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Laboratory of Microbiology and Immunology of Infection, Institute for Molecular and Cell Biology, University of Porto, Porto, Portugal
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. The mechanism involved did not require TNF-
, NO, or the
respiratory burst, and was not dependent on either iron or zinc
withholding. The mycobacteriostatic activity of the macrophages was
associated with the induction of morphological changes that culminated
in apoptosis at day 4 of treatment. PA alone induced apoptosis in
macrophages, and this effect was increased by IFN- treatment.
Apoptosis at day 4 of infection was reduced by inhibiting macrophage
activation with the prostaglandin 15 deoxy-prostaglandin J2
or by treating the cells with the antioxidant
N-acetylcysteine. Mycobacterial growth was partially
restored in macrophages treated with PA and IFN- when 15
deoxy-prostaglandin J2 was added, concomitant with a delay
in apoptosis. N-Acetylcysteine or glutathione could also
completely revert the mycobacteriostatic effects of PA or PA plus
IFN-. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and/or TNF-, mouse macrophages can cause a limited
restriction of the growth of M. avium through a mechanism
that is neither dependent on NO (2) or on reactive oxygen
intermediates (3, 4), but which may involve iron
deprivation (5, 6). In contrast, many researchers have
failed to find such a protective effect of IFN- when studying
M. avium infections in cultured human monocytes (7, 8), despite clinical evidence for a major role of IL-12/IFN-
pathways in the control of infections by mycobacteria in humans
(9). We have previously reported that, in addition to
IFN-/TNF- activation, there may be additional signals required to
lead to complete control of M. avium infection in mice
(10). It is therefore important to study the role of other
potential inducers of macrophage antimycobacterial mechanisms.
Several studies have shown an involvement of tryptophan catabolism in
the antimicrobial mechanisms of the macrophage. During the in vitro
activation of human fibroblast and monocytes with IFN-, the
depletion of tryptophan due to the induction of 2,3-indoleamine
dioxygenase is partially responsible for the inhibition of
intracellular growth of Toxoplasma gondii
(11, 12, 13, 14, 15), Leishmania donovani
(12), Legionella pneumophila (16),
Chlamydia spp. (12, 13, 14, 17), or CMV
(18). Depletion of tryptophan should not affect the
intracellular growth of M. avium because mycobacteria are
able to synthesize all of the amino acids required for growth
(19). The induction of tryptophan catabolism has been
reported in patients with cancer (20), in mice infected
with influenza virus (21), in AIDS patients
(22), and in humans infected with Mycobacterium
tuberculosis and Salmonella (23). However,
the possible role of the catabolites of tryptophan metabolism as part
of the antimicrobial responses has never been seriously considered in
those studies. Picolinic acid
(PA)3 is one of the
naturally occurring degradation products of tryptophan. This molecule
is able to form stable complexes with transition metal ions
(24), and its chelating properties seem to facilitate the
absorption of zinc from the intestine (25) and explain the
inhibitory effect of PA in the axenic growth of Escherichia
coli (26) as well as in the growth of normal kidney
rat cells (27, 28). PA is also tumoricidal in vivo
(29), an effect that is related to the induction of
macrophage-mediated cytostatic activity (30). The idea
that PA plays a role in immune mechanisms is strengthened by the
observation that mice treated with PA have enhanced levels of mRNA for
IL-1 and TNF- and higher resistance to Candida albicans
(31). In vitro studies with macrophages confirmed that PA
is a modulator of macrophage functions because it costimulates
macrophage activation and enhances the antitumor activity of peritoneal
macrophages treated with IFN- (32). In this work, we
have assessed the ability of PA to modulate anti-M.
avium mechanisms in cultured mouse macrophages. We show that
administration of PA to macrophages infected with M. avium
induced mycobacterial growth inhibition strongly synergizing with
IFN- in the induction of complete mycobacteriostasis.
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Materials and Methods |
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A smooth transparent (SmT) variant of M. avium strain 25291 was obtained from the American Type Culture Collection (Manassas, VA). Strain 2-151 SmT and GIR10 SmT were given by J. Belisle (Colorado State University, Fort Collins, CO) and B. Heym (Institut Pasteur, Paris, France), respectively. The mycobacteria were grown in Middlebrook 7H9 broth (Difco, Detroit, MI) containing 10% albumin/dextrose/catalase (ADC) supplement (Difco) and 0.04% Tween 80 (Sigma, St. Louis, MO) at 37°C until mid-log phase. The bacteria were collected by centrifugation, washed in saline containing 0.04% Tween 80, and then resuspended in a small volume of the same solution. The suspension was briefly sonicated until no bacterial clumps were visualized by optical microscopy. The preparation was diluted and frozen in aliquots at -70°C. The aliquots were thawed at 37°C and diluted to the desired concentration before inoculation, according to a previous quantification. For determining numbers of viable mycobacteria, serial dilutions of the suspensions were plated on solid Middlebrook 7H10 agar medium (Difco) supplemented with 10% OADC (5% BSA, 0.06% oleic acid, and 2% dextrose).
Cell cultures
Bone marrow-derived macrophages were obtained by cultivating bone marrow cells from BALB/c or C57BL/6 mice, obtained by flushing the femurs with HBSS (Life Technologies, Paisley, U.K.). In some experiments, mice genetically deficient in the genes for the inducible NO synthase (iNOS-/-, in a C57BL/6 background), the TNF receptor p55 (p55-/-, in a C57BL/6 background), or the p47phox protein (p47phox-/-, in a mixed 129 x C57BL/6 background) were used. To remove fibroblasts, the cells were cultured overnight on cell culture dishes with DMEM (Life Technologies) supplemented with 10 mM HEPES, 1 mM sodium pyruvate, 10 mM glutamine, 10% of heat-inactivated Myoclone calf serum (Life Technologies), and 10% of L929 cell-conditioned medium. The nonadherent cells were collected with warm HBSS medium and culture for 9 days with L929 cell-conditioned medium until they were completely differentiated, as previously described (3). Peritoneal macrophages were obtained from C57BL/6 mice by injecting them i.p. with 1 ml of 4% of thioglycolate. The peritoneal exudate was collected 4 days later by washing the peritoneal cavity with 10 ml of HBSS. The cells were resuspended at the concentration of 2–3 x 106 cells/ml in complete DMEM and cultured for 2 h for cells to adhere. Then the cells were washed with warm HBSS, and the remaining adherent cells were infected. When necessary, the number of macrophages was determined by lysing the cells with 3% cetrimide (Sigma) and 0.25% Pronase (Sigma) and counting the nuclei in a hemocytometer.
Studies in axenic medium
A total of 5 x 105 CFU/ml of M. avium strains 25291 SmT, GIR10 SmT, and 2-151 SmT was inoculated in DMEM or in DMEM plus PA (2, 4, 8, 16, 32 mM). The mycobacteria were cultured in 24-well plates at 37°C in a CO2 incubator. Saponin was added to the wells at day 6 to a final concentration of 0.1%, and the cultures were briefly sonicated to disrupt the clumps before serial dilution and plating on Middlebrook 7H10 medium.
Infection of macrophages and inhibition studies
Each well containing 
0.5 x 106
macrophages was infected with 0.2 ml of DMEM containing 1–5 x
106 CFU of M. avium for 4 h.
After infection, the cultures were washed with HBSS to remove
unphagocytosed bacteria. Macrophages from triplicate wells (time 0)
were immediately lysed in 0.1% saponin, and serial dilutions were
plated in 7H10 solid medium. To test the anti-M. avium
activity of PA (Sigma), bone marrow-derived macrophages were treated
daily after infection with 2 mM PA, 100 U/ml IFN- (R&D Systems,
Minneapolis, MN) or PA (2 mM) plus IFN- (100 U/ml) until day 3 of
infection. Ferric citrate, iron sulfate, zinc sulfate (Merck,
Darmstadt, Germany), apotransferrin (Sigma), IL-4 (R&D), and
anti-TNF mAbs (clone MP6-XT22; DNAX, Palo Alto, CA) were added
after infection, where indicated. Catalase (bovine or erythrocyte;
Sigma or Calbiochem, San Diego, CA) or superoxide dismutase (SOD; Sigma
or Calbiochem) was added during phagocytosis and after washing the
extracellular bacteria in the indicated experiments.
N-Acetylcysteine (NAC; Sigma) or glutathione (Sigma) was
added to macrophages during infection (10 mM), 1 h after infection
at a final concentration of 50 mM, washed away, and then added at days
0 and 1 at a final concentration of 5 mM, where indicated. In the
experiments with peritoneal macrophages, IFN- (100 U/ml) was added
at days 0 and 1, and PA (2 mM) was added until day 3 of infection. The
prostaglandin 15d-PGJ2 (Cayman Chemical, Ann
Arbor, MI) was added after infection at a final concentration of 6
µM. The mycobacterial growth was monitored by lysing macrophages in
triplicate wells up to day 6 of infection. The results are expressed as
CFU ± 1 SD of the means.
Electron microscopy
Macrophages infected with M. avium strain 25291 SmT were processed for electron microscopy at day 4 of infection. Macrophages were fixed for 1 h at room temperature with 2.5% glutaraldehyde in 0.1 M cacodylate buffer containing 0.1 M sucrose, 5 mM Ca2+, and 5 mM Mg2+, pH 7.2. The cells were scraped off the culture dishes with a rubber policeman and treated with 1% osmium tetroxide. Postfixation was done in 1% uranyl acetate. After dehydration in ethanol, the samples were embedded in Epon. Sections were stained with uranyl acetate and lead citrate.
Analysis and quantification of apoptosis
Apoptosis was studied with different assays. For the use of the annexin V-FITC staining, macrophages were cultured on petri dishes and infected with M. avium strain 25291 SmT. At day 4 of infection, nonadherent cells were collected by aspiration of the medium and adherent macrophages were detached by incubating the dishes with cold PBS containing 0.5 mM EDTA during 1 h. Cells were pooled and washed once with binding buffer (10 mM HEPES buffer containing 0.14 mM NaCl and 2.5 mM CaCl2, pH 7.4). A total of 2–5 x 105 cells was incubated in 100 µl of binding buffer containing 2.5 µl of annexin V-FITC (PharMingen, San Diego, CA) for 20 min in ice. Propidium iodide (1 µg/ml) was added before analysis on a FACSorter flow cytometer (Becton Dickinson, Mountain View, CA) to exclude mechanically damaged cells. To evaluate apoptosis by staining DNA with propidium iodide, macrophages were obtained as described above and were permeated with 70% ethanol for 10 min. After centrifugation, the cells were stained for 20 min at 37°C with 50 µg/ml propidium iodide in PBS containing 100 µg/ml RNase (Boehringer Mannheim, Ottweiler, Germany). Cells were analyzed for propidium iodide fluorescence (DNA content) by FACS. To perform DNA labeling with fluorescein dUTP, macrophages were stained with the APO-DIRECT kit, according to the manufacturer’s instructions (Phoenix Flow Systems, San Diego, CA). Finally, cellular viability was also studied by looking at the mitochondrial-dependent reduction of MTT to formazan, as described before (33). Briefly, macrophages were incubated with MTT (0.25 mg/ml) for 1 h at 37°C, and the formazan was dissolved with DMSO (Merck). The extent of reduction of MTT to formazan was determined by measuring the OD at 550 nm.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in controlling mycobacterial proliferation
We tested the ability of PA to enhance the anti-M.
avium activity of IFN- in bone marrow-derived macrophages from
BALB/c mice. Macrophages were infected with the virulent M.
avium strain 25291SmT and treated after phagocytosis with PA (2
mM), IFN- (100 U/ml), or PA plus IFN- daily during the first 3
days of infection. The addition of PA alone to the infected macrophages
inhibited the intramacrophagic growth of M. avium measured
by CFU (Fig. 1
). This decrease was
already significant at day 4 of infection (p <
0.05). When PA was added together with IFN-, mycobacterial
proliferation was completely controlled, as no significant increase
(p > 0.05) in bacterial numbers was observed
during the 6 days of infection. The same bacteriostatic effect was seen
in freshly explanted peritoneal macrophages elicited with either casein
or thioglycolate (results not shown). The induction of macrophage
antimycobacterial activity by PA was also observed with two additional
strains of M. avium (Fig. 1). The effects of PA on the
intramacrophagic growth were independent of the sensitivity of the
different strains to PA in axenic medium. Thus, although M.
avium strain Gir 10 grew 1 log in axenic medium containing 32 mM
PA and was 4 times more resistant to PA in axenic medium
(IC50 = 4 mM) than strains 25291 SmT or 2-151 SmT
(IC50 = 1 mM), the intramacrophagic growth at day
4 of strain Gir10 was reduced by both PA (from 0.37 log to 0.11 log) or
PA plus IFN- (from 0.37 log to -0.04 log) to similar extents as
observed with the two other strains: from 0.52 log to 0.20 log with PA
and from 0.52 log to 0.05 log with PA plus IFN- for strain 2-151
SmT, and from 0.35 log to 0.24 log with PA and from 0.35 log to 0.07
log with PA plus IFN- for strain 25291 SmT, in the same
experiment.
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The arrest of normal rat kidney cell growth by PA (27, 28) is abolished by the addition of either
Zn2+ or Fe3+
(28) because PA interferes with the intracellular levels
of these metal ions (27). On the other hand, it has been
shown that iron is crucial for M. avium growth (5, 6). Therefore, we tested whether the effects of PA or PA plus
IFN- could be blocked by supplementation of the culture media with
excess amounts of these ions. As shown in Fig. 2, the anti-M. avium
mechanism induced by PA was not due to the chelation of Fe or
Zn2+ ions, as the addition of either zinc sulfate
or iron in two different ionic forms (II or III) did not affect the
growth inhibition induced by PA. It is also unlikely that PA acted to
prevent binding of iron to transferrin, as an excess of apotransferrin
did not revert PA growth inhibition (Fig. 2A). Because the
antitumor costimulatory effect of PA is associated with the enhancement
of iNOS activity and with increased levels of TNF- mRNA expression
(34), we investigated the role of NO and of TNF- in the
PA antimycobacterial mechanism. We found that there was no difference
in M. avium growth inhibition when bone marrow-derived
macrophages from iNOS gene-disrupted mice or TNF receptor p55
gene-disrupted mice were used in the experiments and compared with the
respective control macrophages derived from mice with similar
background (C57BL/6, Fig. 3). In
addition, the neutralization of TNF- with a mAb did not affect the
intramacrophagic growth inhibition in macrophages treated with PA or PA
plus IFN- (Fig. 2). Treatment with IL-4, which inhibits the
costimulatory tumoricidal activity of PA (35), did not
revert its bacteriostatic effect (Fig. 2). Finally, the addition of
catalase or SOD plus catalase did not interfere with PA bacteriostatic
activity (Fig. 2), showing that function of the NADPH oxidase is
probably not required for the effects of PA. This result was confirmed
by using bone marrow-derived macrophages from
p47phox-/- mice (Fig. 3), which showed
the same ability of wild-type macrophages (from either the C57BL/6 or
the 129 strains, although the latter data are not shown) to restrict
growth of M. avium after stimulation with PA ±
IFN-.
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We observed that after 3 days of infection, the number of adherent
macrophages, measured by the Pronase-cetrimide method as well as by the
reduction of MTT, decreased when the cultures were treated with PA plus
IFN- (results not shown). PA also induced changes in cell
morphology. At day 4 of infection, PA-treated macrophages showed
numerous small electron-transparent vacuoles in the cytoplasm (Fig. 4B) when compared with
nontreated cells (Fig. 4A), and at day 6 of infection there
was extensive blebbing of the treated cells, which became fragmented
into small vesicles (Fig. 4D), whereas nontreated infected
cells had a normal appearance (Fig. 4C). As these changes
were morphologically compatible with apoptotic modifications, we
measured by FACS analysis the number of apoptotic cells by assessing,
as a marker of an early stage of apoptosis (36), the
exposure of phosphatidylserine at the outer leaflet of the plasma
membrane at day 4 (Fig. 5A)
and, as a marker of a later stage, the fragmentation of DNA by
propidium iodide staining (37) at day 6 (Fig. 5B). Macrophages treated with PA plus IFN- showed clear
signs of apoptosis already at day 4 with an increased exposure of
phosphatidylserine (29.9% ± 6 of positive cells) when compared with
IFN--treated (4.8% ± 0.8), PA-treated (6.9% ± 3.6), or
nontreated cells (1.18% ± 0.2). At day 6, the highest percentage of
cells with fragmented DNA was seen in macrophages treated with PA plus
IFN- (58%). PA alone was also able to induce DNA fragmentation,
with 18% of the cells showing hypoploid DNA when compared with
IFN--treated or nontreated cells. These results were further
confirmed by labeling DNA fragments with fluorescein dUTP (results not
shown).
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Data presented to date suggest that the effects of PA on M.
avium were mediated through the macrophage, by modulating its
function. However, they cannot formally exclude a direct effect of PA
on the mycobacteria. We therefore tested the effects of either a
deactivator of the macrophage, 15d-PGJ2
(38, 39), or an inhibitor of apoptosis, NAC
(39, 40, 41, 42). Peritoneal macrophages elicited with
thioglycolate, known to express the peroxisome proliferator-activated
receptor- (PPAR-) and to respond to
15d-PGJ2 (39), were infected with
M. avium 25291 SmT and stimulated with IFN- (100 U/ml, at
days 0 and 1), with PA (2 mM daily), or with PA plus IFN-. As an
indicator of macrophage activation, we analyzed the levels of nitrites
in the supernatants from the macrophage cultures. When cells were
treated with IFN- or PA plus IFN- in the presence of 6 µM
15d-PGJ2, there was no activation of macrophages
because nitrites were not detectable in the cultures (results not
shown). Macrophages treated with PA plus IFN- in the presence of
15d-PGJ2 did not show the morphological changes
seen in macrophages treated with PA plus IFN- in the absence of the
prostaglandin (results not shown). 15d-PGJ2
treatment also prevented the induction of apoptosis in cells treated
with PA plus IFN-, as evaluated by the maintenance of the ability to
reduce MTT by the mitochondrial respiratory chain (Fig. 6A) or by the reduction in the
exposure of phosphatidylserine (Fig. 6B). The reduction
of apoptosis by 15d-PGJ2 in macrophages treated
with PA plus IFN- was associated with a significant increase in the
number of viable bacteria (Fig. 6C). The prostaglandin did
not induce any significant changes in the number of intracellular
mycobacteria in macrophages treated with PA or IFN-, or in
nontreated macrophages.
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(Fig. 7A). Furthermore, we
completely reverted the effect of PA and PA plus IFN- in the
inhibition of M. avium growth inside the macrophages with
the NAC treatment (Fig. 7B). Addition of glutathione was
also effective in reverting the mycobacteriostatic effect (Fig. 7C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, leading to complete
mycobacteriostasis. All three strains of M. avium tested to
date had the same degree of susceptibility to the mycobacteriostatic
effect of PA and PA plus IFN- in macrophages, independently of the
susceptibility to PA in axenic medium. Also, the growth of the Gir10
strain was not arrested in axenic media with concentrations of PA up to
32 mM, whereas this strain was as easily controlled by activated
macrophages treated with PA as the other two strains of M.
avium. These data strongly argue against a direct effect of PA on
mycobacteria, but suggest an effect mediated through the
macrophage.
PA is able to chelate metal ions. However, iron or zinc withholding was
unlikely the cause of restriction of M. avium growth inside
macrophages treated with PA or PA plus IFN-. Not only was the
addition of the ion metals ineffective in reverting the bacteriostasis,
but also the inhibition of the growth of M. avium induced by
PA plus IFN- was higher than the one obtained with other iron
chelators (4). As previously shown by Varesio and
coworkers (34), PA is also a macrophage agonist leading to
higher secretion of cytokines such as TNF- and of NO. In the system
described in this work, however, TNF- was not involved in modulating
mycobacterial growth inhibition following PA stimulation, as either the
neutralization of the cytokine with specific mAbs or the use of bone
marrow-derived macrophages from TNF receptor
p55-/- mice failed to have an effect in the
macrophages’ anti-M. avium activity induced by PA or PA
plus IFN-. Finally, neither oxygen-reactive species generated by the
NADPH oxidase nor NO was mediating growth inhibition after stimulation
of macrophages with PA or PA plus IFN-. Instead, we have found a new
anti-M. avium macrophage effector mechanism that is
concomitant with the induction of apoptosis and that could only be
reverted by blocking programmed cell death. Others have shown that
human monocytes infected with M. avium and treated with
hydrogen peroxide enter apoptosis after 6 h with concomitant
bacterial killing (44). Furthermore, bacillus
Calmette-Guérin and M. tuberculosis were also killed
in human monocytes a few hours after the apoptotic stimulus given by
ATP (45, 46) or Fas-L (47). The induction of
apoptosis and mycobacteriostasis in murine macrophages by PA plus
IFN- had different features from the previously described. We did
not observe bacterial killing, but rather an inability of M.
avium to grow in treated macrophages. This is more consistent with
what is generally observed in vivo, in which killing of M.
avium is seldom seen and when it occurs it takes place throughout
a protracted period of weeks rather than hours.
While the restriction of M. avium growth was observed from
the early time points of treatment with PA or PA plus IFN-,
apoptotic changes of the phagocytes were only detected later, starting
to become prominent at day 4 of infection. This suggests that the
mechanism leading to mycobacteriostasis occurs in parallel or is the
same that will result in the programmed cell death pathway, and that
mycobacteriostasis is not a consequence of the late cellular apoptotic
changes.
A final argument against a direct role of PA on the inhibition of
M. avium growth inside macrophages comes from the ability to
abolish its effects by preventing apoptosis of the macrophages.
Although the three agents used for that purpose,
15d-PGJ2, NAC, and glutathione, have pleiotropic
effects, their actions are concordant regarding the inhibition of
apoptosis, making it less likely that the effects observed were due to
other biological effects shared by these different molecules. This
strongly suggests a link between the mycobacteriostatic mechanism and
that of programmed cell death. In addition, the bacteriostatic and
apoptotic effects of PA plus IFN- were closely dependent on the
activation of macrophages by IFN- because the mechanism was reverted
by the prostaglandin 15d-PGJ2 in
thioglycolate-elicited macrophages. By binding to PPAR-,
15d-PGJ2 has been shown to block expression of
genes mediated by transcription factors associated to inflammatory
changes such as AP-1, NF-
B, and Stat1 (38). Although
the known consequence of the activation of PPAR- in macrophages is
the inhibition of the expression of genes such as those coding for
gelatinase B, TNF-, and iNOS (38, 39), we observed that
another effect was the inhibition of apoptosis. As a role for TNF-
and NO in the expression of the PA treatment had been excluded, we feel
that the inhibition of apoptosis is the cause of the abolished
anti-M. avium activity. In addition, whenever we failed
to revert the mycobacteriostatic effect induced by PA plus IFN-,
e.g., by supplying metal ions, reactive oxygen scavengers, or cytokine
modulators, or by using the macrophages from the gene-disrupted mouse
strains, we also failed to detect any differences in the morphological
alterations described in this work.
The mechanism whereby PA triggers apoptosis is not clarified, but it
could involve the generation of reactive oxygen species or an
alteration of the cytoplasmic redox potential, as suggested by the
results obtained with NAC and glutathione (48, 49). NAC is
a thiol compound that can act either as a hydroxyl radical scavenger or
by increasing the intracellular levels of glutathione, which protects
the cell against oxidative damage. Although the addition of catalase or
catalase plus SOD did not revert the bacteriostatic effect of PA in our
studies, NAC blocked the mycobacteriostasis induced by PA and PA plus
IFN- probably because of its antiapoptotic effect. A role for NAC in
blocking oxygen-reactive species generated by the NADPH oxidase with
the consequent inhibition of mycobacteriostasis does not seem plausible
because no differences were found when
p47phox-deficient mice were compared with
wild-type controls. Also, no evidence exists to suggest that
oxygen-reactive species generated outside the phagosome (e.g., the
cytosol or the mitochondria) may lead to mycobacterial growth
restriction inside their vacuolar location. It has been reported that
iron (III) picolinate catalyzes the production of hydroxyl radical in
the presence of superoxide radical and hydrogen peroxide
(50). Even though the addition of catalase or SOD plus
catalase did not revert PA or PA plus IFN- effect, it is possible
that the enzymes did not reach the site in which
O2- and
H2O2 are formed, presumably
intracellular. A localized production of the hydroxyl radical could
initiate lipid peroxidation and cell damage with subsequent apoptosis
(46). On the other hand, reactive oxygen species also act
as second messengers in the activation of NF-B (51), a
trancription factor that is associated with transduction of
proapoptotic signals in different cells (52, 53). As
15d-PGJ2 and NAC can inhibit activation of
NF-B (38, 51, 54), we postulate that activation of
NF-B in macrophages treated with PA and PA plus IFN- is central
for both apoptosis and induction of mycobacteriostasis.
IFN- clearly enhanced the apoptotic and bacteriostatic activity of
PA in infected macrophages. That effect is not a combination of two
separate anti-M. avium activities because in
thioglycolate-elicited peritoneal macrophages, the activity of PA plus
IFN- was reverted by 15d-PGJ2 without
disturbing the activities of PA alone. Interestingly, IFN- is a
cytokine that may inhibit cell growth and induce apoptosis (55, 56). Furthermore, a defective Stat1 transcription factor in U3
cells inactivated IFN- signaling and decreased the constitutive
expression of caspases (57). Activation of macrophages
with IFN- could prime the cell to the apoptotic effect of PA. The
dissection of the transduction signals involved in this mechanism could
bring important information to the understanding of the
antimycobacterial mechanisms.
In summary, we describe a novel pathway of mycobacterial growth control
that is triggered by PA in IFN--activated macrophages and that is
related to changes occurring during the programmed cell death of the
host cell. This pathway does not involve previously described effector
mechanisms such as the respiratory burst or NO.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Rui Appelberg, Laboratory of Microbiology and Immunology of Infection, Institute for Molecular and Cell Biology, Rua do Campo Alegre 823, 4150-171 Porto, Portugal. E-mail address:
3 Abbreviations used in this paper: PA, picolinic acid; 15d-PGJ2, 15 deoxy-prostaglandin J2; iNOS, inducible NO synthase; NAC, N-acetylcysteine; PPAR-, peroxisome proliferator-activated receptor-; SmT, smooth transparent; SOD, superoxide dismutase.
Received for publication July 12, 1999. Accepted for publication October 14, 1999.
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M. P. O'Sullivan, S. O'Leary, D. M. Kelly, and J. Keane A Caspase-Independent Pathway Mediates Macrophage Cell Death in Response to Mycobacterium tuberculosis Infection Infect. Immun., April 1, 2007; 75(4): 1984 - 1993. [Abstract] [Full Text] [PDF]
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L. Duan, H. Gan, D. E. Golan, and H. G. Remold Critical Role of Mitochondrial Damage in Determining Outcome of Macrophage Infection with Mycobacterium tuberculosis J. Immunol., November 1, 2002; 169(9): 5181 - 5187. [Abstract] [Full Text] [PDF]
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T. Hayashi, S. P. Rao, K. Takabayashi, J. H. Van Uden, R. S. Kornbluth, S. M. Baird, M. W. Taylor, D. A. Carson, A. Catanzaro, and E. Raz Enhancement of Innate Immunity against Mycobacterium avium Infection by Immunostimulatory DNA Is Mediated by Indoleamine 2,3-Dioxygenase Infect. Immun., October 1, 2001; 69(10): 6156 - 6164. [Abstract] [Full Text] [PDF]
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) or treated daily with IFN-
), PA (2 mM) (
), PA plus IFN-
).
Statistics as in Fig. 1