HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Stober, C. B. Articles by McArdle, C. A. Search for Related Content PubMed PubMed Citation Articles by Stober, C. B. Articles by McArdle, C. A.

ATP-Mediated Killing of Mycobacterium bovis Bacille Calmette-Guérin Within Human Macrophages Is Calcium Dependent and Associated with the Acidification of Mycobacteria-Containing Phagosomes¹

Carmel B. Stober^*, David A. Lammas^2,*, Cheuk M. Li^*, Dinikantha S. Kumararatne^*, Stafford L. Lightman and Craig A. McArdle

^* 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

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We previously demonstrated that extracellular ATP stimulated macrophage death and mycobacterial killing within Mycobacterium bovis Bacille Calmette-Guérin (BCG)-infected human macrophages. ATP increases the cytosolic Ca²⁺ concentration in macrophages by mobilizing intracellular Ca²⁺ via G protein-coupled P2Y receptors, or promoting the influx of extracellular Ca²⁺ via P2X purinoceptors. The relative contribution of these receptors and Ca²⁺ sources to ATP-stimulated macrophage death and mycobacterial killing was investigated. We demonstrate that 1) ATP mobilizes Ca²⁺ in UTP-desensitized macrophages (in Ca²⁺-free medium) and 2) UTP but not ATP fails to deplete the intracellular Ca²⁺ store, suggesting that the pharmacological properties of ATP and UTP differ, and that a Ca²⁺-mobilizing P2Y purinoceptor in addition to the P2Y₂ subtype is expressed on human macrophages. ATP and the Ca²⁺ ionophore, ionomycin, promoted macrophage death and BCG killing, but ionomycin-mediated macrophage death was inhibited whereas BCG killing was largely retained in Ca²⁺-free medium. Pretreatment of cells with thapsigargin (which depletes inositol (1,4,5)-trisphosphate-mobilizable intracellular stores) or 1,2-bis-(2-aminophenoxy)ethane-N, N, N',N'-tetraacetic acid acetoxymethyl ester (an intracellular Ca²⁺ chelator) failed to inhibit ATP-stimulated macrophage death but blocked mycobacterial killing. Using the acidotropic molecular probe, 3-(2,4-dinitroanilino)-3'-amino-N-methyl dipropylamine, it was revealed that ATP stimulation promoted the acidification of BCG-containing phagosomes within human macrophages, and this effect was similarly dependent upon Ca²⁺ mobilization from intracellular stores. We conclude that the cytotoxic and bactericidal effects of ATP can be uncoupled and that BCG killing is not the inevitable consequence of death of the host macrophage.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Macrophages express plasma membrane P2 purinoceptors that are activated by the extracellular nucleotides ATP and UTP. Two mechanistically distinct subfamilies of P2 purinoceptors have been described: 1) G protein-coupled metabotropic P2Y purinoceptors, and 2) ligand-gated ionotropic P2X purinoceptors (1, 2). The presence of at least two P2 purinoceptor subtypes has been demonstrated in macrophages (3, 4, 5, 6), including G protein-coupled P2Y₂ purinoceptors that are equally activated by ATP and UTP (7, 8, 9), and ionotropic P2X₇ purinoceptors (formerly P2Z purinoceptors; Ref. 10), which are ATP activated and UTP insensitive (4, 5, 6, 10, 11). P2Y₂ purinoceptors act via heterotrimeric G proteins to stimulate phospholipase C (PLC),³ which hydrolyzes phosphatidylinositol 4,5 bisphosphate, generating inositol 1,4,5 trisphosphate (Ins (1, 4, 5)P₃) (7, 8, 9). Ins (1, 4, 5)P₃ then increases the cytosolic calcium concentration ([Ca²⁺]_c) following binding to Ins (1, 4, 5)P₃ receptors located on the endoplasmic reticulum. The activation of P2X₇ purinoceptors by low concentrations of ATP (1–100 µM) causes the specific entry of extracellular Ca²⁺ (eCa²⁺) across the plasma membrane (4, 5, 6, 10, 11), but sustained stimulation with high concentrations of ATP results in the formation of large pores in the plasma membrane permeable to molecules of up to 900 Da (10, 11, 12).

We previously demonstrated that the addition of extracellular ATP (ATP_e) 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 ATP_e (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 ATP_e 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.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents

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.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effects of ATP and UTP on [Ca²⁺]_c in human macrophages

ATP_e 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. 1a) 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).

View larger version (23K): [in this window] [in a new window]   FIGURE 1. Effects of UTP (a) and ATP (b) on [Ca2+]c in human macrophages in the absence or presence of eCa2+. Fura-2-loaded human macrophages were stimulated with 1 mM UTP (a) or 1 mM ATP (b) in Ca2+-containing or Ca2+-free PSS. UTP and ATP both caused spike-plateau type increases in [Ca2+]c. The ATP-mediated Ca2+ increase represented by the plateau phase of the response was more substantially reduced than the UTP-stimulated response in Ca2+-free medium, most probably due to ATP promoting the entry of eCa2+ via ionotropic P2X purinoceptors rather than being solely due to the capacitative entry of eCa2+ following the depletion of the iCa2+ store. The data show mean ± SEM from 3–8 independent experiments using human monocyte-derived macrophages derived from the same human donors.

View larger version (19K): [in this window] [in a new window]   FIGURE 2. ATP-mediated increased [Ca2+]c in UTP-desensitized human macrophages. Fura-2-loaded human macrophages were prestimulated with 100 µM UTP in Ca2+-containing PSS (a and b). The cells were then stimulated with either 1 mM ATP in Ca2+-free medium (a) or 1 mM UTP in Ca2+-containing medium (b). Single cell data showed that UTP failed to mobilize Ca2+ in a UTP-prestimulated macrophage (b), whereas ATP promoted a pronounced increase in Ca2+ within a UTP-stimulated macrophage from the same human donor (a). These data suggest that human macrophages possess additional ATP-activated P2Y purinoceptors other than the P2Y2 receptor subtype. These single cell data are representative of numerous observations seen within 3–6 independent experiments with similar results.

View larger version (28K): [in this window] [in a new window]   FIGURE 3. Ca2+ ionophore mediated increased [Ca2+]c in nucleotide prestimulated human macrophages. Fura-2-labeled human macrophages were prestimulated with either medium alone (a), 1 mM ATP (b), or 1 mM UTP (c) (in Ca2+-free PSS) followed by activation with 5 µM of the Ca2+ ionophore, ionomycin, also in Ca2+-free medium. Ionomycin mobilized iCa2+ similarly in human macrophages prestimulated with medium alone or UTP. However, there was a pronounced decrease in the ionomycin-mediated Ca2+ response on prestimulating macrophages with ATP. Therefore, the data demonstrate that P2Y2 purinoceptors (as shown by UTP-mediated Ca2+ mobilization) are desensitized before complete depletion of the iCa2+ store, whereas ATP must activate a P2Y purinoceptor subtype in addition to P2Y2 to prevent iCa2+ mobilization by ionomycin. The data show mean ± SEM from three independent experiments using macrophages derived from the same human donors.

View larger version (19K): [in this window] [in a new window]   FIGURE 4. Thapsigargin pretreatment of human macrophages prevents the ATP-mediated mobilization of iCa2+. Fura-2-loaded human macrophages in Ca2+-free PSS were pretreated with either medium alone (a) or with 1 µM thapsigargin (b) for 7–8 min at 37°C. The macrophages were then stimulated with 1 mM ATP in Ca2+-free PSS. Thapsigargin pretreatment completely prevented the ATP-stimulated increase in [Ca2+]c verifying that ATP and thapsigargin both mobilize Ca2+ from a common Ins (1,4,5)P3-sensitive intracellular pool. The data represent mean ± SEM from three independent experiments using macrophages derived from the same human donors.

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.

View larger version (36K): [in this window] [in a new window]   FIGURE 5. Ca2+ ionophore- and ATP-mediated macrophage death (a) and BCG killing (b) in the absence or presence of eCa2+. Human macrophages labeled with 51Cr and/or infected with BCG were resuspended in Ca2+-containing or Ca2+-free HBSS. The radiolabeled/infected macrophages were stimulated with either ATP (3 mM) or ionomycin (5 µM) in Ca2+-containing or Ca2+-free HBSS for 30 min at 37°C. The macrophages were washed and incubated overnight, supernatants were removed, and pellets lysed for the determination of the specific release of radioactive 51Cr to establish macrophage viability (a) or microcolony-forming unit assessment to examine mycobacterial viability (b). ATP and ionomycin both promoted macrophage death with a concomitant reduction in the viability of the intracellular BCG, but the cytotoxic and bactericidal effects of ionomycin (but not ATP) were blocked (a, macrophage death) or reduced (b, BCG killing) in Ca2+-free medium. The data demonstrate that the mobilization of iCa2+ is sufficient to cause death of the intracellular mycobacteria but not of the host macrophages. The data show mean ± SEM (n = 4) from an independent experiment that was repeated on four occasions with similar results.

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.

View larger version (23K): [in this window] [in a new window]   FIGURE 6. Effects of thapsigargin and BAPTA/AM on ATP-mediated macrophage death (a) and mycobacterial killing (b) in BCG-infected human macrophages. Macrophages labeled with 51Cr and/or infected with BCG were resuspended in Ca2+-containing (BAPTA/AM only) or Ca2+-free HBSS and were preincubated for 30 min at 37°C with 5 µM thapsigargin (which depletes Ins (1,4,5)P3-sensitive Ca2+ stores) or 2 µM BAPTA/AM (a chelator of iCa2+). The macrophages were then stimulated for 30 min at 37°C with 3 mM ATP, the cells washed, incubated overnight, supernatants removed, and pellets lysed for the determination of macrophage viability (specific 51Cr release, a), or mycobacterial viability (microcolony-forming unit assessment, b). ATP-mediated macrophage death was not dependent on the mobilization of iCa2+ as shown by a lack of inhibition imparted by either thapsigargin or BAPTA/AM. However, mycobacterial killing promoted by ATP was dependent on the mobilization of iCa2+ as both thapsigargin and BAPTA/AM inhibited the bactericidal effects of ATP. The data are representative of results (mean ± SEM; n = 4) obtained within individual experiments that were replicated on 3–4 occasions with similar results.

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.

View larger version (23K): [in this window] [in a new window]   FIGURE 7. Bar graphs illustrating the distribution of DAMP (a measure of vacuole acidification) in M. bovis BCG-infected macrophages stimulated with medium alone (control), thapsigargin alone, ATP alone, or thapsigargin plus ATP at 4 (a) or 6 h (b) post stimulation. Human macrophages infected with BCG were resuspended in Ca2+-free HBSS and were preincubated for 30 min at 37°C with 5 µM thapsigargin before stimulation with 3 mM ATP for 30 min at 37°C. The macrophages were washed and replaced in complete medium for 3.5 (a) or 5.5 (b) h post-ATP stimulation, and the samples were processed as described in Materials and Methods. The data are presented as the percentage of BCG-containing vacuoles containing <2 (0–1), <5 (2–4), <10 (5–9), <20 (10–19), or <40 (20–39) gold particles. BCG-containing compartments within control macrophages contained fewer than five gold particles, suggesting that these vacuoles were not acidic. ATP stimulation of BCG-infected macrophages promoted an increase in the acquisition of gold particles to mycobacterial phagosomes, as shown by a right shift in the graphs, indicative of enhanced vacuole acidification. Thapsigargin pretreatment inhibited the ATP-mediated acquisition of gold particles to BCG-containing phagosomes. The data show that ATP stimulation promotes the acidification of mycobacterial phagosomes, which is dependent upon the mobilization of Ca2+ from intracellular stores.

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.

View larger version (203K): [in this window] [in a new window]   FIGURE 8. Electron micrographs of human monocyte-derived macrophages infected with M. bovis BCG and labeled with DAMP to reveal vacuole acidification. a, Electron-dense lysosomes within human macrophages labeled with DAMP and visualized using anti-DNP mAb. b, Mycobacterial phagosomes within ATP-stimulated macrophages at 4 h post stimulation. The BCG show some colocalization with gold particles, indicative of vacuole acidification, in the absence of morphological damage to the mycobacteria. c, Mycobacterial phagosomes within ATP-stimulated macrophages at 6 h post stimulation. The BCG that are colocalized with gold particles (i.e., are acidified) demonstrate pronounced morphological damage, suggesting that vacuole acidification is accompanied by mycobacterial killing and degradation. An electron-dense lysosome (arrow) appears to be fusing with one of the mycobacterial phagosomes (labeled m). Size bars = 200 nm.

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.

View larger version (34K): [in this window] [in a new window]   FIGURE 9. Detection of mRNA for the P2Y11 purinoceptor subtype in human macrophages. THP-1 cells or primary human macrophages were prestimulated with or without 100 U/ml of IFN- for 24 h, total RNA was isolated and used for reverse transcriptase-PCR as described in Materials and Methods. a, The P2Y11 primer pair identified the predicted 234 bp product in THP-1 cells pretreated with IFN-, "high responder" (HR) primary human macrophages in the absence or presence of IFN-, and "low responder" (LR) macrophages with and without IFN- pretreatment. b, -actin reactions were performed as RNA integrity controls and amplified the predicted 309 bp product.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

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 ATP_e 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.

	Acknowledgments

 
We are very grateful for the invaluable advice provided by Dr. David G. Russell (Washington University School of Medicine) and George R. Dubyak (Case Western Reserve University). We thank Lesley Tompkins (University of Birmingham, U.K.) for the electron microscopy studies undertaken and Dr. Susan Searle (University of Cambridge, U.K.) for help with electron micrographs.

	Footnotes

 
¹ These studies were supported by the Glaxo-Wellcome Action Tuberculosis Research Initiative "Investigation of mechanisms of human anti-tuberculous immunity" (01/04/94 to 31/12/98) and the Medical Research Council of the U.K. (GP95-17110PB).

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⁺; ATP_e, 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.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Abbracchio, M. P., G. Burnstock. 1994. Purinoceptors: are there families of P2X and P2Y purinoceptors?. Pharmacol. Ther. 64:445.[Medline]
2. Fredholm, B. B., M. P. Abbracchio, G. Burnstock, J. W. Daly, T. K. Harden, K. A. Jacobson, P. Leff, M. Williams. 1994. Nomenclature and classification of purinoceptors. Pharmacol. Rev. 46:143.[Medline]
3. Dubyak, G. R., C. El-Moatassim. 1993. Signal transduction via P2 purinergic receptors for extracellular ATP and other nucleotides. Am. J. Physiol. 265:C577.[Abstract/Free Full Text]
4. Alonso-Torre, S. R., A. Trautman. 1993. Calcium responses elicited by nucleotides in macrophages: interaction between two receptor subtypes. J. Biol. Chem. 268:18640.[Abstract/Free Full Text]
5. Greenberg, S., F. Di Virgilio, T. H. Steinberg, S. C. Silverstein. 1988. Extracellular nucleotides mediate Ca²⁺ fluxes in J774 macrophages by two distinct mechanisms. J. Biol. Chem. 263:10337.[Abstract/Free Full Text]
6. Falzoni, S., M. Munerati, D. Ferrari, S. Spisani, S. Moretti, F. Di Virgilio. 1995. The purinergic P2Z receptor of human macrophage cells: characterisation and possible physiological role. J. Clin. Invest. 95:1207.
7. Dubyak, G. R., D. S. Cowen, L. M. Meuller. 1988. Activation of inositol phospholipid breakdown in HL60 cells by P2-purinergic receptors for extracellular ATP: evidence for mediation by both pertussis toxin-sensitive and pertussis toxin-insensitive mechanisms. J. Biol. Chem. 263:18108.[Abstract/Free Full Text]
8. Lustig, K. D., A. K. Shiau, A. J. Brake, D. Julius. 1993. Expression cloning of an ATP receptor from mouse neuroblastoma cells. Proc. Natl. Acad. Sci. USA 90:5113.[Abstract/Free Full Text]
9. Erb, L., R. Garrad, Y. Wang, T. Quinn, J. T. Turner, G. A. Weisman. 1995. Site-directed mutagenesis of P2U purinoceptors: positively charged amino acids in transmembrane helices 6 and 7 affect agonist potency and specificity. J. Biol. Chem. 270:4185.[Abstract/Free Full Text]
10. Surprenant, A., F. Rassendren, E. Kawashima, R. A. North, G. Buell. 1996. The cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor (P2X₇). Science 272:735.[Abstract]
11. Rassendren, F., G. N. Buell, C. Virginio, G. Collo, R. A. North, A. Surprenant. 1997. The permeabilising ATP receptor, P2X₇: cloning and expression of a human cDNA. J. Biol. Chem. 272:5482.[Abstract/Free Full Text]
12. Steinberg, T. H., A. S. Newman, J. A. Swanson, S. C. Silverstein. 1987. ATP^4- permeabilises the plasma membrane of mouse macrophages to fluorescent dyes. J. Biol. Chem. 262:8884.[Abstract/Free Full Text]
13. Lammas, D. A., C. Stober, C. J. Harvey, N. Kendrick, S. Panchalingham, D. S. Kumararatne. 1997. ATP-induced killing of mycobacteria by human macrophages is mediated by purinergic P2Z (P2X₇) receptors. Immunity 7:433.[Medline]
14. Molloy, A., P. Laochumroonvorapong, G. Kaplan. 1994. Apoptosis, but not necrosis of infected monocytes is coupled with killing of intracellular BCG. J. Exp. Med. 180:1499.[Abstract/Free Full Text]
15. Lammas, D. A., S. Ragno, C. Stober, I. Estrada, A. Ben-Smith, J. Colston, and D. S. Kumararatne. 1998. ATP-induced death of M. tuberculosis-infected human and murine macrophages results in marked loss of mycobacterial viability. In Clinical Mycobacteriology: Basic Investigations. M. Casal, ed. Prous Science, Philadelphia, p.107.
16. Kusner, D. J., J. Adams. 2000. ATP-induced killing of virulent Mycobacterium tuberculosis within human macrophages requires phospholipase D. J. Immunol. 164:379.[Abstract/Free Full Text]
17. Enarson, D. A., and J. F. Murray. 1996. Global epidemiology of tuberculosis. In Tuberculosis. W.M. Rom and S.M. Garay, eds. Little, Brown, New York, p. 57.
18. Armstrong, J. A., P. D. Hart. 1971. Response of cultured macrophages to M. tuberculosis with observations on fusion of lysosomes with phagosomes. J. Exp. Med. 134:713.[Abstract]
19. Clemens, D. L., M. A. Horwitz. 1995. Characterisation of the Mycobacterium tuberculosis phagosome and evidence that phagosomal maturation is inhibited. J. Exp. Med. 181:257.[Abstract/Free Full Text]
20. Via, L. E., D. Deretic, R. J. Ulmer, N. S. Hibler, L. A. Huber, V. Deretic. 1997. Arrest of mycobacterial phagosome maturation is caused by a block in vesicle fusion between stages controlled by rab 5 and rab 7. J. Biol. Chem. 272:13326.[Abstract/Free Full Text]
21. Xu, S., A. Cooper, S. Sturgill-Koszycki, T. Van Heyningen, D. Chatterjee, I. Orme, P. Allen, D. G. Russell. 1994. Intracellular trafficking in Mycobacterium tuberculosis and Mycobacterium avium- infected macrophages. J. Immunol. 153:2568.[Abstract]
22. Sturgill-Koszycki, S., P. H. Schlesinger, P. Chakraborty, P. L. Haddix, H. L. Collins, A. K. Fok, R. D. Allen, S. L. Gluck, J. Heuser, D. G. Russell. 1994. Lack of acidification in Mycobacterium phagosomes produced by exclusion of the vesicular proton-ATPase. Science 263:678.[Abstract/Free Full Text]
23. Crowle, A. J., R. Dahl, E. Ross, M. H. May. 1991. Evidence that vesicles containing living, virulent Mycobacterium tuberculosis or Mycobacterium avium in cultured human macrophages are not acidic. Infect. Immun. 59:1823.[Abstract/Free Full Text]
24. Malik, Z. A., G. M. Denning, D. J. Kusner. 2000. Inhibition of Ca²⁺ signalling by Mycobacterium tuberculosis is associated with reduced phagosome-lysosome fusion and increased survival within human macrophages. J. Exp. Med. 191:287.[Abstract/Free Full Text]
25. Fazal, N., D. A. Lammas, M. Rahelu, A. D. Pithie, J. S. Gaston, D. S. Kumararatne. 1995. Lysis of human macrophages by CD4⁺ T cells fails to affect survival of intracellular Mycobacterium bovis-Bacille Calmette-Guerin (BCG). Clin. Exp. Immunol. 99:82.[Medline]
26. McArdle, C. A., R. Bunting, W. T. Mason. 1992. Dynamic video imaging of cytosolic calcium in the -T3–1, gonadotrope-derived cell line. Mol. Cell. Neurosci. 3:124.
27. Post, S. R., L. C. Rump, A. Zambon, R. J. Hughes, M. D. Buda, J. P. Jacobson, C. C. Kao, P. A. Insel. 1998. ATP activates cAMP production via multiple purinergic receptors in MDCK-D1 epithelial cells: blockade of an autocrine/paracrine pathway to define receptor prefe