and mycobactericidal effects of ATP following the stimulation of M. bovis BCG-infected human macrophages could be functionally uncoupled. ATP-mediated mycobacterial killing but not macrophage death was dependent on the mobilization of Ca2+ from Ins (1, 4, 5)P3-sensitive iCa2+ stores. Furthermore, ATP-mediated mycobacterial killing was accompanied by the acidification of the mycobacterial phagosome, an event that was observed to be similarly Ca2+-dependent. We present evidence for the existence of ATP-activated P2Y11 purinoceptors on human macrophages, in accordance with the earlier studies of Berchtold et al. (39). These data revealed that intracellular mycobacterial killing could be achieved without concomitant macrophage death and, therefore, established that BCG killing was not simply a consequence of the death of the host cell, but instead reflected a specific and independently regulated event.
The demonstration that mycobacteria could be killed within host macrophages following the mobilization of Ca2+ from intracellular stores was pertinent based on the observation that PL-F has been associated with increased levels of cytosolic calcium in human neutrophils (40) and with endosomal/lysosomal interactions in a number of cell types (41, 42, 43). Jaconi et al. (40) showed that in human neutrophils, PL-F could be inhibited by prior incubation of the cells with the membrane-permeable Ca2+ chelator, BAPTA/AM, and this effect was partly reversed by raising the concentration of eCa2+, as also revealed within the studies described here. Pryor et al. (41) recently proposed that endocytosed Ca2+ released from the lumen of endocytic organelles (such as late endosomes and lysosomes) may be used to mediate membrane fusion events at several stages of the endocytic pathway. There is also evidence demonstrating that lysosomes contain a mobilizable pool of Ca2+ (44) and that endosomal (45) and lysosomal (46) membranes contain Ca2+ transport systems. Furthermore, Malik et al. (24) recently showed that Ca2+ ionophore stimulation of M. tuberculosis-infected human macrophages leads to the demise of the intracellular mycobacteria coincidental with the progression of the bacteria to an intracellular compartment that was lysosomal and acidified. We also found that the stimulation of M. bovis BCG-infected human macrophages with the Ca2+ ionophore, ionomycin, promoted the colocalization of mycobacteria-containing phagosomes with the late endosomal/lysosomal marker, lysosomal-associated membrane glycoprotein-2 (data not shown), which correlated with a decrease in mycobacterial viability. Based on these combined observations, the role of Ca2+ signaling in the survival of mycobacteria within host macrophages warrants further investigation, and the effects of mycobacteria on Ca2+ signaling and subsequent endosomal-lysosomal interactions may prove central to the survival of M. tuberculosis within their host cells.
As UTP failed to promote the killing of M. bovis BCG within infected human macrophages (data not shown) (13, 16), it was unlikely that P2Y2 receptors were the Ca2+-mobilizing purinoceptor involved in the ATP-mediated bactericidal effects described in this study. Therefore, the fact that both ATP and UTP promoted the mobilization of iCa2+ but only ATP stimulated bacterial killing via a mechanism dependent on the mobilization of iCa2+ was paradoxical. However, P2Y2 purinoceptors are down-regulated in macrophages compared with early myeloid progenitors and monocytes (47), and we showed clear differences between the Ca2+-mobilizing effects of ATP and UTP in human macrophages. P2Y2 purinoceptors are rapidly desensitized, and this occurs before complete depletion of the iCa2+ store (31) and studies in cells expressing multiple P2Y receptor subtypes suggest that there are subpools of iCa2+ activated by differential Ins (1, 4, 5)P3-sensitive P2Y receptors (48). With regard to the latter, Haller et al. (44) showed that in MDCK cells, the lysosomal Ca2+ pool could be released by Ins (1, 4, 5)P3-dependent hormones but no such evidence has been reported for P2Y purinoceptor subtypes. Therefore, the lack of effect of UTP on mycobacterial killing may be due to the magnitude of the Ca2+ response promoted by this agonist combined with the Ins (1, 4, 5)P3-sensitive Ca2+ subpool(s) mobilized. Alternatively, the downstream signaling events linked to the activation of differential P2Y purinoceptor subtypes may be central to the mycobactericidal events described here.
Kusner et al. (16) recently showed that ATP-mediated mycobacterial killing was dependent upon the activation of phospholipase D (PLD) in human macrophages, and we have also reported similar findings within our laboratory (49). Studies by Dubyak and colleagues (32, 50) demonstrated that ATP but not UTP activated PLD in murine BAC1.2F5 macrophages and human THP-1 monocytic cells, and the ATP-mediated increased activity of PLD was reduced but not inhibited in Ca2+-free medium in both cell types. However, Gargett et al. (51) reported that ATP-mediated PLD activation in cells derived from individuals with chronic lymphocytic leukemia was absolutely dependent upon the influx of eCa2+. These cells express the P2X7 purinoceptor subtype but lack P2Y purinoceptors; therefore, the ATP-mediated activation of PLD is exclusively due to eCa2+ influx via P2X7 purinoceptors. Based on these observations, PLD activated following either Ca2+ entry (i.e., via the stimulation of ionotropic P2X7 purinoceptors) or due to the mobilization of iCa2+ (i.e., following the activation of metabotropic P2Y purinoceptors) is probably sufficient for the ATP-mediated killing of intracellular mycobacteria described here. Furthermore, PLD activation has also been associated with lysosomal trafficking and the fusion of intracellular organelles (52). An alternative Ca2+-dependent signaling molecule coupled with organelle fusion is calmodulin (53, 54); we found that calmodulin antagonists such as KN-62 and calmidazolium inhibited the bactericidal effects of ATP (data not shown). Additionally, Malik et al. (55) recently revealed that macrophage phagosomes containing live M. tuberculosis contained decreased levels of calmodulin compared with phagosomes encompassing killed tubercle bacilli, suggesting that the defective activation of calmodulin in infected macrophages may be associated with the ability of the intracellular mycobacteria to retard PL-F. Other Ca2+-dependent signaling molecules that may be contributing to ATP-mediated mycobacterial killing and phagosomal-lysosomal interactions in macrophages are currently being investigated in our laboratory.
Consistent with an ATP-activated purinoceptor other than P2X7 as a candidate for mycobacterial killing within host macrophages, Sikora et al. (56) revealed that radicals such as reactive nitrogen intermediates (RNI) were stimulated independently of P2X7 purinoceptors in macrophages derived from P2X7 gene-disrupted mice. Sikora (56) demonstrated that P2 receptor antagonists such as oxidized ATP (oATP) and suramin potently inhibited RNI production and the associated mycobactericidal effects of RNI in inflammatory mediator-stimulated macrophages derived from P2X7 purinoceptor-deficient mice, showing that P2 receptor subtypes other than P2X7 were operating to stimulate the generation of RNI, and that oATP was not a specific P2X7 receptor antagonist. We also reported that oATP and suramin prevented ATP-mediated BCG killing within infected human macrophages (13) and attributed these antagonistic properties to the involvement of P2X7 purinoceptors. However, based on more recent findings described here, the additional involvement of P2 purinoceptors other than P2X7 in ATP-mediated mycobactericidal responses in macrophages is likely. Corroborating evidence for the involvement of a P2X7-independent purinoceptor in ATP-mediated mycobacterial killing was recently provided by Kusner et al. (16), who showed that ATP was more effective than the potent P2X7 agonist, BzATP (10, 11, 50), at promoting killing of virulent M. tuberculosis within human macrophages. This P2X7R-independent mechanism was not due to the stimulation of the P2Y2 purinoceptor subtype as the P2Y2 agonist, UTP, failed to induce this mycobactericidal activity and did not enhance ATP or BzATP-mediated killing of M. tuberculosis (16). In summary, Kusner et al. suggested that P2X7R are necessary, but not sufficient, for maximal ATP-dependent killing of intracellular M. tuberculosis by human macrophages, in agreement with our findings described here.
Recent studies in our laboratory have also examined the effects of ATP on the viability of intracellular mycobacteria contained within macrophages derived from P2X7-/- gene-disrupted mice (data not shown). These studies have shown that although ATP-mediated mycobacterial killing is significantly reduced within such macrophages, a residual level of bactericidal activity remains, suggesting the involvement of an alternative mycobactericidal mechanism. Earlier studies in our laboratory examining mycobactericidal responses within ATP-stimulated hyper P2X7 receptor-expressing J774 murine macrophages (described in Ref. 57) demonstrated that mycobacterial killing was much less effective in these cells when compared with BCG-infected human macrophages, indicating that in murine macrophages, P2X7 purinoceptors are the predominant receptor subtype involved in the ATP-mediated bactericidal response.
Our studies also revealed that a proportion of human donors possessed macrophages that were relatively insensitive to the cytotoxic effects of ATP (13) and these individuals were termed "low responders." The killing of intracellular mycobacteria within low ATP responder macrophages was reduced but not blocked with respect to high responder cells (data not shown), which may reflect a residual P2Y11-like mycobactericidal activity similar to that observed within cells derived from P2X7-/- mice. Recently, Gu et al. (58) described a low ATP response phenotype in human macrophages, resulting from a Glu to Ala polymorphism in the P2X7 carboxyl-terminal tail rendering the receptor nonfunctional. The effect of this polymorphism on the P2X7 receptor-mediated killing of intracellular mycobacteria has not been determined.
The ATP-stimulated acidification of mycobacterial phagosomes described in this study may reflect an increase in the fusogenicity of these vacuoles with organelles containing the vesicular proton pump required for lowering the intraorganellar pH. Schaible et al. (59) recently demonstrated that cytokine activation of M. avium-infected murine macrophages led to the acidification of the bacterial phagosome, and this correlated with an increased accumulation of the vesicular proton pump to these compartments and a concomitant decrease in bacterial viability. An alternative explanation is that within our experiments, the intracellular mycobacteria actively prevented phagosome acidification and/or lysosomal fusion, but these activities ceased when the BCG were killed following ATP stimulation of the host macrophages. Accordingly, the specific mechanism(s) underlying ATP-mediated BCG killing remains undetermined, and it is not yet clear whether the ATP-induced accumulation of M. bovis BCG within an acidified intracellular compartment was a cause or consequence of their demise. However, Gomes et al. (60) showed that the acidification of mycobacterial phagosomes within M. tuberculosis-infected host macrophages led to growth arrest of the bacteria. From our data, it appears that phagosome acidification coupled with lysosomal fusion may provide a suitably noxious environment in which microbicidal mechanisms and acid hydrolases are activated, the net result being the demise of the intracellular mycobacteria.
In summary, we have demonstrated that human monocyte-derived macrophages express both ionotropic (presumably P2X7) and metabotropic (P2Y2 and P2Y11-like) Ca2+-mobilizing purinoceptors, and that ATP acts upon these receptors to mobilize Ca2+ from intracellular stores as well as from the extracellular environment. Activation of these receptors by ATPe stimulates macrophage death and the killing of intracellular mycobacteria, but macrophage death is Ca2+-independent, whereas BCG killing is reliant upon an increase in the [Ca2+]c. Additionally, ATP-mediated bactericidal effects are associated with the acidification of mycobacterial phagosomes, which is similarly a Ca2+-dependent event. We provide evidence for the involvement of a P2X7-independent, ATP-activated Ca2+-mobilizing P2Y purinoceptor in mycobacterial killing but not macrophage death. However, this study does not discount a major role for the P2X7 purinoceptor in ATP-mediated mycobacterial killing but reveals that in the absence of P2X7 receptor activation there is an additional P2Y purinoceptor subtype on host macrophages that can fulfil this role. Thus, mycobacterial killing is not simply the inevitable consequence of death of the host macrophage but is, instead, an independently regulated process.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. David A. Lammas, Medical Research Council Center for Immune Regulation, Division of Immunity and Infection, University of Birmingham, Medical School, Edgbaston, Birmingham, U.K. B15 2TT.
3 Abbreviations used in this paper: PLC, phospholipase C; Ins (1,4,5)P3, inositol 1,4,5 trisphosphate; [Ca2+]c, cytosolic calcium concentration; eCa2+, extracellular Ca2+; ATPe, extracellular ATP; BCG, bacille Calmette-Guérin; PL-F, phagosome-lysosome fusion; iCa2+, intracellular Ca2+; BAPTA/AM, 1, 2-bis-(2-aminophenoxy)ethane-N, N,N',N',-tetraacetic acid acetoxymethyl ester; DAMP, 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine; 51Cr, sodium [51Cr]chromate; PSS, physiological saline solution; BzATP, 3'-O-(4-benzoylbenzoyl)ATP; RNI, reactive nitrogen intermediates; oATP, oxidized ATP.
Received for publication May 5, 2000. Accepted for publication March 15, 2001.
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