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Clinical and Vaccine Immunology, September 2007, p. 1078-1083, Vol. 14, No. 9
1071-412X/07/$08.00+0     doi:10.1128/CVI.00166-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Extracellular ATP Is Cytotoxic to Mononuclear Phagocytes but Does Not Induce Killing of Intracellular Mycobacterium avium subsp. paratuberculosis

Seng-Ryong Woo,¹ Raúl G. Barletta,² and Charles J. Czuprynski^1*

Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin—Madison, Madison, Wisconsin 53706,¹ Department of Veterinary and Biomedical Sciences, University of Nebraska, Lincoln, Nebraska 68583-0905²

Received 18 April 2007/ Returned for modification 6 June 2007/ Accepted 5 July 2007

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Mycobacterium avium subsp. paratuberculosis is the etiologic agent of Johne's disease, a chronic granulomatous enteritis in ruminants. ATP has been reported to induce cell death of macrophages and killing of Mycobacterium species in human and murine macrophages. In this study we investigated the short-term effect of ATP on the viability of M. avium subsp. paratuberculosis-infected bovine mononuclear phagocytes and the bacilli within them. Addition of 5 mM ATP to M. avium subsp. paratuberculosis-infected bovine monocytes resulted in 50% cytotoxicity of bovine monocytes at 24 h. Addition of 2'(3')-O-(4-benzoylbenzoyl) ATP triethylammonium salt (Bz-ATP), which is a longer-lived ATP homologue and purinergic receptor agonist, significantly increased the uptake of YO-PRO, which is a marker for membrane pore activation by P2X receptors. Addition of Bz-ATP also stimulated lactate dehydrogenase release and caspase-3 activity in infected bovine monocytes. Neither ATP nor Bz-ATP reduced the survival of M. avium subsp. paratuberculosis in bovine mononuclear phagocytes. Likewise, addition of ATP or Bz-ATP was cytotoxic to murine macrophage cell lines (RAW 264.7 and J774A.1 cells) but did not affect the intracellular survival of M. avium subsp. paratuberculosis, nor were the numbers of viable Mycobacterium avium subsp. avium or Mycobacterium bovis BCG cells altered in bovine mononuclear phagocytes or J774A.1 cells following ATP or Bz-ATP treatment. These data suggest that extracellular ATP does not induce the killing of intracellular M. avium subsp. paratuberculosis in bovine mononuclear phagocytes.

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ATP can bind to purinergic receptors on the cell surface. Two types of ATP receptors (P2X and P2Y receptors) have been identified. P2Y receptors are metabotropic G-protein-coupled receptors, and P2X receptors are ligand-gated ion channel receptors (9, 27). P2Y receptors do not play a role in ATP-mediated cell death but instead preferentially respond to ADP (8). Activation of the P2X₇ receptor results in the formation of large pores on the cell membrane, leading to cell death (37). Truncation of the final 177 residues of the C-terminal region of P2X₇ prevented ATP-mediated death of HEK293 cells (34). The P2X₁ and adenosine receptors are also reported to be involved in ATP-mediated cell death (2, 3, 6).

Extracellular ATP has been reported to induce the killing of intracellular Mycobacterium tuberculosis or Mycobacterium bovis BCG in murine and human macrophages and to cause the death of the infected cells (10, 18, 19, 21, 25, 33). Bovine monocyte-derived macrophages express mRNA for the bovine P2X₇ receptor, and addition of ATP increased their ability to kill intracellular M. bovis BCG (32). Likewise, it has been reported that the P2X₇ receptor plays a critical role in the ATP-mediated killing of intracellular mycobacteria in human and murine macrophages (10, 31).

Some reports linked the mycobactericidal effect of ATP with the death of the infected macrophages (21, 25, 31). However, other reports suggested that the mycobactericidal effect of ATP and macrophage death are independent events (10, 33). In keeping with the latter scenario, the death of human macrophages following addition of anti-CD95 antibody, anti-CD69 antibody, or anti-major histocompatibility complex class II antibody plus complement or by addition of H₂O₂ did not result in the demise of intracellular M. bovis BCG (21, 25).

In this study, we investigated the short-term effect of ATP addition on the viability of Mycobacterium avium subsp. paratuberculosis-infected bovine mononuclear phagocytes and the bacilli within them. In contrast to previous reports with human mononuclear phagocytes, ATP did not induce the killing of intracellular mycobacteria (i.e., M. avium subsp. paratuberculosis) in bovine mononuclear phagocytes.

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Bacterial culture. Mycobacterium avium subsp. paratuberculosis strain K-10 was grown to a final concentration of 10⁸ CFU/ml in Middlebrook 7H9 broth (Difco Laboratories, Detroit, MI) supplemented with 10% oleic acid-albumin-dextrose-catalase (Becton Dickinson Microbiology System, Sparks, MD), 0.05% Tween 80, and 2 µg/ml of mycobactin J (Allied Laboratories, Ames, IA). After the bacteria were harvested and washed with phosphate-buffered saline (PBS), single-cell suspensions of the bacteria were made by using a motor-driven overhead stirrer and a glass-Teflon homogenizer in a biosafety cabinet. To further remove bacterial clumps, the bacterial suspension was allowed to settle for 30 min and the supernatant was centrifuged at 200 x g for 10 min. The supernatant was resuspended in PBS plus 10% glycerol, aliquoted, and stored at –70°C. The number of viable bacteria in the stock suspension was determined by a radiometric (BACTEC) method or by determination of plate counts (CFU) on 7H10 agar. To differentiate viable and dead bacterial cells microscopically, a Live/Dead Baclight bacterial viability kit (Molecular Probes, Inc., Eugene, OR) was used with the aid of a Petroff-Hauser chamber. Before each assay, an aliquot was thawed, diluted in RPMI 1640 medium without antibiotics, and used to infect monocytes or macrophages in vitro. Mycobacterium avium subsp. avium (ATCC 35712; American Type Culture Collection, Manassas, VA) was prepared as described above. Single-cell suspensions of Mycobacterium bovis BCG (Pasteur strain; Statens Serum Institute) were obtained from M. Sandor (Madison, WI).

Cell culture. To isolate bovine monocytes, blood was collected from the tail veins of healthy donor cows, with sodium citrate (final concentration, 0.38% [vol/vol]) used as the anticoagulant (38). The blood was centrifuged at 400 x g for 30 min, and the plasma was removed. The buffy coat cells were resuspended in 35 ml of Hanks balanced salt solution (HBSS; Mediatech, Inc., Herndon, VA) containing 4 mM EDTA, layered over 15 ml of Ficoll-Histopaque 1083 (Sigma Diagnostic, Inc., St. Louis, MO), and centrifuged at room temperature at 600 x g for 40 min. The mononuclear cells were collected from the interface, and the residual red cells were lysed with lysis buffer (150 mM NH₄Cl and 10 mM Tris, pH 7.5) and washed three times with HBSS. The isolated mononuclear cells were resuspended in RPMI 1640 medium (Mediatech, Inc.) supplemented with 1% fetal bovine serum (FBS) and adjusted to a concentration of 3 x 10⁶ cells/ml. The cells were distributed (1 ml per well) into individual wells of a 24-well tissue culture plate or a 96-well tissue culture plate (Falcon; BD Labware, Franklin Lakes, NJ). The monocytes were allowed to adhere for 2 h at 37°C in 5% CO₂, and the nonadherent cells were removed by washing the plates with warm RPMI 1640 medium. The monocytes were cultured in RPMI 1640 medium with 10% FBS (Atlanta Biologicals, Lawrenceville, GA) without antibiotics. At the time of infection, the estimated number of adherent cells was approximately 2 x 10⁵/well. In some experiments, bovine monocyte-derived macrophages were obtained by culturing the monocytes for 7 days at 37°C. Murine macrophage cell lines RAW 264.7 (ATCC TIB-71) and J774A.1 (ATCC TIB-67) were cultured in Dulbecco's modified Eagle's medium (DMEM/F-12 50/50; Mediatech, Inc.) supplemented with 10% FBS. We did not assess these cultures for the presence of dendritic cells.

Viability of infected monocytes. The viability of infected monocytes was assessed by the CellTiter-Blue cell viability assay (Promega, Madison, WI). Monocytes were infected with M. avium subsp. paratuberculosis as described below and were incubated with ATP (Calbiochem, La Jolla, CA) for 24 h at 37°C in 5% CO_2. The medium was then removed and replaced with 360 µl of RPMI 1640 medium supplemented with 10% FBS, and 40 µl of CellTiter-Blue reagent was added to each well. The plate was incubated at 37°C for 2 h in 5% CO₂, and the fluorescent intensity of each well was measured with a microplate reader (Synergy HT; Bio-Tek, Winooski, VT). Live and dead monocytes were quantified by using a LIVE/DEAD Viability/Cytotoxicity kit (Invitrogen, Carlsbad, CA). Briefly, calcein AM (final concentration, 1 µM) and ethidium homodimer-1 (final concentration, 2 µM) were added to control and ATP-treated monocytes. After incubation at 37°C for 10 min, the stained cells were examined with an inverted fluorescent microscope (Olympus IX70; Leeds Precision Instrument, Minneapolis, MN) with appropriate filter sets. Five different x400 magnification fields per well were examined, and the numbers of live cells (green color) and dead cells (red color) were enumerated. The viability of the ATP-treated monocytes was expressed as a percentage of the number of viable untreated M. avium subsp. paratuberculosis-infected monocytes.

Quantification of intracellular M. avium subsp. paratuberculosis. Freshly isolated monocytes were incubated with M. avium subsp. paratuberculosis at a multiplicity of infection of 10 bacilli per monocyte (10:1) for 3 h in the presence of 10% autologous serum. Uningested bacilli were removed by washing the monocytes three times with warm RPMI 1640 medium. The infected monocytes were incubated with ATP (1 to 5 mM) in RPMI 1640 medium supplemented with 10% FBS for 24 h at 37°C with 5% CO_2. Antibiotics were not added to the medium at any point. After incubation, the conditioned media were removed from infected monocytes, centrifuged at 2,000 x g for 30 min, and lysed with 0.05% sodium dodecyl sulfate (SDS) to release any bacilli within detached cells. The adherent monocytes were also lysed with 0.05% SDS, and these lysates were combined with the conditioned medium lysate from the same well and inoculated into BACTEC 12B vials. The numbers of viable bacilli were evaluated by a radiometric method, as described previously (20, 38).

Uptake of YO-PRO. Isolated monocytes (10⁵/well) were cultured in a 96-well tissue culture plate (Falcon; BD Labware) and treated with 10 µM YO-PRO, which is an impermeant nuclear dye (Molecular Probes, Inc.). Various concentrations of ATP or 2'(3')-O-(4-benzoylbenzoyl) ATP triethylammonium salt (Bz-ATP; Sigma, St. Louis, MO) were added to the wells. After a 15-min incubation at 37°C in 5% CO₂, the fluorescent intensities of the wells were measured with a microplate reader (Synergy HT; Bio-Tek) with excitation and emission filters (485 and 528 nm, respectively). A 0.1% solution of Triton X-100 (Calbiochem, La Jolla, CA) was added to separate wells and served as a positive control. The uptake of YO-PRO was expressed as a percentage of the signal for the positive control.

Measurement of LDH release. The release of lactate dehydrogenase (LDH) was measured by using a CytoTox 96 nonradioactive cytotoxicity assay kit (Promega). Bovine monocytes (10⁵/well) were incubated with M. avium subsp. paratuberculosis (at a multiplicity of infection of 10:1) in the presence of 10% autologous serum for 3 h at 37°C with 5% CO₂. Uningested bacilli were removed by washing the monocytes three times with warm RPMI 1640 medium, and the infected monocytes were incubated with various concentrations of ATP or Bz-ATP in RPMI 1640 medium at 37°C with 5% CO₂. After a 4-h incubation, the conditioned media were collected and centrifuged at 250 x g for 4 min. A sample (50 µl) of each supernatant was transferred to separate wells of a 96-well plate, and substrate solution (50 µl) was added to the wells. After a 30-min incubation at room temperature, the absorbance was measured at 490 nm with a microplate reader (µQuant; Bio-Tek Instruments). The release of LDH was expressed as the percentage of the signal for the positive control cells (monocytes to which lysis solution was added).

Caspase-3 assay. Bovine monocytes were infected with M. avium subsp. paratuberculosis as described above and cultured in a 96-well plate. Infected monocytes were treated with various concentrations of ATP, Bz-ATP, or 400 nM staurosporine (a positive control for apoptosis) and incubated in RPMI 1640 medium supplemented with 10% FBS at 37°C in 5% CO₂ for 4 h. After the plates were washed, 100 µl of Apo-ONE Caspase-3/7 reagent was added to the wells and mixed by shaking the plate on a plate shaker for 30 s. The plates were incubated for a further 18 h in the dark at room temperature, and the fluorescent intensities were measured with a microplate reader (Synergy HT; Bio-Tek Instruments) with excitation and emission filters (499 and 521 nm, respectively). The fold change in caspase-3 activity was expressed by comparing the absorbance of the untreated control cells with that of the treated cells.

Statistical analysis. Data are presented as the means ± the standard errors of the means (SEMs) and were analyzed for statistical significance by Student's t test with the Prism 4 statistical software package (GraphPad, San Diego, CA).

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ATP is cytotoxic for bovine monocytes. As expected, ATP was cytotoxic for bovine monocytes. The cytotoxic effects were similar for M. avium subsp. paratuberculosis-infected and uninfected monocytes at 24 h of incubation (data not shown). Addition of 1 mM ATP to M. avium subsp. paratuberculosis-infected bovine monocytes resulted in 20% cytotoxicity, which increased to 50% at 5 mM ATP. There was no further significant increase in cytotoxicity at 10 mM ATP (Fig. 1A). To confirm the cytotoxic effect of ATP on bovine monocytes, we also performed live/dead staining of bovine monocytes. These data indicated that the total numbers of live cells decreased by 18%, 57%, and 69% after a 24-h incubation with 1 mM, 5 mM, and 10 mM ATP, respectively, compared to the numbers of untreated control monocytes. We also observed a decreased total number of cells and an increased proportion of dead cells following ATP treatment (Fig. 1B).

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FIG. 1. ATP decreases the viability of M. avium subsp. paratuberculosis-infected bovine monocytes in a dose-dependent manner. Bovine monocytes were infected with M. avium subsp. paratuberculosis and then incubated with 1 to 10 mM ATP for 24 h. (A) Monocyte viability was measured by using the CellTiter-Blue cell viability assay reagent. The conditioned media were removed, and 360 µl of RPMI 1640 medium supplemented with 10% FBS and 40 µl of CellTiter-Blue cell viability assay reagent was added to the wells. After a 2-h incubation, the fluorescent intensities of individual wells were measured with a microplate reader by using the appropriate filter sets. (B) Monocytes were incubated with calcein AM (final concentration, 1 µM) and ethidium homodimer-1 (final concentration, 2 µM) for 10 min. The stained cells were examined with an inverted fluorescent microscope with appropriate filter sets. Five different x400 magnification fields per well were examined, and the numbers of live cells (green color) and dead cells (red color) were enumerated (B). The results are the means ± SEMs of three independent experiments. **, P < 0.01.

ATP treatment does not affect intracellular survival of M. avium subsp. paratuberculosis. We next assessed the number of viable M. avium subsp. paratuberculosis cells after ATP treatment. Addition of ATP (Fig. 2A) or Bz-ATP (Fig. 2B) to M. avium subsp. paratuberculosis-infected bovine monocytes did not affect the survival of intracellular bacilli. The absence of an effect of ATP or Bz-ATP was also true if the monocytes were first incubated with recombinant bovine gamma interferon (IFN-) or were allowed to mature into monocyte-derived macrophages (7 days in culture) before being infected with M. avium subsp. paratuberculosis (data not shown). These observations are in contrast to those described in previous reports, in which ATP decreased the survival of mycobacteria in human or murine mononuclear phagocytes (10, 18, 19, 21, 25).

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FIG. 2. ATP or Bz-ATP treatment of M. avium subsp. paratuberculosis-infected bovine monocytes does not affect the number of viable bacilli within a 24-h incubation period. Bovine monocytes were incubated with M. avium subsp. paratuberculosis at a multiplicity of infection of 10:1 (bacilli:monocytes) for 3 h in the presence of 10% autologous serum. After the uningested bacilli were removed, the monocytes were incubated with 1 mM to 10 mM ATP (A) or 1 mM to 5 mM Bz-ATP (B) in RPMI 1640 medium with 10% FBS for 24 h. The conditioned media were collected, centrifuged at 2,000 x g for 30 min, and lysed with 0.05% SDS to release any bacilli within detached cells. The adherent monocytes were similarly lysed with 0.05% SDS, and the combined lysates were inoculated into BACTEC 12B vials. The numbers of viable M. avium subsp. paratuberculosis cells were assessed by a radiometric method, as described previously (20, 38). The results are the means ± SEMs of three independent experiments.

YO-PRO uptake, LDH release, and caspase-3 activity of ATP or Bz-ATP treated bovine monocytes. High concentrations of ATP (mM) are reported to induce pore formation in cells via activation of the P2X₇ receptors on the cell membrane (1, 5, 9, 14-16, 26, 30, 35). We investigated pore formation after ATP treatment of bovine monocytes, using a YO-PRO uptake assay. Addition of 1 to 5 mM ATP decreased YO-PRO uptake by bovine monocytes, whereas addition of Bz-ATP, which is a more potent agonist for purinergic receptors, increased YO-PRO uptake by bovine monocytes (Fig. 3A). Similar to these results, addition of Bz-ATP, but not ATP, induced LDH release from bovine monocytes (Fig. 3B). We also investigated whether ATP or Bz-ATP might induce apoptosis via activation of caspase-3. Only 5 mM Bz-ATP significantly increased caspase-3 activation in bovine monocytes (an approximately 60% increase in activity; P < 0.05); addition of ATP had no effect (data not shown).

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FIG. 3. Bz-ATP but not ATP stimulates YO-PRO uptake, LDH release, and caspase-3 activity in bovine monocytes. (A) Bovine monocytes were incubated with 10 µM YO-PRO (Molecular Probes, Inc.) and various concentrations of ATP or Bz-ATP for 15 min at 37°C in 5% CO2. After incubation, the fluorescent intensities of the wells were measured with a microplate reader with excitation and emission filters (485 and 528 nm, respectively). Monocytes permeabilized with 0.1% Triton X-100 and uninfected monocytes served as positive (100%) and negative (0%) controls, respectively. The uptake of YO-PRO by Bz-ATP- and ATP-treated M. avium subsp. paratuberculosis-infected monocytes was expressed as a percentage of that for the positive control. (B) M. avium subsp. paratuberculosis-infected bovine monocytes were incubated with various concentrations of ATP or Bz-ATP for 4 h at 37°C in 5% CO2 in RPMI 1640 medium without FBS. After incubation, the conditioned media were collected and LDH release was determined as described in the Materials and Methods. The release of LDH was expressed as a percentage of that for the positive control (cells lysed with the lysis solution contained in the kit). The results are the means ± SEMs of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

ATP is cytotoxic to but does not affect survival of M. avium subsp. paratuberculosis in murine macrophage cell lines RAW 264.7 and J774A.1. For comparative purposes, we next investigated the effect of ATP on the viability of RAW 264.7 and J774A.1 murine macrophage cell lines using the CellTiter-Blue cell viability assay. RAW 264.7 cells and J774A.1 cells were more susceptible to ATP-induced cytotoxicity than bovine monocytes after a 4-h or a 24-h incubation with 5 mM ATP (Fig. 4). Because monocytes undergo morphological and functional changes into macrophage-like cells (i.e., monocyte-derived macrophages) after 4 to 7 days of culture in vitro (39), we also compared monocyte-derived macrophages with monocytes. ATP had similar cytotoxic effects on bovine monocytes and monocyte-derived macrophages at 4 and 24 h in culture (Fig. 4).

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FIG. 4. ATP (5 mM) is cytotoxic for uninfected bovine monocytes, monocyte-derived macrophages (MDM), and the RAW 264.7 (RAW) and J774A.1 cell lines. Cells were cultured as described in the Materials and Methods, and viability was measured after 24 h incubation by using the CellTiter-Blue cell viability assay reagent. The viability of ATP-treated cells was expressed as the percentage of the fluorescent intensity for untreated control cells (100% viability) at the same time points (data not shown). The results are the means ± SEMs of three independent experiments.

Likewise, neither ATP nor Bz-ATP reduced the number of viable M. avium subsp. paratuberculosis in RAW 264.7 (Fig. 5) or J774A.1 (data not shown) murine macrophage cell lines. In subsequent experiments, we observed that neither ATP nor Bz-ATP induced the killing of M. avium subsp. avium in bovine monocytes (Fig. 6A), nor did ATP or Bz-ATP affect the survival of M. bovis BCG in bovine monocytes or monocyte-derived macrophages or M. bovis BCG in RAW 264.7 cells (Fig. 6B).

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FIG. 5. Neither ATP nor Bz-ATP stimulates killing of M. avium subsp. paratuberculosis in the RAW 264.7 murine macrophage cell line RAW 264.7 cells were infected with M. avium subsp. paratuberculosis in the absence of serum and incubated with DMEM/F-12 50/50 supplemented with 10% FBS. Infected cells were incubated with the indicated concentrations of ATP or Bz-ATP for 24 h, and the number of viable M. avium subsp. paratuberculosis cells was assessed as described in the legend to Figure 2. The results are the means ± SEMs of three independent experiments.

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FIG. 6. Neither ATP nor Bz-ATP stimulates killing of M. avium subsp. avium in bovine monocytes (A) or M. bovis BCG in bovine monocytes, monocyte-derived macrophages (MDM), or RAW 264.7 (RAW) cells after a 24-h incubation at 37°C (B). The number of viable M. avium subsp. avium or M. bovis BCG cells in the cell lysates was assessed by determination of plate counts on 7H10 agar supplemented with 10% oleic acid-albumin-dextrose-catalase. The results are the means ± SEMs of two independent experiments.

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Johne's disease is a chronic granulomatous enteritis of ruminants (4). Acid-fast bacilli can be prominent within mononuclear phagocytes in granulomatous lesions in the intestinal wall of cattle with clinical Johne's disease (7, 29). It is possible that some of the cells in these foci of granulomatous inflammation are undergoing apoptosis or necrosis and, consequently, release ATP (22). Extracellular ATP is cytotoxic to human and murine macrophages and is reported to induce the intracellular killing of Mycobacterium tuberculosis and M. bovis BCG in human and murine macrophages (10, 18, 19, 21, 25, 33). We found that ATP exerted a dose-dependent (1 to 5 mM) cytotoxic effect on bovine monocytes and that M. avium subsp. paratuberculosis infection did not significantly increase the cytotoxicity of ATP. This finding is similar to that described in a previous report that ATP is no more cytotoxic to M. bovis BCG-infected human monocytes than uninfected monocytes (25). Based on prior reports, we expected the ATP-mediated cytotoxicity for bovine monocytes to be accompanied by the decreased survival of M. avium subsp. paratuberculosis. However, after ATP treatment the number of viable intracellular M. avium subsp. paratuberculosis cells was not reduced compared to the number of untreated bovine monocytes.

ATP can bind to several different purinergic receptors, including the P2X₇, P2X₁, and adenosine P1 receptors (3, 6). The P2X₇ receptor plays a major role in the cytotoxic effect of ATP for several cell types (12, 30, 34-36). The P2X₇ receptor was also reported to play a major role in the mycobactericidal effect of ATP for human and murine macrophages (10, 31). One of the characteristics of the P2X₇ receptor is its ability to create a membrane pore that allows the entrance of large molecules, such as lucifer yellow and YO-PRO, after the addition of ATP (9). Although ATP treatment did not result in the increased uptake of YO-PRO by bovine monocytes, addition of Bz-ATP, which is a more potent P2X₇ agonist (28), enhanced the uptake of YO-PRO in bovine monocytes. Likewise, Bz-ATP stimulated LDH release and caspase-3 activation in bovine monocytes in a dose-dependent manner, whereas ATP did not. Caspase activation was previously reported to be partially involved in ATP-induced cell death (8, 13, 23, 24). Overall, our data suggest that the ATP-mediated cytotoxicity might be P2X₇ independent and that the Bz-ATP-mediated cytotoxicity might be P2X₇ dependent in bovine monocytes.

Although Bz-ATP was cytotoxic for bovine monocytes, it did not induce the killing of M. avium subsp. paratuberculosis. Because macrophages express more P2X₇ receptors than monocytes (11, 14, 15), we compared bovine monocytes and monocyte-derived macrophages for their responses to ATP and Bz-ATP. Neither ATP nor Bz-ATP induced the killing of intracellular M. avium subsp. paratuberculosis by bovine monocyte-derived macrophages. IFN- is reported to enhance the expression of P2X₇ during the differentiation of monocytes into macrophages (14). However, prior activation of bovine monocyte-derived macrophages with IFN- followed by incubation with ATP or Bz-ATP did not increase the killing of intracellular M. avium subsp. paratuberculosis (data not shown). We found that 5 mM ATP was cytotoxic to RAW 264.7 and J774A.1 cells but did not cause the intracellular killing of M. avium subsp. paratuberculosis, nor did ATP or Bz-ATP induce the killing of M. avium subsp. avium or M. bovis BCG in bovine monocytes, monocyte-derived macrophages, or RAW 264.7 cells. These findings are in contrast to those of a previous report that incubation of the J774A.1 murine macrophage cell line with ATP increased the killing of intracellular M. bovis BCG (10).

It was previously suggested that the ability of ATP to increase the killing of intracellular mycobacteria in human macrophages (10, 18, 19, 33) may have been due to increased intracellular calcium concentrations and phospholipase D activity that in turn enhanced phagosome-lysosome fusion (18, 19). In our hands, ATP treatment did not increase intracellular calcium levels in bovine monocytes, nor did addition of calcium ionophore alter the intracellular fate of M. avium subsp. paratuberculosis in bovine monocytes (data not shown). These findings are somewhat at odds with those described in previous reports that apoptosis, but not necrosis, of infected human macrophages is associated with the killing of intracellular M. bovis BCG (25) and that ATP-dependent killing of M. bovis BCG is P2X₇ dependent (21). These findings highlight the differences and similarities in the responses of bovine mononuclear phagocytes compared with those of human or murine mononuclear phagocytes to purinergic stimulation, which might account for the differences in their responses to mycobacteria. One possible explanation for the discrepancy between monocyte death and mycobacterial survival might include differences in cell signaling via the P2X₇ receptor in human and bovine mononuclear phagocytes. For example, KN-62, which is an antagonist of the P2X₇ receptor, blocks the cation current and the uptake of ethidium after ATP treatment in human cells but not rat cells (17). It has also been shown that a polymorphism in the human P2X₇ receptor reduces the killing of intracellular M. bovis BCG in human macrophages (31). However, it is also possible that the differences in the methods used in our study compared to those used in earlier studies (i.e., radiometric versus plate counts, multiplicity of infection, etc.) might also contribute to the different outcomes for intracellular mycobacteria that were observed.

In summary, the results of this study demonstrate that M. avium subsp. paratuberculosis-infected bovine mononuclear phagocytes respond differently to ATP than was previously reported for human and murine mononuclear phagocytes. Although ATP was cytotoxic to bovine mononuclear phagocytes, it did not reduce the number of viable M. avium subsp. paratuberculosis cells recovered from infected cells. One might speculate that ATP-mediated local destruction of M. avium subsp. paratuberculosis-infected mononuclear phagocytes would remove a favorable site for bacillary multiplication and, hence, might play a beneficial role in the host defense against bovine paratuberculosis. Although the present study did not directly address this possibility, our in vitro findings would be compatible with the situation in which ATP-mediated cytotoxicity reduces M. avium subsp. paratuberculosis multiplication at sites of infection.

 
This work was supported by funds from the Wisconsin Agricultural Experimental Station (grant WIS 00770), the U.S. Department of Agriculture National Research Institute (grant CSREES-NRI 2004-35204-14231), and the Walter and Martha Renk Endowed Laboratory for Food Safety.

 
* Corresponding author. Mailing address: Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin—Madison, 2015 Linden Drive, Madison, WI 53706. Phone and fax: (608) 262-8102. E-mail: czuprync{at}svm.vetmed.wisc.edu

Published ahead of print on 18 July 2007.

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Clinical and Vaccine Immunology, September 2007, p. 1078-1083, Vol. 14, No. 9
1071-412X/07/$08.00+0     doi:10.1128/CVI.00166-07
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