Induction of Mycobacterium avium growth restriction and inhibition of phagosome–endosome interactions during macrophage activation and apoptosis induction by picolinic acid plus IFN{gamma}

Teresa F. Pais{dagger} and Rui Appelberg{ddagger}

Laboratory of Microbiology and Immunology of Infection, Institute for Molecular and Cell Biology, Rua do Campo Alegre 823, 4150-180 Porto, Portugal

Correspondence
Rui Appelberg
rappelb{at}ibmc.up.pt


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Treatment of mouse macrophages with picolinic acid (PA) and {gamma}-interferon (IFN{gamma}) led to the restriction of Mycobacterium avium proliferation concomitant with the sequential acquisition of metabolic changes typical of apoptosis, mitochondrial depolarization, annexin V staining and caspase activation, over a period of up to 5 days. However, triggering of cell death by ATP, staurosporine or H2O2 failed to affect mycobacterial viability. In contrast to untreated macrophages where extensive interactions between phagosomes and endosomes were observed, phagosomes from treated macrophages lost the ability to acquire endosomal dextran. N-Acetylcysteine was able to revert both the anti-mycobacterial activity of treated macrophages as well as the block in phagosome–endosome interactions. The treatment, however, induced only a minor increase in the acquisition of lysosomal markers, namely Lamp-1, and did not increase to any great extent the acidification of the phagosomes. These data thus suggest that the anti-mycobacterial activity of PA and IFN{gamma} depends on the interruption of intracellular vesicular trafficking, namely the blocking of acquisition of endosomal material by the microbe.


Abbreviations: FACS, fluorescence-activated cell sorting; Jc-1, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide; IFN{gamma}, {gamma}-interferon; MTT, methylthiazoletetrazolium; PA, picolinic acid; PS, phosphatidylserine; TMR, tetramethylrhodamine

{dagger}Present address: Instituto Gulbenkian de Ciência, Oeiras, Portugal.

{ddagger}Present address: ICBAS, Instituto de Ciências Biome'dicas Abel Salazar, Porto, Portugal.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Mycobacterium avium is an opportunistic pathogen that infects patients suffering from local or systemic deficiencies of the immune system (Falkinham, 1996). Resistance to infection is dependent on both innate mechanisms and acquired immunity through the development of antigen-specific CD4+ T cells (Appelberg, 1994; Holland, 1996). M. avium replicates inside the macrophage and is particularly resistant to the oxidative antimicrobial mechanisms of this phagocyte (Gomes & Appelberg, 2002; Gomes et al., 1999a). Like other pathogenic mycobacteria, M. avium has evolved survival strategies that interfere with the normal intracellular trafficking of vesicular compartments. After being internalized by macrophages, mycobacteria inhibit the fusion of the vacuole they inhabit with lysosomes, thus blocking maturation of the phagosomes into phagolysosomes and proliferating in vacuoles with early endosomal characteristics (Armstrong & d'Arcy-Hart, 1971; Clemens & Horwitz, 1995; Frehel et al., 1986; Russell et al., 1997). Several mechanisms associated with this inhibition have been described, such as reduced phagosomal acidification (Crowle et al., 1991; Sturgill-Koszycki et al., 1994; de Chastellier et al., 1995) due to exclusion of the vacuolar H+-ATPase (Sturgill-Koszycki et al., 1994), altered signalling pathways (Malik et al., 2001; Fratti et al., 2003), interference with the recycling of Rab proteins (Via et al., 1997), retention of the actin-binding coronin/TACO protein (Ferrari et al., 1999; see also Schuller et al., 2001, for a critical reappraisal of this issue), disorganization of the actin filament network (Guérin & de Chastellier, 2000) and tight apposition of the membrane vacuole to the surface of the pathogen, leading to altered exchange of fusion factors (de Chastellier & Thilo, 1997). However, mycobacterial phagosomes keep their ability to interact extensively with endosomes that may carry solutes from the extracellular milieu (Clemens & Horwitz, 1996; Frehel et al., 1986; Russell et al., 1996; Sturgill-Koszycki et al., 1996; Xu et al., 1994). In activated macrophages that control the intracellular growth of mycobacteria, this pattern of phagosomal trafficking changes. Thus when treated with {gamma}-interferon (IFN{gamma}) plus lipopolysaccharide (LPS) macrophages show increased phagosome acidification (Schaible et al., 1998). Acidification in itself does not explain the anti-mycobacterial activity of macrophages (de Chastellier et al., 1999; Gomes et al., 1999b) and more likely reflects an enhancement in the maturation of the phagosome (Schaible et al., 1998). This would promote contact of the pathogen with a lower pH, a reducing environment and acid hydrolases as well as conditions favouring the generation of oxidative radicals (Schaible et al., 1998). Furthermore, the co-stimulation of macrophages with IFN{gamma} and LPS inhibited the fusion between the mycobacterial phagosome and early endosomes loaded with transferrin, suggesting that withdrawal from the recycling endosomal pathway might lead to starvation from nutrients (Schaible et al., 1998).

Picolinic acid (PA) is one of the naturally occurring degradation products of L-tryptophan detected in several biological fluids. The in vivo induction by IFN{gamma} of the activity of one of the enzymes that catalyses the oxidative catabolism of tryptophan, indoleamine 2,3-dyoxygenase, illustrates the immune regulation of tryptophan availability and of its catabolites in the organism (Burke et al., 1995). In addition, there is an increase in the catabolism of tryptophan in humans infected with HIV, Mycobacterium tuberculosis and Salmonella (Fuchs et al., 1990; Wannemacher, 1977) and high levels of PA have been detected in the cerebrospinal fluids of children with cerebral malaria (Medana et al., 2003). We have previously described that the treatment of mouse macrophages with PA in the presence of IFN{gamma} makes these phagocytes able to completely inhibit the replication of virulent M. avium (Pais & Appelberg, 2000). The induction of anti-M. avium activity was closely associated with the induction of apoptosis which occurred over a protracted period of a few days. PA is also a co-stimulator of macrophage tumoricidal and antimicrobial activities (Blasi et al., 1993; Leuthauser et al., 1982; Ruffmann et al., 1984; Varesio et al., 1990).

It has been described before that macrophage apoptosis is a pathway that may lead to killing of mycobacteria. Thus, macrophages treated with agents such as ATP (Lammas et al., 1997; Molloy et al., 1994), CD95 ligand (CD178) (Oddo et al., 1998) or H2O2 (Laochumroonvorapong et al., 1996) undergo apoptosis and, simultaneously cause the restriction of growth or even killing of ingested mycobacteria. However, the basis of the anti-mycobacterial effects of macrophage apoptosis is not clear. Stober et al. (2001) have shown that the killing of M. bovis BCG induced by ATP treatment of the infected macrophages is associated with enhanced phagosomal acidification in the apoptotic macrophages. Curiously, the anti-mycobacterial effects could be uncoupled from macrophage death. In their model of ATP-mediated killing of BCG, the mycobactericidal activity and the phagosomal acidification were calcium-dependent, whereas the triggering of apoptosis did not depend on calcium. On a follow up of this work (Fairbairn et al., 2001), the same group showed that ATP treatment of infected macrophages led to an increase of fusion between phagosomes and lysosomes and further confirmed the ability to dissociate anti-mycobacterial activity from apoptosis. Similarly, Kusner & Barton (2001) found that killing of M. tuberculosis by ATP-treated macrophages was associated with an increase in the fusion between phagosomes and lysosomes which was calcium-dependent. Here we have addressed the issue of the relationship between apoptosis and the induction of anti-mycobacterial activity and started dissecting the mechanisms induced by PA and IFN{gamma} responsible for the restriction of the proliferation of M. avium.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Mycobacteria.
A smooth transparent variant (SmT) of M. avium strain 25291 was obtained from the American Type Culture Collection (Manassas, VA, USA). Frozen aliquots of M. avium prepared as described before (Pais & Appelberg, 2000) were thawed and diluted to the desired concentration to perform the macrophage infection experiments. Quantification of viable mycobacteria was done by plating serial dilutions of the suspension on solid Middlebrook 7H10 agar medium (Difco) supplemented with 10 % OADC (5 % BSA, 0·06 % oleic acid, 2 % glucose).

Cell culture.
Bone-marrow-derived macrophages were obtained by cultivating bone marrow cells from BALB/c mice with 10 % L929 cell-conditioned medium in DMEM (Life Technologies) supplemented with 10 mM HEPES, 10 mM glutamine, 10 % heat-inactivated Myoclone calf serum (Life Technologies) as described previously (Pais & Appelberg, 2000). The cells were used at day 9 of culture. To follow the intramacrophagic growth of M. avium, cells were cultured in 24-well tissue culture plates (0·5x106 cells per well). For fluorescence-activated cell sorting (FACS) analysis, the cells were cultured in 6-well bacteriologic plates to prevent strong adherence to the plastic (5x106 cells per well). For the co-localization studies, bone marrow cells (0·5x106 cells per well) were cultured on glass coverslips pre-treated with nitric acid to remove any endotoxin contaminant.

Infection of macrophages and induction of apoptosis.
After 9 days in culture, macrophages were incubated with a mycobacterial suspension of M. avium strain 25291. After 4 h, the cultures were washed with HBSS (Life Technologies) to remove extracellular mycobacteria. To quantify the number of intracellular mycobacteria, macrophages from triplicate wells were immediately lysed (time 0 of infection) in 0·1 % saponin (Sigma) and serial dilutions were plated on 7H10 solid medium. Cells were treated daily after infection with PA (2 mM) (Sigma) and IFN{gamma} (100 U ml–1) (Life Technologies) until day 3 of infection or were left untreated. In other experiments, ATP (Sigma), staurosporine (Sigma) or H2O2 (Merck) were added to the macrophages at day 5 of infection. The cells were treated for 6 h with ATP (3 mM) and staurosporine (3 µM) or for 19 h with H2O2 (2 mM).

Analysis of apoptotic parameters
1. MTT (methylthiazoletetrazolium) reduction.
The mitochondrial-dependent reduction of MTT to formazan was analysed as described by Zingarelli et al. (1996). The macrophages were incubated with DMEM containing MTT (0·25 mg ml–1) (Sigma) for 1 h at 37 °C. The medium was removed and the formazan was dissolved in 1 ml DMSO (Merck). The extent of reduction of MTT to formazan was given by measuring OD550.

2. Mitochondrial membrane potential ({Delta}{Psi}m).
Mitochondrial membrane potential was analysed by means of the lipophilic cation 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) (Molecular Probes). At high mitochondrial membrane potential, JC-1 forms J-aggregates that have high fluorescence emission on both FL1 and FL2 channels (Smiley et al., 1991). A decrease in the mitochondrial potential favours the monomeric form with a consequent decrease in emission in FL2 (Zamzami et al., 1995). Macrophages were infected on different days so cells at different points of infection could be collected on the same day for mitochondrial potential analysis. For each time point, bone-marrow-derived macrophages either not treated or treated with PA plus IFN{gamma} were detached from 6-well plates with cold PBS containing 0·5 mM EDTA as described previously (Pais & Appelberg, 2000). When cells were treated with H2O2, the drug was added at different times so cells could be collected, stained and analysed by FACS at the same time. The protocol followed was based on the work of Polla et al. (1996), although the time and temperature of incubation of cells with JC-1 were optimized for this system. Cell suspension was adjusted to a density of 0·5x106 cells ml–1 and incubated in complete DMEM (1 ml) containing 10 µg JC-1 ml–1 for 20 min in the dark at 37 °C in a CO2 incubator. Cells were then washed twice in PBS and resuspended in 400 µl PBS before FACS analysis. Cells treated with H2O2 (2 mM) for 2 h were used as a positive control for the decrease in mitochondrial membrane potential as shown by Polla et al. (1996).

3. Phosphatidylserine (PS) exposure.
PS externalization was quantified by annexin V staining as described previously (Pais & Appelberg, 2000). Briefly, at day 4 of infection, non-adherent and adherent macrophages were pooled and 2x105–5x105 cells were incubated in 100 µl binding buffer (10 mM HEPES buffer containing 0·14 mM NaCl and 2·5 mM CaCl2, pH 7·4) containing 2·5 µl annexin V-FITC (BD Biosciences) for 20 min in ice. Propidium iodide (1 µg ml–1) was added before analysis on a FACSorter flow cytometer (Becton Dickinson) to exclude dead cells from cells which were undergoing apoptosis and stained only for annexin V-FITC.

4. Caspase 3 activity.
Cell extracts were prepared as described by Macen et al. (1998). Briefly, cells were detached with PBS containing EDTA (0·5 mM), washed in PBS and then washed in cold extract preparation buffer (EPB): 50 mM PIPES, pH 7·0) (Sigma), 50 mM KCl (Merck), 5 mM EGTA (Sigma), 2 mM MgCl2 (Merck), 1 mM DTT (Sigma), 20 µM cytochalasin B (Calbiochem-Novabiochem). Cells were resuspended in cold EPB containing a cocktail of proteinase inhibitors (PMSF, 0·2 mM; chymostatin, 20 µg ml–1; leupeptin, 5 µg ml–1; antipain, 20 µg ml–1; pepstatin A, 5 µg ml–1; all from Sigma). The cells were then lysed by four cycles of freezing and thawing. The cytoplasmatic extract was obtained after centrifugation at 10 000 g for 15 min. Protein concentration was determined with the Bio-Rad Protein assay reagent. Caspase 3 activity was assayed in 60 µg protein by measuring the colorimetric cleavage product (p-nitroaniline) of the caspase 3 substrate I (Ac-DEVD-{rho}NA) (Calbiochem) and following the manufacturer's instructions. To test the specificity of the caspase 3 substrate I, hydrolysis-positive samples were incubated with the caspase 3 inhibitor I (Asp-Glu-Val-Asp-CHO) (Calbiochem) before adding the substrate. Colour development was followed over 24 h. The results shown correspond to 5 h incubation with the substrate.

Co-localization studies.
Tetramethylrhodamine (TMR)-labelled 10 000 Da lipophilic dextran (Molecular Probes) was used to assess the ability of M. avium vacuoles to fuse with endocytosed markers. Bone marrow cells were cultured on coverslips and at day 9 they were infected with 5x106 c.f.u. M. avium. Cells were left untreated, treated with PA plus IFN{gamma} or incubated with 50 mM N-acetylcystein as described previously (Pais & Appelberg, 2000). Macrophages were loaded with 1·5 mg TMR-dextran ml–1 for 6 h (Heinzen et al., 1996) at different time points of infection. The coverslips were extensively washed with PBS and fixed in a PBS solution with 4 % paraformaldehyde (Merck) containing 120 mM sucrose as described by Ojcius et al. (1996). Before immunostaining, the cells were blocked in PBS containing 2 % BSA (Sigma), 0·05 % saponin (Sigma) and an mAb (clone 2·4G2) against the Fc receptor to block irrelevant binding of the antibodies to the macrophages. Localization of LAMP-1 was performed with the rat mAb 1D4B labelled with fluorescein (BD Biosciences) at day 3 of infection. To study phagosome acidification, macrophages were incubated after infection with the acidotropic dye LysoTracker Red DND-99 (Molecular Probes) in DMEM (50 nM). At day 3 of infection the cells were washed with PBS and fixed. Mycobacteria were stained with immune serum from 2-months-infected mice followed by incubation with anti-mouse IgG labelled with fluorescein (Vector) or with Alexa 488 (Molecular Probes). Co-localization was analysed by laser-assisted confocal microscopy (Bio-Rad).

To estimate the percentage of phagosome/endosome fusion, the green, red and merged spots were counted in infected macrophages and the percentage co-localization was determined. At least 100 bacilli were analysed for co-localization with dextran in triplicate coverslips. Phase contrast microscopy was used to confirm that bacteria were within the contour of the cells. No mycobacterial staining was observed when permeabilization was not performed and no cell lysis occurred during the culture or the preparation of the cells. All these data strongly support the intracellular nature of the mycobacteria. Similar results were obtained with shorter pulsing periods (2 h) with the tracer. To control the amount of internalized tracer, macrophages were pulsed for 2 h with FITC-labelled 10 000 Da lipophilic dextran (Molecular Probes), collected and run through a Becton Dickinson FACSorter. Analysis of horseradish peroxidase (HRP; Sigma) uptake was performed as described by de Chastellier et al. (1995). At day 4 of infection, bone-marrow-derived macrophages were incubated at 37 °C for 1 h with 25 µg HRP ml–1 in DMEM. The cells were washed in cacodylate buffer (0·1 M, pH 7·3) and fixed for 1 h at room temperature with 2·5 % glutaraldehyde in 0·1 M cacodylate buffer, pH 7·3, containing 0·1 M sucrose. After being washed overnight at 4 °C with sucrose-containing cacodylate buffer, the cells were incubated for 30 min at room temperature with 0·05 % 3,3'-diaminobenzidine tetrachlorhydrate (DAB; Sigma) in cacodylate buffer, pH 6·9, followed by incubation for 1 h with 0·05 % DAB in cacodylate buffer, pH 6·9, containing 0·01 % H2O2. Cells were washed twice in 0·1 M cacodylate buffer and scraped off the culture wells with a rubber policeman, then treated with 1 % osmium tetroxide. After dehydration in ethanol, the samples were embedded in Epon. Co-localization of the HRP reaction product and phagosomes was assessed by electron microscopy. Whenever co-localization occurred, HRP was distributed as an electron-dense ring around the bacteria.

Statistics.
Each point represents the mean of triplicate determinations±SD. Statistically significant growth inhibition is labelled * for P<0·05, ** for P<0·01 and *** for P=0·001 according to Student's t-test.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Relationship between the bacteriostatic effect induced by PA plus IFN{gamma} and the emergence of markers of programmed cell death
We have previously demonstrated that the control of M. avium growth induced by PA plus IFN{gamma} was associated with activation of a cell death program in macrophages (Pais & Appelberg, 2000). However, the mycobacteriostatic effect was present prior to the appearance of late apoptotic changes such as DNA fragmentation. We therefore analysed other metabolic alterations triggered by programmed cell death during treatment of macrophages with PA plus IFN{gamma} and their correlation with the induction of bacteriostasis (Fig. 1). Bone-marrow-derived macrophages were infected with the M. avium strain 25291 and treated daily with PA (2 mM) plus IFN{gamma} (100 U ml–1) or cultured in medium. At day 2 of infection, a reduction in the transmembrane potential of mitochondria was observed in a small but significant (P<0·05) percentage of treated macrophages (Fig. 1). At day 3 the mitochondrial depolarization observed in cells treated with PA plus IFN{gamma} was similar to the one induced by H2O2 (2 mM) after 2 h of treatment (Fig. 1, bottom panel). At day 4 approximately 75 % of the macrophages had activated the cell death program as measured by the disruption of the mitochondrial potential, although only a lower percentage was positive for annexin V staining (Fig. 1). At this time point the number of bacteria had doubled in control macrophages, although no significant growth was observed in cells treated with PA plus IFN{gamma} (Fig. 1). Activation of the effector caspase 3 was only clearly detected at day 5 of infection in treated macrophages (Fig. 1) when the activity measured was comparable to the values detected in cytosolic extracts of macrophages treated for 6 h with 3 mM ATP (A405=0·109). The activity of caspase 3 was blocked when the extracts of the macrophages treated with PA plus IFN{gamma} were pre-incubated with the caspase 3 inhibitor I (DEVD-CHO) (A405=0·049). M. avium failed to proliferate to any extent in macrophages treated with PA and IFN{gamma}, confirming our previous observations (Pais & Appelberg, 2000). When we analysed the development of apoptosis in uninfected macrophages treated with PA plus IFN{gamma} we found a much lower percentage of annexin V-positive cells as compared with macrophages infected with M. avium (7·3±2·3 % in uninfected cells versus 25·5±5·3 % in cells infected for 4 days with an m.o.i. of 2·2).



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Fig. 1. Kinetics of acquisition of apoptotic markers by infected macrophages treated with PA plus IFN{gamma}. Macrophages were infected with M. avium and treated daily with PA (2 mM) and IFN{gamma} (100 U ml–1) until day 3 of infection. At different time points, cells cultured in medium (open squares) and PA plus IFN{gamma} treated cells (filled squares) were analysed for different apoptotic markers. Macrophages were collected for staining with annexin V to measure PS exposure and JC-1 to evaluate the decrease in the mitochondrial potential ({Delta}{Psi}m), and analysed by FACS. Cell lysates were assayed for caspase 3 activity by a colorimetric assay (Caspase 3) and data are presented as the raw absorbance results for the enzyme assay. Parallel cultures of macrophages were used for mycobacterial growth evaluation (Growth). The bottom panel shows a representative FACS density plot of cells labelled with JC-1. The experiment was repeated once with the same overall result. Statistically significant growth inhibition is labelled * for P<0·05, ** for P<0·01 and *** for P=0·001 according to Student's t-test.

 
Dissociation between induction of apoptosis and anti-mycobacterial activity in infected macrophages
We studied whether additional known inducers of apoptosis had any effect on M. avium viability in infected macrophages. Bone-marrow-derived macrophages were infected with M. avium and at day 5 of infection were either left untreated or treated with H2O2 (2 mM), ATP (3 mM) or staurosporine (3 µM). Both macrophage apoptosis and M. avium viability were followed for 19 h in infected macrophages treated with H2O2 (2 mM) (Fig. 2). A decrease in the mitochondrial potential ({Delta}{Psi}m) analysed by the quenching of JC-1 fluorescence at 590 nm was observed in nearly 50 % of macrophages after 1 h of treatment with H2O2 (Fig. 2, left graph). Nineteen hours later, more than 50 % of the macrophages stained positive for annexin V and the whole population of cells showed disruption of the {Delta}{Psi}m (Fig. 2, left graph). The induction of apoptosis by H2O2 had no significant effect on mycobacterial viability (Fig. 2, right panel). Likewise, ATP (3 mM) or staurosporine (3 µM) markedly decreased the ability of mitochondria from infected macrophages to reduce MTT after 6 h of treatment: A550 values were 0·40±0·03 for untreated macrophages, 0·18±0·01 for staurosporine-treated cells and 0·20±0·04 for ATP-treated cells (P<0·01 for both treatments). The induction of apoptosis by ATP or staurosporine in the macrophages infected with M. avium had no significant effect on mycobacterial viability 6 h post-treatment: the mean log(c.f.u.) values were 6·74±0·04 for untreated macrophages, 6·80±0·07 for staurosporine-treated cells and 6·69±0·04 for ATP-treated cells (P>0·05 for both treatments).



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Fig. 2. Effect of induction of apoptosis on the viability of M. avium. At day 5 of infection macrophages were either not treated or treated with H2O2 (2 mM) as an inducer of apoptosis. The percentage of apoptotic cells was determined by analysing PS exposure (circles) or depolarization of mitochondria (squares) which were followed for 19 h in macrophages treated with H2O2 (filled symbols) and in non-treated cells (open symbols) (left panel). The number of viable mycobacteria was determined at the same time points after treatment with H2O2 (right panel). Results are representative of four different experiments.

 
Inhibition of the interactions between mycobacterial phagosomes and the endosomal compartment are associated with induction of bacteriostasis by PA plus IFN{gamma}
The maturation arrest of the mycobacterial phagosome seems to be crucial for the successful growth of mycobacteria inside macrophages. The mycobacterial phagosome interacts with the endocytic compartment, although it does not acidify (Sturgill-Koszycki et al., 1994). We therefore assessed the effect of the treatment with PA plus IFN{gamma} on the interaction of the M. avium phagosome with endosomes and late endosomes. At different days of infection macrophages were loaded for 6 h with 1·5 mg TMR-labelled dextran ml–1 (Heinzen et al., 1996). Co-localization of the label and the mycobacteria was analysed by laser-assisted confocal microscopy after staining mycobacteria with immune serum followed by incubation with anti-mouse IgG labelled with fluorescein. At day 3 of infection the fusion events between the phagosome and the vesicles loaded with dextran were significantly reduced when compared to untreated macrophages (P<0·05) (Fig. 3 and Fig. 4). Beyond day 3 of infection, treated macrophages started to lose adherence to the coverslips and the extensive washes after incubation with dextran detached most of the cells, preventing further co-localization studies. This was not a problem with mycobacteria quantification where the whole culture medium is used, nor with the FACS analysis where both adherent and non-adherent cells can be collected. However, confocal microscopy requires that cells remain associated with the slide.



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Fig. 3. Decreased interaction of M. avium phagosomes with endocytosed dextran in macrophages treated with PA and IFN{gamma}. (a) Fusion events between phagosomes and endosomes loaded with dextran were quantified during infection in macrophages cultured in medium (open squares), in macrophages incubated with PA plus IFN{gamma} (filled squares) or in treated macrophages incubated with N-acetylcysteine (open circles) (b). Parallel cultures were used to assess the number of intracellular mycobacteria. Statistically significant growth inhibition is labelled * for P<0·05, ** for P<0·01 and *** for P=0·001 according to Student's t-test.

 


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Fig. 4. Decreased interaction of M. avium phagosomes with endocytosed dextran in macrophages treated with PA and IFN{gamma}. Macrophages infected with M. avium were (a) not treated, (b) treated daily with PA (2 mM) plus IFN{gamma} (100 U ml–1) as described in Fig. 1, or (c) treated with N-acetylcysteine before and during stimulation with PA plus IFN{gamma}. At day 3 of infection the cells were incubated with 1·5 mg TMR-dextran ml–1 for 6 h. The cells were fixed and M. avium bacilli were vizualized with immune serum and with anti-mouse IgG labelled with FITC. Co-localization of M. avium phagosomes with FITC-dextran-loaded vesicles was assessed by confocal microscopy and appears in the figure as yellow spots after overlaying the green and red images. Bar, 20 µm. Results are representative of three different experiments.

 
We next studied the effect of N-acetylcysteine, an inhibitor of the bacteriostasis induced by PA plus IFN{gamma} (Pais & Appelberg, 2000) on the interactions between phagosomes and endosomes. N-Acetylcysteine restored the fusion pattern between M. avium vacuoles and TMR-dextran-containing vesicles in treated macrophages (Fig. 3 and Fig. 4), concomitant with a recovery in the intracellular mycobacterial growth at day 4 (Fig. 3). The percentage of co-localization in macrophages only pre-treated with N-acetylcysteine was 89·5 % at day 4 of infection. To exclude the possibility that the dextran label was being captured non-specifically by dead cells, we repeated the assays in the presence of chemical or physical inhibitors of endocytosis. Internalization of TMR-dextran by macrophages, namely by those treated with PA plus IFN{gamma}, was abrogated in the presence of cytochalasin D (0·5 µg ml–1), an inhibitor of actin polymerization (Parlato et al., 2000), and by incubation on ice (data not shown). The same results were obtained when a shorter exposure to the endocytic tracer was used. Thus, infected macrophages were treated for 2 h with 1·5 mg TMR-dextran ml–1 and then washed and fixed to analyse co-localization. The patterns of internalization and co-localization were similar to those obtained with a longer time of incubation. As observed previously there was fusion inhibition in treated macrophages. In an independent experiment, the effects of PA alone and IFN{gamma} alone on phagosome–endosome interactions were compared to those of the combination of PA and IFN{gamma} and the control untreated cells. In the control cells, there were 64 % dextran-positive phagosomes, whereas those values were 37 % for PA-treated cells, 33 % for IFN{gamma}-treated cells and 15 % for PA plus IFN{gamma}-treated cells.

The effects of the treatment with PA and IFN{gamma} on the amount of tracer internalized was studied by flow cytometry. Macrophages infected for 3 days with M. avium and either not treated or treated daily with PA plus IFN{gamma} were incubated with FITC-dextran for 2 h. After washing, the cells were detached and analysed by FACS. As shown in Fig. 5, treatment of infected macrophages reduced the amount of label internalized.



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Fig. 5. Decreased internalization of lipophilic dextran by infected macrophages treated with PA and IFN{gamma}. Macrophages infected with M. avium were either not treated or treated daily with PA (2 mM) plus IFN{gamma} (100 U ml–1). At day 3 of infection the cells were incubated with 1·5 mg FITC-dextran ml–1 for 2 h. After washing, the cells were detached and analysed by FACS. The histogram shows the intensity of fluorescence per macrophage containing lipophilic dextran. The amount of internalized dextran was compared between not treated macrophages (bold line) and cells treated with PA plus IFN{gamma} (dotted line). The solid-line histogram on the left shows the fluorescence intensity of macrophages not incubated with dextran.

 
We also assessed phagosome–endosome fusion using a different tracer, HRP. At day 4 of infection, macrophages pulsed with HRP revealed that the HRP reaction product was in vesicles and in M. avium vacuoles in non-treated macrophages (Fig. 6a, arrows). In contrast, in those macrophages that were treated with PA plus IFN{gamma}, even though there was HRP product in cytoplasmic vesicles, none of the vacuoles clearly co-localized with the enzyme (Fig. 6b).



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Fig. 6. Decreased interaction of M. avium phagosomes with endocytosed HRP. Macrophages infected with M. avium were either (a) not treated or (b) treated daily with PA (2 mM) plus IFN{gamma} (100 U ml–1) as described in Fig. 1. At day 4 of infection the cells were incubated with 25 µg HRP ml–1 for 1 h and the cells were processed for HRP cytochemistry and electron microscopy. The enzyme reaction rims the membranes of the phagosomes, showing discharge of the endosomal marker into the phagosome (arrows) (a), while in cells treated with PA plus IFN{gamma} most intact mycobacteria fail to exhibit an HRP reaction product (arrows) (b). Bars, 0·6 (a) and 0·8 µm (b).

 
To assess the effects of the treatments on the maturation of the phagosomes, namely the possible increase in lysosomal markers suggestive of an override of the inhibition of phagosome–lysosome fusion, we studied the acquisition of LAMP-1 by phagosomes as well as their acidification. The results are presented in Table 1 and show that the increase in LAMP1 staining is present with all treatments and that neither of the treatments leads to an extensive increase in acidified phagosomes.


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Table 1. Co-localization of M. avium in phagosomes with the LAMP-1 marker or the pH-sensitive label Lyso-Tracker

Macrophages were infected with M. avium for 3 days and processed as indicated in Methods. Triplicate slides were scored for the co-localization of the fluorescent labels with the mycobacteria stained with fluorescently labelled antibodies.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
We have previously reported that macrophages treated with PA and IFN{gamma} acquired a strong mycobacteriostatic activity against M. avium which occurred concomitantly with the induction of apoptotic changes, such as the emergence of annexin V staining and, at later stages, DNA degradation (Pais & Appelberg, 2000). We also showed that we could not dissociate the two phenomena when using the compounds N-acetylcysteine and prostaglandin J2, both able to inhibit apoptosis and anti-mycobacterial activity. Here we readdressed the issue of the relationship between apoptosis induction and anti-mycobacterial activity in macrophages. We observed that the induction of macrophage apoptosis per se was not an anti-M. avium mechanism, since different apoptotic stimuli, such as ATP, H2O2 or staurosporine, did not have any effect on M. avium viability despite inducing extensive cell death. Given that the kinetics of apoptosis induction was very different in cells treated with PA plus IFN{gamma} compared to cells treated with the other stimuli, we were limited in the time frame allowed for the study of mycobacterial proliferation. This limit in time for the assay during treatment with ATP, H2O2 or staurosporine did not allow the long-term analysis of mycobacteriostasis as extensive extracellular growth would take place in the cultures following macrophage death. However, it showed that killing of M. avium was not associated with apoptosis as reported for other mycobacteria. Thus, a peculiar form of apoptosis was induced by the treatment with PA and IFN{gamma}, leading to macrophage alterations resulting in mycobacterial stasis.

Our results show that M. avium, as opposed to BCG (Molloy et al., 1994) and M. tuberculosis (Kusner & Adams, 2000), is resistant to the downstream events triggered by ATP upon binding to the P2Z receptors on macrophages. In addition, induction of apoptosis by H2O2, which was shown to mediate the killing of M. avium in human macrophages (Laochumroonvorapong et al., 1996), did not lead to the induction of a bactericidal activity in our model. Although we observed a decrease in the number of bacilli when macrophages were treated with higher doses of H2O2 (5 mM, results not shown), a similar effect was seen when mycobacteria were incubated in culture medium alone containing the oxidant at the same concentration. Therefore, a direct effect of H2O2 on mycobacteria rather than an apoptosis-mediated mechanism should be considered in the case of H2O2.

Several metabolic pathways induced during the cell death program were activated by PA and IFN{gamma} treatment, namely the depolarization of mitochondria, the activation of caspases and the flipping of PS into the outer leaflet of the cytoplasmic membrane. Curiously, these phenomena took place over a protracted period of a few days instead of the normal time frame of a few hours classically seen in apoptosis. It can be argued that the sequence of events seen at the level of the population, which takes place over several days, does not correspond to a sequence of events at the single cell level. However, in our macrophage cultures there was no cell proliferation and the total number of cells did not decrease during the first 4 days of the study. Thus, the sequence of acquisition of the studied markers occurred in the same population of cells, strongly suggesting that they took place in a similar sequence in individual macrophages.

From our kinetic studies, we found that the bacteriostatic effect was preceded by mitochondrial depolarization and that it paralleled the exposure of PS on the outer leaflet of the plasma membrane. Moreover, the activation of caspase 3 was only observed at the end of the experimental period, suggesting that the induction of bacteriostasis may not be dependent on this effector caspase, although we cannot exclude a possible involvement of low levels of caspase 3 activity.

Although the reports on the anti-mycobacterial activity of apoptotic macrophages are already numerous, little is known about the mechanisms underlying the killing or the control of the proliferation of the different mycobacterial species analysed so far. Recently, two groups have suggested that an increase in the acidification brought about in the phagosomes of macrophages undergoing apoptosis might be the cause of the death of the mycobacteria (Fairbairn et al., 2001; Kusner & Barton, 2001). Given that a common hallmark of pathogenic mycobacteria is their ability to manipulate the intracellular vacuolar trafficking, leading to arrested maturation of the phagosome, we analysed the ability of M. avium-containing vacuoles of control and treated macrophages to acquire endosomal molecules. These studies were not performed to thoroughly characterize the vacuolar compartment where mycobacteria reside, but rather to test the fusogenicity of such vacuoles. Although mycobacteria-containing phagosomes fail to mature into phagolysosomes, extensive interactions between these phagosomes and early endosomes can be observed. Thus, Frehel et al. (1986) have shown that 80 % of M. avium-containing phagosomes acquired the fluid-phase endosomal marker HRP over 1 week of in vitro infection of bone-marrow-derived macrophages. Clemens & Horwitz (1996) showed that 60 % of M. tuberculosis-containing phagosomes acquired transferrin from the endosomes in macrophages pulsed with this ligand for 1 h. Similar data were obtained by Sturgill-Koszycki et al. (1996) with M. avium-infected macrophages (50 % of transferrin labelling after a 2 h pulse). Using the B subunit of cholera toxin to label GM1 gangliosides from the plasma membrane, Russell et al. (1996) have shown that mycobacteria-containing phagosomes readily fuse with membrane vesicles originating from the plasma membrane. These authors provided data showing that around 80 % of the M. avium-containing phagosomes had received plasma membrane components within 1 h of labelling. The nature of the tracker used for following the endocytic compartment has, however, dramatic influences on the data generated. We have found that lipophilic dextran reaches the phagosome more effectively than normal dextran. In contrast, mannosylated BSA or acetylated low-density lipoprotein failed to reach the phagosomes at all (our unpublished observations). Xu et al. (1994), using immunoelectron microscopy, failed to show any transfer of mannosylated BSA into phagosomes containing M. avium. Additionally, the transfer of 10 kDa dextran into phagosomes harbouring pathogenic mycobacteria was very modest. It is therefore clear that there is a sorting mechanism at the level of the early endosomal compartment screening through which solutes may find their way into the phagosomes. This sorting involves more than the use of specific receptors since both HRP and mannosylated BSA use the mannose receptor but only the former was able to reach the M. avium-containing phagosome. On the other hand some endosomal markers can stay longer or are more stable in the phagosome and therefore are more easily detected. We confirmed those findings showing that about 80 % of M. avium-containing phagosomes received endosomal material and interacted extensively with the endosomes that had been labelled with dextran. Furthermore, we have provided evidence that macrophages that had been treated with PA plus IFN{gamma} showed a dramatic reduction in such phagosome–endosome fusion events which could be reverted by exposure to the anti-oxidant N-acetylcysteine with concomitant loss of mycobacteriostatic activity. Additionally, a reduction in the overall uptake of the tracer was observed in treated macrophages. Therefore, we suggest that PA together with IFN{gamma} can regulate signalling events that interfere with the phagosomal intracellular traffic and which results in mycobacterial growth inhibition. The interference with phagosome–endosome interactions induced by the treatments and assessed with the use of labelled dextrans was confirmed in one time point using electron microscopic analysis of HRP trafficking. The mechanism whereby N-acetylcysteine inhibits the action of PA plus IFN{gamma} is not yet clear, but its antioxidant activity suggests that oxygen radicals may play a role in the induction of apoptosis or may constitute part of the signalling cascade in this pathway.

Maturation into a phagolysosome has been shown not to interfere with the proliferation of M. avium (Gomes et al., 1999b). Therefore, increased vacuolar maturation induced by PA plus IFN{gamma} treatment does not seem to explain the observed results. This was confirmed here by studying either the acquisition of the Lamp-1 marker as well as the acidification of the phagosomes. Roughly half of the phagosomes in untreated macrophages already showed staining with Lamp-1, consistent with previous reports (Sturgill-Koszycki et al., 1994; Xu et al., 1994). Both PA and IFN{gamma}, alone or in combination, induced a slight increase in the acquisition of Lamp-1. This increase could be related to acquisition from the trans-Golgi network rather than from fusion with lysosomes (Sturgill-Koszycki et al., 1994; Xu et al., 1994). On the other hand, the treatments failed to affect the acidification of the phagosomes to any major degree. Overall, it appears that the maturation of the phagosomes towards a more mature phagolysosome did not occur. We therefore favour the view that the blocking of endosome trafficking of molecules into the phagosome is the main reason for the proliferation arrest. Our data strengthen the idea (Clemens & Horwitz, 1995; Russell et al., 1997) that mycobacteria could take advantage of their residence in an early endosome-type vacuole which does not mature into a phagolysosome as it would allow the bacteria to get essential nutrients by fusion with other endosomes. Based on our results showing that PA plus IFN{gamma} treatment prevented the access of M. avium vacuoles to the endosomal material, we propose a new bacteriostatic mechanism of the macrophage that relies on blocking access to the nutrients required by the mycobacteria. Also, we propose that the modulation of the phagosome–endosome interaction in the macrophage should be considered as a target for the control of mycobacterial growth.


   ACKNOWLEDGEMENTS
 
This work was supported by contract 32629/99 from the Foundation for Science and Technology (FCT, Lisbon, Portugal) and contract SDH.IC.I.01.15 from the Calouste Gulbenkian Foundation. We thank Dr Paula Coelho for the assistance on the confocal microscopy and Paula Macedo for help with electron microscopy.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 3 October 2003; revised 26 January 2004; accepted 2 February 2004.



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