Extracellular ATP is cytotoxic to mononuclear phagocytes but does not induce killing of intracellular Mycobacterium avium subsp. paratuberculosis

Abstract

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.

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 ×400 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.

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 × 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.

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% CO_2. 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.

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.

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.

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.