From the National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India
Received for publication, February 25, 2003 , and in revised form, April 21, 2003.
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ABSTRACT |
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INTRODUCTION |
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Intracellular pathogen survival depends upon the efficiency of host-defense mechanisms like the respiratory burst (16, 17) that involves generation of large amounts of superoxide anion ()1 and related reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), nitric oxide (NO), hydroxyl radicals, and singlet oxygen (18, 19). ROS is known to induce apoptosis in metazoans (20) and in unicellular organisms existing outside the host cell (4, 9). Therefore, it is possible that ROS generation might have a direct bearing on apoptotic death of intracellular parasites, but this phenomenon has not been examined adequately. ROS is also generated by anti-leishmanial drugs like antimonials in blood (21), but it is not known whether this is directly linked to apoptotic death of amastigote forms of Leishmania spp. It remains to be shown whether apoptotic pathway of death is an inherent feature of intracellular parasitic life style because the features and mechanisms of apoptotic death in intracellular parasitic forms have not been sufficiently described.
The idea that, while existing in closely knit groups, unicellular organisms acquire a new level of individuality and can express features of apoptosis under situations of stress prompted us to explore whether or not, while living in close association inside the macrophages, the parasites have the capability to undergo apoptosis. Also, if there is apoptotic death, what are the associative mechanisms of the process? Earlier studies from this laboratory have shown that the promastigotes of L. donovani undergo apoptosis in response to H2O2 via a Ca2+-mediated mechanism induced by oxidative stress (4, 9); therefore, we asked whether intracellular amastigote death involves similar features or uses different mechanisms. To address this issue, we used a model of drug-induced intracellular amastigote death to study the changes associated with amastigote clearance. The findings project a new possibility that, under conditions of drug exposure, nonselective cation channels in both the host and the parasite are important modulators of drug efficacy. Importantly, it lends acceptance to the suggestion that, in situations of close existence of the parasites, such as within the host cell, apoptosis is the preferred mode of death.
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EXPERIMENTAL PROCEDURES |
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Cells
PromastigotesCulture of promastigotes of L. donovani of URL6 strain was carried out as described previously (4, 9). Briefly, the cultures were grown on slants containing 1% glucose, 5.2% brain heart infusion agar extract, and rabbit blood (6%, v/v) at 25 °C.
MacrophagesJ774A.1 macrophage cell line (ATCC no. TIB-67) was maintained in Dulbecco's modified Eagle's medium (DMEM) with 4 mM L-glutamine adjusted to contain 1.5 g/liter sodium bicarbonate, 4.5 g/liter glucose, and 10% fetal calf serum (FCS) at 37 °C in an atmosphere of 5% CO2 and air.
Infected MacrophagesJ774A.1cells (5 x 105 cells/ml) were seeded on coverslips and incubated overnight using DMEM containing 10% FCS. After 24 h, these cells were incubated with L. donovani promastigotes (5 x 106) in the log phase of growth in serum-free media for 26 h. The infected cells were then maintained in DMEM containing 10% FCS from 118 h at 37 °C with 5% CO2 and air. Before use, loosely adherent promastigotes were removed from the macrophages by incubation of the cells for 3 min in cold distilled water.
Purified AmastigotesJ774A.1 cells grown in 125-mm tissue culture flasks infected with L. donovani promastigotes (as per procedures described above) were used to purify amastigotes by the method of Chang and Hendricks (22) with a few modifications. Briefly, flasks containing infected J774A.1 were flushed, and cells were harvested and centrifuged. The cells were then subjected to freezing in liquid nitrogen with subsequent thawing at 37 °C for four cycles. The released amastigotes and disrupted macrophages were spun at 400 x g for 5 min, and the pellet was suspended in DMEM containing 10% FCS. This suspension was loaded onto a Percoll gradient (2040-90%) and spun in a swing rotor (S40) in a Jouan 4i (Saint Herblain, France) centrifuge at 1200 x g for 1 h. Amastigotes were collected using a Pasteur pipette from the appropriate layer and viewed under a phase contrast microscope for purity check.
Treatments
To rule out any toxic effect of the drug on the host cell, potassium antimony tartrate (PAT) was used at different doses (1050 µg/ml) to incubate uninfected macrophages and cell viability was determined at 36 h. For assessment of anti-leishmanial activity of the drug, infected macrophages were incubated with selected doses of PAT from 3 to 36 h. For cell treatments in Ca2+-related studies, nifedipine was used at a concentration of 100 µM and flufenamic acid (FFA) was used at a concentration of 200 µM. For Ca2+ chelation, 0.3 and 3 mM EGTA was used in the extracellular media. Antioxidants, NAC and MnTBAP were used at concentrations of 20 mM and 100 µM, respectively. Aminoguanidine, known to be a fairly specific inhibitor of cytokine inducible nitric-oxide synthase (iNOS) (23), was used as a nitric oxide inhibitor at the doses of 50 and 100 µM.
Cytotoxicity Measurements
To visualize the infection rate of the macrophages, coverslips containing infected J774A.1 cells were treated with cold water for 3 min with mild shaking to dislodge loosely bound promastigotes. Coverslips were then washed thoroughly with phosphate-buffered saline and stained with propidium iodide (PI) (7.5 µg/ml) containing digitonin (0.05%) for permeabilization for 5 min so that both the macrophage and amastigote nuclei take up PI to make counting comfortable. The coverslips were mounted in 10% glycerol and infection was checked either with a Nikon Optiphot fluorescence microscope or a Zeiss LSM 510 confocal microscope (Zeiss Inc, Thornwood, NY). For confocal microscopy, a 488-nm argon ion laser was used to excite the cells and a 560-nm long pass filter was used to collect images for the PI stain. Phase contrast images were collected in the transmission mode, both images were overlapped when necessary, and the number of amastigotes counted. Only well rounded nuclei showing PI stain were counted as surviving amastigotes because irregular structures may represent disrupted nuclei of cells undergoing death. At least 200 cells of four independent experiments were counted. All counts were carried out on coded samples. Measures of both the number of infected cells and the number of amastigotes/100 cells were taken at different intervals to arrive at a time point that could be used as time 0 for drug treatment. Following drug treatment, drug activity was estimated by calculating percentage of growth inhibition by estimating the number of amastigotes/100 infected macrophages in treated wells divided by the number of amastigotes/100 infected macrophages in untreated wells x 100.
Detection of DNA Fragmentation
Terminal Deoxynucleotidyltransferase Enzyme (TdT)-mediated dUTP Nick End Labeling (TUNEL)Detection of DNA fragmentation by TUNEL was carried out using a TUNEL assay kit according to instructions from the manufacturer and as described previously (4, 9). Briefly, cells were fixed in 4% formaldehyde and postfixation permeabilization was carried out with 0.2% (v/v) Triton X-100 for 10 min at room temperature followed by incubation with buffer containing nucleotide mix (50 µM fluorescein-12-dUTP, 100 µM dATP, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 7.6) for 1 h at 37 °C. Detection was carried out by both flow cytometry and microscopy.
Flow CytometryFormaldehyde fixed cells (106) stained for TUNEL were analyzed on an Elite ESP flow cytometer (Beckman Coulter, Fullerton, CA) using an argon ion laser tuned to 488 nm. Green fluorescence gated on forward and side light scatter was collected using a band-pass filter (525 ± 10 nm) and displayed using logarithmic amplification.
MicroscopyTUNEL labeling was visualized with a Zeiss LSM 510 confocal microscope using plan-neofluor objectives of 40x or 100x with numerical apertures of 0.75 and 1.3, respectively. Pinhole was set at 100 µm. For detection of green TUNEL-stained nuclei and red PI counterstain, cells were simultaneously illuminated with 488- and 563-nm laser lines and fluorescence was collected with a 500550-nm band pass filter for green fluorescence detection and a 560-nm long pass filter for red fluorescence. Individual frames were averaged over four to eight frames. Laser was used at 13% power. Serial z-sections of each field were captured sequentially, and the sections were collected at 0.5-µm intervals in the z axis through a 40x objective.
Agarose Gel DNA AnalysisIsolation of DNA from amastigotes and agarose gel electrophoresis was carried out as described previously (4, 9).
Visualization of Phosphatidylserine Exposure
Amastigotes harvested from infected macrophages and subsequently Percoll purified were incubated with annexin V conjugated to fluorescein isothiocyanate (1:50) for 30 min at room temperature in dark. Cells were then lightly permeabilized and stained by a solution containing digitonin (0.01%) and PI (7.5 µg/ml), respectively. The staining was visualized through a confocal microscope by simultaneously illuminating the cells with 488- and 563-nm laser lines, and fluorescence was collected with a 500550-nm band pass filter for green fluorescence and a 560-nm long pass filter for red fluorescence detection.
Measurement of Reactive Oxygen Species
To monitor the level of ROS, the cell permeant probe H2DCFDA was used as described previously (9). H2DCFDA is a nonpolar compound that readily diffuses into cells, where it is hydrolyzed to the nonfluorescent derivative dichlorodihydrofluorescein and trapped within the cells. In the presence of a proper oxidant, dichlorodihydrofluorescein is oxidized to the highly fluorescent 2,7-dichlorofluorescein. Cells from different treatment groups were suspended in DMEM and incubated with H2DCFDA (2 µg/ml) for 15 min in the dark. Relative fluorescence was monitored in a PerkinElmer LS50B Spectrofluorometer (PerkinElmer Life Sciences) set at an excitation wavelength of 507 nm and emission wavelength of 530 nm with a slit width of 5 nm. Data were normalized to values obtained from vehicle-treated controls. For each experiment, fluorometric measurements were performed in triplicate and expressed as fluorescence intensity units (FIU). For microscopy, cells were illuminated with a 488-nm laser and images were collected using a band pass filter (505550 nm) with the pinhole set at 100 µm. Laser was used at 1315% power, and individual frames were averaged over four to eight frames. Collecting the fluorescent image with a single rapid scan minimized the effects of dichlorodihydrofluorescein photo-oxidation. Identical parameters were used for different groups.
For measurement of , lucigenin was used as an indicator of
(24). Typically, 1 x 106 cells were suspended in 0.5 ml of buffer. 50 µl of suspended cells was mixed with 50 µl of 2 mM lucigenin (dissolved in reaction buffer, phosphate-buffered saline, 1 g of glucose/liter, and 2 mM MgCl2). The reaction was placed in the dark for 10 min and then read using a luminometer (Lumicount, Packard, CT). Data are expressed as luminometer units.
NO detection was carried out as per protocol from the manufacturer. Briefly, treated and untreated cells were incubated with the fluorescence probe DAF-FM (1 µM) for 30 min and washed two times, and NO generation was measured at excitation of 495 nm and emission at 515 nm with a LS-50B spectrofluorometer. Data are expressed as FIU at 515 nm.
Reduced glutathione (GSH) was measured as described previously (9).
Measurement of Mitochondrial Potential
Mitochondrial membrane potential (m) was measured using JC-1 probe as described previously (9). JC-1 is a cationic mitochondrial vital dye that is lipophilic and becomes concentrated in the mitochondria in proportion to the membrane potential; more dye accumulates in mitochondria with greater
m and ATP generating capacity. Therefore, the fluorescence of JC-1 can be considered as an indicator of relative mitochondrial energy state. The dye exists as a monomer at low concentrations (emission, 530 nm, green fluorescence) but at higher concentrations forms J-aggregates (emission, 590 nm, red fluorescence). Briefly, cells after different treatments were collected and incubated for 7 min with 10 µM JC-1 at 37 °C, washed, resuspended in media, and measured for fluorescence at two different wavelengths as mentioned above. The ratio of the reading at 590 nm to the reading at 530 nm (590/530 ratio) was considered as the relative
m value.
Measurement of Cytosolic Free Ca2+ Concentrations
Changes in intracellular Ca2+ concentration, [Ca2+]i, were monitored with the fluorescent probe fluo-3/AM as described previously (9). Briefly, purified amastigotes or infected macrophages were loaded for 30 min at 25 °C with 5 µM fluo-3/AM containing 1 µM pluronic acid F-127 for proper dispersal and 0.25 mM sulfinpyrazone, an organic anion transport inhibitor, to inhibit the leakage of the fluo-3 dye. Just before use, a sample of loaded cells was washed with medium to remove nonhydrolyzed fluo-3/AM. Fluorescence measurements were performed at 25 °C with excitation at 488 nm and emission at 522 nm. To convert fluorescence values into absolute [Ca2+]i, calibration was performed at the end of each experiment. [Ca2+]i was calculated using the equation: [Ca2+]i = Kd[(F Fmin)/(Fmax F)], where Kd is the dissociation constant of the Ca2+·Fluo 3 complex (400 nM) and F represents the fluorescence intensity of the cells. Fmax represents the maximum fluorescence (obtained by treating cells with 10 µM calcium ionophore A23187
[GenBank]
), and Fmin corresponds to the minimum fluorescence (obtained from ionophore-treated cells in the presence of 3 mM EGTA). Fluorescence intensities were expressed as the increase in fluorescence with respect to base-line fluorescence intensity before stimulation.
For microscopic detection of fluo-3 staining in infected macrophages, fluo-3-labeled cells were probed by a single rapid scan with a 488-nm argon ion laser and images collected by using a 530-nm filter. Nomarski images were collected in the transmission mode and overlapped with the fluo-3 images.
Statistical Analysis
Paired comparisons were conducted using a paired t test, and all data are presented as mean values ± S.E. Differences were considered significant at a 0.05 level of confidence.
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RESULTS |
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For combating visceral leishmaniasis, the most commonly used anti-leishmanicidal drug is sodium stibogluconate, a pentavalent antimonial that is targeted to destroy parasites within the PVs (26). We chose PAT, a trivalent antimonial, because pentavalent antimony is converted to trivalent antimony inside macrophages before it can be cytotoxic to the parasites; therefore, trivalent antimony is the active form of pentavalent antimony (27, 28). Prior to assessing the anti-leishmanicidal activities of PAT toward L. donovani amastigotes residing within J774A.1 cells, toxicity of the drug (10, 15, 20, 25, and 50 µg/ml) on uninfected J774A.1 cells was evaluated to rule out doses that would affect the host cell. With 50 µg of PAT, within 12 h 45% macrophages were dead, a feature reported for pentavalent antimony that is toxic at high doses to THP-1 cells (8). Although 20 and 25 µg/ml amounts of the drug did not have significant effect on macrophages in terms of cell death, the adhering capacity of the macrophages was compromised by 36 h. The doses of 10 and 15 µg/ml PAT were neither cytotoxic to macrophages nor did they reduce their adhering capacity by 36 h. Therefore, we tested these two doses for their cytotoxic activity on amastigotes to arrive at a suitable time frame within which the effects of the drug on the amastigotes could be studied. To determine the rate of clearance, permeabilized PI-stained treated and untreated infected macrophages were used for counting the number of amastigotes in the host cell by visualization using both phase contrast and fluorescence images (Fig. 1, inset ii). The dose of 15 µg/ml was found to be the appropriate dose because that could reduce the number of amastigotes significantly within a 24-h treatment period (Fig. 1). Percentage of growth inhibition by the dose of 15 µg/ml PAT is represented in Fig. 1 (inset i). Thus, treatment of the infected macrophages with 15 µg/ml PAT provided a time frame of 24 h for mechanistic studies on changes associated with amastigote clearance.
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Amastigote Nuclear DNA Fragmentation Occurs when Infected Macrophages Are Treated with PATHaving established the dose of the drug and the time required for its cytotoxic effect on amastigotes, we checked amastigote DNA after exposure of the macrophages to PAT. DNA fragmentation, recognized as a hallmark of metazoan apoptosis (29), is reported to occur in free-swimming protozoan parasites and intracellular amastigotes after exposure to various apoptosis-inducing stimuli (4, 5, 15). Flow cytometric analysis of amastigotes probed for TUNEL isolated from treated and untreated macrophages showed an increase in the number of cells staining positive for TUNEL, represented by increased forward scatter in the drug-treated groups indicating greater degree of staining for fragmented DNA with advancing time of exposure (Fig. 2, A (a), B (a), and C (a)). Analysis of optical sections through the TUNEL-labeled treated and untreated macrophages with a laser-scanning confocal microscope showed insignificant numbers of TUNEL-positive amastigote nuclei in untreated macrophages (Fig. 2A (b)). The number of TUNEL-positive cells gradually increased with time of exposure to the drug (Fig. 2, B (b) and C (b)). Late time points like 12 h showed larger masses of green (Fig. 2C (b)), possibly representing large communal PVs harboring multiple TUNEL-positive amastigotes. Macrophage nuclei did not stain positive for TUNEL in any of the groups, and the cells showed intact morphology demonstrating that they were not affected during the treatment. It is pertinent to mention here that, while scanning a sizable number of infected macrophages not exposed to drug, 1 in 50 macrophages would show occasional green nuclei of 1 or 2 amastigotes. Therefore, it is possible that, during normal growth within the macrophages, a few cells do undergo death that is preceded by DNA fragmentation. Actual counts of the TUNEL-positive amastigotes within the macrophages in different treatment groups showed a significant increase of TUNEL-positive parasites by 12 h of drug treatment (Fig. 3A (a)). Analysis of the DNA from amastigotes recovered from treated (Fig. 3A (b)) and untreated macrophages on agarose gels showed a clear breakdown in the treated groups (Fig. 3A (c), lane 2) as compared with controls (Fig. 3A (c), lane 1) although no oligonucleosomal ladder was visible. This observation differs from previous studies with free-swimming kinetoplastid parasites, where DNA fragmentation in multiples of oligonucleosomal fragments were reported during apoptotic death induced by various stimuli (1, 5, 4, 30). Our observations are similar to DNA fragmentation in staurosporine-induced apoptotic death of L. major, where no DNA breakage into oligonucleosomal fragments was detected (11). PAT is known to induce DNA fragmentation in axenically grown amastigotes of L. infantum (8), but its effect on intracellular amastigote DNA is not known. Therefore, the data presented in this part of the study provide clear evidence that intracellular protozoan parasites respond to appropriate doses of antimonial treatment by expressing the apoptotic phenotype of DNA fragmentation.
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Externalization of Phosphatidylserine Occurs in the Amastigotes after Exposure to PATExternalization of phosphatidylserine is a feature of metazoan apoptosis (1) that has also been demonstrated in staurosporine-treated promastigotes of Leishmania spp. (11). In our experiments, when amastigotes were harvested from treated and untreated infected macrophages and stained with annexin V conjugated to FITC, a significant number of amastigotes recovered from treated cells stained positive for annexin V (Fig. 3B (ac)). The percentage cells staining positive for annexin-V was significantly high (6 h, 30 ± 4, n = 3) (12 h, 82 ± 10, n = 3), as compared with controls (0 h, 3 ± 0.2, n = 3). Therefore, externalization of phosphatidylserine occurs in the amastigotes when exposed to anti-leishmanial drugs while residing within the PVs of the host cell.
PAT Induces an Increase in the Generation of ROS within the MacrophagesBecause DNA fragmentation is a relatively late event in apoptotic death, changes upstream to DNA fragmentation induced by the drug would then have occurred at a much earlier time point. To investigate early changes induced by PAT, we checked ROS generation within the macrophages as these cells can ingest and kill invading microorganisms using microbicidal mechanisms that include generation of ROS (31) and PAT is known to induce ROS production in blood (21). Three different fluorescent probes were used for the investigations, namely H2DCFDA, which primarily detects H2O2 and hydroxyl radical, and lucigenin and DAF-FM, which are probes for and NO, respectively. Increase in signals was detected with all three probes, indicating an augmentation in H2O2, hydroxyl radical,
, and NO with increasing time of exposure to the drug (Fig. 4A (ac)). Note that DAF-FM- and H2DCFDA-sensitive fluorescence increased significantly within the first 6 h. Studies at earlier time points on the level of H2DCFDA-sensitive fluorescence showed that as early as 1 h after drug treatment there was an increase in ROS (FIU at 530 nm; control, 22 ± 3; 1 h, 75 ± 3; 3 h, 127 ± 24; n = 3). This was further checked by microscopy where cells labeled with H2DCFDA were probed with a laser-scanning confocal microscope and a rise in the total number of cells showing H2DCFDA fluorescence, with an enhancement in general ROS levels with increasing time of exposure to the drug, was detected (Fig. 4B (ah)). It was therefore clear that PAT was inducing an increase in ROS levels. The next question was whether the site of generation of ROS was within the macrophage cytoplasm or within the PVs harboring the parasites, as that would indicate the possible site of action of the ROS. It is known that Leishmania-containing phagosomes fuse with lysosomes or late endosomes of the macrophages to form PVs (32); therefore, to pinpoint the site of ROS generation, we used a lysosome-specific lysotracker red dye to identify the PVs, whereas H2DCFDA was used to detect ROS. Overlapping of two images with differentially colored labels showed that ROS was primarily localized within the PVs, although some basal staining with H2DCFDA was present within the cytosol (Fig. 4C (ac)). Similar phenomenon has been shown in compartments harboring Staphylococcus aureus (33). Our studies with uninfected macrophages show that PAT is able to generate ROS in uninfected J774A.1 cells as well (FIU at 530 nm: control, 9.25; drug-treated, 57.1; result is representative of three replicates); however, the rate of generation was much less as compared with infected macrophages. The high generation of ROS within the PVs may have collectively contributed to high ROS observed in infected cells. Therefore, it can be inferred that PAT induces ROS generation within the infected macrophages with maximal concentration occurring within the PVs.
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Preincubation of Cells with Antioxidants Reduces Intracellular Parasite Death Induced by PATTo explore further the role of ROS in the induction of apoptotic death, we studied the impact of a thiol and a non-thiol antioxidant like NAC and MnTBAP on amastigote death induced by PAT. Arguably, if ROS were the cause of death, then scavenging the ROS with antioxidants would reduce the clearance of amastigotes from the macrophage. NAC, an aminothiol, is a unique compound that can raise intracellular GSH levels by acting as a synthetic precursor to intracellular cysteine and GSH and thereby protect cells from adverse effects of ROS. In addition, the chemical properties of the cysteinyl thiol of NAC provide antioxidant properties (34). MnTBAP, a stable metalloporphyrin that is a cell-permeable low molecular weight superoxide dismutase mimetic and peroxynitrite scavenger (35), also has the capability to scavenge H2O2 in certain cell types (36).
Total cell death percentages as determined by PI staining at 12 h showed that both NAC and MnTBAP were able to provide protection against the cytotoxic effects of PAT (Fig. 5A). The efficacy of protection was significantly more when cells were preincubated with a combination of MnTBAP and NAC, the percentage of cell death being only 15% (percentage of cell death: drug-treated, 51 ± 4; drug + MnTBAP + NAC, 8 ± 1) as compared with independent treatments where the percentage of cell death varied between 30 and 40% (Fig. 5A). This suggested that the scavenging action of the two antioxidants was partially independent of each other. Flow cytometric analyses of TUNEL-positive amastigotes in groups treated with or without NAC showed a decrease in the percentage of TUNEL-positive cells (percentage of TUNEL-positive cells: infected macrophages treated with drug (0 h), 15%; infected macrophages treated with drug (3 h), 62%; infected macrophages treated with drug (6 h), 70.3%; infected macrophages pretreated with 20 mM NAC + drug (0 h), 8%; infected macrophages pretreated with 20 mM NAC + drug (3 h), 27%; infected macrophages pretreated with 20 mM NAC and drug (6 h), 41%; results are representative of four experiments) showing that apoptotic death was reduced by NAC known to interfere with the efficacy of PAT to eliminate intracellular parasites (14). Even though the protective effect of NAC on kinetoplastid parasite apoptosis is not known, it protects several types of mammalian cells from undergoing apoptosis (37, 38). Therefore, there was a clear relationship between cell survival and antioxidant treatment. To ensure that the effects of the antioxidants observed were actually through the scavenging of ROS, the capability of the above compounds to reduce ROS levels was checked. Both MnTBAP and NAC were able to reduce H2DCFDA-sensitive fluorescence during the period of 6 h monitored (Fig. 5B). Next, the possibility of NAC acting through the increase in cellular GSH was probed and we found a decrease in GSH induced by PAT, which could be inhibited if NAC was present (nmol of GSH/106 cells: control, 0.680; PAT (6 h), 0.262; PAT (6 h) + NAC pretreatment (20 mM), 0.546; data are representative of three experiments). Even a partial depletion of GSH, an important molecule for protecting organisms including kinetoplastids and metazoans from ROS and other toxic chemicals, would be detrimental to the cell (39). Therefore, one of the possible mechanisms by which NAC provided protection could be through an increase in GSH synthesis that would alter the redox state of the cell; however, the relative contribution of the different mechanisms of NAC action is not possible to specify. Taken together, the ability of the antioxidants to reduce ROS and inhibit amastigote death by PAT links drug-induced ROS generation to the cytotoxic effect of the drug and indicates the existence of a regulated mechanism used for the clearance of L. donovani-infected macrophages stimulated to produce ROS by oxidative stress generating drugs like PAT.
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Preincubation with Aminoguanidine Reduces Cell Death The significant increase in DAF-FM-sensitive fluorescence after drug treatment indicating generation of NO suggested the involvement of the reactive species in amastigote death. NO generated in vitro by NO donating compounds have been shown to induce DNA fragmentation (15). To investigate whether NO was actually responsible in PAT-induced killing, we used aminoguanidine, an inducible nitric-oxide synthase inhibitor (23) during treatment. There was no significant improvement of amastigote growth within the macrophages with 50 µM aminoguanidine; however, with 100 µM amount of this drug there was a significant increase in amastigote survival within the drug-treated macrophages (Fig. 6A). A reduction in DAF-FM-sensitive fluorescence (Fig. 6B) by aminoguanidine indicated a decrease in NO levels. This observation, coupled with the partial prevention of amastigote death observed, suggests that inhibition of NO is in part responsible for prevention of parasite death.
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Isolated Amastigotes Show Increased Cell Death when Exposed to PAT and a Decrease in Mitochondrial Membrane PotentialHaving shown that intracellular amastigote apoptosis was linked to ROS, we wanted to further look at changes downstream to ROS generation and upstream of DNA fragmentation. Our earlier studies have established a close link between ROS-induced apoptosis in promastigotes and the maintenance of m (9); however, measurement of amastigote mitochondrial potential was not possible, as macrophage mitochondria would have interfered with the measurements. Conversely, the other possibility was to isolate the amastigotes from macrophages after drug treatment and measure
m; however, in this case the preparation time would have contributed to alterations in the
m. Therefore, amastigotes were isolated and purified (Fig. 7, inset a) to carry out the studies on PAT-induced cellular changes. Treatment of PAT to isolated amastigotes had a significant effect on
m by 3 h (Fig. 7A) that could be rescued if these cells were preincubated with either MnTABP or NAC (Fig. 7A).
20% fall in
m occurred by 1 h after drug treatment (ratio at 590/530 nm: control, 9.7; drug 1 h, 7.79; results are representative of three experiments) reaching to above 40% by 3 h as shown in Fig. 6A. One might reasonably assume that, if mitochondria undergo a major depolarization, the cells will proceed to die, and if this depolarization is linked to death, prevention of depolarization should prevent death. An increase in amastigote death was recorded after treatment with PAT, and this drug-induced death of the amastigotes was preventable if the cells were pretreated with the antioxidants (Fig. 7B). Therefore, drug-induced mitochondrial depolarization was a possible step leading to the death of the isolated amastigotes. Because PAT was inducing an apoptotic type of death in the amastigotes inside the macrophages, the next question was whether the death in isolated amastigotes occurred in a similar manner or not. It was necessary to do this because, to link mitochondrial depolarization to amastigote death, we had to be sure that isolated amastigote death showed similar features to those inside macrophages. TUNEL labeling studies with isolated amastigotes and analysis of DNA on agarose gels showed that DNA fragmentation occurred in response to PAT in isolated forms as well (Fig. 7B, inset a, b, and c). Experiments described in the earlier section indicate that the generation of ROS was high inside the PVs. To find out whether the amastigotes could themselves generate ROS or whether ROS observed in the PVs was a contribution from the macrophage, we determined whether PAT was able to induce ROS generation in the isolated amastigotes. A significant increase in H2DCFDA fluorescence that could be salvaged by antioxidants was noted (Fig. 7C). However, DAF-FM-positive fluorescence signifying NO levels showed an increase with drug but no significant quench with MnTBAP (Fig. 7D). This is understandable because MnTBAP is primarily a scavenger of superoxide and peroxinitrite; therefore, it would effectively not reduce the NO levels. Because MnTBAP was able to salvage cell death, its primary action was possibly through scavenging of the peroxinitrite or reduction of
level. Although intracellular amastigote forms have not been directly shown to be susceptible to PAT in vitro, axenic amastigotes of L. donovani grown in vitro without any contact with macrophages are known to be killed by PAT (27). As the amastigotes were susceptible to drug action even when they were outside the macrophages, it raises the possibility that the drug action inside the macrophages could possibly be host cell-independent. Taken together, studies with isolated amastigotes recovered from macrophages demonstrate that
m loss was an upstream event to DNA fragmentation induced by PAT.
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Amastigotes Show an Increase in Cytosolic Ca2+ in Response to PAT, and Inhibition of Non-selective Cation Channels Could Block the Increase in Ca2+In the metazoans it is established that ROS can impair Ca2+ transport systems (40), and our studies with promastigotes of L. donovani show that they undergo apoptosis by a Ca2+-mediated mechanism when exposed to oxidative stress (9). In this part of the study, we used both the isolated amastigotes and amastigotes residing within the macrophages to define the role of Ca2+. When intact infected macrophages were exposed to PAT, a clear increase in fluo-3 fluorescence representing increase in Ca2+ was recorded at 3 h. This could be inhibited in the presence of MnTBAP and NAC and a combination of both (Fig. 8A). This was evidence that antioxidants were able to inhibit events leading to increase of intracellular Ca2+ levels. To see whether the changes in Ca2+ translated into apoptotic death, the number of apoptotic cells under manipulated Ca2+ conditions was examined and a reduction in apoptotic cells was observed (no. of TdT-positive cells: control, 2%; PAT-treated cells, 62%; PAT-treated cells in the presence of 3 mM EGTA, 9%; data are representative of three experiments), thus linking Ca2+ to apoptotic death. When isolated amastigotes were exposed to PAT, there was an increase in cytosolic Ca2+. Presence of extracellular EGTA prevented the cytosolic Ca2+ increase, showing that Ca2+ was entering the cells from extracellular sources (Fig. 8B). Because the above data clearly indicated that the primary source of increased Ca2+ was from extracellular sources, experiments were conducted to establish the route of entry. We used nifedipine, a blocker of voltage gated L-type Ca2+ channel, which could reduce the Ca2+ increase by 10% only and was unable to reduce drug-induced cell death (PAT treatment, 50% at 12 h) significantly (PAT + nifedipine, 43% at 12 h). Because entry of Ca2+ through the non-selective cation channels was a possibility (9), isolated amastigotes were incubated with FFA prior to exposure to PAT and an increase in amastigote viability was recorded in the presence of FFA (Fig. 8C (a)). The question was whether non-selective cation channels were important in isolated amastigote death: were these channels active when the amastigotes were residing within the macrophages? To address this question, we pre-incubated infected macrophages with FFA prior to exposure to PAT, and a count of intracellular amastigotes at different time points after drug exposure was estimated. There was a significant reduction in the percentage of amastigotes dying within the macrophages if the macrophages were preincubated with FFA (Fig. 8C (b)). To relate whether this was because of a decrease in Ca2+ levels within the macrophages, we measured cytosolic Ca2+ levels of the FFA-treated infected macrophages and found that there was an inhibition of PAT-induced increase in cytosolic Ca2+ within the macrophages (nmol of Ca2+: control, 83.2 ± 19; drug-treated, 419 ± 32; FFA + drug-treated, 153.4 ± 7; data ± S.E. of three experiments, p < 0.05, drug-treated versus FFA + drug-treated), thus linking decreased Ca2+ influx to increased survival of the amastigotes. However, it was not clear in this scenario whether a decrease of Ca2+ occurred within the macrophages or a decline in the amastigotes was responsible for improved survival. Therefore, to delineate whether FFA was affecting the macrophage Ca2+ or not, which would imply the involvement of macrophage channels, we labeled drug-treated infected macrophages with fluo-3 and subsequently lysed the cells, following which separation of macrophage cytosol, cell debris, and amastigotes was carried out. The results show that Ca2+ levels in both the cell cytosol of the macrophages and the amastigotes increase after PAT treatment; however, if EGTA or FFA was present, the increase in both cells was inhibited (Fig. 8D). The fact that FFA was able to decrease macrophage Ca2+ made it clear that FFA-sensitive channels were also operative in macrophages. Because spectrofluorometric results represent an averaged or integrated estimate, we analyzed Ca2+ localization by confocal microscopy. Visual examination of macrophages labeled with fluo-3 showed that fluo-3 labeling was strong in the intracellular amastigotes exposed to drug (Fig. 9B) as compared with controls (Fig. 9A). Amastigotes appeared as distinctive strong fluo-3 spots (Fig. 9B) because the amastigote cell bodies being small concentrated Ca2 in small areas as compared with macrophage cytosol where Ca2 was distributed over a much larger surface area. Presence of FFA reduced the number of fluo-3-positive cells (Fig. 9C). This experiment confirmed that there was a significant increase in Ca2+ in the macrophages as well as the amastigotes induced by PAT that was inhibited by FFA. In combination, these observations confirm the importance of non-selective cation channels in modulation of drug activity in both the parasite and the host. It appears that PAT opens non-selective cation channels in infected macrophages and in the amastigotes as well. The presence of FFA inhibits Ca2+ increase in the cytosol of both cell types that translates into increased amastigote survival.
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DISCUSSION |
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The J774A.1 cells infected with the L. donovani amastigotes was an ideal system to demonstrate drug-induced changes in the parasites because resident macrophages, which fail to restrict Leishmania growth, do not mount a significant anti-leishmanicidal response like ROS generation as compared with activated macrophages from mouse peritoneum (41). Therefore, the J774A.1 cells harboring Leishmania promastigotes would have minimal background ROS generation, and this was suitable because measurement of ROS was a significant component of the study. Fragmentation of DNA is a hallmark of apoptosis that occur in metazoans (29) as well as unicellular organisms (4, 5) in response to various apoptosis-inducing stimuli. PAT-induced fragmentation of amastigote DNA provided evidence that amastigote death was a consequence of induction of apoptosis-like death process. Interestingly, we observed few occurrences of DNA fragmentation in amastigotes existing within untreated macrophages, showing that death induced in the continuing struggle between the parasite and the host cell also occurs through apoptosis. Unpublished data2 from this laboratory demonstrate that there is a larger number of TUNEL-positive amastigotes within the untreated activated mouse peritoneal macrophages as compared with infected J774A.1 cells, suggesting that the normal response of macrophages toward the pathogens with oxidative burst (31) also induce pathogen death accompanied by DNA fragmentation. Therefore, the very low rate of amastigote DNA fragmentation observed in the untreated infected J774A.1 macrophages may be the result of low reactivity of the host cell toward the parasites. The apoptotic nature of death was further confirmed by the exposure of phosphatidylserine (1) in the amastigotes recovered from treated macrophages, a feature detected during staurosporine-induced death of Leishmania promastigotes (11). Certainly, a mode of death similar to apoptosis would be preferable for intracellular parasites because products generated by necrotic death would adversely affect other parasite members residing in close proximity and the host cell as well.
To illustrate the mechanistic details leading to DNA fragmentation, several cellular parameters were checked. Even though there is no clear consensus in the literature as to how the ROS brings about pathogen death inside the host cell, it is known that anti-leishmanicidal drugs generate ROS in blood (21). Therefore, it was checked whether the cellular response to the drug was generation of ROS. The increase in several species of ROS after exposure to PAT followed by amastigote clearance suggested drug action via the ROS. Interestingly, higher level of ROS was actually being generated within the PVs, a site where amastigotes were lodged. This indicated two possibilities; either the macrophage-generated ROS (31) was being concentrated in the PVs because host-generated ROS can enter the PVs (32), or the drug was able to generate ROS within the PVs or in the parasites located within the PVs. Because PAT could induce ROS in isolated amastigotes, it was possible that ROS generation in response to the drug in the PVs was a macrophage-independent event. The difference between the amount of ROS generated by the uninfected and infected macrophages treated with PAT was possibly caused by the additive effect of the ROS generated by the amastigotes residing within the PVs. In this connection, it is instructive to note that several reactive oxygen intermediates have been implicated in Leishmania killing including , H2O2, OH, and NO (41, 42). Because PAT was able to generate all the above species, on the basis of existing literature they can be linked to amastigote death via ROS generation. ROS are known to induce apoptosis in mammalian cells (43, 44); therefore, it was entirely possible that, when amastigotes were exposed to appropriate levels of these species, apoptotic death ensued. This concept of ROS-induced amastigote death was further validated by the use of antioxidants. Arguably, if oxidants were responsible for the cytotoxic effect of the drug, antioxidants should have protective action. The ability of one thiol and one non-thiol antioxidant and an iNOS inhibitor to significantly inhibit parasitic death provided clues to a direct link between ROS generation and amastigote death. The ability of NAC to reduce DAF-FM- and H2DCFDA-sensitive fluorescence and apoptotic death indicated that it either acted via inhibition of NO through interference with iNOS activation (45) or through direct scavenging of the radicals. NO has a significant role in amastigote killing, as shown by data with iNOS knockout mice (19) and its ability to induce DNA fragmentation in intracellular amastigotes (15). It is pertinent to mention that the possibility of the protective effect of NAC through the increase in GSH levels cannot be ruled out because the depletion of GSH that occurred concurrently with the increase in ROS (46) was restored by NAC. Therefore, the ability of NAC to act as an intracellular antioxidant (37), coupled with its capability to function as a modifier of intracellular thiol levels (38), was important. Involvement of NO in this particular system was evident from the ability of a NO inhibitor, aminoguanidine (23), to provide protection against cell death through inhibition of NO. MnTBAP was ineffective in reducing NO levels because it does not scavenge NO but it scavenges peroxinitrite, a product generated by reaction between NO and superoxide (47). Our earlier studies have shown that promastigotes were susceptible to ROS at high doses (9); similarly, when we compared the susceptibility of the promastigotes and amastigotes to withstand PAT-induced stress, we found that amastigotes isolated from the macrophages were far more susceptible than the promastigotes (percentage of promastigote death at 24 h: 15 µg/ml, 12 ± 1; 25 µg/ml, 17.3 ± 4.5; 50 µg/ml, 16 ± 2; 100 µg/ml, 22 ± 3), confirming that susceptibility to PAT differed between the two forms of the parasites. Taken together, the data displayed that antimonial compound-induced ROS generation was closely related to amastigote apoptotic death.
Having established that ROS were actually the effectors for cytotoxic effects of PAT, we probed for cellular changes associated with oxidative stress. Our earlier studies showed that Ca2+ was an important cation in the promastigote apoptotic death (9). To test the possibility that Ca2+ was also involved in amastigote death, we checked the level of the cation in both the drug-treated infected macrophages and isolated amastigotes. Significant changes in Ca2+ homeostasis were noted in both. This could be linked to cell death, as chelation of extracellular Ca2+ resulted in the diminution of Ca2+ levels and reduced amastigote death. It was evident from the above data that the major source of intracellular free Ca2+ was from extracellular sources. There were several possible routes through which Ca2+ influx within the cell may have occurred. Therefore, to identify the channels involved in Ca2+ entry, we used nifedipine, a voltage-gated L-type Ca2+ channel blocker, during drug treatment because unpublished data3 from this laboratory show that, under certain conditions, nifedipine can block Ca2+ entry in the L. donovani promastigotes. Nifedipine was unable to inhibit cell death and Ca2+ entry, indicating non-involvement of L-type Ca2+ channels in the amastigotes. Based on our earlier observations in promastigotes (9), we used FFA, a nonselective cation channel blocker (48), to see whether these channels were involved in the Ca2+ entry. The ability of FFA to reduce Ca2+ influx (49) and restore cell viability clearly provided two pieces of evidence, first that Ca2+ was involved in the cell death process and second that non-selective cation channels were involved. At this point, the primary question was whether the intracellular Ca2+ increase took place only within the amastigotes or in the macrophages as well. Both the lysed macrophage supernatants and amastigotes showed increased Ca2+ levels, providing evidence that Ca2+ increase took place in both the cell types. The ability of FFA to reduce Ca2+ increase in both the macrophage and the parasite suggested that the amastigotes were drawing Ca2+ from the macrophage cytosol and the macrophage was in turn drawing Ca2+ from the extracellular media, both via non-selective cation channels. Because ROS generation occurred prior to Ca2+ increase and antioxidants could prevent Ca2 entry into the cell, it can be concluded that Ca2 increase was a consequence of drug-induced ROS action. To examine the relationship of ROS and Ca2+ further, we measured ROS generation after FFA treatment and found that FFA did not alter ROS generation (data not shown). Therefore, there was no secondary ROS increase after Ca2 influx. These results suggest that Ca2+ plays a central role in the mechanism of PAT-induced death. This is the first demonstration that an anti-leishmanicidal drug induces the opening of FFA-sensitive channels in both the host and the parasite.
Our earlier studies have implicated the involvement of promastigote mitochondria in H2O2-induced death (9); therefore, the generation of ROS by PAT suggested that amastigote mitochondria might be a possible target. Because it was not possible to measure amastigote m while they were lodged inside the macrophages, this was confirmed in studies with isolated amastigotes, where an actual loss of potential was observed after drug treatment and this loss could be salvaged by antioxidants that reduced ROS levels and cell death. The 20% potential fall that occurred within 1 h showed that this early loss was indicative of considerable cellular changes taking place after application of oxidative stress. Evidently, PAT did not interfere with macrophage mitochondria, as our tests with uninfected J774A.1 cells did not show any potential fall (data not shown). Because we do not know that exact ROS content per cell in the macrophages versus the amastigotes, we are unable to comment on the susceptibility of the macrophage mitochondria versus the amastigote mitochondria. Because macrophages use oxidative stress to kill pathogens, their mitochondria may be comparatively more resistant to oxidative stress than the parasite mitochondria. Thus, the temporal relationship between increase in ROS generation, reduction in
m, and augmentation of Ca2+ levels followed by DNA fragmentation clearly shows that amastigote apoptosis is a consequence of changes in Ca2+ homeostasis brought about by ROS generation induced by the antimonial.
Interpreting the above results, we propose that influx of Ca2+ through FFA-sensitive channels in both the macrophages and amastigotes represents a novel way by which pathogen death occurs in response to an anti-parasitic drug. Because opening of these channels brought about by ROS generated by the drug could be a critical event in drug efficacy, it would be interesting to design drugs with improved ability to open these channels to precipitate apoptotic death. Finally, the identification of parasite-specific modulators of Ca2+, effective in the phagosomal environment, may lead to improved methods for inducing parasite clearance.
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FOOTNOTES |
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To whom correspondence should be addressed. Fax: 11-2616-2125; E-mail: cshaha{at}nii.res.in.
1 The abbreviations used are: , superoxide anion; PV, parasitophorous vacuoles; ROS, reactive oxygen species; JC-1, 5,5',6,6'-tetrachloro1,1',3,3'-tetraethylbenzimidazole carbocyanide iodide; fluo-3/AM, fluo-3 acetoxymethyl ester; DAF-FM, 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate; H2DCFDA, 2'-7'-dichlorodihydrofluoresceindiacetate; MnTBAP, Mn(III) tetrakis(4-benzoic acid)porphyrin chloride; FITC, fluorescein isothiocyanate; NAC, N-acetylcysteine; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; PAT, potassium antimony tartrate; FFA, flufenamic acid; iNOS, inducible nitric-oxide synthase; PI, propidium iodide; TdT, terminal deoxynucleotidyltransferase enzyme; TUNEL, terminal deoxynucleotidyltransferase enzyme-mediated dUTP nick end labeling; FIU, fluorescence intensity unit(s);
m, mitochondrial membrane potential; GSH, glutathione.
2 G. Sudhindharan and C. Shaha, unpublished observations.
3 S. B. Mukherjee and C. Shaha, unpublished observations.
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ACKNOWLEDGMENTS |
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REFERENCES |
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