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
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ABSTRACT |
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Present address: Instituto Gulbenkian de Ciência, Oeiras, Portugal.
Present address: ICBAS, Instituto de Ciências Biome'dicas Abel Salazar, Porto, Portugal.
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INTRODUCTION |
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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 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
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
responsible for the restriction of the proliferation of M. avium.
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METHODS |
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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 (100 U ml1) (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 ml1) (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 (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
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 ml1 and incubated in complete DMEM (1 ml) containing 10 µg JC-1 ml1 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 2x1055x105 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 ml1) 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 ml1; leupeptin, 5 µg ml1; antipain, 20 µg ml1; pepstatin A, 5 µg ml1; 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-
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 or incubated with 50 mM N-acetylcystein as described previously (Pais & Appelberg, 2000
). Macrophages were loaded with 1·5 mg TMR-dextran ml1 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 ml1 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.
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RESULTS |
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The effects of the treatment with PA and IFN 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
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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DISCUSSION |
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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 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
showed a dramatic reduction in such phagosomeendosome 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
can regulate signalling events that interfere with the phagosomal intracellular traffic and which results in mycobacterial growth inhibition. The interference with phagosomeendosome 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
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
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
, 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
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 phagosomeendosome interaction in the macrophage should be considered as a target for the control of mycobacterial growth.
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ACKNOWLEDGEMENTS |
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Received 3 October 2003;
revised 26 January 2004;
accepted 2 February 2004.
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